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■ 所長挨拶・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・１分子生体膜研究所

所長

井ノ口

仁一

■ 箱守仙一郎糖鎖科学シンポジウム開催報告・・・・・・・・・・・・・・・・・・・４■ 箱守仙一郎賞（Sen-itiroh Hakomori Glycoscience Award）
・・・・・・・・・・・６
東北医科薬科大学分子生体膜研究所「箱守仙一郎賞」規約
平成 29 年度 箱守仙一郎賞
奨励賞 山本 勇人（弘前大学医学部）
奨励賞 郷 慎司（東北医科薬科大学）
優秀論文賞 周 穎（東北医科薬科大学）■研究報告・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・１５機能病態分子部門「スフィンゴ糖脂質の病態生理学的意義の解明に向けて」
井ノ口 仁一、稲森 啓一郎、永福 正和、狩野 裕考

生体膜情報部門「生活習慣病に関連した G タンパク質共役受容体の機能と糖鎖および受容体間相互作用による調節」
東 秀好、中川 哲人、黒田 喜幸

細胞制御部門「がん細胞におけるN-型糖鎖の機能解析と臨床への応用に向けて」
顧 建国、福田 友彦、伊左治 知弥

分子認識部門「レクチンのがん薬物治療への応用」
細野 雅祐、菅原 栄紀、立田 岳生■学会発表・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・２７■学術論文・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・３１

ĺ箱守仙一郎糖鎖科学シンポジウム開催報告
東北医科薬科大学附属分子生体膜研究所名誉所長であります恩師・箱守仙一郎先生におかれましては、2017 年 2 月 13 日をもって 88 歳の米寿を迎えられました。箱守先生は、1959 年（昭和 34年）に東北大学の医学部から東北薬科大学附属癌研究所第一部（当時）教授として赴任され、研究所の充実発展に尽くされた後渡米し、糖脂質研究の世界的権威となられました。以来、多くの優れた門下生を世に送り出しつつ、一貫して癌研究所の指導にもご尽力下さいました。米国シアトルのワシントン州立大学、フレッドハッチンソン癌研究所、バイオメンブレンインスティテュートおよびパシフィックノースウェスト研究所において先生のご指導を受けた研究者が、本邦に限らず世界
で糖鎖生物学をリードする存在となっていることは周知の事実であります。2006 年 4 月、癌研究所が発展的に改組され分子生体膜研究所（以下分生研）となって以降も顧問・指導教授として研究のご指導を頂いております。
分生研では、米寿を迎えられた恩師箱守仙一郎先生の、多年にわたる世界の糖鎖生物学への比類のないご貢献ならびに私どもへの温かいご指導に対し、限りない尊敬と報恩の念を込めまして、箱守先生が長年追求してこられた糖鎖生命科学に関する最新の知識を共有できる場として箱守仙一
郎糖鎖科学シンポジウム（Glycoscience Symposium with Gratitude to Dr. Sen-itiroh Hakomori）
を開催いたしました。
2017 年 11 月 10 日（金）13:30 から東北医科薬科大学 70 周年記念講堂において開催されたシンポジウムには、門下生を中心に国内外から多くの参加者が訪れ盛況を博しました。また、箱守先生のご功績を長く後世に伝承すべく、分生研では後進研究者の育成を目的とした「箱守仙一郎賞（Sen-itiroh Hakomori Glycoscience Award）」を創設いたしました。シンポジウム懇親会会場において第一回の受賞式が執り行われ、受賞者には箱守先生より顕彰楯と副賞が贈られました。

Ļ#*$-!497538
Glycoscience Symposium with
Gra4tude to Dr. Sen-i4roh Hakomori291110:,;13:30=17:00
 !'!70 () 22

Opening remark

13:30-13:35

221111111111
Roger A Laine (University of Louisiana)
Windows Opened to Many Research Direc4ons byExperiencing Postdoctoral Training with Hakomori-sensei

13:35-14:00

Reiji Kannagi (Academia Sinica)

14:00-14:25

Kiyotoshi Sekiguchi (Osaka University)

14:25-14:50

Glycan markers for embryonic stem cells and human cancers

An odyssey in the extracellular world:
From ﬁbronec4n to matrixome researchCoﬀee breakYasuyuki Igarashi (Hokkaido University)

15:10-15:35

Alessandro Prine> (University of Milano)

15:35-16:00

Kazuhisa Iwabuchi (Juntendo University)

16:00-16:25

Masahiro Hosono

16:25-16:50

Run aWer Hakomori-sensei:
ten years in SeaXle and 20 years aWerward

GEMs from the Emerald City

A paradigm shiW in innate immunity has been the recent
evidence of carbohydrate-carbohydrate interac4ons(Tohoku Medical and Pharmaceu4cal University)
A Half-century Story of the Frog Lec4n:from CRI to IMBG with Dr. Hakomori
Closing

Chair>Jin-ichi Inokuchiļ!'!.&"222222222222222222
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箱守イ山一郎先生の米寿を祝う会平成29年11月10日於江陽グランドﾎﾃﾙ

東北医科薬科大学分子生体膜研究所「箱守仙一郎賞」（Sen-itiroh Hakomori Glycoscience Award）規約

平成 29 年 11 月 10 日
東北医科薬科大学分子生体膜研究所
井ノ口 仁一名称
箱守仙一郎賞
授与機関
東北医科薬科大学分子生体膜研究所
目的
生物系化学系を問わず広く糖鎖科学を専攻し、日夜努力を続けている東北エリアの研究者を顕彰することにより、日本の糖鎖科学研究の増進を図る。
賞の種別
奨励賞
優秀論文賞

1名
1名

顕彰楯および副賞
顕彰楯および副賞対象（１）奨励賞：東北エリア（新潟県および群馬県を含む）で研究に従事する 45 歳未満の研究者（２）優秀論文賞：同地域で研究に従事し，申請時点で大学院生もしくは博士研究員である者＊いずれも指定された期間内に発表（accepted でも可）された学術論文に対して審査・授与する。

選考方法
自薦および他薦によるものとし、東北糖鎖研究会世話人が審査する。
評価を点数化（次項参照）して決する。
授賞方法
東北糖鎖研究会開催時に授賞および受賞講演を行う。
運用方法
顕彰楯および副賞の購入費は、箱守賞基金を原資とし、分子生体膜研究所がこれを支弁する。
その他
事務局は分子生体膜研究所内に置く。

Ľ審査（１）応募資格および応募方法
東北 6 県、新潟県および群馬県（東北糖鎖研究会エリア）で研究を行っている 45 歳未満の糖鎖科学研究者（大学院生、博士研究員は自動的に優秀論文賞へのノミネートとなる）。
対象論文は、その年度内（ただし応募締切以前）に Impact factor が付与されている英文学
術雑誌（査読有）に出版、または掲載が決定されている原著論文（accepted でも可）とし、応募者が筆頭著者となっているものとする。要旨和訳と論文 PDF を添えて既定のエントリーフォームに記入してメールにて応募する。化学系・生物系は問わない。自薦および他薦どちらでも受け付ける。応募期間等についてはその都度決定し、周知する。
書類送付先：東北医科薬科大学 分子生体膜研究所
メールアドレス：jin@tohoku-mpu.ac.jp

所長 井ノ口 仁一（２）エントリーフォーム（別添）（３）審査委員および審査方法
１）分子生体膜研究所所長が審査員長を務める。
２）東北糖鎖研究会世話人が審査員となる。ただし、世話人が推薦者または自薦者の所属責
任者あるいは論文の共著者である場合は審査に参加しない。
３）期限内に提出された論文について事務局が一次審査（下記基礎点による）を行い、奨励賞および優秀論文賞それぞれ上位３報を選定する。
４）上位３報について審査員が下記評価項目を採点し、全員（その都度人数は異なる）の評
価平均点＋基礎点（100 点満点、1 点未満は四捨五入）から最上位者を受賞者として決定する。
５）最高点が複数出た場合は審査員長の判断に委ねる。
基礎点
Impact factor

2.0 未満 10 点
4.0 未満 20 点
6.0 未満 30 点
8.0 未満 40 点
10 未満 50 点
10 以上 60 点

評価最高点
独創性
インパクト（重要性）
論文構成
将来性

ľ

10 点
10 点
10 点
10 点

平成 29 年度 箱守仙一郎賞（Sen-itiroh Hakomori Glycoscience Award）
奨励賞
山本 勇人 弘前大学医学部 附属病院泌尿器科 助教
新規ヒアルロン酸分解酵素 transmembrane protein 2 (TMEM2) の発見
A mammalian homolog of the zebrafish transmembrane protein 2 (TMEM2) is thelong-sought-after cell-surface hyaluronidase
郷慎司
東北医科薬科大学 機能病態分子学 助教（現：川崎医科大学 病態代謝学 講師）
ガングリオシドの微細構造変化が制御する筋分化機構
Altered expression of GM3 molecular species during myoblast differentiation, and its
biological significance

優秀論文賞
周 穎
東北医科薬科大学 細胞制御学（D3）Inhibition of fucosylation by 2-fluorofucose suppresses human liver cancer HepG2cell proliferation and migration as well as tumor formation
Scientific Reports 7(1):11563, 2017, doi:10.1038/s41598-017-11911-9

左より、井ノ口所長、山本勇人博士、箱守先生、郷慎司博士

奨励賞：郷

慎司博士

優秀論文賞：周

Ŀ

穎さん

奨励賞
新規ヒアルロン酸分解酵素transmembrane protein 2 (TMEM2) の発見
A mammalian homolog of the zebrafish transmembrane protein 2 (TMEM2)
is the long-sought-after cell-surface hyaluronidase山本勇人（弘前大学医学部 附属病院泌尿器科・助教）

ヒアルロン酸（HA）は、細胞の様々な機能に関与する高分子多糖類である。HA の代謝に関しては、まず、細胞外または細胞表面の HA 分解酵素により細胞外にある高分子（HMW）HA が切断される。次に切断された中分子量の HA が細胞内に取り込まれ、リソソーム内で単糖へ分解されると考えられている。細胞内における HA 分解酵素に関しては、HYAL1 と HYAL2 は主にリソソー
ムに存在する HA 酸分解酵素であり、近年同定された CEMIP / KIAA1199 は、細胞内のクラスリ
ン被覆小胞の経路において HA を分解すると考えられている。細胞外における HA 酸分解酵素についても様々研究されているが、依然として詳細は不明瞭なままである。今回我々は心内膜の発生において重要な役割を果たす Transmembrane protein 2（TMEM2）が細胞表面に存在するである
HA 分解酵素であることを証明した。まず Live staining および surface biotin assay を用いてTMEM2 が II 型膜貫通型タンパク質として細胞表面に発現していることを確認した。次に TMEM2が、HMW-HA を約 5kDa の HA に分解すること、コンドロイチン硫酸またはデルマタン硫酸分解2+
活性を持たないこと、Ca 依存性の HA 分解酵素であること、至適 pH が約 6〜7 であることを確認した。さらには細胞内のクラスリン被覆小胞内で活性を有する CEMIP / KIAA1199 とは異なり、
TMEM2 は細胞を必要とせずタンパク質のみで活性を有することを証明した。最後に in situ HAdegradation assay を用いて、TMEM2 が細胞表面に接触している細胞外 HA を分解していることを確認した。これらの結果は、TMEM2 が細胞外において HMW-HA を中分子量の HA に切断する
HA 分解酵素であることを示唆している（1）。
(1) Hayato Yamamoto, Yuki Tobisawa, Toshihiro Inubushi, Fumitoshi Irie, Chikara
Ohyama,Yu Yamaguchi. J. Biol. Chem. , 292 (18), 2017. DOI 10.1074
【経歴】
平成１５年
平成１９年
平成２３年

弘前大学医学部医学科卒業
弘前大学大学院泌尿器科学講座入局
弘前大学大学院医学研究科卒業（膀胱癌の浸潤機構における invdopodia の意義について研究）平成２７〜２９年５月
Sanford Burnham Prebys MEDICAL DISCOVERY INSTITUTE, Yamaguchi
Laboratory へ客員研究員として研修。
（新規ヒアルロン酸分解酵素 TMEM2 について研究）平成２９年６月 弘前大学医学部附属病院泌尿器科配属

ŀ【受賞の感想と抱負】
この度、箱守仙一郎賞奨励賞を賜りましたこと大変光栄に存じます。箱守先生はじめ東北糖鎖研究会論文賞審査委員の先生方に深く御礼申し上げます。私の所属している弘前大学泌尿器科は、箱守先生の教室におられた福田穣、道子先生より指導を仰ぎ、現在の糖鎖研究に至っております。今回の受賞まで私自身、箱守先生とお会いする機会はありませんでしたが、箱守先生の多くのご功績を拝見しておりました。その箱守仙一郎先生のお名前がついた賞をいただけること、また今回は第一回記念として箱守先生の米寿を祝う会にお招きいただき、箱守先生とお会いすることができたこと、誠に身に余る光栄です。
受賞論文は２０１７年５月まで留学していた Sanford Burnham Prebys Institute、山口祐教授の教室での研究内容となります。当教室ではてんかん発作、自閉症などの脳神経疾患や多発性外骨腫（MHE）など多岐にわたる疾患と糖鎖との関連性について研究しておりますが、以前よりヒアルロン酸の代謝に関して興味を持っておりました。今回我々は細胞膜に存在するヒアルロン酸分解酵素に着目し、心内膜の発生において重要な役割を果たす Transmembrane protein 2（TMEM2）が細胞表面に存在するヒアルロン酸分解酵素であることを証明しました。論文内容は以下の通りです。①TMEM2 が II 型膜貫通型タンパク質として細胞表面に発現している。②TMEM2 はヒアルロ
2+ン酸を約 5kDa に分解し、コンドロイチン硫酸またはデルマタン硫酸分解活性を持たない。③Ca
依存性の分解酵素である。④至適 pH が約 6〜7 である。⑤実際に TMEM2 が細胞表面に接触している細胞外 HA を分解していることを確認。詳細などご興味のある方は J Biol Chem. 2017
May;292(18):7304-7313 を参照いただけると幸いです。
この研究が今後のヒアルロン酸代謝の研究の一助になればこれ以上の喜びはありません。また、
TMEM2 が臓器の発生や悪性腫瘍の浸潤転移に関与していると考えており、泌尿器科の領域で更にヒアルロン酸の研究を進めていきたいと考えております。
最後に山口教授、大山教授をはじめ、両研究室のメンバーの支えなくして、今回の僥倖に恵まれる
ことはなかったので、この場を借りまして御礼を申し上げさせて頂きます。今後受賞者として恥じることのないよう、より一層精進して参りたいと思います。
山本

ĸķ

勇人

奨励賞
ガングリオシドの微細構造変化が制御する筋分化機構
Altered expression of GM3 molecular species during myoblast differentiation,and its biological significance郷 慎司（川崎医科大学 病態代謝学・講師）

スフィンゴ糖脂質は、セラミドにグルコース，ガラクトース，N-アセチルグルコサミン，N-アセチ
ルガラクトサミン，シアル酸などの糖が酵素により段階的に付加された糖鎖構造を有した両親媒性の細胞膜構成脂質である。とくに糖鎖構造中にシアル酸を含むスフィンゴ糖脂質は「ガングリオシ
ド」とよばれている。ガングリオシドは、細胞膜上で様々な分子と特異的に相互作用し機能的微小膜領域「マイクロドメイン（ラフト）」を形成し様々な生命現象を制御していることが報告されている。当研究室では、ガングリオシドの一つ「GM3」に注目し、GM3 がインスリンシグナル、免疫機能、聴覚機能、摂食行動などを制御していることを新たに見出してきた（1）。
近年、スフィンゴ糖脂質のセラミドの構造の多様性とその機能にも注目が集まってきている。セラミド構造中のアシル鎖およびスフィンゴイド塩基の構造（鎖長、水酸化などの修飾、不飽和度）の組み合わせにより、約 40 種の多様な構造が天然で見出されている。我々はヒト血清中の GM3分子種を LC-MS/MS を用いて解析した結果、炭素数 24 で水酸化修飾されたアシル鎖を持つ GM3
分子種の量が生活習慣病の様々なリスクファクターと強く相関していることを見出した（2）。この報告を含め、糖鎖部分とセラミド部分の両者の多様性の組み合わせによる「スフィンゴ糖脂質分子種の超多様性」が複雑に生命現象を制御していることが示唆されてきている。
筋細胞はその分化において、隣接する細胞間での認識・情報交換を行い、細胞膜融合を起こし多核の筋管細胞を形成する。この筋分化過程では細胞膜の劇的な変化がおこるため、細胞膜におけるスフィンゴ糖脂質およびマイクロドメインの機能を考えるうえで興味深い対象である。そこで本研
究では、筋細胞分化モデルとして汎用されるマウス筋芽細胞 C2C12 細胞の分化過程におけるスフィンゴ糖脂質を解析した。分化前の筋芽細胞の段階では GM3 のシアル酸は「Neu5Ac」であるのに対し、分化進行（筋管形成）に伴い、「Neu5Gc」へと変化した。また、GM3 のセラミドアシル
鎖部分の構造は分化前には炭素数 24 のものが主要であったが、分化に伴い炭素数 16 のものが増加し主要なものへと変化した。見出したシアル酸分子種変化およびセラミドのアシル鎖構造変化は筋分化過程の様々なステップに重要であることが示唆された（3）。
つまり筋細胞は、同じ GM3 であっても、そのセラミド構造および糖鎖中のシアル酸の構造を変化させることで、その性質を変え、筋分化を適切に、繊細に制御していると考えられる。
シアル酸は一括りで考えられがちであるが、炭素 5 位の構造、各位の水酸基に対する多様な修飾によって多様な分子種が存在する。シアル酸の構造多様性も加味するとスフィンゴ糖脂質の多様性
はさらに広がる。今後、これらスフィンゴ糖脂質構造多様性の発現制御機構、各構造の性質・機能、およびスフィンゴ糖脂質構造多様性の生物学的意義に関して解明を目指す。
(1) Inokuchi J. (2011) Proc. Jpn. Acad., Ser. B. 87, 179-198(2) Veillon L, Go S, Matsuyama W, Suzuki A, Nagasaki M, Yatomi Y, Inokuchi J.,(2015) PLoSĸĸOne.,10(6):e0129645(3) Go S, Go S, Veillon L, Ciampa MG, Mauri L, Sato C, Kitajima K, Prinetti A, Sonnino S,
Inokuchi J. (2017) J. Biol. Chem., 292, 7040-7051【経歴】
名古屋大学 農学部 卒業
名古屋大学大学院 生命農学研究科 博士課程修了 （博士（農学））（生物機能開発利用研究センター 北島健教授のもと、シアル酸の代謝に関する研究）東北薬科大学（現・東北医科薬科大学）分子生体膜研究所 学術フロンティア研究員
東北薬科大学（現・東北医科薬科大学） 機能病態分子学教室 助教（分子生体膜研究所 機能病態分子学 井ノ口仁一教授のもと、ガングリオシドの代謝制御機構および機能に関する研究）2017 年 3 月より、川崎医科大学 病態代謝学教室 講師【受賞の感想と抱負】
この度、平成２９年度箱守仙一郎賞 奨励賞を賜りましたこと、大変光栄に存じます。学生の時
代、糖鎖研究を始めてからずっと教科書・論文で目にし続けてきた、箱守先生の御名前を冠した賞であることに恐縮しつつ、糖鎖科学の発展に寄与できる知見を得られるよう、研鑽を積んでまいりたいと存じます。
名古屋での学生時代、北島健先生、佐藤ちひろ先生のもとシアル酸の代謝制御の研究に取り組み、卒業後、東北薬科大学 分子生体膜研究所で井ノ口仁一先生のご指導のもとガングリオシドの機能および代謝制御機構に関する研究に取り組んできました。今回の受賞対象となった論文は、ガング
リオシドの新機能解明に向け、新たに立ち上げた筋分化の系で思いがけず偶然見つかってきた現象です。ガングリオシド“GM3”のセラミド部分の構造とシアル酸部分の構造が筋分化に伴い大きく変化するという現象で、糖脂質の構造多様性の発現が厳密に制御されており、繊細に生命現象を制御し得る可能性を示唆したものでした。
マニアックな部分ではありますが、名古屋時代に見ていたシアル酸分子種と、仙台で見てきた糖脂質がつながった研究でもあります。諸先生方、先輩方、同僚の皆様、学生さんとの出会い、そして皆様のご指導・ご協力・ご支援のおかげであると大変感謝しております。
東北ですごした約10年は大変すばらしい期間でした。東北糖鎖研究会にもほぼ毎回参加させていただき、様々な方々と活発で有意義な議論をさせていただきました。
縁あって2017年3月に岡山県にある川崎医科大学に移籍しました。東北での経験を生かし、糖鎖の生合成・分解の代謝バランス制御機構、そして機能に関して新たな視点で研究を展開しようと考えております。環境もこれまでとは大きく異なり、悪戦苦闘の日々が続いておりますが、できれば近い将来、大きな成果をもって東北の地を訪れることができるように…、今回の受賞を励みに研鑽を重ね、糖鎖科学研究に尽力しようと思っております。今後ともご指導ご鞭撻のほどよろしくお願い申し上げます。
郷ĸĹ慎司

優秀論文賞「Inhibition of fucosylation by 2-fluorofucose suppresses human liver cancer
HepG2 cell proliferation and migration as well as tumor formation」
Scientific Reports 7(1):11563, 2017, doi:10.1038/s41598-017-11911-9

周穎（東北医科薬科大学 •薬科学専攻博士後期課程(D3)）

【論文要旨】
α1,6 フコース（コアフコース）転移酵素(Fut8)により生合成されるコアフコシル化糖鎖は、肝がんのバイオマーカーとして非常に特異性が高いことで注目されている糖鎖構造である。一方、我々は肝がんの発生・増殖にもコアフコシル化糖鎖の発現が重要であることを明らかにしている。しかし、治療を考える上では、Fut8 遺伝子の欠失やその酵素活性の阻害という方法は困難である。本
研究では、コアフコシル化をがん組織特異的に抑制することを目的に、L-フコースのアナログである 2-fluorofucose (2FF)の有用性を検討した。2FF は細胞内に取り込まれて、GDP-fucose 合成のde novo 経路を阻害すると知られている。2FF の添加によりヒト肝がん由来 HepG2 細胞のコアフ
コシル化が劇的に抑制されることをレクチン検出や質量分析で確認した。細胞表面の重要な受容体である EGFR や β1 インテグリンなど個別の分子でもコアフコシル化が消失することを確認した。
また、これらの受容体を介したシグナルが減弱、細胞増殖や細胞遊走、軟寒天上でのコロニー形成能も有意に抑制された。一方、in vivo では、マウス皮下へ HepG2 細胞を移植し、定期的に移植部
位近傍へ 2FF を投与したところ、腫瘍組織のコアフコシル化糖鎖の発現が強く抑制された。さらに、
腫瘍サイズ並びに細胞内のシグナル伝達経路も有意に抑制されることがわかった。以上のことから、肝がん組織におけるコアフコシル化の重要性を示すと共に、2FF のような化合物が肝がんの新規治療薬になる可能性が強く示唆された。

ĸĺ【受賞の感想と抱負】
Itʼs really a great honor for me to receive the Sen-itiroh Hakomori glycoscience award. This isthe best affirmation and encouragement for my three years studying in Tohoku Medical andpharmaceutical University.

Here, I would like to show my heartfelt gratitude to Prof. Hakomori as well as the Prizecommittee members. Prof. Hakomori is an outstanding scientist in the world, who has made agreat contribution to the glycobiology, the biochemistry and cell biology. As a student, I wasvery lucky to have several chances to listen to his lectures, and very impressed with his talk.
Under the guidance of Prof. Hakomori, I had deeper understanding of glycobiology andbecame more and more interested in the glycobiology.

Time flies, I have been studying in Tohoku Medical and pharmaceutical University for nearly
3 years. I owe my deepest gratitude to President Motoaki Takayanagi to provide theopportunity to study in Tohoku Medical and Pharmaceutical University. I feel grateful to ouruniversity for providing us such a high-quality academic environment, a good academic
atmosphere, plenty of academic resource and an extensive communication platform. During
the 3-year staying, I have learned a lot of things, not only about science, but also Japanese
habits, lifestyle and cultures.

I would also like to express my sincere thanks to Prof. Jianguo Gu, Dr. Tomohiko Fukuda andDr. Tomaya Isaji. Their rigorous academic attitude, extensive academic knowledge as well as
their enthusiasm for science deeply infected me and triggered my love for these marvelous
experiments. Under their help, I gradually clarified my research direction with confidence,and finally I understood the value and charm of the glycanʼs world.

After graduation, I plan to go back to China to continue scientific research on glycobiology. I
want to apply my basic knowledge that I have learned here to translational research, and try
my best to introduce the importance of glycans to more and more researchers and clinicaldoctors. And also I would like to appreciate your advices and comments on my research in the
future.
Thank you very much!
周ĸĻ穎（Zhou Ying）

研究報告

ĸļ

スフィンゴ糖脂質の病態生理学的意義の解明に向けて
機能病態分子部門 井ノ口 仁一
機能病態分子学教室の 2017 年最初のトピックは、11 年間の研究生活を共にした郷慎司
助教が 3 月に川崎医科大学の松田純子教授が新たに開設された病態代謝学教室の講師として栄転したことである。郷博士との仕事は、ガングリオシド GM3 と聴覚、筋分化とガン
グリオシド、シアル酸代謝、o-GlcNAc とスフィンゴ糖脂質代謝、などの多岐にわたっており現在も共同研究を継続している。１０年間の研究成果をまとめた 2 編の総説「ガングリオシド GM3 合成酵素欠損症から見るスフィンゴ糖脂質機能」(1)および「Gangliosides
and Hearing」(2)を出版した。また、最近の研究成果である「筋分化とガングリオシドの生理的意義」に関する原著論文を JBC に報告した(3)。マウス筋芽細胞 C2C12 細胞の分化過程におけるスフィンゴ糖脂質を解析したところ、分化前の筋芽細胞の段階では GM3 のシアル酸は「Neu5Ac」であるのに対し、分化進行（筋管形成）に伴い、「Neu5Gc」へと変化した。また、GM3 のセラミドアシル鎖部分の構造は分化前には炭素数 24 のものが主要であったが、分化に伴い炭素数 16 のものが増加し主要なものへと変化した。見出した
シアル酸分子種変化およびセラミドのアシル鎖構造変化は筋分化過程の様々なステップに重要であることが示唆された(3)。つまり筋細胞は、同じ GM3 であっても、そのセラミド構造および糖鎖中のシアル酸の構造を変化させることで、その性質を変え、筋分化を適切に、繊細に制御していると考えられる。この研究で郷博士は、第一回箱守仙一郎賞（奨励賞）を受賞したのは大変喜ばしいことである。
郷博士の川崎医大転出に伴い、当研究室の博士研究員であった狩野裕考博士
Inflammatory GM3 Anti-inflammatory
GM2, GM1, and
HMGB1 (24:0, 22:0, h24:0)
GM3
が 2017 年 4 月より助手として着任した。
(24:1, 16:0, 18:0) Polysialo species
LPS(GD1a, GT1b, …)
狩野博士は、私が北大薬学部の助教授時代の学部学生であり、今は亡き稲垣冬彦
Siglecs,
CLECs
TLR4/MD2Other TLRs教授の下で構造生物学を学んで修士号を取得し、その後、東北大学薬学部の倉田教授の研究室でショウジョウバエの自然免疫で学位を取得した後、縁あってTNF-α, IL-6, IL-1β
NF-κB
わたしの研究室に所属した。現在、自然免疫系におけるスフィンゴ糖脂質の病
図１ ガングリオシドを介した炎症制御メカニズムの仮説
態生理的意義について鋭意研究を進めている。我々の主研究課題である「メタボリック症候群におけるスフィンゴ糖質病態生理学的意義」の解明において、自然免疫系の異常な活性化にもとづく炎症反応の誘導に、ガングリオシド GM3 分子種発現バランスの異常が原因であることを証明しつつある。図１に現在の知見に基づく作業仮説を示す。
研究員の宍戸は、医学部医化学教室の上村講師と共同でガングリオシド GM2 合成酵素
におけるゴルジ体繋留機構の解析についての原著論文を発表した(4)。本研究では、GM2合成酵素（GM2S）が新規 mRNA バリアントをもち、この新規 mRNA からは逆行輸送シ
グナルとして機能する R-based motif をもつ M1-GM2S が翻訳されることを示した。さらに、R-based motif をもたない既知のアイソフォームである M2-GM2S と M1-GM2S がヘĸĽGolgiテロダイマーを形成することで、M2-GM2S ホモ
Lysosome
ダイマーよりも高い安定性をもつことが示された。興味深いことに、この安定性の向上は小胞Degradation
体への逆行輸送ではなくゴルジ体繋留に依存すEndosome
るものであり、GM2S において R-based motifがゴルジ体繋留シグナルとして機能することがtrans
示唆された (図２)。また、GM3 合成酵素（GM3S）の Exon3 ノックアウトマウスを用いた解析で、Retrograde transport
Golgi retention!
between Golgi!
= stabilization!
他組織では GM3 合成活性が消失するが、肝臓でmedialGM3 合成活性が残存することを見出した。これ
R-based motif!
cis
まで、野生型マウスの解析において、肝臓組織
Retrograde transport!
From Golgi to ER!
で高い GM3 合成活性が認められるにも関わらER
ず、mRNA の発現量が他臓器と比較して著しく
M1/M1 M1/M2 M2/M2
低いことが示されていた。肝臓のみで高発現しGM2S
ている新規 mRNA バリアント（c1-type）を同
M1-GM2S の細胞質領域 N 末端に存在する R-based motif は、定し、肝臓での高い GM3 合成活性は c1-type の 図２
ゴルジ体から小胞体への逆行輸送とゴルジ体繋留に関与する発現に由来することを示した。また、c1-type はExon3 がスプライシングされても、新たに人工的なスプライシングアイソフォームが合成される。これが GM3S-Exon3-KO マウスの肝臓で GM3 合成活性が残存していた理由であることを示した(5)。
東京医科歯科大の横山博士との共同研究で、非常に興味深い結果が得られた(6)。セラミ
ドアナログである 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) はス
フィンゴ糖脂質の合成阻害剤として開発されたが、リソソームや小胞体の構造にも影響を与えることが知られていた。本研究では、PDMP が mTORC1 の細胞内局在を変化させることで mTORC1 の活性を制御する可能性に着目し、その検証を試みた。その結果 PDMP
処理により mTORC1 の小胞体との共局在率が有意に増加した。さらに mTORC1 をリソ
ソーム膜に動員する足場となる Ragulator の構成要素 LAMTOR1 も、PDMP 処理によりリソソームから小胞体へと局在が変化することが判明した。これらの結果から、PDMP 処
理により mTORC1 がリソソームから小胞体へ移行することが示された(6)。本研究は細胞内局在の変化という、今までに全く知られていない作用機序による mTORC1 阻害剤の開発にもつながると期待される。
2017年に発表した論文
1. ガングリオシド GM3 合成酵素欠損症から見るスフィンゴ糖脂質機能 郷慎司、井ノ口
仁一 「生化学」2017
2. Gangliosides and hearing. Inokuchi J., Go S., Yoshikawa M. and Strauss K.
Biochim. Biophys. Acta, 1861, 2485-2493, 2017
3. Altered expression of ganglioside GM3 molecular species and a potential
regulatory role during myoblast differentiation. Go S., Go S., Veillon L., Ciampa
M.G., Mauri L., Soto C., Kitajima K., Prinetti A., Sonnino S. and Inokuchi J. J. Biol.
Chem., 292, 7040-7051, 2017
4. Identification of a new B4GalNAcT1 (GM2/GD2/GA2 synthase) isoform, andregulation of enzyme stability and intracellular transport by arginine-based motifĸľShishido F., Uemura S., Kashimura M. and Inokuchi J. Biochim. Biophys. Acta ,1859, 2001-2011, 2017
5. Identification of a new liver-specific c-type mRNA transcriptional variant formouse ST3GAL5 (GM3/GM4 synthase). Shishido F., Uemura S., Niita T. andInokuchi J. Glycoconj. J ., 34, 651-659, 2017
6. PDMP, a ceramide analogue, acts as an inhibitor of mTORC1 by inducing its
translocation from lysosome to endoplasmic reticulum. Ode T., Podyma-Inoue
K.A., Terasawa K., Inokuchi J., Kobayashi T., Watabe T., Izumi Y. andHara-Yokoyama M. Exp. Cell Res. 350, 103-114, 2017

ĸĿ

生活習慣病に関連した G タンパク質共役受容体の機能と糖鎖および受容体間相互作用による調節生体膜情報部門 東 秀好
1. 肥満関連 GPCR の機能とそのネットワーク
我々が以前から注目しているリガンド不明の G タンパク質共役受容体（GPCR）の一つである GPRC5B は、中枢神経系や脂肪細胞、膵臓 β 細胞に発現し、その多コピーが BMI
値と相関する肥満関連受容体である。mGluR 等と同じ Class C に属する GPRC5B は、高脂肪食摂取時の脂肪細胞の増大やインスリン分泌、血糖値の制御に関与する。膵臓 β 細胞の生存性やインスリン分泌が、GPRC5B を発現することで低下することが分かっており、糖尿病患者の膵臓 β 細胞では GPRC5B 発現が亢進している。しかし、GPRC5B がどのようなシグナルを介して、これらの現象を制御しているのかは明らかでない。そこで我々は、
GPRC5B の発現により、細胞内でどのようなシグナル伝達が引き起こされるのかを検討した (1)。まず、HEK293 細胞に GPRC5B を発現させると、発現した細胞の増殖が亢進していることが分かった。次に、シグナル伝達分子の阻害剤を用いて、GPRC5B の発現による細胞増殖の亢進に関わるシグナル伝達経路を検討した。その結果、ラパマイシン標的シグ
ナル（mTOR）とプロテインキナーゼ C（PKC）の阻害剤により、GPRC5B による細胞増殖が抑制された。さらに、三量体 G タンパク質の α サブユニットの阻害ペプチドにより、
GPRC5B に結合する Gα タンパク質を検討したところ、Gαq の阻害により細胞増殖が抑制された。これらの結果から、GPRC5B のシグナルは、Gαq および PKC を介して mTOR
を活性化すると考えられた。mTOR シグナルは、脂肪細胞の増殖亢進により肥満を誘導する。また、mTOR シグナルが膵臓 β 細胞からのインスリン分泌を促進する一方、慢性的な活性化はインスリン分泌を低下させ、糖尿病の発症につながると考えられている。
GPRC5B による脂肪細胞の増大やインスリン分泌の制御といった現象は、mTOR シグナルを制御することで引き起こされていると考えられた。
そこで、我々は、GPRC5B の過剰発現がマウスインスリノーマ（MIN6）細胞のインス
リン分泌に及ぼす影響を検討したところ、GPRC5B の過剰発現によって MIN6 細胞から培
地中に放出されるインスリン量が上昇することを見出した (2)。次に、このインスリン分
泌亢進のメカニズムを明らかにするため、インスリン遺伝子の発現をリアルタイム PCR により測定すると、GPRC5B を過剰発現した MIN6 ではインスリン遺伝子の発現が上昇していた。インスリン遺伝子は、転写因子の PDX-1 により転写が調節される。また、PDX-1の発現は mTOR シグナルにより調節される。従って、GPRC5B の発現による MIN6 のイ
ンスリン分泌の亢進には、GPRC5B から mTOR シグナルを介したインスリンの増産が寄与していることが示唆された。
β2-アドレナリン受容体（β2AR）は、カテコラミンをリガンドとする G タンパク質共役受容体（GPCR）で、気管支や血管等の拡張・収縮、グリコーゲン分解を制御する。また、β2AR は脂肪細胞にも発現し、脂質代謝に関与する。近年、酵母の系を用いた解析結果から、β2AR と GPRC5B が相互作用することが報告された。我々は、GPRC5B が関与する現象が、β2AR と GPRC5B の相互作用により引き起こされているのではないかと考えた。そこで、HEK293T に β2AR と GPRC5B を発現させ、免疫沈降をおこなったところ、両 GPCR は共沈降されたので、これらが哺乳動物細胞においても相互作用しているこĸŀ

とが明らかになった (3)。次に、活性化した GPCR は β アレスチンが結合することで脱感作されることから、β2AR と β アレスチンの結合を β ガラクトシダーゼの α 相補を利用して検出した。その結果、GPRC5B の存在により、β2AR と β アレスチンの結合が低下した。さらに、β2AR が持つ 2 本の N 結合型糖鎖が欠損した β2AR は、アゴニスト刺激により脱感作されやすいが、GPRC5B が共存すると、糖鎖欠損 β2AR と β アレスチンの結合も顕著に低下しており、脱感作が起こりにくくなると考えられた。このことから、
β2AR と GPRC5B の相互作用は、アゴニスト刺激による β2AR の脱感作を起こりにくくし、β2AR の G タンパク質経路の活性化が維持される可能性が示唆された。また、GPRC5Bとの相互作用に、β2AR の N 結合型糖鎖は関与しないと考えられた。特に、GPRC5B とβ2AR は脂肪細胞において共発現しているので、両分子が相互作用し β2AR のシグナルを増強し、脂質代謝を亢進させることによって、脂肪細胞の増大に繋がっている可能性がある。
2. GPCR の N 結合糖鎖とジスルフィド架橋の機能、脂質ラフトとの関わり
上記のごとく、β2AR では N 結合糖鎖は脱感作の制御に関与していた。我々の研究室ではブラジキニン B2 受容体(B2R)が GT1b ガングリオシドとコンドロイチン硫酸 C の連続刺激により不応化されることと ATP/UTP 受容体 P2Y2 (P2Y2R)と共役することを見出した (4)ので、この現象における N 結合糖鎖の役割を含めた分子機構を解析している。B2R と P2Y2R
は共に Gαq/11 を共役 G タンパク質とする Class A の GPCR で、炎症誘発、血圧低下、発痛に関与する。両者のヘテロオリゴマーはジスルフィド架橋による共有結合によるものと推定している。そこで、まず、両受容体の細胞における局在を調べた。両者は ER から
ゴルジ装置での糖鎖付加を経て細胞膜表面に発現されるが、細胞膜上でのマイクロドメイ
ンへの分布は少し異なり、P2Y2R がほぼすべて脂質ラフトの画分に分画されるのに対して
B2R はラフトと非ラフトの両画分にほぼ等量分画された。P2Y2R の N 結合糖鎖は N 末端ドメインに２ヶ所あり、２ヶ所の糖鎖を欠損させた変異体は細胞膜表面に輸送されずに ERに留まったが、脂質ラフト画分には分画された (6)。興味深いことに、P2Y2R を共発現す
ると B2R はラフト画分へ多く分画され、両受容体の密な相互作用が示唆された (5)。一方、
B2R はアゴニスト刺激によりホモダイマーを形成し、このホモダイマー形成やアレスチン
リクルートには N 末端ドメインが不可欠である。我々は、P2Y2R についてアゴニスト刺激とは無関係にホモダイマーが形成されることを見出し、それはジスルフィド架橋によるものであることを証明した (7)。P2Y2R には N 末端ドメイン、第１、第 2、第 3 細胞外ループにそれぞれ１ヶ所ずつ Cys 残基が存在する。細胞膜表面発現や活性化に必須な中央の 2
ヶ所の Cys 残基に加え、外側の 2 ヶ所の Cys 残基間の架橋がホモダイマー形成に関与することを明らかにした。
B2R の N 結合糖鎖はホモダイマー形成に必要とされているが、P2Y2Rとのヘテロオリゴマー形成には糖鎖の小さい B2R が優先的に関わっていた (4)。P2Y2R の
場合、N 結合糖鎖が小さい方がホモダイマー・オリゴマーの形成率が高かった (8,9)。前
述のごとく、２ヶ所の N 結合糖鎖を欠失すると、細胞膜表面に発現されないが、2 ヶ所とも、あるいは、どちらか１ヶ所の糖鎖欠損変異体は野生型よりもホモダイマー・オリゴマーの形成率が高かった。さらに、Asn9 は欠失しても活性を保持したが、Asn13 の欠失は活性をほぼ失った。このように、同じ Class A に属する B2R と P2Y2R であるが、これらの N 型糖鎖の機能は大きく異なっていた。

ĹķREFERENCES

1. 黒田 喜幸, 中川 哲人, 東 秀好 (2017) 肥満と関連する受容体 GPRC5B による mTOR シグ ナルの制御. 日本薬学会第 137 回年会，仙台
2. 小松 伊織, 黒田 喜幸, 中川 哲人, 東 秀好 (2017) β2-アドレナリン受容体と GPRC5B の相互作
用による脱感作の制御. 日本薬学会第 137 回年会，仙台
3. 黒田 喜幸, 中川 哲人, 東 秀好 (2017) オーファン GPCR, GPRC5B によるインスリン分泌亢進
とそのメカニズム. 第 36 回日本糖質学会年会，旭川
4. Shimazaki, A., Nakagawa, T., Mitoma, J., and Higashi, H. (2012) Gangliosides and chondroitin sulfate
desensitize and internalize B2 bradykinin receptors. Biochem. Biophys. Res. Commun. 420, 193-198
5. Nakagawa, T., Takahashi, C., Matsuzaki, H., Kuroda, Y., and Higashi, H. Membrane raft recruitment ofbradykinin B2 receptor by ATP/UTP receptor P2Y2 via their close association. Manuscript in
preparation.
6. Nakagawa, T., Takahashi, C., Matsuzaki, H., Takeyama, S., Sato, S., Sato, A., Kuroda, Y., and Higashi,
H. (2017) N-glycan-dependent cell-surface expression of the P2Y2 receptor and N-glycan-independent
distribution to lipid rafts. Biochem. Biophys. Res. Commun. 485, 427-431
7. Abe, M., Watanabe, K., Kuroda, Y., Nakagawa, T., and Higashi, H. (2018) Homodimer formation by the
ATP/UTP receptor P2Y2 via disulfide bridges. J. Biochem., Tokyo. in press.
8. 矢口 沙也子，黒田 喜幸，中川 哲人，東 秀好 (2017) ATP/UTP 受容体 P2Y2 の N 結合糖鎖の２量体形成に及ぼす効果：ブラジキニン B2 受容体との比較.
第 11 回東北糖鎖研究会，桐生
9. 矢口 沙也子，阿部 真耶，黒田 喜幸，中川 哲人，東 秀好 (2017) ATP/UTP 受容体 P2Y2 の細胞外 Cys 残基と N 結合糖鎖の機能. 第 90 回日本生化学会大会，神戸Ĺĸがん細胞におけるN-型糖鎖の機能解析と臨床への応用に向けて
細胞制御部門 顧 建国
糖タンパク質は糖鎖と蛋白質が共有結合した生体分子である。蛋白質上の糖鎖の結合様式は一般
的にはN-型と0-型という２種類がある。N-型糖鎖は、ポリペプチド上にAsn-X-Ser/Thrモチーフのアスパラギン（Asn）のアミド基(-NH2)にC-N結合でN-アセチルグルコサミン(GlcNAc)を還元末端とする糖鎖の総称である。糖鎖はタンパク質の物性に影響を与えるとともにタンパク質の機能、そして分子間の認識や細胞内シグナル伝達に大きな役割を持つ。私達は、図1に示すような分岐型糖鎖の生合成に関わるN-アセチルグルコサミン転移酵素III(GnT-III)、N-アセチルグルコサ
ミン転移酵素V(GnT-V)、α1,6 フコース転移酵素(Fut8)の機能に注目して研究してきた。近年の研究によりそれら
図 1 .注 目す る 分岐 型 N-型 糖 鎖の 生 合成 と主 な 機能の糖鎖は、細胞機能に密接に関わっていることが明らかになりつつある。
I. 細 胞 接 着 お よ び 移 動 に お け る イ ン テ グ リ ン α 5上 の N-型 糖 鎖 の 重 要 性 に つ い て細胞接着分子であるインテグリンはα鎖とβ鎖から成るヘテロ二量体で、ECMへの細胞の接着を媒介する主要な糖蛋白質で、細胞内蛋白質のチロシンリン酸化や低分子量GTP結合蛋白質の活性化を通じて細胞内骨格系の再編成や増殖･分化のシグナル伝達に深く関わる(図２，Ａ)。また、インテ
グリンと細胞増殖因子受容体との相互作用は、細胞の増殖・移動・生存などの生理的な過程のみならず、がん細胞の増殖・転移･浸潤など病理的な過程にも重要な役割を担うと考えられている。図2のようにインテグリンα5に14カ所、β1鎖には１２カ所の糖鎖付加部位があるが、これまで我々はそれぞれ３カ所の糖鎖がインテグリンのαとβ鎖ヘテロダイマー形成や機能発現に重要である

図 2.

A , イ ンテ グ リン の機 能 ； B, α 5β 1 に N-型 糖鎖 付 加サ イト と 機能 の 特異 性ĹĹ

ことを同定した（Isaji,T., et al. JBC. 2009）。最近、我々はさらにインテグリンα5のN-型糖鎖に焦点を絞り、分子間複合体の形成および細胞内シグナル伝達の制御を解析し(図２，Ｂ)、α5がlipid
raftにおいてEGFRと相互作用し、EGFRを介した細胞内シグナル伝達を負に制御することを明らかにした(Hang, Q., et al. JBC. 290, 29345-60, 2015)。また、部位特異的変異導入法を駆使してα5の11番目の糖鎖がα5とEGFRとの相互作用に重要であり、その相互作用はEGFRの負の制御に重要であることを立証した(Hang, Q., et al. Sci. Rep. doi: 10.1038/srep33507, 2016)。その11番目サ
イトには他のサイトより分岐型糖鎖が多く付加されたことも分かった。また、インテグリンβ1に対しても解析を行った。β1がEGFRと相互作用において、β1に付加されている糖鎖の中でも特に膜近傍の糖鎖が重要であることを明らかにした。さらに、それらの糖鎖構造にα2,6シアリル化が必要である (Hou, S., FASEB J., 30, 4120-31, 2016)。それらの知見から、N-型糖鎖または特定の糖鎖構造が細胞膜上での分子間の相互作用に重要な役割を演じていると考えられる。
最近、私達は、細胞移動を制御するインテグリンα5上の重要な糖鎖付加部位（1と2番目）の同定に成功した（１）。その分子機序としては、1と2番目に付加された糖鎖が細胞膜上syndecan-4分子とα5との複合体形成に重要で、その糖鎖を無くすとα5のエンドサイトーシスが抑制され、そして細胞移動が低下すると考えられる。さらに興味深いとこに、その1と2番目の糖鎖の欠損でインテグリンからのシグナル伝達に重要な働きをするFocal adhesion kinase (FAK)が過剰に活性化された。実はFAKの活性化はインテグリンを介する細胞遊走に大事であるが、過剰な活性化は逆に細胞移動を阻害する。今後、これらの結果をin vivoでも検討し、インテグリンと他の受容体を介するシ
グナル伝達のCross-talkの分子機序の解明や糖鎖による抗がん剤の開発に貢献する新知見の提供を目指す。
II. コ ア フ コ ー ス に よ る が ん 性 変 化 の 制 御 に つ い て
一方、コアフコースは図1に示すようにフコース転移酵素(Fut8)により生合成され、肝がんの特異的バイオマーカーや抗体医療とも深く関わっており、注目されている糖鎖の一つである。そのFut8遺伝子が欠損させると、マウスは様々な病態を生じる。一方、我々は肝がんの発生・増殖にもコアフコシル化糖鎖の発現が重要であることを明らかにしている（Wang, et al., Sci Rep, 2015；
FASEB J, 2015）。しかし、治療を考える上では、Fut8遺伝子の欠失やその酵素活性の阻害という方法は困難である。最近、コアフコシル化をがん組織特異的に抑制することを目的に、L-フコースのアナログである2-fluorofucose (2FF)の有用性を検討した（６）。2FFは細胞内に取り込まれて、
GDP-fucose合成のde novo経路を阻害すると知られている。 2FFの添加によりヒト肝がん由来
HepG2細胞のコアフコシル化が劇的に抑制されることをレ
クチン検出や質量分析で確認した。細胞表面の重要な受容体であるEGFRやβ1インテグリ
ンなど個別の分子でもコアフコシル化が消失することを確認した。また、これらの受容体を介したシグナルが減弱、細胞
増殖や細胞遊走、軟寒天上でのコロニー形成能も有意に抑制さ
図 3 . 2 FF に よる 腫 瘍形 成と 細 胞内 シ グナ ル の抑 制Ĺĺ

れた。一方、in vivoでは、マウス皮下へHepG2細胞を移植し、定期的に移植部位近傍へ2FFを投与したところ、腫瘍組織のコアフコシル化糖鎖の発現が強く抑制された。さらに、腫瘍サイズ並びに細胞内のシグナル伝達経路も有意に抑制されることがわかった（図3）。以上のことから、肝がん
組織におけるコアフコシル化の重要性を示すと共に、2FFのような化合物が肝がんの新規治療薬になる可能性が強く示唆された。
2017年に発表した論文
1. A key regulator of cell adhesion: Identification and characterization of important N-glycosylation sites
on integrin α5 for cell migration. Hang, Q., Isaji, T., Hou, S., Wang, Y., Fukuda, T. and Gu, J. Mol. Cell.
Biol. 37: e00558-16, 2017
2. Alteration of N-glycan expression profile and glycan pattern of glycoproteins in human hepatoma cells
after HCV infection. Xiang, T., Yang, Q., Liu, X., Zhou, Y., Lu, F., Gu, J., Taniguchi, N., Tan, Z., Chen,
X., Xie, Y., Guan, F. and Zhang, X. Biochim. Biophys. Acta, 1861: 1036-1045, 2017
3. Chitosan
oligosaccharides
inhibitepithelialcellmigration
throughblockadeofN-acetylglucosaminyltransferase V and branched GlcNAc structure. Xu, Q., Wang, W., Qu, C., Gu, J.,
Yin, H., Song, L. and Du, Y. Carbohydrate Polymers, 170:241-246, 2017
4. Differential expression of ST6GAL1 in the tumor progression of colorectal cancer. Zhang, S., Lu, J., Xu,
Z., Zou, X., Sun, X., Xu, Y., Shan, A., Lu, J., Yan, X., Cui, Y., Yan, W., Du, Y., Gu, J., Zheng, M., Feng,
B., Zhang, Y. Biochem Biophys Res Commun. 486:1090-1096, 2017
5. Specific N-glycan alterations are coupled in EMT induced by different density cultivation of MCF 10A
epithelial cells. Xu, Q., Niu, X., Wang, W., Yang, W., Du, Y., Gu, J., Song, L. Glycoconj J. 34:219-227,
2017
6. Inhibition of fucosylation by 2-fluorofucose suppresses human liver cancer HepG2 cell proliferation andmigration as well as tumor formation. Zhou, Y., Fukuda, T., Hang, Q., Hou, S., Isaji, T., Kameyama, A.,
and Gu, J. Sci Rep. doi: 10.1038/s41598, 2017
7. Roles of Integrin α6β4 Glycosylation in Cancer. Kariya, Y., Kariya, Y and Gu, J. Cancers 9: doi:
10.3390/cancers9070079, 2017ĹĻ

レクチンのがん薬物治療への応用
分子認識部門 細野 雅祐（１）ナマズ卵レクチン（SAL）による抗がん剤排出抑制機構の解明
SAL は細胞膜表面の非還元末端 Galα-結合糖鎖，実際にはグロボトリアオシルセラミド（Gb3）を主とする，を特異的に認識して結合するレクチンである．
SAL は，ヒト子宮頸がん由来細胞株 HeLa のスニチニブ（スーテント，主として腎がんに適用される分子標的薬）に対する感受性を高め，単独処理に比べ細胞生存率を著しく減少させるが，この機序として，SAL の Gb3 を介した結合により，①スニチニブの細胞内取り込み量が増加すること，②SAL 処理により細胞内に形成される油滴状の膜構造物の周囲にスニチニブが集積する結果，この分子の排出が抑制されること，が分かっている．
本年度は，SAL により形成される膜構造物について解析を行った．この構造物は SAL 単独処理
でも明視野像で明確に観察することができ，SAL と Gb3 との特異的相互作用により出現する．Nile
Red では染色されないことから，いわゆる脂肪滴ではなく，ブレフェルディン A の前処理によって形成が減少した．また，膜部分が 抗 LAMP1 抗体で染色され，かつ取り込まれたスニチニブと
LAMP1 が共局在したことから，この構造物はリソソームである可能性が考えられた．一方，この構造物は，リソソームマーカーである Lysotracker では染色されず，ニュートラルレッドで染色された．したがって SAL は，HeLa 細胞内にゴルジ体由来で内部が中性のリソソーム様構造物の形成を促進し，その膜部分に細胞内に取り込まれたスニチニブがトラップされることで排出が抑制されている可能性が示唆された（投稿準備中）．
（２）SAL 受容体としての細胞膜 Gb3 分子の機能
バーキッリンパ腫細胞 Raji の Gb3 欠損株（Gb3-KO-Raji）および Gb3 の発現を誘導した慢性骨髄性白血病細胞株 K562（Gb3-K562）細胞を作製し，SAL 受容体分子としての Gb3 について検討した．Gb3-KO-Raji は，親株と比較して細胞増殖スピードが遅くなるが，このとき細胞周期を負に制御する p21 と p27 の発現上昇およびリン酸化 ERK 量が増加していることが示された．Gb3-K562
では，逆にわずかではあるが増殖速度が速くなり，p21 の発現低下およびリン酸化 ERK 量の低下が認められた．また，Gb3-KO-Raji および Gb3-K562 を SAL 処理しても，Raji で見られるような変化は観察されなかった．以上の結果から，SAL の効果発現には，受容体としての Gb3 分子が必要ではあるもののそれだけでは十分ではなく，Raji で見られる細胞応答には Gb3 以外の分子が関与している可能性が示唆された．現在，この分子の探索を行っている．
（３）ウシガエル卵レクチン（cSBL）の悪性胸膜中皮腫（MPM）に対する他剤併用効果
cSBLは，レクチン活性およびリボヌクレアーゼ (RNase) 活性を併せ持つ抗腫瘍性タンパク質である．cSBLはRNAをターゲットとした新規抗腫瘍メカニズムにより，MPMを含む多くのがん細胞株に対して，アポトーシスの誘導を介した細胞傷害作用を示すことを明らかにしている．
本年度は，in vitro における数種のMPM細胞株に対するcSBLの有効性，既存治療薬との併用効
果ならびにその作用機序について解析を行った．その結果，既存治療薬と比較してcSBLは強い細胞増殖抑制効果および高いがん細胞選択性を示すことを明らかにした．また，H28細胞を用い，2剤併用時の効果について検討したところ，pemetrexed + cSBL が，既存レジメンと同等以上の抗腫瘍作用および相乗性を持つことが示唆された．この相乗効果は，pemetrexedの持つcyclin Aの持続Ĺļ的活性化による細胞周期の停止作用とcSBLの持つcaspase-3の活性化を介したアポトーシスの誘導作用が協働することで引き起こされると考えられる．
次に異種移植可能なH2452およびMSTOを用いて異種移植モデルマウスを作成し，in vivo におけるcSBLの抗腫瘍効果の検討を試みた．まず，in vitro における殺細胞メカニズムの解析を行った
ところ，caspase-9，-8，-3 が順次活性化され，アポトーシスが誘導されることが示唆された．in vivo
におけるcSBLの有効性の検討では，通常動物実験に用いられる濃度のpemetrexedと比較すると，
cSBL処理群でより早期に腫瘍の増殖抑制が引き起こされ，同時にこの濃度においてマウスに有意な体重変化は生じず，有害作用は小さいことが示唆された．
さらに，MPMの治療障壁の一つとなっているpemetrexed耐性株への各薬剤の効果を比較した．H28およびH2452に対する効果では，pemetrexedおよびcisplatinと比較し，cSBL，TRAIL，およびEGFR-TKIs（チロシンキナーゼ阻害薬）はいずれも中皮腫細胞に対して高い選択性を示した．また，cSBLおよびEGFR-TKIsは，細胞株の違いによる増殖抑制効果にほとんど差がないことが明らかとなった．また併用効果の検討では，耐性細胞においてpemetrexed + cSBLが既存レジメンと比較しpemetrexedのDRIが2倍以上高く，耐性細胞株に対しても有効なレジメンとなる可能性が示された．3剤併用では，Combination Index (CI, 薬剤併用効果の指標) およびDRIの観点から，pemetrexed + cisplatin +cSBLが最も有効な組み合わせであることが示された．
2017年に発表した原著論文(1) Catfish rhamnose-binding lectin induces G0/1 cell cycle arrest in Burkitt's lymphoma cells via
membrane surface Gb3
Shigeki Sugawara, Changhun Im, Tasuku Kawano, Takeo Tatsuta, Yasuhiro Koide, Daiki Yamamoto,
Yasuhiro Ozeki, Kazuo Nitta, Masahiro Hosono
Glycoconj. J., 34, 127-138 (2017)(2) Lissoclibadin 1, a Polysulfur Aromatic Alkaloid from the Indonesian Ascidian Lissoclinum cf.
badium, Induces Caspase-Dependent Apoptosis in Human Colon Cancer Cells and Suppresses Tumor
Growth in Nude Mice
Takeo Tatsuta, Masahiro Hosono, Henki Rotinsulu, Defny S. Wewengkang, Deiske S. Sumilat, Michio
Namikoshi, Hiroyuki Yamazaki
J. Natl. Product., 80, 499-502 (2017)(3) Synergistic anti-tumor effect of bullfrog sialic acid-binding lectin and pemetrexed in malignant
mesothelioma
Toshiyuki Satoh, Takeo Tatsuta, Shigeki Sugawara, Akiyoshi Hara, Masahiro Hosono
Oncotarget., 8, 42466-42477 (2017)(4) Sialidase NEU3 defines invasive potential of human glioblastoma cells by regulating
calpain-mediated proteolysis of focal adhesion proteins
Kohta Takahashi, Sergei Proshin, Kazunori Yamaguchi, Yoji Yamashita, Ryuichi Katakura, Koji Yamamoto,
Hiroshi Shima, Masahiro Hosono, Taeko Miyagi
Biochim. Biophys. Acta., 1861, 2778-2788 (2017)ĹĽ■学会発表記録<機能病態分子部門>

・ガングリオシド欠損による肥満モデルマウスの病態改善および受容体機能に与える影響
稲森 啓一郎，伊藤 英樹，田村 有美，楊 燕華，二瓶 渉，宍戸 史，突田 壮平 a，山田 哲也 a，片桐 秀樹 a，井ノ口 仁
一（東北大・院医 a）
第 59 回日本脂質生化学会，京都，2017 年 6 月，要旨集 p.140-141
・ガングリオシド GM3 のアシル鎖構造に基づく新たな自然免疫応答と慢性炎症
狩野 裕考，郷 慎司，新田 昴大，Lucas Veillon，藤居 真優 a，樺山 一哉 a，下山 敦史 a，深瀬 浩一 a，安藤 弘宗 b，
石田 秀治 b，名取 良浩 c，吉村 祐一 c，鈴木 明身，井ノ口 仁一（大阪大・院理 a，岐阜大・応用生物科学 b，東北医薬大・薬・分子薬科学 c）
第 59 回日本脂質生化学会，京都，2017 年 6 月，要旨集 p.137-139・胸腺細胞の分化におけるスフィンゴミエリン発現とその機能的役割
豊島 かおる，永福 正和，岡崎 俊郎 a，井ノ口 仁一（金沢医科大・血液免疫内科 a）
第 59 回日本脂質生化学会，京都，2017 年 6 月，要旨集 p.244-246
・ガングリオシド分子種による慢性炎症応答の新機軸
井ノ口 仁一，狩野 裕考
第 36 回日本糖質学会年会，旭川，2017 年 7 月，要旨集 p.103
・ガングリオシドは NPC1L1 を介した腸管からのコレステロール吸収を制御する
二瓶 渉，永福 正和，狩野 裕孝，稲森 啓一郎，新井 健太 a，樺山 一哉 a, 深瀬 浩一 a,(大阪大・院理 a)
第 36 回日本糖質学会年会，旭川，2017 年 7 月，要旨集 p.107

井ノ口 仁一・New Insights into Ganglioside Functions from GM3S Knockout in Obese Mouse Models
Kei-ichiro Inamori
Muscle Membrane Serendipity: Past, Present and Future Conference, Iowa City, IA, USA, July 2017, Program p.3
・New Paradigm for Chronic Inflammation Mediated by Ganglioside Molecular Species.
Jin-ichi Inokuchi, Hirotaka Kanoh
24th International Symposium on Glycoconjugates, Korea, August 2017, p.61
・Gangliosides and Hearing
Jin-ichi Inokuchi
International Conference on the Glycobiology of Nervous System: From the Genome Research Era to the Glycome,Korea, September 2017
・肥満糖尿病モデルにおけるガングリオシド GM3 欠損マウスから得られた新知見
稲森 啓一郎，伊藤 英樹，田村 有美，新田 昂大，楊 燕華，二瓶 渉，宍戸 史，突田 壮平 a，山田 哲也 a，片桐 秀
樹 a，井ノ口 仁一（東北大・院医 a）
東北糖鎖研究会・東京糖鎖研究会 合同シンポジウム，桐生，2017 年 11 月，要旨集 p.52
・ガングリオシド分子種による慢性炎症制御の新機軸
井ノ口 仁一東北糖鎖研究会・東京糖鎖研究会 合同シンポジウム，桐生，2017 年 11 月，要旨集 p.22-24
・ガングリオシドのアシル鎖構造が制御する新たな慢性炎症メカニズム
狩野 裕考，郷 慎司，新田 昴大，Lucas Veillon，藤居 真優 a，樺山 一哉 a，下山 敦史 a，深瀬 浩一 a，安藤 弘宗 b，
石田 秀治 b，名取 良浩 c，吉村 祐一 c，鈴木 明身，井ノ口 仁一Ĺľ（大阪大・院理 a，岐阜大・応用生物科学 b，東北医薬大・薬・分子薬科学 c）
東北糖鎖研究会・東京糖鎖研究会 合同シンポジウム，桐生，2017 年 11 月，要旨集 p.87
・細胞膜スフィンゴミエリンは胸腺における自己反応性 T 細胞の除去に関与するか？
豊島 かおる，永福 正和，岡崎 俊郎 a，井ノ口 仁一（金沢医科大・血液免疫内科 a）
東北糖鎖研究会・東京糖鎖研究会 合同シンポジウム，桐生，2017 年 11 月，要旨集 p.142
・慢性炎症によるガングリオシド分子種の発現変化
新田 昂大, 狩野 裕考, 郷 慎司, Lucas Veillon, 鈴木 明身, 井ノ口 仁一
東北糖鎖研究会・東京糖鎖研究会 合同シンポジウム，桐生，2017 年 11 月，要旨集 p.134
・ガングリオシドによる NPC1L1 を介した腸管からのコレステロール吸収制御
二瓶 渉，永福 正和，狩野 裕孝，稲森 啓一郎，新井 健太 a，樺山 一哉 a, 深瀬 浩一 a, 井ノ口 仁一(大阪大・院理 a)
東北糖鎖研究会・東京糖鎖研究会 合同シンポジウム，桐生，2017 年 11 月，要旨集 p.79
・New Paradigm for Chronic Inflammation Mediated by Ganglioside Molecular Species.
Jin-ichi Inokuchi, Hirotaka Kanoh
9th Asian Community of Glycoscience and Glycotechnology Conference, Hong Kong, December 2017
・ガングリオシドの糖鎖・アシル鎖構造が制御する新たな慢性炎症メカニズム
狩野 裕考
ConBio2017，神戸，2017 年 12 月，ワークショップ 3PW24-4
・New Paradigm for Chronic Inflammation Mediated by Ganglioside GM3 Molecular Species.
Jin-ichi Inokuchi
2018 Gordon Research Conference on “Glycolipid and Sphingolipid Biology”, Galveston Texas USA, February2018<生体膜情報部門>

・オーファン GPCR, GPRC5B によるインスリン分泌亢進とそのメカニズム
黒田 喜幸, 中川 哲人, 東 秀好
第 36 回日本糖質学会年会，旭川，2017 年 7 月，演題番号 3B-03
・ATP/UTP 受容体 P2Y2 の N 結合糖鎖の２量体形成に及ぼす効果：ブラジキニン B2 受容体との比較
矢口 沙也子，黒田 喜幸，中川 哲人，東 秀好
第 11 回東北糖鎖研究会，桐生，2017 年 11 月
・ATP/UTP 受容体 P2Y2 の細胞外 Cys 残基と N 結合糖鎖の機能
矢口 沙也子、阿部 真耶、黒田 喜幸、中川 哲人、東 秀好
第 90 回日本生化学会大会，神戸，2017 年 12 月

<細胞制御部門>

・細胞接着における N-型糖鎖の機能発現と細胞内シグナルの制御
杭 慶雷，伊左治 知弥，侯 思聡，福田 友彦，顧 建国第 69 回日本細胞生物学大会，2017 年 6 月，仙台，要旨集 p61
・PI4KIIα による N-型糖鎖の調節とインテグリン α3β1 の機能制御細胞接着における N-型糖鎖の機能発現と細胞内シグナルの制御伊左治 知弥，福田 友彦，顧 建国第 69 回日本細胞生物学大会，2017 年 6 月，仙台，要旨集 p96
・脳における α1,6 フコース転移酵素 Fut8 の機能とその役割

ĹĿ福田 友彦
第 16 回生物化学若手研究者セミナー，仙台，2017 年 7 月
・インテグリン α5 の糖鎖が細胞増殖•運動の鍵となる
杭 慶雷，伊左治 知弥，侯 思聡，福田 友彦，顧 建国第 36 回日本糖質学会年会，旭川，2017 年 7 月，要旨集 p83
・コアフコシル化を欠損した肝細胞由来の細胞外小胞では，インテグリン α3β1 が増加した
新居 瞳 a，鎌田 佳宏 a，赤松 佑香 a，前田 恵 a，福田 友彦，顧 建国，三善 英知 a
（大阪大学保健学科 a）
第 76 回日本癌学会学術総会，横浜，2017 年 9 月，要旨集 p109
・2-フルオロフコースによるフコシル化の阻害はヒト肝がん細胞の細胞増殖・移動および腫瘍形成を抑制する
周 穎，福田 友彦，杭 慶雷，侯 思聡，伊左治 知弥，亀山 昭彦，顧 建国
第 11 回東北糖鎖研究会・GlycoTOKYO 2017，群馬，2017 年 11 月，要旨集 p111
・N-glycosylation in cell adhesion of cancer cells
顧 建国
システム糖鎖生物学と展望国際シンポジウム，和光市，2017 年 11 月，要旨集 p30
・N-glycosylation in cancer cells and towards application
顧 建国
第 9 回南京腫瘍診断治療国際会議，南京市，2017 年 11 月，要旨集 p102
・Importance of N-glycosylation in cell adhesion and EMT
顧 建国
IUBMB Special Meeting Frontiers in Glycoscience II: Oncology，台北市，2017 年 12 月，要旨集 p45-46
・Function of N-glycans in cancer cells and toward clinical application
顧 建国
中国科学院 生物化学細胞研究所・国家重点研究拠点セミナー，上海市，2018 年 3 月
・Importance and complexity of N-glycosylation and its possibilities for clinical application
顧 建国
復旦大学 中国衛生部複合糖質重点研究拠点セミナー，上海市，2018 年 3 月<分子認識部門>

・SAL 処理による抗がん剤取り込み促進に関与する Gb3 分子種の解析菅原 栄紀，石川 幼尋，立田 岳生，藤村 務，細野 雅祐
第 36 回日本糖質学会年会, 旭川，2017 年 7 月, 要旨集 p.117
・悪性中皮腫に対するレクザイムの in vivo における抗腫瘍効果およびペメトレキセドとの併用効果
立田 岳生，佐藤 稔之，細野 雅祐
第 76 回日本癌学会学術総会，横浜，2017 年 9 月，演題番号 P-1408
・ナマズ卵レクチン処理バーキットリンパ腫細胞において発現が変化する分子の探索
石川 幼尋，菅原 栄紀，立田 岳生，細野 雅祐
第 56 回日本薬学会東北支部大会, 青森，2017 年 10 月, 要旨集 p.42
・ナマズ卵レクチンは腎癌細胞において sunitinib の効果を増強する
伊藤 淳，岩村 大径，阿南 剛，海法 康裕，立田 岳生，菅原 栄紀，細野 雅祐，佐藤 信
第 11 回東北糖鎖研究会, 桐生，2017 年 11 月, P.31
・ナマズ卵レクチン処理細胞における薬物取り込みおよび排出機構について
本田 捷太，菅原 栄紀，石川 幼尋，立田 岳生，伊藤 淳，佐藤 信，細野 雅祐

Ĺŀ第 11 回東北糖鎖研究会, 桐生，2017 年 11 月, P.47
・ナマズ卵レクチンによる抗がん剤排出抑制機構の解明
菅原 栄紀，石川 幼尋，本田 捷太，立田 岳生，細野 雅祐
2017 年度生命科学系学会合同年次大会, 神戸，2017 年 12 月, 1P-0046
・悪性中皮腫に対するシアル酸結合性レクチン (cSBL) と他剤との併用効果
佐藤 稔之，立田 岳生，菅原 栄紀，原 明義，細野 雅祐
2017 年度生命科学系学会合同年次大会 (第 90 回日本生化学会大会)，神戸，2017 年 12 月，演題番号 2P-0914
・シアル酸結合性レクチン（cSBL）の EGFR pathway への影響
佐藤 稔之，立田 岳生，菅原 栄紀，原 明義、細野 雅祐
日本薬学会第 138 年会, 金沢，2018 年 3 月，演題番号 27J-am12S
・ナマズ卵レクチンの処理による細胞増殖抑制にかかわる分子の発現調節機構について
石川 幼尋，菅原 栄紀，立田 岳生，細野 雅祐
日本薬学会第 138 年会, 金沢，2018 年 3 月，演題番号 27J-pm16

ĺķExperimental Cell Research 350 (2017) 103–114

Contents lists available at ScienceDirect

Experimental Cell Research
journal homepage: www.elsevier.com/locate/yexcr

PDMP, a ceramide analogue, acts as an inhibitor of mTORC1 by inducing its
translocation from lysosome to endoplasmic reticulum

MARK

Takashi Odea,b, Katarzyna A. Podyma-Inouec, Kazue Terasawac, Jin-ichi Inokuchid,⁎Toshihide Kobayashie,f, Tetsuro Watabec, Yuichi Izumia, Miki Hara-Yokoyamac,aDepartment of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyoku, Tokyo 113-8510, Japanb
Research Fellow of the Japan Society for the Promotion of Science (JSPS), 5-3-1 Kojimachi, Chiyoda-ku, Tokyo 102-0083, Japanc
Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku,
Tokyo 113-8510, Japand
Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima,
Aoba-ku, Sendai, Miyagi 981-8558, Japane
Lipid Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japanf
CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch, France

A R T I C L E I N F O

A BS T RAC T

Keywords:
PDMP
mTOR
Subcellular localization
LysosomeER
Osteoblast

Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth,
metabolism, and cell diﬀerentiation. Recent studies have revealed that the recruitment of mTORC1 to lysosomes
is essential for its activation. The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol(PDMP), a well known glycosphingolipid synthesis inhibitor, also aﬀects the structures and functions of variousorganelles, including lysosomes and endoplasmic reticulum (ER). We investigated whether PDMP regulates themTORC1 activity through its eﬀects on organellar behavior. PDMP induced the translocation of mTORC1 fromlate endosomes/lysosomes, leading to the dissociation of mTORC1 from its activator Rheb in MC3T3-E1 cells.
Surprisingly, we found mTORC1 translocation to the ER upon PDMP treatment. This eﬀect of PDMP was
independent of its action as the inhibitor, since two stereoisomers of PDMP, with and without the inhibitor
activity, showed essentially the same eﬀect. We conﬁrmed that PDMP inhibits the mTORC1 activity based on
the decrease in the phosphorylation of ribosomal S6 kinase, a downstream target of mTORC1, and the increase
in LC3 puncta, reﬂecting autophagosome formation. Furthermore, PDMP inhibited the mTORC1-dependent
osteoblastic cell proliferation and diﬀerentiation of MC3T3-E1 cells. Accordingly, the present results reveal anovel mechanism of PDMP, which inhibits the mTORC1 activity by inducing the translocation of mTOR fromlysosomes to the ER.

1. Introduction

has been identiﬁed as the major intracellular site where the multiple
signals are integrated to activate mTORC1 [10]. In the presence ofamino acids, mTORC1 is recruited to LE/Ly membranes and activatedby the mTORC1 activator Rheb, which is constitutively located on LE/
Ly membranes [11]. The Ragulator complex and Rag GTPase are
essential for the recruitment of mTORC1 to LE/Ly, and the Ragulator
complex acts as a scaﬀold for mTORC1 at LE/Ly membranes [11–13].
Whereas the tethering of mTORC1 to LE/Ly membranes is required for
mTORC1 activation, the inactivation of mTORC1 occurs through thedissociation of mTORC1 from LE/Ly and the recruitment of TSC2, anegative regulator of Rheb, to LE/Ly membranes in the absence ofamino acids [14] or growth factors [15]. Thus, the association ofmTORC1 with LE/Ly is essential for cell growth, proliferation, andMammalian (mechanistic) target of rapamycin (mTOR) is a serine/
threonine kinase that functions as the master regulator of cell
metabolism and growth [1]. mTOR forms two distinct complexes,mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).
mTORC1 regulates protein synthesis, cell proliferation, diﬀerentiation,
and autophagy [1–3]. mTORC2 controls cytoskeleton organization andcell survival [1,2,4]. Recent studies revealed that mTORC1 also plays
an important role in osteoblast diﬀerentiation, both in vitro and invivo[5–9].
mTORC1 is activated in response to growth factors, amino acids,
and energy status [1–3]. Recently, late endosome/lysosome (LE/Ly)⁎

Corresponding author.
E-mail address: m.yokoyama.bch@tmd.ac.jp (M. Hara-Yokoyama).

http://dx.doi.org/10.1016/j.yexcr.2016.11.011
Received 2 August 2016; Received in revised form 3 November 2016; Accepted 15 November 2016
Available online 16 November 2016
0014-4827/ © 2016 Elsevier Inc. All rights reserved.

Experimental Cell Research 350 (2017) 103–114

T. Ode et al.

diﬀerentiation.
Ceramides, composed of a sphingosine base linked to a fatty acid,are the precursors of all sphingolipids, including sphingomyelin andglycolipids. In addition to their structural role, ceramides act as
bioactive molecules that regulate various cellular responses, including
metabolism, apoptosis, and diﬀerentiation [16–19]. Many studies havedemonstrated the biological actions of ceramides on the “plasma”
membrane. For example, studies on ceramides in insulin resistancesuggested that ceramides suppress the activation of Akt/protein kinase
B (PKB) on the plasma membrane, which plays a crucial key role in cell
growth, proliferation, survival, and metabolism [20,21]. The ceramideinduced inhibition of the Akt/PKB signaling may also underlie theapoptosis-inducing eﬀects of ceramides in numerous types of cancer
cells [22]. One part of ceramides is generated from sphingomyelin byneutral sphingomyelinase 2 (nSMase2), localized in the plasma membrane. The deﬁciency of nSMase2 reportedly causes impaired bone andtooth mineralization in mice [23,24], suggesting the importance of theceramides on the plasma membrane for osteogenesis [25].
Endogenous ceramides generated by de novo synthesis are rapidly
converted into sphingolipid species, whereas exogenously added ceramide analogues that cannot be metabolized are accumulated within
intracellular “organelles”. Ceramide analogues reportedly act on the
mitochondrial membrane to induce apoptosis [26]. However, thebiological actions of ceramides or ceramide analogues on otherorganelle membranes have not been suﬃciently investigated.
The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino1-propanol (PDMP) inhibits glucosylceramide synthase, leading to the
suppression of glycosphingolipid biosynthesis [27]. PDMP prevents
atherosclerosis and cardiac hypertrophy [28,29] and augments theactivities of several anti-cancer agents, including the MEK/ERK
inhibitor AZD-6244 [30,31]. Apart from the inhibitory eﬀect on
glucosylceramide synthase, PDMP has also been reported to alter theorganization of LE/Ly [32,33] and the endoplasmic reticulum (ER)[34]. This action of PDMP on the organellar morphology can be clearly
distinguished from that on the glycosphingolipid biosynthesis by theuse of two distinct stereoisomers of PDMP. D-threo-PDMP (D-PDMP),
but not its stereoisomer L-threo-PDMP (L-PDMP), inhibits glucosylceramide synthase [27], whereas both PDMP isomers aﬀect organellarorganization [33,34]. In addition, the inhibitory eﬀect of PDMP on themTORC1 activity has been reported [30]. Therefore, we considered
PDMP to be an excellent material to evaluate the biological potential of
ceramide analogues on intracellular organelles.
The objective of the present study was to clarify whether theceramide analogue PDMP regulates the mTORC1 activity by aﬀecting
its subcellular localization. We used the pre-osteoblastic cell line
MC3T3-E1, in which PDMP suppressed the cell proliferation and theosteoblastic diﬀerentiation. We successfully linked the eﬀects of PDMP
on the mTORC1 activity and the intracellular organelles: PDMP causes
the translocation of mTOR from LE/Ly membranes to the ER and thesegregation of mTOR from its activator Rheb, leading to the inhibitionof the mTORC1 activity. The present study provides a new perspectiveon the biological actions of ceramide analogues on LE/Ly membranes.
2. Materials and methods
2.1. Compounds
The syntheses of D-PDMP and L-PDMP, were performed as
described previously [27]. D-threo-1-phenyl-2-benzyloxycarbonylamino-3-pyrrolidino-1-propanol (PBPP), a newly developed glucosylceramide synthase inhibitor, was synthesized according to our previous
report [35]. D-PDMP, L-PDMP, and PBPP were dissolved in water. The
structures of D-PDMP, L-PDMP, and PBPP are shown in Fig. 1A.
Rapamycin was purchased from Focus Biomolecules (Pennsylvania,USA).
2.2. Cell culture
The mouse pre-osteoblastic cell line (MC3T3-E1) was obtained
from the RIKEN Bioresource Center (RCB1126). The cells were seeded
and maintained in growth medium consisting of α-modiﬁed MEM(Sigma-Aldrich Japan, Tokyo, Japan), supplemented with 10% fetal
bovine serum (Sigma-Aldrich Japan, Tokyo, Japan), 4 mM L-glutamine, L-penicillin (50 U/ml), and streptomycin (50 μg/ml), at 37 °C in
a 5% CO2 atmosphere. To induce osteoblastic diﬀerentiation, MC3T3E1 cells were cultured in osteogenic medium, consisting of growth
medium supplemented with β-glycerophosphate (10 mM), ascorbic
acid (50 μg/ml), and dexamethasone (10 nM).
2.3. Immunocytochemistry
MC3T3-E1 cells were cultured on 15-mm round glass coverslips in
12-well plates (4×104 cells/well). After 24 h, the medium was exchanged from growth medium to osteogenic medium in the absence(control) or presence of the compounds. The cells were washed oncewith PBS and ﬁxed with 3 or 4% paraformaldehyde (PFA) in PBS,

D-PDMP

L-PDMP

OH

OH

PBPP
OH

O N

O N
CH3

HN
O

N
CH3

HN

Control

D-PDMPO

HN
OO

L-PDMP

PBPP

20 μm

KDEL

Fig. 1. D-PDMP and L-PDMP, but not PBPP, aﬀect the ER morphology. (A) The structures of ceramide analogues. D-PDMP is an inhibitor of glucosylceramide synthase, but L-PDMP, a
stereoisomer of D-PDMP, lacks this inhibitory activity. PBPP is a newly developed, more potent inhibitor of glucosylceramide synthase. (B) The ER morphology is altered by treatmentswith D-PDMP and L-PDMP, but not PBPP. MC3T3-E1 cells were seeded on 15-mm round glass coverslips in 12-well plates (4×104 cells/well). After 24 h, the cells were treated withoutor with 40 μM D-PDMP, 40 μM L-PDMP, or 40 μM PBPP in osteogenic medium for 24 h. Untreated cells cultured in osteogenic medium for 24 h were used as the control. The cells were
ﬁxed with 4% PFA for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and then labeled with an anti-KDEL antibody. Cells are outlined with white dashed lines.

104Experimental Cell Research 350 (2017) 103–114

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followed by permeabilization with 0.1% Triton X-100 or digitonin (50or 100 μg/ml) in PBS containing 0.1 M glycine. The cells were thenwashed with PBS, blocked with 1% bovine serum albumin (BSA) in PBS
for 30 min at RT, and then stained with the primary antibodies for 1 h
at RT. The primary antibodies used in the present study were against
the ER marker KDEL (sc-58774, 1:400; Santa Cruz Biotechnology, CA,USA), mTOR (2983, 1:400; Cell Signaling Technology, MA, USA), theLE/Ly marker LBPA (1:20; prepared as described [36]), LAMTOR1(8975, 1:100; Cell Signaling Technology, MA, USA), and the autophagosome marker LC3 (PM036, 1:500; MBL, Nagoya, Japan).
Subsequently, the cells were washed three times with 0.1% BSA in
PBS and incubated with an Alexa Fluor 488-conjugated anti-rabbit
antibody (A11008, 1:200; Invitrogen, OR, USA) and/or an Alexa Fluor
555-conjugated anti-mouse antibody (A21422, 1:200; Invitrogen, OR,
USA). Images were captured using a FLUOVIEW FV10i laser confocal
microscope (Olympus, Tokyo, Japan).
Co-localization analysis was performed using the FV10-ASW software (Olympus, Tokyo, Japan). The co-localization ratio was quantiﬁed
as described [15,37,38]. Brieﬂy, the ratio of the number of mTOR
pixels co-localized with LBPA or KDEL pixels to the total number ofmTOR pixels was calculated, as expressed as the percentage. At least 20
cells were randomly chosen for co-localization analysis for eachexperimental condition.

Western Blotting Detection Reagent (GE Healthcare Japan, Tokyo,
Japan). The band intensities of S6K and P-S6K were determined using
the ImageJ software (NIH, MD, USA), and the intensities of the P-S6K
bands relative to those of the S6K bands were calculated.
2.6. Cell proliferation assay
MC3T3-E1 cells were seeded in 96-well plates (2×103 cells/well)and treated without or with D-PDMP, L-PDMP, PBPP, or rapamycin in
osteogenic medium for 1, 2, and 3 days. The cell proliferation assay was
performed using a WST-8 assay (Cell Counting Kit-8, Dojindo,
Kumamoto, Japan), according to the manufacturer's instructions withminor modiﬁcations. The absorbance was measured at 450 nm using a
microplate reader (Model 680, Bio-Rad, CA, USA).
2.7. Alkaline phosphatase (ALP) staining
The cells were plated in 12-well plates (1×104 cells/well). After
24 h, the medium was exchanged from growth medium to osteogenic
medium in the absence or presence of D-PDMP, L-PDMP, PBPP, or
rapamycin, and the cells were then incubated for 12 days. The
osteogenic medium without or with these compounds was changed
every 3 days. The cells were washed three times with PBS and ﬁxed
with 4% PFA in PBS for 10 min at RT. Subsequently, the cells wereincubated with ALP staining solution, consisting of 0.2 M Tris-HCl, pH
8.6, 50 mM MgCl2, 0.01% naphthol AS-MX phosphate (Sigma-Aldrich
Japan, Tokyo, Japan), and 0.06% Fast Blue BB Salt (Sigma-Aldrich
Japan, Tokyo, Japan), for 30 min at RT, and then washed three times
with MilliQ water.

2.4. Co-immunoprecipitation
MC3T3-E1 cells were transfected with pRK7-Rheb (15888,
Addgene, MA, USA), the plasmid encoding FLAG-tagged Rheb, using
the FuGENE6 transfection reagent (Promega, WI, USA). The cells were
washed with PBS and lysed with 1% Triton X-100 in buﬀer A (20 mM
Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM iodoacetamide, 2.5 mM NaF,
2.5 mM sodium pyrophosphate, 10 mM EDTA, 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl
ﬂuoride, 5 μg/ml leupeptin, 10 μg/ml pepstatin, and 10 μg/ml aprotinin, pH 7.5) for 60 min on ice. The lysates were then centrifuged for
15 min at 15,000g, and the supernatants were incubated with antiFLAG M2 aﬃnity gel (Sigma-Aldrich Japan, Tokyo, Japan) for 120 min
at 4 °C with rotation. The immune-complexes were washed with PBS
and subjected to immunoblotting. A biotinylated polyclonal rabbit antiFLAG antibody (GTX77459, 1:1,000; GeneTex, CA, USA), a monoclonal anti-FLAG M2 antibody (F1804, 1:2,000; Sigma-Aldrich Japan,
Tokyo, Japan), and the mTOR antibody (1:1,000) were used for the
immunoblotting of immunoprecipitants (IP), cell lysates, and both,respectively.

2.8. Real-time RT-PCR
The cells were seeded in 6-well plates (2.25×104 cells/well). After
24 h, the medium was changed from growth medium to osteogenic
medium in the absence or presence of D-PDMP, L-PDMP, PBPP, or
rapamycin (Day 0), and the cells were then incubated for 14 days (Day14). Total RNA was isolated using an RNAqueous-4PCR Kit (Ambion,
TX, USA), according to the manufacturer's instructions. The cDNAs
were obtained from 1 μg of RNA, using a High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, CA, USA). Quantitative PCR
was performed using SYBR Premix Ex Taq II (TaKaRa, Shiga, Japan)
and a Thermal Cycler Dice Real-Time System II (TaKaRa, Shiga,
Japan). The primers used were 5′-GCGTCAACACCATCATTCTG-3′
and 5′-CAGACCAGCAGCACTCCATA-3′ (Runx2), 5′-AAGGCGGTTGGCAATAGTGG-3′ and 5′-CCGCTCTAGCTCCTGACAGT-3′ (Osx), and5′-AGGACCAGGTTGTCTCCTGT-3′ and 5′-TTACTCCTTGGAGGCCATGT-3′ (GAPDH). Ampliﬁcation conditions were as follows: 95 °C for
5 s, 55 °C for 10 s, and 72 °C for 20 s, with 45 cycles. The mRNA
expression levels of the target genes were normalized to those of
GAPDH, and the relative ratios of the expression levels in the Day 14
group to those in the Day 0 group were calculated.

2.5. Immunoblotting
The cells were lysed with 1% Triton X-100 in buﬀer A for 15 min on
ice, and then centrifuged for 10 min at 15,000g. The supernatants were
subjected to 10% SDS-PAGE, and then transferred to PVDF membranes.
For the detection of ribosomal S6 kinase (S6K), phosphorylated S6K (PS6K), or mTOR, the membranes were incubated with 5% BSA in TBS-T
for 1 h, followed by an incubation with primary antibodies overnight at
4 °C. The membranes were then washed three times with TBS-T andincubated with secondary antibodies for 1 h. When the polyclonal rabbit
biotinylated anti-FLAG antibody or the monoclonal anti-FLAG M2
antibody was used, the membranes were incubated with 1% BSA in
TBS-T or with 4% Block Ace (Yukijirushi, Tokyo, Japan), respectively, for
1 h, and incubated with each primary antibody for another 1 h at RT.
The primary antibodies against S6K (9202, 1:500; Cell Signaling
Technology, MA, USA), P-S6K (9205, 1:500; Cell Signaling
Technology, MA, USA), and α-tubulin (ab4074, 1:10,000; Abcam,
Tokyo, Japan) were used. The secondary antibodies were an HRPconjugated anti-rabbit antibody (1858415, Pierce, IL, USA) and an HRPlinked anti-biotin antibody (7075 S, Cell Signaling Technology, MA,
USA). The immunoblot signals were visualized using the ECL Prime

2.9. Statistical analysis
Diﬀerences between diﬀerent groups were analyzed using theStudent's t-test or Welch's t-test. All experiments were repeated atleast twice, with triplicate samples.
3. Results
3.1. The alteration of ER morphology in MC3T3-E1 cells induced by
PDMP is independent of its inhibitory action related toglycosphingolipid synthesis
PDMP has been widely used as an inhibitor of glucosylceramide
synthase. In addition, PDMP aﬀects the morphology of the ER in HeLa
105

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T. Ode et al.

cells [34] and the LE/Ly in human skin ﬁbroblasts [33]. In the present
study, we focused on the eﬀects of PDMP on the organellar morphology.
In order to discriminate between the inhibitor activity-dependent and-independent eﬀects, we used three ceramide analogues, D-PDMP and
its stereoisomer L-PDMP, as well as the newly developed glucosylceramide synthase inhibitor PBPP. D-PDMP and PBPP, but not L-PDMP,
showed inhibitory activity against glucosylceramide synthase [27,35].
The glucosylceramide levels in the D-PDMP- and PBPP-treated MC3T3E1 cells were signiﬁcantly lower than that in the untreated control cells,
while the glucosylceramide level in the L-PDMP-treated cells was similar
to that in the control cells (Supplementary Fig. S1). In our system,
MC3T3-E1 cells were treated without or with D-PDMP, L-PDMP, or
PBPP, followed by an immunocytochemical analysis with the anti-KDEL(an ER marker) antibody. The anti-KDEL antibody recognizes thesequence Lys-Asp-Glu-Leu (KDEL) at the carboxy-terminus of the ERresident proteins [39]. The staining pattern of KDEL in the D-PDMP- or
L-PDMP-treated cells was altered, as compared with that in control cells,
and the intensity was appreciably increased in the presence of PDMP
(Fig. 1B), likely due to the PDMP-dependent restructuring of the ER[34]. In contrast, PBPP had no obvious eﬀect on the KDEL-staining
pattern. These results suggested that PDMP aﬀects the ER architecture in
MC3T3-E1 cells, and this eﬀect is independent of the inhibition of
glucosylceramide synthase.

experiments revealed the presence of mTOR in the membrane fraction,rather than the cytosolic fraction, under normal conditions(Supplementary Fig. S2). Since PDMP reportedly aﬀects the function
and morphology of LE/Ly [32,33], we investigated the eﬀects ofceramide analogues on the subcellular localization of mTOR. MC3T3E1 cells were co-stained with anti-mTOR and anti-LBPA (an LE/Ly
marker) antibodies. In control cells, both mTOR and LBPA exhibited
punctate staining patterns (Fig. 2A). In contrast, a non-punctate
ﬂuorescence pattern was observed in both the D-PDMP- and L-PDMPtreated cells. No appreciable changes were seen in the PBPP-treated cells,
as compared to control cells. A co-localization analysis of mTOR and
LBPA revealed that mTOR was speciﬁcally co-localized with LBPA in thecontrol group (Fig. 2B). The co-localization ratio was signiﬁcantly
decreased in the D-PDMP-treated group and the L-PDMP-treated group,
while no signiﬁcant diﬀerence was observed between the control groupand the PBPP-treated group, suggesting that PDMP induces the dissociation of mTOR from LE/Ly. Similar results were obtained in a mousebone marrow-derived stromal cell line (ST2) and a mouse embryonicﬁbroblast cell line (NIH3T3) (Supplementary Fig. S3).
In order to study the kinetic actions of D-PDMP, we performed atime-course analysis of the decrease in the co-localization between
mTOR and LBPA upon D-PDMP treatment. The decrease occurredsigniﬁcantly 12 h after D-PDMP treatment (Fig. 3). We also investigated the eﬀects of the culture medium composition on the subcellular
localization of mTOR, and found that the co-localization between
mTOR and LBPA remained unchanged in MC3T3-E1 cells cultured
in growth medium or osteogenic medium without D-PDMP at any time
point (Supplementary Fig. S4).
As shown in Fig. 2A and Supplementary Fig. S3 A and C, the intensity

3.2. Dissociation of mTOR from LE/Ly induced by PDMP
The recruitment of mTOR to LE/Ly membranes is required for its
activation [11]. mTOR is dissociated from LE/Ly in the absence of aminoacids [14] or growth factors [15]. In the present study, cell fractionation

mTOR

LBPA

Merge
100

Co-localization ratio (%)

90

Control
20 μm

D-PDMP

80
70
60
50

***

40

***

30
20
10

PBPP

IP:FLAG

PBPP

Lysate

+

P
M
PD

+

L-

−

−

-P

−

D

Inhibitors:
FLAG-Rheb:

D

M

L-PDMP+PB
PPControl D-PDMP L-PDMP

P

0

+

mTOR
FLAG-Rheb
mTOR
FLAG-Rheb

Fig. 2. mTOR is dissociated from LE/Ly by D-PDMP and L-PDMP treatments. (A) The co-localization of mTOR with LBPA is decreased in the presence of D-PDMP and L-PDMP.
MC3T3-E1 cells were seeded on 15-mm round glass coverslips in 12-well plates (4×104 cells/well). After 24 h, the cells were treated without or with 40 μM D-PDMP, 40 μM L-PDMP, or
40 μM PBPP in osteogenic medium for 24 h. Untreated cells cultured in osteogenic medium for 24 h were used as the control. The cells were ﬁxed with 3% PFA for 20 min, permeabilizedwith 50 μg/ml digitonin for 5 min, and then labeled with an anti-mTOR antibody (green) and an anti-LBPA antibody (magenta). Cells are outlined with white dashed lines. (B)Quantiﬁcation of the co-localization between mTOR and LBPA in MC3T3-E1 cells. (C) The interaction between FLAG-tagged Rheb (FLAG-Rheb) and endogenous mTOR is impaired by
D-PDMP and L-PDMP treatments. MC3T3-E1 cells were seeded in 10-cm dishes (3×105 cells/dish). After 24 h, the cells were treated without or with 40 μM D-PDMP, 40 μM L-PDMP,
or 40 μM PBPP for 24 h, and then transfected or mock-transfected with the plasmid encoding FLAG-Rheb. Based on the results of preliminary experiments, we adjusted the amount of
the plasmid encoding FLAG-tagged Rheb, so that comparable amounts of the FLAG-Rheb protein were expressed under the four conditions (control, D-PDMP, L-PDMP, and PBPP).
After the immunoprecipitation of FLAG-Rheb, the co-immunoprecipitated mTOR was detected by immunoblotting. Data are expressed as means ± SD. ***P < 0.001 compared with
control group.

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T. Ode et al.

of the LBPA ﬂuorescence was lower in the D-PDMP-treated cells than in
the control cells. Using immunocytochemistry for LAMP-1, anotherlysosome marker, we analyzed whether this phenomenon was due tothe decreased number of LE/Ly or the reduced reactivity of LBPA against
the anti-LBPA antibody. The number of LAMP-1-positive dots was notdecreased in the D-PDMP-treated cells (Supplementary Fig. S5), suggesting that D-PDMP does not aﬀect the LE/Ly biogenesis. PDMP
associates with the acidic compartment due to its positively charged
amino group, where LBPA (a unique acidic phospholipid) is enriched[36]. Since PDMP induces the formation of multilamellar structures of
the LBPA-containing membranes [33], PDMP may cause the decreased
accessibility or reactivity of the anti-LBPA antibody to LBPA.
Furthermore, the staining for ICDH and GOLGA5, markers of
mitochondria and Golgi, respectively, revealed that D-PDMP does notalter the subcellular localizations of mitochondria and Golgi(Supplementary Figs. S5 and S6). Considering the characteristic
morphologies of mitochondria and Golgi, it is apparent that mTOR is
not present on these organelles.

GM

OM

24 h

24 h

Since the localization of mTOR on the LE/Ly membranes isrequired for the interaction of mTORC1 with its activator Rheb
[10,11], we investigated whether PDMP weakens the interaction
between mTOR and Rheb. Co-immunoprecipitation assays of mTOR
and Rheb in MC3T3-E1 cells demonstrated that the level of coimmunoprecipitated mTOR was decreased in the presence of DPDMP or L-PDMP (Fig. 2C), suggesting that PDMP causes thedissociation of mTORC1 from LE/Ly and reduces the interaction
between mTOR and Rheb.

3.3. mTOR translocation to ER upon PDMP treatment
D-PDMP treatment causes the dissociation of mTOR from LE/Ly,
as shown in Figs. 2 and 3 and Supplementary Fig. S3. However, the cell
fractionation experiments demonstrated that mTOR is not translocatedto the cytosol and remained localized at the membrane after D-PDMP
treatment (Supplementary Fig. S2). To identify the subcellular localization of the mTOR that dissociated from LE/Ly in ceramide analogue-

OM + D-PDMP
6h

12 h

18 h

24 h

mTOR
20 μm

LBPA

Merge

100

Co-localization ratio (%)

90

ns

ns

80

***
***

70
60

ns

50
***

40

***

30
20
10
0

24 h

24 h

GM

OM

6h

12 h

18 h

24 h

OM + D-PDMP

Fig. 3. Kinetics of mTOR dissociation from LE/Ly by D-PDMP. (A) Time-course analysis of the co-localization of mTOR with LBPA. MC3T3-E1 cells were seeded on 15-mm round glass
coverslips in 12-well plates (4×104 cells/well). After 24 h, the cells were treated without or with 40 μM D-PDMP in growth medium or osteogenic medium for the indicated times. The
cells were ﬁxed with 3% PFA for 20 min, permeabilized with 50 μg/ml digitonin for 5 min, and then labeled with an anti-mTOR antibody (green) and an anti-LBPA antibody (magenta).
Cells are outlined with white dashed lines. (B) Quantiﬁcation of the co-localization between mTOR and LBPA in MC3T3-E1 cells. Data are expressed as means ± SD. ***P < 0.001; ns(not signiﬁcant), P > 0.05 compared with OM 24-h group. Abbreviations: GM, cells cultured in growth medium; OM, cells cultured in osteogenic medium.

107

Experimental Cell Research 350 (2017) 103–114

T. Ode et al.

mTOR

KDEL

Merge
100

20 μm

Co-localization ratio (%)

90

Control

D-PDMP

***

***

80
70
60
50
40
30
20
10
0

Control D-PDMP L-PDMP

PBPP

L-PDMP

PBPP

Fig. 4. mTOR is translocated to the ER by D-PDMP or L-PDMP treatment. (A) The co-localization of mTOR with KDEL is increased in the presence of D-PDMP and L-PDMP. MC3T3E1 cells were seeded on 15-mm round glass coverslips in 12-well plates (4×104 cells/well). After 24 h, the cells were treated without or with 40 μM D-PDMP, 40 μM L-PDMP, or 40 μM
PBPP in osteogenic medium for 24 h. Untreated cells cultured in osteogenic medium for 24 h were used as the control. The cells were ﬁxed with 4% PFA for 15 min, permeabilized with
0.1% Triton X-100 for 5 min, and then labeled with an anti-mTOR antibody (green) and an anti-KDEL antibody (magenta). Cells are outlined with white dashed lines. (B) Quantiﬁcationof the co-localization between mTOR and KDEL in MC3T3-E1 cells. Data are expressed as means ± SD. ***P < 0.001 compared with control group.

treated cells, the cells were co-stained with anti-mTOR and anti-KDEL
antibodies, because the mTOR-staining pattern resembled the KDELstaining pattern when the cells were treated with D-PDMP or L-PDMP,
as shown in Figs. 1B and 2A. The immunoﬂuorescence double staining
and the subsequent co-localization analysis revealed that both mTOR
and KDEL showed the non-punctate ﬂuorescence patterns in D-PDMPtreated cells and L-PDMP-treated cells, and that the co-localization
ratios between mTOR and KDEL were signiﬁcantly increased in the DPDMP-treated and L-PDMP-treated groups, as compared with thecontrol group (Fig. 4), clearly indicating the translocation of mTOR
from LE/Ly to the ER. Consistent with our mTOR and LBPA double
staining results, PBPP had no apparent eﬀect on mTOR localization.
Similar eﬀects of D-PDMP on the co-localization between mTOR and
KDEL were also observed in ST2 cells and NIH3T3 cells(Supplementary Fig. S7). The increase in the co-localization between
mTOR and KDEL upon D-PDMP treatment occurred signiﬁcantly 12 hafter D-PDMP treatment (Fig. 5), which inversely corresponds to thedecreased co-localization of mTOR with LE/Ly. The co-localization
between mTOR and KDEL remained unchanged in MC3T3-E1 cells
cultured in growth medium or osteogenic medium in the absence of DPDMP at any time point (Supplementary Fig. S8).
We noticed that the signal intensity of mTOR in the mTOR/LBPA
staining decreased upon the PDMP treatment (Figs. 2 and 3), whereasthat in the mTOR/KDEL staining increased (Figs. 4 and 5). The
immunocytochemistry methods for the two staining procedures are
diﬀerent. Since the total surface area of the ER (~60%) is much larger
than that of LE/Ly (~1%), the distribution of mTOR can become
broader, when mTOR is translocated from LE/Ly to the ER. If theaccessibility of the anti-mTOR antibody to mTOR is unchanged, thenthe signal intensity of mTOR per unit area should be reduced. In
contrast, if the accessibility of the antibody to a sparse antigen issigniﬁcantly improved, then the signal intensity of mTOR per unit area
would be increased. The diﬀerence in the signal intensities of mTOR
may be explained by the possibilities that the accessibility of theantibody is unchanged for the mTOR/LBPA staining and increased forthe mTOR/KDEL staining upon PDMP treatment.
3.4. Changes of LAMTOR1 subcellular localization upon PDMP
treatment
LAMTOR1, also called p18, is an essential anchor protein of the
Ragulator complex. It functions as a scaﬀold to recruit mTORC1 to LE/
Ly [13,40]. The subcellular localization analysis of LAMTOR1 demonstrated that the LAMTOR1-ﬂuorescence signals that were co-localizedwith the LBPA-ﬂuorescence signals were signiﬁcantly decreased in the
D-PDMP-treated cells, as compared to control cells (Fig. 6 A and B). In
contrast, the D-PDMP treatment led to a signiﬁcant increase in the colocalization ratio between LAMTOR1 and KDEL (Fig. 6 C and D),
suggesting that not only mTOR but also LAMTOR1 is translocatedfrom LE/Ly to the ER by the treatment with PDMP.
Since LAMTOR1 is reportedly associated with lipid rafts [13], we
further analyzed the eﬀects of PDMP on the raft-localization of
LAMTOR1. Flotillin-1 was used as the raft marker of LE/Ly [41]. An
analysis of detergent-resistant membranes (DRMs) demonstrated thatthe lipid rafts were dramatically aﬀected by the D-PDMP treatment(Supplementary Fig. S9A), and that the proportion of LAMTOR1 in
DRMs was increased upon the D-PDMP treatment (Supplementary Fig.
S9 B and C). These results imply that the PDMP treatment exerts its
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A

GM

OM

24 h

24 h

OM + D-PDMP
6h

12 h

18 h

24 h

mTOR
20 μm

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Merge

B

ns
100

***

Co-localization ratio (%)

90
80

***

***

18 h

24 h

70
60
***

50
40
30
20

ns

ns

10
0

24 h

24 h

GM

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12 h

OM + D-PDMP

Fig. 5. Kinetics of translocation of mTOR to the ER by by D-PDMP. (A) Time-course analysis of the co-localization of mTOR with KDEL. MC3T3-E1 cells were seeded on 15-mm roundglass coverslips in 12-well plates (4×104 cells/well). After 24 h, the cells were treated without or with 40 μM D-PDMP in growth medium or osteogenic medium for the indicated times.
The cells were ﬁxed with 4% PFA for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, and then labeled with an anti-mTOR antibody (green) and an anti-KDEL antibody(magenta). Cells are outlined with white dashed lines. (B) Quantiﬁcation of the co-localization between mTOR and KDEL in MC3T3-E1 cells. Data are expressed as means ± SD. ***P <0.001; ns (not signiﬁcant), P > 0.05 compared with OM 24-h group. Abbreviations: GM, cells cultured in growth medium; OM, cells cultured in osteogenic medium.

eﬀects on LAMTOR1 by modulating lipid rafts.

increased in a time-dependent manner in the cells cultured inosteogenic medium (Fig. 7E, left panel). In contrast, in the D-PDMPand L-PDMP-treated groups, the mTORC1 activity increased after ashort-term treatment (6 h) and then decreased (Fig. 7E, middle andright panels, respectively), which implies that the inhibitory eﬀect of
PDMP is not one-step process, but requires several intermediate steps
to take place. The mechanisms of the transient and stimulatory eﬀects
should be elucidated in future work. In the present study, the timecourse experiments conﬁrmed that the inhibition of the mTORC1
activity by D-PDMP and L-PDMP was evident after treatments for
24 h.
The inactivation of mTORC1 induces autophagy [1–3]. Hence, we
assessed the activity of mTORC1 by monitoring the punctate formationof LC3 (an autophagosome marker). The results indicated that DPDMP and L-PDMP, but not PBPP, induced the LC3 puncta formation(Fig. 7F). Taken together, PDMP inhibits the mTORC1 activity,
regardless of the presence or absence of the glucosylceramide

3.5. PDMP inhibits mTORC1 activity
The mTORC1 activity depends on its recruitment to the LE/Ly
membranes [11,14,15]. Since PDMP caused the translocation ofmTORC1 from LE/Ly to the ER (Figs. 2 and 4), we monitored theeﬀects of PDMP on the mTORC1 activity based on the phosphorylationlevel of S6K, a downstream target of mTORC1. MC3T3-E1 cells weretreated with D-PDMP, L-PDMP, or PBPP. Rapamycin was used as apositive control of mTORC1 inhibition. As shown in Fig. 7A, the DPDMP and L-PDMP treatments, but not the PBPP treatment, dramatically decreased the level of P-S6K, indicating the inhibition ofmTORC1 activity. The level of P-S6K was decreased in a dosedependent manner in the D-PDMP- and L-PDMP-treated groups. In
contrast, the PBPP-treated group showed no signiﬁcant change even ata high concentration (50 μM) (Fig. 7 B–D). The mTORC1 activity
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LAMTOR1

LBPA

Merge
100

20 μm
Co-localization ratio (%)

90

Control

D-PDMP

80
70
60
50
40
30
20

***

10
0

LAMTOR1

KDEL

Control D-PDMP

Merge

Co-localization ratio (%)

60

Control
20 μm

D-PDMP

50

***

40
30
20
10
0

Control D-PDMP

Fig. 6. LAMTOR1 is translocated from LE/Ly to ER by D-PDMP treatment. (A) The co-localization of LAMTOR1 with LBPA is decreased in the D-PDMP-treated MC3T3-E1 cells.
MC3T3-E1 cells were seeded on 15-mm round glass coverslips in 12-well plates (4×104 cells/well). After 24 h, the cells were treated without or with 40 μM D-PDMP in osteogenic
medium for 24 h. Untreated cells cultured in osteogenic medium for 24 h were used as the control. The cells were ﬁxed with 3% PFA for 20 min, permeabilized with 50 μg/ml digitoninfor 5 min, and then labeled with an anti-LAMTOR1 antibody (green) and an anti-LBPA antibody (magenta). Cells are outlined with white dashed lines. (B) Quantiﬁcation of the colocalization between LAMTOR1 and LBPA in MC3T3-E1 cells. (C) The co-localization of LAMTOR1 with KDEL is increased in the D-PDMP-treated cells. MC3T3-E1 cells were seededon 15-mm round glass coverslips in 12-well plates (4×104 cells/well). After 24 h, the cells were treated without or with 40 μM D-PDMP in osteogenic medium for 24 h. Untreated cells
cultured in osteogenic medium for 24 h were used as the control. The cells were ﬁxed with 3% PFA for 20 min, permeabilized with 100 μg/ml digitonin for 10 min, and then labeled withan anti-LAMTOR1 antibody (green) and an anti-KDEL antibody (magenta). Cells are outlined with white dashed lines. (D) Quantiﬁcation of the co-localization between LAMTOR1 andKDEL in MC3T3-E1 cells. Data are expressed as means ± SD. ***P < 0.001 compared with control group.

synthase-inhibitory activity.

We evaluated the eﬀects of PDMP on the osteoblastic diﬀerentiationof MC3T3-E1 cells, using ALP staining as an indicator of osteogenesis.
Control cells, cultured in osteogenic medium without any treatmentswith compounds for 12 days, exhibited strongly positive ALP staining(Fig. 9A). In contrast, ALP staining was negative in the cells treatedwith rapamycin for 12 days, suggesting the importance of the mTORC1
activity in the diﬀerentiation process. In addition, the ALP staining
intensity was decreased by the treatment with D-PDMP or L-PDMP,
but not PBPP (Fig. 9A), and in a dose-dependent manner with DPDMP (Fig. 9B). Similar results were obtained in ST2 cells cultured inosteogenic medium for 21 days (Supplementary Fig. S11).
We measured the mRNA levels of two transcription factors, Runx2and Osx, early-stage markers of osteoblastic diﬀerentiation, in MC3T3E1 cells. Unlike the expression of the late-stage markers, the expressionof the early-stage markers does not exclusively depend on the mTORC1
activity, since the expression is not aﬀected by the deletion of Raptor(an essential component of mTORC1) [8]. Consistent with this notion,
rapamycin failed to abolish the expression of Runx2 and Osx.

3.6. Inhibition of mTORC1 activity by PDMP aﬀects osteoblastic
proliferation and diﬀerentiation
The contribution of mTORC1 inactivation to the inhibition of
osteoblastic cell proliferation has been conﬁrmed by several recent
studies [7,8]. Thus, we investigated the eﬀects of PDMP on osteoblastic
cell proliferation and diﬀerentiation, using MC3T3-E1 cells. The
proliferation rates of MC3T3-E1 cells treated with D-PDMP, LPDMP, or rapamycin for 3 days were dramatically decreased, as
compared with the control group, while no signiﬁcant diﬀerence wasobserved in the PBPP-treated group (Fig. 8A). Both D-PDMP and LPDMP inhibited the cell proliferation in dose-dependent manners(Fig. 8B). The decreased cell proliferation by the PDMP treatment is
not due to cell death, as the amounts of LDH release in all treatmentgroups were not signiﬁcantly diﬀerent from that in the untreatedcontrol group (Supplementary Fig. S10).
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R

ap

a

P
M

PB
PP

PD
L-

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D

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on
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M

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T. Ode et al.

P-S6K

P-S6K

S6K

S6K

α-tubulin

α-tubulin

P-S6K/S6K:

1.00

0.29

0.39

0.90

0.25

P-S6K/S6K:

0

10

20

30

40

50

Rapa

1.00

0.76

0.73

0.67

0.24

0.24

0.23

L-PDMP (μM)
0

10

20

30

PBPP (μM)
40

50

Rapa

P-S6K

P-S6K

S6K

S6K

α-tubulin

α-tubulin

P-S6K/S6K:

1.00

0.96

0.84

0.57

0.41

0.34

0.27

OM

Treatment
period (h) :P-S6K/S6K:

0

10

20

30

40

50

Rapa

1.00

1.01

1.00

0.85

0.86

0.89

0.21

Rapa

OM + D-PDMP

Rapa

OM + L-PDMP

6

12

18

24

6

12

18

24

24

6

12

18

24

24

0.30

0.45

0.74

1.00

0.90

0.82

0.47

0.32

0.23

1.07

0.41

0.37

0.32

0.17

P-S6K
S6K
α-tubulin
P-S6K/S6K:

Control

D-PDMP

L-PDMP

PBPP

Rapa

LC3

20 μm

Fig. 7. The mTORC1 activity is inhibited by D-PDMP and L-PDMP. (A) D-PDMP and L-PDMP, but not PBPP, decrease the mTORC1 activity in MC3T3-E1 cells. MC3T3-E1 cells wereseeded in 6-well plates (1.8×105 cells/well). After 24 h, the cells were treated without or with 40 μM D-PDMP, 40 μM L-PDMP, 40 μM PBPP, or 100 nM rapamycin in osteogenic
medium for 24 h. Untreated cells cultured in osteogenic medium for 24 h were used as the control. P-S6K/S6K is the ratio of the band intensity of P-S6K versus the total amount of S6K.
Data are expressed as the relative values to the control group. The phosphorylation levels of S6K (P-S6K), a downstream target of mTORC1, were decreased in the D-PDMP- and LPDMP-treated groups, as compared with the control group. (B–D) D-PDMP and L-PDMP, but not PBPP, decrease the mTORC1 activity in dose-dependent manners. MC3T3-E1 cells
were seeded in 6-well plates (1.8×105 cells/well). After 24 h, the cells were treated without or with the indicated concentrations of D-PDMP, L-PDMP, or PBPP in osteogenic medium for
24 h. Rapamycin (100 nM for 24 h) was used as a positive control. Each P-S6K/S6K shows the relative ratio to that in the absence of drugs. (E) D-PDMP and L-PDMP time-dependently
reduce the levels of P-S6K. MC3T3-E1 cells were seeded in 6-well plates (1.8×105 cells/well). After 24 h, the medium was exchanged from growth medium to osteogenic medium in theabsence or presence of 40 μM D-PDMP, 40 μM L-PDMP, or 100 nM rapamycin for the indicated times. P-S6K/S6K shows the relative ratio to the OM 24-h group. (F) D-PDMP and LPDMP, but not PBPP, induce the formation of LC3-positive puncta (autophagosomes). MC3T3-E1 cells were seeded on 15-mm round glass coverslips in 12-well plates (4×104 cells/well). After 24 h, the cells were treated without or with 40 μM, D-PDMP, 40 μM L-PDMP, 40 μM PBPP, or 100 nM rapamycin in osteogenic medium for 24 h. Untreated cells cultured inosteogenic medium for 24 h were used as the control. The cells were ﬁxed with 4% PFA for 10 min and then permeabilized with 100 μg/ml digitonin for 10 min. To detect theendogenous LC3 protein, the cells were labeled with an anti-LC3 antibody (green). Abbreviations: OM, cells cultured in osteogenic medium; Rapa, rapamycin.

Nonetheless, the expression levels of Runx2 and Osx were signiﬁcantly
reduced in the D-PDMP- and L-PDMP-treated groups, as comparedwith the control group (Fig. 9 C and D), suggesting that PDMP
suppresses the early stage of osteoblastic diﬀerentiation by an
mTORC1-independent mechanism.
It is noteworthy that L-PDMP tended to inhibit cell proliferationand osteoblastic diﬀerentiation more than D-PDMP (Figs. 8 and 9),
whereas the apparent diﬀerence between these compounds was notobserved in the P-S6K levels (Fig. 7A) (We will discuss this point
later.). These results suggested that PDMP inhibits not only cellproliferation but also the early stage of osteoblastic diﬀerentiation,
through the inactivation of mTORC1.
Collectively, PDMP suppresses the proliferation and osteoblastic
diﬀerentiation of MC3T3-E1 cells in an mTORC1 activity-dependent
manner. PDMP also exerts the mTORC1-independent action to inhibit

the early stage of osteoblastic diﬀerentiation.
4. Discussion
Accumulating evidence has revealed that the LE/Ly surface is a
crucial site for the activation of mTORC1 [10]. In the present study,
using an MC3T3-E1 model, we demonstrated that the ceramide
analogue PDMP induces the translocation of both mTORC1 and its
scaﬀold protein LAMTOR1 from LE/Ly to the ER, and suppresses theactivation of mTORC1. The time-course experiments strongly support
the proposal that mTOR is translocated from LE/Ly to the ER in thepresence of PDMP, which is closely related to the inhibition of themTORC1 activity (Figs. 3, 5, and 7E). We also found that PDMP
impairs the association of mTORC1 with its activator Rheb, suggesting
the PDMP-dependent spatial control of the mTORC1 signaling by the111

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1.4
Control
D-PDMP
L-PDMP
PBPP
Rapamycin

1.2
1.0
0.8
0.6

Absorbance (450 nm)

Absorbance (450 nm)

1.4

***
***

0.4
0.2
*

0.0

***

*
**
**

1

2

1.2
1.0

*

*

0.8
***

0.6

***

D-PDMP
L-PDMP

0.4

***

0.2
0.0

3

0

5

Time (days)

10

20

30

40

Concentration (μM)

Fig. 8. MC3T3-E1 cell proliferation is inhibited by D-PDMP and L-PDMP. (A) Treatments with D-PDMP and L-PDMP, but not PBPP, for 3 days reduce the proliferation of MC3T3-E1
cells. MC3T3-E1 cells were seeded in 96-well plates (2×103 cells/well). After 24 h, the cells were treated without or with 40 μM D-PDMP, 40 μM L-PDMP, 40 μM PBPP, or 10 nM
rapamycin in osteogenic medium for the indicated periods of time. Untreated cells cultured in osteogenic medium for the indicated periods of time were used as the control. (B) D-PDMP
and L-PDMP inhibit the proliferation of MC3T3-E1 cells in dose-dependent manners. MC3T3-E1 cells were seeded in 96-well plates (2×103 cells/well). After 24 h, the cells were treatedwith the indicated concentrations of D-PDMP or L-PDMP in osteogenic medium for 3 days. Untreated cells cultured in osteogenic medium for 3 days were used as the control. The cellproliferation was determined by a Cell Counting Kit-8 assay. Data are expressed as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 compared with control group.

GM

Control

D-PDMP

L-PDMP

PBPP

Rapa

20

30

OM

0

GM

10

Runx2

35

7
6
5
4

***

3

**
***
***

2
1
0

40

OM + D-PDMP (μM)

Relative expression level (fold)

Relative expression level (fold)

8

5

GM

Control D-PDMP L-PDMP

PBPP

30
25
*

20
15
10

***
***

5
0

Rapa

Osx

GM

***

Control D-PDMP L-PDMP

PBPP

Rapa

OM

OM

Fig. 9. The osteoblastic diﬀerentiation of MC3T3-E1 cells is inhibited by D-PDMP and L-PDMP. (A) ALP staining in MC3T3-E1 cells. MC3T3-E1 cells were seeded in 12-well plates(1×104 cells/well). After 24 h, the medium was exchanged from growth medium to osteogenic medium in the absence or presence of 40 μM D-PDMP, 40 μM L-PDMP, 40 μM PBPP, or
10 nM rapamycin for 12 days. Untreated cells cultured in osteogenic medium for 12 days were used as the control. Treatments with D-PDMP and L-PDMP, but not PBPP, decreased the
ALP staining intensity, as compared with control groups. (B) MC3T3-E1 cells were seeded in 12-well plates (1×104 cells/well). After 24 h, the medium was exchanged from growth
medium to osteogenic medium in the absence or presence of the indicated concentrations of D-PDMP for 12 days. Untreated cells cultured in osteogenic medium for 12 days were usedas the control. D-PDMP decreased the ALP staining intensity of MC3T3-E1 cells in a dose-dependent manner. (C, D) mRNA expression of osteoblastic diﬀerentiation markers (Runx2and Osx) in MC3T3-E1 cells. MC3T3-E1 cells were seeded in 6-well plates (2.25×104 cells/well), cultured in growth medium for 24 h (Day 0), and then treated without or with 40 μM DPDMP, 40 μM L-PDMP, 40 μM PBPP, or 10 nM rapamycin in osteogenic medium for 14 days (Day 14). Untreated cells cultured in osteogenic medium for 14 days were used as thecontrol. mRNA expression levels were determined by a quantitative real-time RT-PCR analysis. Target gene expression was normalized to GAPDH mRNA, and shown as relative ratios of
the gene expression in the Day 14 group to that in the Day 0 group. The expression levels of the osteoblastic diﬀerentiation markers were decreased by the D-PDMP and L-PDMP
treatments. Data are expressed as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 compared with control group. Abbreviations: GM, cells cultured in growth medium; OM, cells culturedin osteogenic medium; Rapa, rapamycin.

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segregation of mTOR from Rheb. Furthermore, the present study alsosuggests that the eﬀects of PDMP treatment on LAMTOR1 aremediated via lipid rafts (Supplementary Fig. S9). Thus, we propose anovel mechanism of action for ceramide analogues that target mTORC1
on the LE/Ly membrane. This mechanism adds new insights to ourcurrent understanding of the biological actions of ceramides andceramide analogues, which is mainly based on the Akt/PKB signalingon the plasma membrane.
PDMP reportedly induces the formation of an ER tubular network
in a glycosphingolipid-independent manner [34]. Sprocati et al. proposed that PDMP induces ribosome clustering on the ER and leaves alarge area available for tubular network formation. The exclusion ofribosomes from the ER membrane is a prominent feature of themembrane contact sites between the ER and other organelles [42].
Recent studies suggested the importance of the contact sites between
the ER and LE/Ly in endosome traﬃcking and endosome ﬁssion, as
well as the transport of cholesterol and ceramide [42,43]. Combining
these studies, as an underlying mechanism for the translocation ofmTORC1 from LE/Ly to the ER, we hypothesize that PDMP promotes
the contacts between the ER and LE/Ly.
Impaired intracellular cholesterol transport is associated with the
inhibition of the mTORC1 activity, in the cases of ezetimibe in humanhepatocytes [41] and itraconazole in human endothelial cells andglioblastoma cells [44,45]. Coincidentally, ezetimibe also signiﬁcantly
increases the proportion of LAMTOR1 in DRMs [41], similar to PDMP.
The oxysterol-binding protein ORP1L senses the cholesterol level in
LE/Ly and interacts with the ER integral protein VAP (vesicleassociated membrane protein) [46], which may link the inhibition of
intracellular cholesterol transport with the formation of ER-LE/Ly
contact sites. We speculate that the molecular machinery of cholesteroltransport, the ER-LE/Ly contact sites, and the association of
LAMTOR1 to DRMs are converged for the translocation of mTORC1
from LE/Ly to the ER.
According to the results shown in Figs. 2 and 4, the translocation ofmTORC1 from LE/Ly to the ER was induced by PDMP, but not PBPP.
Morpholino and decanoylamino groups are present in PDMP, whereas
pyrrolidino and benzyloxycarbonylamino groups exist at the corresponding positions in PBPP, respectively (Fig. 1A). The presence of theacyl chain in PDMP may be involved in its aﬃnity to membrane lipids.
It is also possible that an oxygen atom in the morpholino group has anaﬃnity to a hydroxyl or amino group in the vicinal PDMP molecules,
which allows the lateral association of the PDMP molecules to form
distinct membrane domains similar to lipid rafts. Further studies are
required to identify whether PDMP exerts its eﬀects though alterationsof local membrane structures or associations with distinct target
proteins.
In the present study, we demonstrated the PDMP-dependenttranslocation of mTORC1 and its scaﬀold protein LAMTOR1 to theER, and the PDMP-dependent dissociation of mTORC1 from Rheb. In
the presence of PDMP, Rheb likely remains on the LE/Ly membrane,
thus leading to the segregation of mTORC1 from Rheb. Then, thequestion arises: why is LAMTOR1, but not Rheb, translocated to the
ER? The diﬀerence in the membrane association modes, such as thefatty acid modiﬁcation of LAMTOR1 and the isoprenoid modiﬁcation ofRheb, is likely to be responsible for the distinct eﬀects of PDMP on
LAMTOR1 and Rheb.
PDMP is a promising target for preventing atherosclerosis andcardiac hypertrophy [28,29] and for suppressing osteoclastogenesis[47,48] by inhibiting glycosphingolipid synthesis. PDMP is reportedly
potent in combination therapies for cancer treatment [30,31]. Since themTORC1 activity is enhanced in cancer cells to potentiate theirsurvival, the inhibitory eﬀect of PDMP on the mTORC1 activity could
explain the sensitization of cancer cells to anti-cancer drugs. In thepresent study, we showed that PDMP suppresses osteoblastic proliferation and pre-osteoblastic cell diﬀerentiation by inhibiting themTORC1 activity. The underlying mechanism is distinct from theinhibition of glycosphingolipid synthesis. However, the eﬀect of LPDMP on osteoblastic diﬀerentiation was more potent than that of DPDMP. Since glycosphingolipids such as GM3 inhibit osteoblastic
diﬀerentiation [49,50], the eﬀect of D-PDMP may have relieved theglycosphingolipid-mediated inhibition of diﬀerentiation. It is conceivable that the various eﬀects of PDMP are opposing or additive inmembrane property-dependent manners. Accordingly, the present
study adds an important rationale to extend the utility of PDMP.
5. Conclusions
Our study revealed that PDMP induces the translocation of mTOR
from LE/Ly to the ER through the eﬀects on the organelles, which is
independent of its well-known action as an inhibitor of glycosphingolipid synthesis. The translocation of mTOR results in the impaired
interaction between mTOR and Rheb, an mTOR activator, leading tomTORC1 inactivation. The inactivation of mTORC1 leads to reductionsin the proliferation and osteoblastic diﬀerentiation of MC3T3-E1 cells.
Acknowledgements
This study was supported by a Grant-in-Aid for the Japan Society
for the Promotion of Science (JSPS) Fellows 16J07413 (to T.O.) and
Grants-in-Aid for Scientiﬁc Research 22592060 and 25463072 (toM.H.) and 23650319 and 26463087 (to K.A.I.). T.O. was a recipient of
the Iwadare Scholarship Foundation.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.yexcr.2016.11.011.
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Contents lists available at ScienceDirect

BBA - General Subjects
journal homepage: www.elsevier.com/locate/bbagen

Gangliosides and hearing
a,⁎

a,b

Jin-ichi Inokuchi , Shinji Go

0$5.
a,c

, Misato Yoshikawa , Kevin StraussdaDivision of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku,
Sendai, Miyagi 981-8558, Japanb
Kawasaki medical school, 577, Matsushima, Kurashiki, Okayama 701-0192, Japanc
School of Pharmacy, Shujitsu University, 1-6-1 Nishigawara, Naka-ku, Okayama-shi, Okayama 703-8516, Japand
Clinic for Special Children, 535 Bunker Hill Road, Strasburg, PA 17579, United States

A R T I C L E I N F O

A B S T R A C T

Keywords:
Gangliosides
GM3 synthase
Auditory system
Deafness
Hair cells
Stereocilia

Severe auditory impairment observed in GM3 synthase-deﬁcient mice and humans indicates that glycosphingolipids, especially sialic-acid containing gangliosides, are indispensable for hearing. Gangliosides associate with glycoproteins to form membrane microdomains, the composition of which plays a special role inmaintaining the structural and functional integrity of hair cells. These microdomains, also called lipid rafts,
connect with intracellular signaling and cytoskeletal systems to link cellular responses to environmental cues.
During development, ganglioside species are expressed in distinctive spatial and temporal patterns throughout
the cochlea. In both mice and humans, blocking particular steps of ganglioside metabolism produces distinctive
neurological and auditory phenotypes. Thus each ganglioside species may have speciﬁc, non-overlapping
functions within the cochlea, central auditory network, and brain.

1. Background
Deafness is the most common hereditary disability in humans, affecting 1 per 1000 newborns, and more than 40% of people older than
65 years suﬀer from age-related hearing loss. Cell-surface glycoconjugates—proteoglycans, glycoproteins, and glycosphingolipids (GSLs)—havespeciﬁc spatial and temporal patterns of expression in the developing
cochlea and appear to subserve important roles in hearing [1]. Gangliosides (sialic acid-containing GSLs) are abundant in mammalian brains,
where they typically reside in the outer leaﬂet of cell membranes andcluster in microdomains (lipid rafts) specialized for adhesion and signaling[3,4]. They comprise 0.5% of mature human brain (~1.8% of non-water
brain weight) [5], have 3-fold greater abundance in gray relative to whitematter, and are requisite for the normal growth, diﬀerentiation, andmaintenance of neural tissues [2].
GM3 synthase (a.k.a lactosylceramide sialyltransferase; SIAT9,ST3GAL5) is encoded by ST3GAL5 and mediates the sialylation of lactosylceramide (LacCer) to form GM3, the root structure for all downstream a- and b-series gangliosides (Fig. 1A) [6–8]. B4GALNT1 subsequently transfers N-acetylgalactosamine to LacCer, GM3, or GD3 to⁎Corresponding author.
E-mail address: jin@tohoku-mpu.ac.jp (J.-i. Inokuchi).

http://dx.doi.org/10.1016/j.bbagen.2017.05.025
Received 25 April 2017; Received in revised form 25 May 2017; Accepted 25 May 2017
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form GA2, GM2 or GD2, respectively. Based on this enzymology, various patterns of systemic ganglioside deﬁciency have been modeled intransgenic mice [9].
St8sia1−/− (GD3 synthase null) mice lack b-series gangliosides (GD3
and its downstream derivatives) and suﬀer from thermal hyperalgesia andmechanical allodynia [10]. B4galnt1−/− mice express only two ganglioside species (GM3 and GD3) and manifest delayed nerve conduction,axonal degeneration, and ataxia [11,12]. B4galnt1−/−: St8sia1−/−
double knockout animals express only GM3 and have fatal audiogenic
seizures and peripheral neuropathy [7,13]. Knocking out both St3gal5 andB4galnt1 produces mice deﬁcient of GM3 as well as all downstream a-, b-,
and o-series gangliosides; these animals have lethal neurodegenerative
disease characterized by micrencephaly, aberrant axon-glial interactions,and axonal degeneration [14].
We generated St3gal5−/− mice lacking GM3 as well as a- and bseries gangliosides [9]. These animals have no overt neurodegenerative
phenotype, perhaps owing to compensatory B4galnt1-mediated production of o-series ganglisosides [14]. St3gal5−/− mice do not startle inresponse to sound but startle normally to puﬀs of air, which suggests
hearing impairment [15]. This is corroborated by electrophysiological and%%$*HQHUDO6XEMHFWV  ²

J.-i. Inokuchi et al.

Fig. 1. Marked increase of GSLs in cochlea during postnatal maturation period. (A) Schematic presentation of GSL biosynthesis. (B) High performance-thin-layer chromatograms (HPTLC
analysis of gangliosides in cochlea of wild-type mice during the postnatal maturation period. Upper panel is neutral glycolipids and lower panel is acidic glycolipids. Cochlea were isolated
and subjected for the puriﬁcation of GSL analysis as described in ref. [16]. Each puriﬁed fraction was spotted as 2 mg protein per lane. (C) HPTLC analysis of gangliosides in whole brainof wild-type mice during the postnatal maturation period (D) Summary of stage-speciﬁc expression of GSLs during postnatal maturation of cochlea based on the information of Fig. 1B and
LC-MS/MS analysis of ganglioside species reported in ref. [16]. Comparative development of the cochlea of both humans and mice are also shown.

2. Scope of review

histological analyses of the auditory system, which reveal selective degeneration of the organ of Corti [15,16]. Interestingly, auditory impairment is the herald sign of disease in newborns with ST3GAL5 deﬁciency[16], indicating that GM3 may be indispensible for murine and humanhearing.

We provide an overview of glycoconjugate expression in the murine
auditory system and consider the association between ganglioside deﬁciency and hearing, including recent auditory ﬁndings in ganglioside%%$*HQHUDO6XEMHFWV  ²

J.-i. Inokuchi et al.
Table 1
Phenotypes of hearing loss in mice depleted enzymes related to glycoconjugate metabolism.
Mouse

Type of hearing loss

Symptom, histology

Ref.

Neu1 knockout (Lysosomal sialidase)

Conductive

Inﬂammation in middle ear
Thickened mucosa
Reduce of endolymphatic potential
Degradation of supporting cells
Degradation of outer hair cells

[51]

[52]
[53]

No obvious cell loss

[54]

Degeneration and loss of outer and inner hair cells
Hypertrophy of hair cells and dieters cells Loss of outerhair cells
Degeneration of spiral ganglions[15,16][55]

Fbs2 knockout (Ubiquitin ligase of glycoproteins)
Cmah knockout (Sialic acid metabolism)
Mucopolysaccharidosis type VII model (Lysosomal
glucuronidase)
ST3GAL5 knockout (GM3 synthase)
Prosaposin knockout (Glycosphingolipid catabolism)
SaposinB knockout (Glycosphingolipid catabolism)

SensorineuralSensorineural (progressive)
Sensorineural(reducing hearing ability
with age)
Hearing loss with age
SensorineuralSensorineural (progressive)
Sensorineuraldeﬁcient mice and humans.

Plasma of aﬀected children had undetectable GM3 and downstream aand b-series derivatives accompanied by elevations of LacCer, globoside, and paragloboside species. All ST3GAL5 c.694C > T homozygotes
had the characteristic phenotype of slow postnatal head growth, intractable epileptic encephalopathy, severe psychomotor delay, visual
impairment, and hearing loss; the latter manifesting as a failed newborn
hearing screen.
Auditory brainstem responses (ABRs) had abnormal thresholds in allears tested and cochlear microphonics (CM) were consistently absent(Table 3). One child had no reproducible ABR waves. Wave I was observed in one subject, questionable in another, and absent in remaining
patients. In six children, only Waves III and V could be clearly deﬁned.
Wave latencies were normal (80 dB) in the majority of patients. In mostears, comparison of condensation and rarefaction responses revealed
waveform phase reversals (Fig. 2). Although cortical auditory-evoked
potentials (CAEPs) were detectable in all 8 ST3GAL5 c.694C > Thomozygotes (Table 3), their morphology was abnormal in 7 (88%) andP2/N2 latency was delayed in 6 (75%).

3. Major conclusions
3.1. Hearing loss in human and animals with disorders of glycoconjugate
metabolism
Abundant glycans (carbohydrate portions of glycoconjugates) incochlear tissue have been reported from 1980s using various stainings
such as reagents, lectins and antibodies against speciﬁc glycan[15–18,36–50]. Further insights about the relationship between glyconjugates and hearing emerged from studies of transgenic mice. Deletion of enzymes that synthesize (Cmah and St3Gal5) and catabolize(Neu1, Fbx2, glucuronidase, and Saposins) glycoconjugates cause
hearing loss in mice, and each enzymopathy entrains a particular pattern and mechanism of hearing loss (Table 1) [15,16,51–56].
Mixed type (conductive and sensorineural) hearing loss is observedin humans with lysosomal storage diseases (LSDs) and congenital disorders of glycosylation (CDGs), which result from mutations that interfere with the catabolism and assembly of glycoconjugates, respectively. Nerve defects and middle ear disease are observed in thesepatients, but the molecular mechanisms of hearing loss may be more
speciﬁcally related to alterations of glycoconjugate expression within
the cochlea (Table 2, [57–66]).
The study of autosomal recessive ST3GAL5 (GM3 synthase) deﬁciency provides the strongest link between ganglioside metabolism andauditory function in humans. Biallelic damaging ST3GAL5 variants
cause infantile-onset epileptic encephalopathy, slow brain growth,stagnant psychomotor development, growth failure, blindness, dyspigmentation, and deafness [19–23]. We examined eight children (ages
4.1 ± 2.3 years) from the Old Order Amish community who were
homozygous for pathogenic ST3GAL5 c.694C > T variants [16].

3.2. Gangliosides and mammalian auditory development
3.2.1. Signiﬁcant increase of glycosphingolipids during postnatal
maturation of murine cochlea
We investigated the expression of GSLs in wild-type murine cochlea
during the postnatal period of auditory system maturation. Healthy
mice begin to recognize sound by postnatal day 12 (P12), designatedthe ‘onset of hearing’. As shown in Fig. 1B, GM3 is the dominant cochlear GSL at P1. After P3, there is marked increase of cochlear GM3 aswell as other GSLs, including glucosylceramide, complex gangliosides(GM1, GD1a, GD3, GD1b, GT1b), and sulfatides (SM3 and SM4)
(Fig. 1B). In contrast, expression of cerebral gangliosides reaches astable, mature pattern as early as P3 (Fig. 1C).
Signiﬁcant alterations of ganglioside expression in murine cochlea
after P3 reveal the vital role these molecules play in hearing onset [16].
After auditory maturation, GM3 and GM1 are discretely distributed
among inner hair (IHC), outer hair (OHC), Dieters, and pillar cells of theorgan of Corti (Table 4). Both IHCs and OHCs express GM3, which ispreferentially distributed to the apical surface, cuticular plate, andstereocilia. GM1 is the primary IHC ganglioside and predominates on
the apical surface of supporting cells, but is largely absent from OHCs
(Fig. 3) [16].

Table 2
Hearing loss in human LSDs and CDGs.
Human diseases

Causative gene

Ref.

Lysosomal storage disorders
・Gaucher disease・Fabry disease・Mucopolysaccharidosis
・Pompe disease・Sialidosis
・GM2 gangliosidosis
・Mannosidosis

Glucocerebrosidase
Alpha-galactosidasehydrolases of glycosaminoglycans
Alpha-glucosidase
Neuraminidase
Beta-hexosaminidase
Mannosidases

[57]
[58]
[59]
[60]
[61]
[62]
[63]

Congenital disorder of glycosylation
・Type Ig
ALG12・Type II
SLC35A2 (UDP-Gal transporter)
SLC39A8 (Manganese transporter)

[64]
[65]
[66]

[56]

3.2.2. Glycocalyx integrity and membrane cycling in GM3 synthasedeﬁcient mice
Apical membranes of stereocilia are covered with a glycocalyx
composed of sialic acid-containing glycoproteins and glycolipids (including gangliosides) [24] to create a dense negative charge ﬁeld that
normally prevents fusion of adjacent stereocilia. In experimental animals, aminoglycoside administration reduces expression of sialo%%$*HQHUDO6XEMHFWV  ²

J.-i. Inokuchi et al.
Table 3
Auditory brainstem responses and CAEPs in children homozygous for ST3GAL5 c.694C > T.
Age

Auditory brainstem responses(years)

CM

1.6
2.0
2.8
3.1
3.6
4.5
7.2
7.9

CAEPs

Morphology

Waves (80 dB)

Thresholds

Latency (80 dB)

Phase reversal

Morphology

Latency

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Abs
Abs
Abs
Abs
Abs
Abs
Abs
Abs

Abs
Abs
Abs
Abs
Abs
Abs
Abs
Abs

–
Abs
Abs
Abs
Abs
Abs
Abs
Abs

–
Abs
Abs
Abs
Abs
Abs
Abs
Abs

Abs
I, III, V
III, V
III, V
III, VIa, III, V
III, V
IIIa, Va

Abs
I, III, V
III, V
III, V
III, VIa, III, V
III, V
IIIa, Va

–
50
70
80
80
DNT
60
80

–
50
70
80
80
DNT
50
80

–
N
D
N
N
N
N
N

–
N
D
N
N
N
N
D

–
No
Yes
No
Yes
Yes
No
No

–
Yes
Yes
Yes
Yes
Yes
Yes
a

Abn
Abn
Abn
Abn
Abn
Abn
N
Abn

Abn
Abn
Abn
Abn
Abn
Abn
N
Abn

D
D
D
D
D
N
N
D

D
D
D
D
D
N
N
D

Abn, abnormal; Abs, absent; CM, cochlear microphonic; D, delayed; N, normal; DNT, did not test.
aEquivocal result.

Fig. 2. Neurological and auditory phenotype of ST3GAL5 (GM3synthase) deﬁciency in humans. (A) From left to right, T2, ﬂuidattenuated inversion recovery, diﬀusion-weighted, and 1H-MR
spectroscopy at 1.5 T show normal cortical and subcortical structure, but severe hypomyelination and increased water diﬀusion
throughout the corona radiata, as well as elevated choline (Cho)relative to creatine–phosphocreatine (Cr) and N-acetylaspartate(NAA) 1H signals (single voxel PRESS, TE 144/TR 1500). (B)Electroencephalogram of a 2 year-old child homozygous for
ST3GAL5 c.694C > T shows a slow, chaotic background, absent
posterior rhythm and no discernable change through a sleep–wakecycle. Beginning at the dashed red line, there is a burst of generalized, frontal predominant, 3 Hz spike-slow wave discharges in excess of 450 µV. (C) In the majority of aﬀected children, auditory
brainstem responses show waveform reversals [16].

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J.-i. Inokuchi et al.
Table 4Comparison of GM3 and GM1 expression of speciﬁc regions in the organ of Corti at P14.
OHC

GM3
GM1

IHC

DC

PC

Stereocilia

Cuticular plate

Cell body

Stereocilia

Cuticular plate

Cell body

Cell surface

Cell body

Cell body

++
–

+
–

–
–

++
++

+
+

–
+

–
++

+
+

–
+

OHC; outer hair cell, IHC; inner hair cell, DC; Deiters cell, PC; pillar cell.

glycoconjugates in the OHC glycocalyx and precipitates fusion of stereocilia [25,26].
To better deﬁne the role of various ganglioside species in hearing,
we compared auditory function between St3gal5 −/− and B4galnt1 −/− mice, which have distinctive patterns of GSL expression within theorgan of Corti (Fig. 4A). Murine B4galnt1 −/− cochlea has rich GM3
content, intact hair bundles, and normal auditory function whereas
St3gal5 −/− cochlea, devoid of GM3, are associated with deafness(Fig. 4B). Using confocal laser microscopy with phalloidin staining, we
observe normal IHC and OHC morphology in 4 week-old B4galnt1 −/−mice (Fig. 4C and D). In contrast, St3gal5 −/− OHCs have blebs andintracellular vesicles, the latter composed of membranous structures(Fig. 4E), indicative of unbalanced endocytosis and exocytosis [16]. As
noted above, GM3 is expressed in both IHCs and OHCs of wild-type
mice, whereas only IHCs express GM1 (Table 4). This may explain inpart why OHCs degenerate in advance of IHCs in St3gal5 −/− mice.

3.2.3. GM3-enriched membrane organization, PTPRQ-myosin VI complex
localization and hair cell morphology
Hair cells are specialized to mediate auditory and vestibular transduction. Projecting from their apical surface are ﬁlopodial processes
called stereocilia, each ﬁlled with hundreds of cross-linked actin ﬁlaments. Certain proteins—e.g. unconventional myosins, Usher proteins,and some deafness-related gene products (e.g. PTPRQ, cadherin23,
protocadherin15, Usherin, and VLGR1)–are expressed predominantly
or exclusively in stereocilia to form a structured, interactive networkthat supports mechanoelectrical transduction (Fig. 5) [27].
In the normal organ of Corti, functional complexes of PTPRQ andmyosin VI appear critical to maintaining the integrity of this network[28]. PTPRQ, a shaft connector at the tapered base of stereocilia, iscomposed of an extracellular domain containing 18 ﬁbronectin III(FNIII) repeats, a membrane spanning domain, and a cytoplasmic domain with both phosphatidylinositol and tyrosine phosphatase activities [29,30]. Myosin VI is an actin-based motor protein that associates
Fig. 3. Comparison of gangliosides expression in hair cells ofmice aged 4 weeks. (A) Whole-mount immunostaining was
performed. GM3 (upper panel, green) and GM1 (lower
panel, green) of OHC stereocilia. Only GM3 was highly enriched in OHC stereocilia without staining of GM1. On theother hand, the expression levels of GM1 but not GM3 wasespecially high on the surface of supporting cells. (B)Schematic images of distinct expression of GM3 and GM1 in
OHC.

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Fig. 4. Identiﬁcation of GM3 as an essential molecule for hair cell function. (A) HP-TLC of acidic (left) and neutral (right) GSLs from the cochlea of 4-week-old wild-type (WT),
B4galnt1 −/− and St3gal5 −/− mice. The cochlea of B4galnt1 −/− mice were mainly expressed GM3 and GD3. Each puriﬁed fraction was spotted 2 mg protein per lane. (B) Auditoryevoked brainstem response (ABR) of wild-type, B4galnt1 −/− and St3gal5 −/− mice at 4 weeks old. (C and D) Comparison of gangliosides expression in hair cells among wild-type,B4galnt1 −/− and St3gal5 −/− mice aged 4 weeks. Whole-mount immunostaining was performed. GM1 (C, green) and GM3 (D, green) of IHC (a–c) and OHC (d–f) stereocilia in wildtype (a and d), B4galnt1 −/− (b and e) and St3gal5 −/− (c and f) mice. In IHC, both GM1 and GM3 were expressed along the stereocilium from the taper region to top (Ca and Da). On
the other hand, only GM3 was highly enriched in OHC stereocilia (Dd) without staining of GM1 (Cd). The stereocilia of IHC and OHC in B4galnt1 −/− mice were expressed GM3 but not
GM1 (Cb and Db). In St3gal5 −/− mice, fused stereocilia was observed in IHC (Cc and Dc) and irregular phalloidin staining was observed in OHC (Cf and Df). Notably, the expression
levels of GM1 but not GM3 was especially high on the surface of supporting cells (Deiters cell) (Cd and Dd). (E) TEM images of OHC in St3gal5 +/+ and −/− mice at P17 [16].

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IHCA

Myosin VI

OHC
Merge

Myosin VI Merge+/+

Fig. 5. Dislocalization of PTPRQ and myosinVI inthe stereocilia of St3gal5 null mice. Confocal
images showing stereocilia of IHCs and OHCs of
St3gal5 +/+ and −/− mice stained for myosinVI(A) PTPRQ (B) (green) and F-actin (phalloidin,
magenta). (C) Schematic images for dislocalizationof PTPRQ and myosin VI in the IHC stereocilia of
St3gal5 −/− mouse [16].

-/5 m

B

PTPRQ

5 m

Merge

PTPRQ

Merge

+/+
-/5 m

5 m

B

with PTPRQ to form a complex mediating interaction between theplasma membrane and cytoskeleton [31]. Mice lacking PTPRQ are deaf
and lack tapering at the base of stereocilia, which become fused [28].
Myosin VI-deﬁcient mice are also deaf and have comparable structuralchanges of hair cells accompanied by maldistribution of PTPRQ along
the length of stereocilia [28,32,33].
In bull frogs, PTPRQ colocalizes with ganglioside-rich membrane
domains in basal stereocilia [34]. We used immunostaining to further
investigate the relationship between gangliosides and the basal PTPRQmyosin VI complex (Fig. 5). In wild-type murine IHCs, PTPRQ localizes
exclusively to the stereocilia base, whereas PTPRQ is maldistributed
along shafts of fused St3gal5 −/− stereocilia (Fig. 5B) and myosin VI ispresent from base to midshaft but absent from distal regions (Fig. 5A).
Such structural disturbances likely reﬂect loss of normal ciliary motor
action. In St3gal5 −/− OHCs, myosin VI expression is concentrated at
the surface of the cuticular plate, close to vestigial kinocilia (Fig. 5A).
Together, these observations suggest that membrane microdomains

enriched with GM3 are crucial to the formation and proper localizationof PTPRQ-myosin VI complexes in hair cells. In the absence of GM3,
aberrant expression of PTPRQ-myosin VI complexes has important
structural consequences for stereocilia and likely impairs their ability totransduce auditory signals (Fig. 5C).
4. Conclusions
Our results implicate a speciﬁc and indispensable role for GM3 inthe development, function, and viability of cochlear hair cells. Althoughhearing is already impaired at birth in humans with GM3 synthasedeﬁciency, the murine St3gal5 −/− auditory phenotype indicates thatganglioside repletion might partially rescue auditory function during a
critical postnatal window. These ﬁndings inform our understanding of
hearing loss in humans with ST3GAL5 deﬁciency, but do not explain the
broader neurological phenotype associated with this condition (Fig. 2).
Thus future studies should more carefully delineate the role of lipid

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rafts generally, and GM3 speciﬁcally, in the development and physiology of the central nervous system.
New concepts and techniques will allow us to determine novel
therapies for hearing impairment. In a recent report, functional haircell-like cells were generated from embryonic and induced pluripotent
stem cells [35]. This and other strategies will give rise to new frontiersof hearing research, further advancing studies of glycoconjugate-richmicrodomains and their auditory actions. We hope such research will
engender new and eﬀective therapies to restore hearing in humans.
Transparency document
The http://dx.doi.org/10.1016/j.bbagen.2017.05.025 associatedwith this article can be found, in the online version.
Acknowledgements
This work was supported by research grants for a Grant-in-Aid for
Scientiﬁc Research (B) (to J.-i.I.) and for Scientiﬁc Research on
Innovative Areas (no. 23110002, Deciphering sugar chain-based signals
regulating integrative neuronal functions) from MEXT, Japan, theMizutani Research Foundation for Glycoscience (J.-i.I.), The Naito
Foundation (J.-i.I.), the ONO Medical Research Foundation (J.-i.I.), theUehara Memorial Foundation (J.-i.I.), and by Grant-in-Aid for Japan
Society for the Promotion of Science Fel-lows (M.Y.). This work wasalso supported by MEXT-Supported Program for the Strategic Research
Foundation at Private Univer-sities. The Clinic for Special Children is
supported by generous charitable contributions from the Amish andMennonite commu-nities it serves.
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ARTICLE

Altered expression of ganglioside GM3 molecular species anda potential regulatory role during myoblast differentiation
Received for publication, December 7, 2016, and in revised form, February 27, 2017 Published, Papers in Press, March 8, 2017, DOI 10.1074/jbc.M116.771253

Shinji Go‡1, Shiori Go‡§1, Lucas Veillon‡, Maria Grazia Ciampa¶, Laura Mauri¶, Chihiro Sato§, Ken Kitajima§,
Alessandro Prinetti¶, Sandro Sonnino¶, and Jin-ichi Inokuchi‡2
From the ‡Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical andPharmaceutical University, Sendai 981-8558, Japan, §Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601,
Japan, and ¶Department of Medical Biotechnology and Translational Medicine, University of Milan, 20090 Segrate Milano, Italy
Edited by Gerald W. Hart

Glycosphingolipids (GSLs)3 are constituents of eukaryotic
cell membranes located exclusively on the outer leaflet of theplasma membrane. Gangliosides, a subgroup of GSLs having
one or more sialic acid residues, are involved in regulation ofnumerous cell biological events, including development, trafficking, signaling, and cellular interactions. Gangliosides havepathophysiological functions in diseases such as cancer, neurodegenerative disorders, and diabetes (1, 2). Sialic acid plays aThis work was supported by a Grant-in-aid for Scientific Research (B) (to J.-i. I.)
and for Scientific Research on Innovative Areas (23110002, Decipheringsugar chain-based signals regulating integrative neuronal functions) from
MEXT, Japan and by MEXT-Supported Program for the Strategic Research
Foundation at Private Universities. The authors declare that they have noconflicts of interest with the contents of this article.
1
Both authors contributed equally to this study.
2
To whom correspondence should be addressed: Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku
Medical and Pharmaceutical University, 4-4-1, Komatsushima, Sendai
981-8558, Japan. Tel.: 81-22-727-0117; Fax: 81-22-727-0076; E-mail:jin@tohoku-mpu.ac.jp.
3
The abbreviations used are: GSL, glycosphingolipid; Cer, ceramide; CerS,
ceramide synthase; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; EGFR, EGF receptor; MyHC, myosin heavy chain; HPTLC,
high performance thin layer chromatography; CMAH, CMP-Neu5Ac
hydroxylase; Elovl, expression levels of fatty acyl-CoA elongase; DM, differentiation medium; GM3, Sia!2,3Gal"1,4Glc-ceramide; GM2, GalNAc"1,
4(Sia!2,3)Gal"1,4Glc-ceramide; GM1, Gal"1,3GalNAc"1,4(Sia!2,3)Gal"1,4Glcceramide; GD1a, Sia!2,3 Gal"1,3GalNAc"1,4(Sia!2,3)Gal"1,4Glc-ceramide.

key role in the biological activities of gangliosides (3). For example, we demonstrated that localization of insulin receptor incaveolae is disrupted by elevated levels of endogenous GM3
during the state of insulin resistance. This effect is due to electrostatic interaction between the lysine residue of insulin receptor (Lys-944) and the carboxyl group of sialic acid of GM3(4 – 8).
The structural diversity of GSLs arises from both the ceramide (Cer) moiety and glycan portion (Fig. 1). Cer is composedof sphingosine and a single acyl chain (Fig. 1B). Cer acyl chainsvary in length of carbon backbone, degree of saturation, and thepresence/absence of !-hydroxylation (9, 10).
Additional structural diversity of gangliosides arises fromsialic acid (acidic sugar molecule with characteristic ninecarbon backbone) in the glycan portion. The most common
sialic acid in mammals is N-acetylneuraminic acid (Neu5Ac).
The other common sialic acid, N-glycolylneuraminic acid
(Neu5Gc), differs from Neu5Ac by the presence of an additional oxygen atom in the acyl group at position C5 (Fig. 1B).
Further structural diversity of sialic acids results from combinations of this variation at position C5 with modifications ofhydroxyl groups at positions C4, C7, C8, and C9 by acetate,lactate, sulfate, phosphate esters, or methyl ethers. Sialic acidmodifications greatly alter the size, hydrophobicity, net charge,
and enzymatic susceptibility of the parent compound (11).
Such structural divergence generated by different combinations of Cer and sialic acids in gangliosides (Fig. 1B) results in ahuge numbers of “molecular species.” The identity of sialic acidspecies in GM3 affects its biological properties. GM3 inhibits
EGFR-tyrosine kinase to differing degrees depending on its
sialic acid species. Such a difference apparently results fromdifferential carbohydrate-carbohydrate interactions between
EGFR and GM3 (Neu5Ac) or GM3 (Neu5Gc) (12). We havealso demonstrated relationships between various metabolic
diseases and levels of serum ganglioside GM3 “acyl chain”
molecular species. In particular, levels of GM3 with hydroxylated acyl chain species in human serum show strong correlations with several risk factors for metabolic diseases (13).
Taken together, the above findings indicate that differing
molecular species of a given ganglioside have distinct biologicalproperties and regulate specific and distinct biological events.
Elucidation of the function of each molecular species is important and will provide new insights into ganglioside biology.

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Gangliosides (sialic acid-containing glycosphingolipids) help
regulate many important biological processes, including cellproliferation, signal transduction, and differentiation, via formation of functional microdomains in plasma membranes. The
structural diversity of gangliosides arises from both the ceramide moiety and glycan portion. Recently, differing molecularspecies of a given ganglioside are suggested to have distinct biological properties and regulate specific and distinct biologicalevents. Elucidation of the function of each molecular species isimportant and will provide new insights into ganglioside biology. Gangliosides are also suggested to be involved in skeletal
muscle differentiation; however, the differential roles of ganglioside molecular species remain unclear. Here we describe striking changes in quantity and quality of gangliosides (particularly
GM3) during differentiation of mouse C2C12 myoblast cells andkey roles played by distinct GM3 molecular species at each stepof the process.

GM3 molecular species and myoblast differentiation

Regulation of skeletal muscle formation is an essential process in both normal development and repair of damaged muscle
tissue. Differentiation of skeletal muscle cells (myocytes) is amultistep process; the steps include signal transduction, celladhesion, and cell-cell fusion (14, 15). Gangliosides are known
to modulate various plasma membrane functions, e.g. cell-cellinteractions, signal transduction, and membrane ion channel
activity, through formation of functional membrane microdomains termed “rafts.” Gangliosides are presumably involved inthese processes in myocytes (16 –19); however, direct evidence
for such involvement is limited and fragmentary. In the present
study we evaluated expression of gangliosides in myoblasts during differentiation. We observed striking changes in gangliosidemolecular species composition during differentiation andassessed their differential functions. GM3 (Neu5Gc) and 16:0
acyl chain species, in particular, appear to play essential roles indifferentiation of myoblasts to myotubes.

Results
Changes in ganglioside quantity and quality during myogenic
differentiation
C2C12 is a myoblast line established from normal adult C3H
mouse leg skeletal muscle and is a well established model of invitro differentiation. C2C12 cells were cultured to !90% confluence in growth medium and then cultured in differentiation
medium (DM; see “Experimental procedures”). Upon differentiation stimulus, the cells gradually fused to form multinucleated fibers (myotubes) (Fig. 2A) as expected. Differentiation to
myotubes was confirmed by Western blotting. Levels of differentiation marker proteins myogenin and myosin heavy chain(MyHC) increased during differentiation (Fig. 2B). HPTLC
revealed the appearance of bands corresponding to GM3, GM2,
GM1, and GD1a mobilities during differentiation (Fig. 2C).
Amounts of all acidic GSLs increased gradually during differentiation (Fig. 2C). In particular, GM3 content increased strikJ. Biol. Chem. (2017) 292(17) 7040 –7051

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Figure 1. Structural diversity of gangliosides. A, ganglioside biosynthetic pathways. B, structures of ganglioside GM3 and sialic acid molecular species.

GM3 molecular species and myoblast differentiation

ingly, with three major bands. Through day 2, the top band
(black pointer) and middle band (gray pointer) were the major
GM3 bands. From day 3 onward, the top band decreased, andthe middle band and bottom band (white pointer) became themajor bands. These findings revealed clear changes in quantityand quality of GM3 molecular species, reflecting their importance in myogenic differentiation.
Changes of sialic acid species in gangliosides during myogenic
differentiation
GM3 synthase expression increased during differentiation(Fig. 3A), consistent with the observed increase of GM3 levels(Fig. 2A). To evaluate ganglioside changes during differentiation, we used TLC immunostaining and fluorometric HPLC toassess the quantity and quality of lipid-linked sialic acids.

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Changes of sialic acid species of GM3 were confirmed by TLC
immunostaining using specific anti-GM3 mAbs. For anti-GM3(Neu5Ac) mAb GMR6, positive staining was detected at day 0,
and its intensity increased until day 3 and decreased thereafter(Fig. 3B). In contrast, for anti-GM3 (Neu5Gc) mAb GMR8 (20),positive bands were very weak until day 2 then increased gradually after day 3 (Fig. 3B). High GM3 (Neu5Gc) expression wasmaintained even on day 8.
Quantity and quality of lipid-linked sialic acids were next
assessed by fluorometric HPLC. In C2C12 myoblasts before
differentiation, almost all gangliosides contained Neu5Ac assialic acid in the glycan portion (Fig. 4A, f). After differentiation induction, the total amount of lipid-linked sialic acidincreased (Fig. 4A, Œ), consistent with TLC immunostaining
results (Fig. 2C). The amount of lipid-linked Neu5Ac increased
gradually through day 3 and declined thereafter. In contrast,
lipid-linked Neu5Gc was barely detectable until day 2, increased markedly on day 3, and by day 5 was the major sialic
acid species (Fig. 4A, Œ), again consistent with TLC results (Fig.
3, B and C). Taken together, these findings demonstrate thatsialic acid species of gangliosides change from Neu5Ac to
Neu5Gc during myogenic differentiation of C2C12 cells.

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Figure 2. Changes of ganglioside expression during myoblast C2C12 differentiation. Differentiation of C2C12 cells was induced by culture in DM (see“Experimental procedures”). A, morphology of proliferating cells (day 0) andmyotube formation on days 2– 8, as revealed by phase-contrast microscopy.
Scale bar, 200 !m. B, expression of differentiation marker proteins myogenin
and MyHC on various days, revealed by Western blotting. "-actin: loading
control. Shown are representative Western blot images. C, GSL fraction was
prepared as described under “Experimental procedures.” Acidic GSLs
extracted from cells (0.2 mg protein basis) were spotted and separated on
HPTLC. GSLs were visualized by orcinol/sulfuric acid staining. Pointers indicate GM3 species with differing polarity (see “Changes in ganglioside quantity and quality during myogenic differentiation” under “Results”). Shown is a
representative HPTLC image.

Figure 3. Alteration of sialic acid species of GM3 during C2C12 differentiation. A, the GM3 synthase mRNA expression levels on the indicated days,
determined by quantitative RT-PCR. Data represent the relative gene expression in cells of each day compared with that in cells of day 0 (set to 1.0 separately for each target gene). Average -fold change was obtained from threeexperiments, shown as the means ! S.D. B, GM3 (Neu5Ac) expression,
detected by TLC immunostaining with specific mAb GMR6. C, GM3 (Neu5Gc)
expression, detected by TLC immunostaining with specific mAb GMR8. Pointers indicate the position of the GM3 band on HPTLC in Fig. 2.

GM3 molecular species and myoblast differentiation

Changes of acyl chain structures in the Cer portion of GM3
during myogenic differentiation

Figure 4. Quantitative and qualitative analysis of lipid-linked sialic acidspecies during C2C12 differentiation. A, lipid-linked sialic acid species inC2C12 were quantified and qualified by fluorometric HPLC. Œ, total lipidlinked sialic acids. f, lipid-linked Neu5Ac. #, lipid-linked Neu5Gc. Detectedsialic acids: sum of sialic acids in GM3, GM2, GM1, and GD1a. B, lipid-linked
sialic acid species in growth and differentiation medium were quantified byfluorometric HPLC. Shown is growth medium containing 10% FBS (black bar)and differentiation medium containing 2% HS (gray bar). C, expression levelsof Cmah mRNA, determined by agarose gel electrophoresis and quantitative
RT-PCR. Data represent the relative gene expression in cells of each day compared with that in cells of day 0 (set to 1.0 separately for each target gene).
Average -fold change obtained from three experiments is shown as themeans $ S.D.

Structural diversity of GM3 derived from acyl chain structures in C2C12 cells was evaluated by LC-MS/MS after differentiation induction. On day 0 (Fig. 5A, black bars), the threemost abundant GM3 species detected were Neu5Ac-GM3(d18:1–24:0), Neu5Ac-GM3 (d18:1–24:1), and Neu5Ac-GM3(d18:1–16:0). Species with acyl chain carbon number 24
accounted for !50% of total GM3 (Fig. 5B, green and blue colors). Species that were abundant on day 0 contained primarily
Neu5Ac as sialic acid, consistent with TLC immunostaining
and HPLC results (Figs. 3 and 4).
On day 7, when differentiation was completed, Neu5GcGM3 (d18:1–16:0) was the most abundant GM3 molecular species, and Neu5Ac-GM3 (d18:1–16:0) was the second mostabundant (Fig. 5A). GM3 species with 16:0 acyl chainsaccounted for "70% of the total. Proportions of Neu5Ac species were lower, and those of Neu5Gc species were higher on
day 7, but total amounts of 24:0 and 24:1 species showed little
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Neu5Gc residues on glycans are synthesized from CMPNeu5Gc through catalytic activity of sialyltransferases. There
are two CMP-Neu5Gc synthesis pathways in mammalian cells.
(i) In the salvage pathway, cells take up extracellular Neu5Gccontaining glycoconjugates derived from foods such as meat,
fish, and eggs (or from serum in the case of cultured cell lines).
Subsequently, Neu5Gc-containing glycoconjugates are hydrolyzed to Neu5Gc monosaccharides in lysosomes. Neu5Gcmonosaccharides are transported to cytosol, and Neu5Gc is
activated to CMP-Neu5Gc by CMP-sialic acid synthetase in the
nucleus (21–23). (ii) In the de novo synthesis pathway, sialic
acid Neu5Ac is synthesized in cytosol and activated to CMPNeu5Ac in the nucleus, and CMP-Neu5Ac is subsequently converted to CMP-Neu5Gc by the enzyme CMP-Neu5Ac hydroxylase (CMAH) in cytosol (24, 25). CMP-Neu5Gc is transported
into the Golgi apparatus, where it undergoes transfer to glycans
through catalytic activity of various sialyltransferases. Reversetranscription polymerase chain reaction (RT-PCR) analysis of
the CMAH encoding gene (Cmah) revealed that expression
level of Cmah mRNA was very low during days 0 –2 but
increased thereafter (Fig. 4C). Expression of Cmah mRNA wascorrelated with GM3 (Neu5Gc) level during C2C12 differentiation (Figs. 3 and 4).
It is known that fetal bovine and horse serum contain
Neu5Gc glycoconjugates. Therefore, we analyzed sialic acid ingrowth (10% FBS) and differentiation (2% HS) medium andconfirmed the existence of Neu5Gc in both mediums. Lipidlinked Sia in the media is shown in Fig. 4B. Growth medium
contained large amounts of sialyl glycosphingolipids comparewith differentiation medium. Both mediums contained large
amounts of Neu5Ac compare with Neu5Gc. Although Neu5Gc
glycosphingolipids, especially Neu5Gc-GM3 in medium, may
be taken up and utilized by differentiating cells, the amounts
would be small. Because the expression levels of both GM3
synthase and Cmah increased after the induction of differentiation (Figs. 3A and 4C), we could conclude that the increase of
Neu5Gc-GM3 during differentiation may be due to the increased de novo synthesis.

GM3 molecular species and myoblast differentiation

change (Fig. 5A). The proportion of 16:0 species increased
markedly during differentiation (Fig. 5, A and B).
Expression of Cer synthases
Ceramide synthase (CerS) enzymes regulate synthesis of thevarious Cer species. Six types of mammalian CerS have beenreported. Each CerS has a preference for a unique range ofacyl-CoA groups and, therefore, controls a particular subset of
Cer species. We used real-time PCR to determine expression
levels of CerS1 through CerS6 in C2C12 cells. The predominant
forms detected in these cells were CerS1, -2, -5, and -6. CerS2,

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which catalyzes synthesis of ceramide with very long acyl chainssuch as C24, was most abundant in C2C12. CerS2 was slightly
increased after the induction of differentiation, but it was
decreased sharply during the differentiation process. On theother hand the levels of CerS5 and -6, which catalyze C16-Cer
synthesis, remained constant (Fig. 6A). Therefore, the increaseof GM3 C16 species after differentiation (Fig. 5) could be attributed to the marked decrease of CerS2 expression during thedifferentiation process (Fig. 6A).
We also examined changes in mRNA expression levels of
fatty acyl-CoA elongases (Elovls) during differentiation. Levels

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Figure 5. LC-MS/MS analysis of GM3 molecular species during C2C12 differentiation. A, levels of GM3 molecular species in C2C12 cells. The eight mostabundant species on day 0 (black bars) and day 7 (gray bars) are shown. The amount of each species was quantified by LC-MS/MS using Neu5Ac GM3(d18:1-[13C]16:0) as the internal standard, as described under “Experimental procedures.” y axis, amount of GM3 species (ng) present per 50 !g of cell protein.
B, proportions of GM3 molecular species on days 0 and 7. Amounts of individual species were determined, and their proportions were calculated.

GM3 molecular species and myoblast differentiationof all five Elovls examined were lower on day 8 than on day 0(Fig. 6B). This trend may also explain in part the increase of
shorter acyl-chain Cer species.
Effects of exogenous sialic acid and GM3 species on cellmorphology
Sialic acid and acyl chain structures of GM3 showed notablechanges during C2C12 cell differentiation. The potential role ofsuch changes was investigated by adding various sialic acid andGM3 molecular species to culture medium.
Sialic acid monosaccharide (Neu5Ac or Neu5Gc) was added
during medium change each day to change the sialic acid species of sialylglycoconjugates. Treatment with 2 mM Neu5Ac or
Neu5Gc efficiently increased intracellular Sia monosaccharide(Fig. 7A, left panel), so exogenous sialic acids were taken up by
C2C12 cells. After 2 days the amount of lipid-linked Neu5Gc
exceeded that of Neu5Ac in cells treated with 2 mM Neu5Gc,
although Neu5Ac was the major sialic acid in normal andNeu5Ac-treated cells (Fig. 7A, right panel). Thus, we were able
to change the ratio of sialic acid species in gangliosides by treatment with Neu5Gc. Cell morphology on day 6 is shown in Fig. 7,
B and C.

Discussion
Skeletal muscle differentiation comprises a chain of complex
processes in which cell plasma membranes play a crucial role.
Cell-to-cell adhesion and recognition are essential for the differentiation process. It is necessary for progenitor cells to contact numerous similar cells and respond to inductive signals inorder to undergo a coordinated process of differentiation. In
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Figure 6. Changes in expression of Cer synthase mRNAs during C2C12
differentiation. A, expression levels of CerS mRNAs, determined by quantitative RT-PCR. -Fold change was calculated as 2!""#CT with tubulin used as
the endogenous control. B, expression levels of Elovl mRNAs during differentiation, determined by quantitative RT-PCR. Data represent the relative gene
expression in cells of each day compared with that in cells of day 0 (set to 1.0
separately for each target gene). Average -fold change obtained from threeexperiments is shown the means # S.D.

Sialic acid species differentially affected morphology of myoblasts and/or myofibers. The ratio of long fibers was increased
by treatment with Neu5Gc (Fig. 7C, right panel). And theformed fiber width was small (Fig. 7C, left panel). Therefore,
cells treated with Neu5Gc monosaccharide formed longer, narrower myofibers. Neu5Ac or Neu5Gc treatment had no effecton the expression of differentiation marker proteins myogenin
and MyHC (Fig. 7D, left panel). However, phosphorylation of
paxillin, which regulates adhesion state, was increased by treatment with Neu5Gc (Fig. 7D, right panel). These findings suggest that sialic acid affects cell morphology via control of cytoskeleton formation or myofiber adhesion state.
Although Neu5Ac was taken up by cells during Neu5Ac
treatment, the quality and quantity of glycosphingolipids was
not drastically changed (Fig. 7A). The ratio of shorter length
myotubes was slightly increased by Neu5Ac treatment. Thesechanges may not be an effect of only change in GLSs, althoughthese results suggest that extracellular sialic acid affected myoblast differentiation.
Exogenous gangliosides can be inserted into the outer leafletof plasma membranes and be partially taken up and metabolized (26). We confirmed the uptake and increase of GM3 incells by TLC and fluorometric HPLC (Fig. 8A). 25 !M exogenous GM3 efficiently increased cellular gangliosides (Fig. 8A,
left panel) and lipid linked sialic acids (Fig. 8A, right panel).
Therefore, we added GM3 species, shorter (16:0) species, which
are the major species after differentiation, and longer (24:0 or18:0) species, which are the major species before differentiation
to evaluate their properties. In experiments with different GM3
molecular species, d18:1–16:0 species were well aligned andformed numerous myotubes, whereas cells treated with GM3
d18:1–18:0 or d18:1–24:0 species were poorly aligned andformed fewer myotubes (Fig. 8B); the ratio of cells with shorter
length and larger width was increased when treated with GM3
d18:1–18:0 or d18:1–24:0 species (Fig. 8C). Although expression level of myogenin was relatively unaffected by treatment
with different GM3 molecular species, the level of MyHC wasnotably reduced by treatment with GM3 d18:1–18:0 or d18:1–
24:0 species (Fig. 8D). Cells treated with GM3 d18:1–16:0
formed relatively longer myofibers.
These findings indicate that increased longer acyl-chain
GM3 species inhibit some signaling in differentiation processes such as fusion process and/or formation of myotubes,
whereas shorter acyl-chain GM3 species do not inhibit, or
might, enhance.
In this study we found an association of GM3 lipid specieschange and differentiation in myoblast. The results shown in
Fig. 7 and in Fig. 8 are consistent with the hypothesis that myoblasts may regulate their differentiation by changing GSLs
species.

GM3 molecular species and myoblast differentiation

the final steps of differentiation, plasma membranes fuse witheach other (14, 15, 27). Biochemical characteristics of plasma
membranes thus have important effects on skeletal muscle differentiation. GSLs, particularly gangliosides, help determine
plasma membrane properties and play essential roles in regulating many biological processes, including cell-cell interactions and signal transduction (1, 2). Expression of GM3, GM2,
GM1, and GD1a has been reported in myoblast cell line C2C12(16 –18); however, changes in ganglioside expression and functions of specific ganglioside species during myogenic differentiation have not been studied. We analyzed changes in gangli-7046 J. Biol. Chem. (2017) 292(17) 7040 –7051

oside species during myogenic differentiation using C2C12
cells as a model. We demonstrated for the first time striking
changes in quantity and quality of gangliosides during the differentiation process. Alterations of molecular species of GM3,
the major ganglioside in C2C12 cells, were particularly notable.
Sialic acid species in GM3 were changed from Neu5Ac to
Neu5Gc through elevated expression of hydroxylase CMAH.
GM3 species with 16:0 acyl chains were greatly increased. To
elucidate the function of this GM3 molecular species change,
we added sialic acid monosaccharide or synthetic GM3 acyl
chain species to culture medium. Our findings (Figs. 6 and 7)

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Figure 7. Effects of sialic acid monosaccharide supplementation on C2C12 differentiation. A, uptake and utilization of exogenous sialic acids in C2C12
cells. 2 mM Neu5Ac or Neu5Gc was added to the differentiation medium. After 2 days Sia monosaccharide in cytosol (left panel) and lipid-bound Sia (right panel)
were measured as described under “Experimental procedures.” B, phase-contrast microphotographs on day 6 of cells treated with sialic acid (2 mM) in DM. C,
effects of exogenous sialic acids on morphology of myofibers. The ratio of each fiber length (right panel) and width (left panel) are shown (control and Neu5Gc;
n ! 100, Neu5Ac; n ! 89). D, expression levels, on day 6 of differentiation, of marker proteins myogenin and MyHC in cells treated with sialic acid in DM (rightpanel). Shown are the effects of sialic acids on the phosphorylation state of paxillin on days 1and 2 (left panel). Shown are representative Western blot images.

GM3 molecular species and myoblast differentiation

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Figure 8. Effects of GM3 acyl chain structure on C2C12 differentiation. A, uptake of exogenous GM3. Shown is TLC analysis (right panel), and the amountsof lipid-linked sialic acids (left panel) of C2C12 cells treated with 25 mM GM3 for 24 h. B, phase-contrast microphotographs on day 6 of cells treated with three
GM3 species (25 !M). C, effects of exogenous sialic acids on morphology of myofibers. The ratio of each fiber length (right panel) and width (left panel) are shown(n ! 20). D, expression levels on day 6 of differentiation marker proteins myogenin and MyHC in cells treated with three GM3 species. Shown are representative
Western blot images.

indicate that sialic acid species affect cell morphology andthat GM3 acyl chain structures play key roles in regulating
differentiation.
In regard to GM3 function in myogenic differentiation, previous studies indicate that sialidase (Neu3), which is localized in
the plasma membrane and hydrolyzes GM3 and other gangliosides, affects differentiation of C2C12 cells. Neu3 overexpression delayed myoblast differentiation and induced hypertrophic myotube formation, whereas differentiation was

inhibited by Neu3 gene silencing (16 –18). The Neu3 protein
per se presumably modulates certain cell functions, but thesestudies suggest that GM3 in appropriate amounts also plays animportant role in normal C2C12 differentiation, most likely via
modulation of EGFR signaling. We used a GSL synthesis inhibitor to confirm that GSLs in appropriate amounts are importantin myofiber formation (data not shown). Thus, proper amounts
of GM3 are required at each step in the process for normal
myogenic differentiation. We observed here for the first time
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acyl-chain GM3 species inhibited differentiation, whereas
shorter acyl-chain GM3 did not result in inhibition (Fig. 8, B
and C). These results suggest that myoblasts regulate some signaling processes during differentiation, such as fusion processes and/or formation of myotubes, by changing the metabolism of ganglioside acyl chain species.
Besides changes in acyl chains of GM3 molecular species, thepresent study demonstrates for the first time changes of sialic
acid species from Neu5Ac to Neu5Gc during differentiation.
We also observed morphological changes in myotubes after
treatment with Neu5Gc (Fig. 7, B and C). This phenomenon
suggests a function for Neu5Gc glycoconjugates in the processof myogenic differentiation, especially in the regulation of cellmorphology. Although during the exogenous treatment we
employed, the ratio of Neu5Gc was increased in both glycoproteins and glycolipids (data not shown); therefore, we cannot
concluded the effects observed were the result of Neu5Gc GM3
alone. Several groups have described changes of sialic acid species on glycans during various biological processes (40 – 43). It
appears that Neu5Gc expression can also be affected by cell
activation and disease status. Cmah mRNA expression isaltered during lymphocyte activation in mice (41, 42), and
Neu5Gc treatment modifies interactions between glycans andtheir binding proteins. Taken together, these past and present
findings indicate that the change of sialic acid species from
Neu5Ac to Neu5Gc is crucial for myogenic differentiation in
mice. Deletion of Cmah in mice increased the severity of muscular dystrophy (44, 45), suggesting that this gene is a genetic
modifier of muscle homeostasis. Taken together, our results
suggest that myoblasts regulate their differentiation by changing the metabolism of sialic acids, ceramide, and, consequently,
gangliosides.
We want to elucidate the function of specific GM3 molecularspecies, which have their expression levels affected before andafter myogenic differentiation, especially focusing on NeuGc
GM3 with a C16 acyl chain as shown in Fig. 5A. We confirmed
the change in expression of some genes related to the synthesisof this molecule. Therefore, we tried to modify the expressionof these genes (such as GM3 synthase, Cmah, and CerSs) toshow their function in myogenic differentiation. However, we
could not establish gene-modified C2C12 cells while maintaining differentiation potency. Therefore, we will report further onthese investigations in our next manuscript.
In conclusion, the present results demonstrate that quantityand quality of GM3 change greatly during myoblast C2C12 differentiation and suggest that these changes play a key role inregulation of the differentiation process. These findings help
clarify the significance of GSL structural diversity in mammalian cells. Our ongoing studies, using genetically modified miceand cells, are focused on roles of GM3 molecular species infunction and regeneration of muscle cells and tissues.

Experimental procedures
Materials
Hybridoma cells producing anti-GM3 (Neu5Gc) monoclonal
antibody (mAb) GMR8 were kindly donated by Ikuo Kawashima(20), RIKEN BioResource Center. Anti-GM3 (Neu5Ac) mAb

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striking changes in GM3 quality during differentiation; i.e.
major alterations in sialic acid species and acyl chain structures (Figs. 2– 4). Our findings suggest that ganglioside quality as well as quantity is important at each step for normal
myogenic differentiation.
Sialic acid species and the acyl chain structures of gangliosides are involved in maintaining cell membrane properties andlipid microdomain structures. Hashimoto and co-workers (28,29) observed that disruption of lipid rafts by cholesterol depletion directly blocked the plasma membrane fusion that normally occurs as part of myogenic cell fusion. The molecularmechanisms that control cell fusion and other steps of myogenic differentiation remain to be elucidated; however, plasma
membrane rafts clearly play an important role. A number ofmembrane molecules have been implicated in regulation of
myogenic differentiation. Extracellular matrix receptor integrins (30) and adhesion molecules such as cadherins, NCAM,
and CD9 (31–33) are involved in regulation of the recognition
and adhesion steps in myoblast differentiation and maintenance of myofibers. Crucial molecules (e.g. BAI1, myomaker)
involved in regulation of initiation signals for myogenic differentiation and plasma membrane fusion were discovered in2013 (34, 35). Such transmembrane proteins are localized in
specific regions of the plasma membrane during differentiation; it is thus possible that various GM3 molecular species
interact specifically with the proteins, directly or indirectly, toregulate their functions and localization. Membrane fluidity
affects myogenic differentiation, particularly the plasma membrane fusion process (36 –38); thus, another possibility is thatconcentrations of various GSL molecular species in myoblasts
help regulate membrane fluidity and are strictly controlled forthis purpose.
Our knowledge of molecular mechanisms that regulate distribution and expression of GSL molecular species and otherplasma membrane molecules remains fragmentary. The CerS
enzymes provide one regulatory mechanism for expression of
GSL acyl chain molecular species. Six mammalian CerSs havebeen described, each utilizing fatty acyl CoAs with differing
chain lengths for N-acylation of the sphingoid long chain base.
CerS5 and CerS6 catalyze synthesis of C16 acyl chain Cer species (9, 10). Most studies to date focused on roles of CerSs inregulation of sphingolipids have relied on CerS overexpression
to increase Cer synthesis. Therefore, it remains unclear how
individual CerSs contribute to a steady-state sphingolipid poolin mammalian cells, and whether maintenance of sphingolipids
with specific acyl chains depends on specific CerSs (39). C16
GSL species were strikingly increased during C2C12 differentiation in the present study (Fig. 5). The increase of GM3 C16
species after differentiation could be attributed to the marked
decrease of CerS2 expression during the process of differentiation (Fig. 6A). We also observed that expression levels of Elovls
declined during myogenic differentiation, which may help
account for increased levels of shorter acyl-chain species (Fig.
6B). Future studies may reveal unknown mechanisms for regulation of GSL molecular species by inter-regulation of CerSs
and Elovls.
By treatment with exogenous GM3 species, cellular gangliosides were changed (Fig. 8). We found that increased longer

GM3 molecular species and myoblast differentiation
GMR6 was from Seikagaku Co. (Tokyo, Japan). Anti-myogenin
and myosin heavy chain antibodies were from e-Bioscience (Santa
Clara, CA). GM3 molecular species were synthesized according toprevious reports (46, 47). Neu5Ac GM3 (d18:1-[13C]16:0), aninternal standard for quantification by LC-MS/MS, was from
Tokyo Chemical Industry (Tokyo, Japan).
Cell culture

Analysis of GSLs
Analysis of GSLs by TLC was performed as described originally in Refs. 48 and 49 and slightly modified (50). In brief, total
cell lipids were extracted using chloroform/methanol, andextracts were applied to DEAE-Sephadex A-25 column equilibrated with chloroform/methanol/water (30:60:8, v/v/v). The
column was washed with the same solvent, and acidic GSL fraction was eluted with chloroform/methanol/1 M sodium acetate(30:60:8, v/v/v). Acidic and neutral lipids were subjected to mild
alkaline hydrolysis and desalted using a Sep-Pak C18 cartridge.
Acidic GSLs were spotted on a TLC plate and developed withchloroform/methanol/0.2% CaCl2 (60:25:4, v/v/v). GSLs werevisualized by orcinol/sulfuric acid staining.
TLC immunostaining
Acidic GSLs were spotted on a TLC plate and developed withchloroform/methanol/0.2% CaCl2 (60:25:4, v/v/v). The driedplate was dipped in cyclohexane containing 0.1% (w/v) poly(isobutyl methacrylate) for 1 min, blocked by incubation in PBS
containing 1% BSA at room temperature for 1 h, incubated with
anti-GM3 mAb GMR6 (GM3(Neu5Ac) specific) or GMR8
(GM3(Neu5Gc) specific) in PBS containing 1% BSA at room
temperature for 2 h, washed 5 times with PBS, and incubatedwith anti-mouse IgM horseradish peroxidase (HRP)-conjugated antibody at 37 °C for 1 h. HRP was detected by the addition of appropriate substrates following the manufacturer’s
instructions.
Sialic acid analysis by fluorometric HPLC
Sialic acids were hydrolyzed from acidic GSL fractions bytrifluoroacetic acid. An aliquot of the supernatant was lyophilized and then incubated with 1,2-diamino-4,5-methylene
dioxybenzene as described previously (51). 1,2-Diamino-4,5methylene dioxybenzene-labeled sialic acids were separated
and detected using an HPLC system (JASCO; Tokyo, Japan)
equipped with a reversed-phase C18 column (Wakopak
Handy-ODS (4.6 mm " 250 mm); Wako Pure Chemical, Osaka,
Japan).

Gene
CerS1
CerS2
CerS3
CerS4
CerS5
CerS6
Cmah
St3gal5(GM3 S)
TubulinElovl 1
Elovl 2
Elovl 3
Elovl 4
Elovl 5
Elovl 6
Elovl 7

Primer sequence
Forward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
Reverse5#-TGACTGGTCAGATGCGTGA-3#
5#-TCAGTGGCTTCTCGGCTTT-3#
5#-TCATCATCACTCGGCTGGT-3#
5#-AGCCAA AGAAGGCAGGGTA-3#
5#-ATCTCGAGCCCTTCTTCTCC-3#
5#-CTGGACGTTCTGCGTGAAT-3#
5#-TGCGCATGCTCTACAGTTTC-3#
5#-CTCGAGCCATCCCATTCTT-3#
5#-TCCATGCCATCTGGTCCTA-3#
5#-TGCTGCCAGAGAGGTTGTT-3#
5#-GGGTTGAACTGCTTCTGGTC-3#
5#-TTTCTTCCCTGGAGGCTCT-3#
5#-GAAAGGCCTGTGTTTTGGAA-3#
5#-TCATGAACCGCAGACTTTTG-3#
5#-GTGGACCCTGACCGGATAAAG-3#
5#-AACAGAGCCATAGCCGTCTTC-3#
5#-CACTACACCATTGGCAAGGA-3#
5#-TGTGGAAAACCAAGAAGCCC-3#
5#-GGTGGGGGATAAAAATTGCT-3#
5#-CCAAGGGCAGACAATCCATA-3#
5#-GACGCTGGTCATCCTGTTCT-3#
5#-GCTTTGGGGAAACCATTCTT-3#
5#-TTTGCCATCTACACGGATGA-3#
5#-CGTGTCTCCCAGTTCAACAA-3#
5#-TTTGGTGGAAGCGATACCTG-3#
5#-ATGTCCGAGTGTAGAAGTTG-3#
5#-CTCTCGGGTGGCTGTTCTT-3#
5#-AGAGGCCCCTTTCTTGTTGT-3#
5#-ACAATGGACCTGTCAGCAAA-3#
5#-GTACCAGTGCAGGAAGATCAGT-3#
5#-ATGGGACCAGCCTACCAGAA-3#
5#-TTGCAGTCCTCCATGAAGAA-3#

RT-PCR
Total RNAs were prepared from cells using TRIzol reagent.
Reverse transcription of total RNA was performed to generate
first-strand cDNA. For detection of cDNA of mouse CMPsialic acid hydroxylase, we performed RT-PCR assays using aprimer set based on sequences of CMP-sialic acid hydroxylase(5#-ctgatcccaggtctcctgaa-3#, 5#-agcctctccaaccagtcaga-3#) and
GAPDH (5#-acaaaatggtgaaggtcggt-3#, 5#-tccagggtttcttactcctt3#). For quantitative real-time PCR, cDNA was prepared fromtotal RNA using a PrimeScript™ RT Reagent kit (Takara Bio).
RT-PCR was performed and analyzed using SsoFast™ EvaGreen
Supermix on a Real-Time PCR system (Bio-Rad). Expression
values were normalized to tubulin, and relative mRNA expression was calculated as described by Livak and Schmittgen (52).
Primers used are listed in Table 1.
Mass spectrometric analysis
Neu5Ac GM3 (d18:1-[13C]16:0) was added to acidic GSL
samples as the internal standard. GM3 molecular species were
quantified using HPLC coupled with electrospray ionization
tandem mass spectrometry (MS/MS) in multiple reactionmonitoring negative ionization mode. The triple-stage quadrupole (TSQ) Vantage AM instrument (Thermo Fisher, Waltham, MA) was calibrated by directly infusing a mixture ofGM3 species extracted from milk, and all ion source parameters and ionization conditions were optimized to improve sensitivity. GSLs were dissolved in methanol, injected onto an
HPLC pump (Accela 1250, Thermo Fisher), and separatedusing a Develosil carbon 30 column (C30-UG-3–1 " 50 mm,
Nomura Co.; Aichi, Japan). The gradient program started with
100% solvent A (20% H2O, 50% 2-propanol, 30% methanol conJ. Biol. Chem. (2017) 292(17) 7040 –7051

7049

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C2C12 myoblast cells were obtained from RIKEN BioResource Center (Tsukuba, Japan) and cultured in Dulbecco’s
modified Eagle’s medium (DMEM; Nacalai; Kyoto, Japan) containing 10% heat-inactivated fetal bovine serum at 37 °C in 5%
CO2 atmosphere. To induce differentiation, growth medium
was replaced with DMEM containing 2% horse serum (DM)when cells reached !90% confluency. Cells were replenishedwith fresh DM every 24 h. To evaluate the effects of exogenous
sialic acids, Neu5Ac or Neu5Gc (2 mM) were added to DM. To
evaluate the effects of exogenous GM3, synthetic GM3 acyl
chain species (25 !M) were added to DM.

Table 1
Primers used to specifically enhance each mRNA fragment

GM3 molecular species and myoblast differentiation
Table 2Mass spectrometer settings and MRM transition pairs
Q, quadrupole; CE, collision energy; S-lens, stacked-ring ion guide; RF, radio frequency.
Molecular
species (Sia)GM3 (Neu5Ac) [M-H]!

Q1

Q3

S-lens RF
CE amplitude

m/z

m/z

eV

d18:1–16:1
d18:1–16:0
d18:1-h16:1
d18:1-[13C]16:0
d18:1–18:1
d18:1–18:0
d18:1-h18:1
d18:1-h18:0
d18:1–20:1
d18:1–20:0
d18:1–21:1
d18:1–21:0
d18:1-h20:0
d18:1–22:1
d18:1–22:0
d18:1-h21:0
d18:1–23:1
d18:1–23:0
d18:1-h22:0
d18:1–24:1
d18:1–24:0
d18:1-h23:0
d18:1-h24:1
d18:1-h24:0
d18:1–16:1

1149.71151.71165.71167.91177.71179.71193.71195.71205.71207.71219.71221.71223.71233.71235.71237.71247.71249.71251.71261.81263.81265.81277.81279.81165.7

289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
289.9
305.9

53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53

276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276

d18:1–16:0
d18:1-h16:1
d18:1–18:1
d18:1–18:0
d18:1-h18:1
d18:1-h18:0
d18:1–20:1
d18:1–20:0
d18:1–21:1
d18:1–21:0
d18:1-h20:0
d18:1–22:1
d18:1–22:0
d18:1-h21:0
d18:1–23:1
d18:1–23:0
d18:1-h22:0
d18:1–24:1
d18:1–24:0
d18:1-h23:0
d18:1-h24:1
d18:1-h24:0

1167.71181.71193.71195.71209.71211.71221.71223.71235.71237.71239.71249.71251.71253.71263.71265.71267.71277.81279.81281.81293.81295.8

305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9
305.9

53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53

276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276
276

taining 0.1% acetic acid and 0.1% ammonia) for 5 min, thenramped to 100% solvent B (2% H2O, 50% 2-propanol, 48%
methanol containing 0.1% acetic acid, and 0.1% ammonia) over
30 min. 100% solvent B was maintained for 4 min, then thesolvent was returned to 100% solvent A over 1 min and held
there for 10 min. Flow was 50 !l/min throughout the chromatographic run. !2500 V potential was applied between ion
source and electrospray needle. The carrier gas was nitrogen.
Relative abundances of molecular species were assessed basedon relative percentage of internal standard. All GM3 molecularspecies may not necessarily have identical ionization efficiencies; however, because of limited availability of pure molecularspecies standards, we assumed that all species have ionization
efficiencies comparable with that of the internal standard.
Thus, in evaluating relative abundances of molecular species,
detected amounts are compared that may not necessarily rep-

7050 J. Biol. Chem. (2017) 292(17) 7040 –7051

Author contributions—Shinji G. and J.-i. I. designed the study andwrote the paper. Shiori G. and Shinji G. performed and analyzed theexperiments shown in Figs. 2– 8. V. L. performed and analyzed theexperiments shown in Fig. 5 by using LC-MS/MS. M. G. C., L. M.,
A. P., and S. S. synthesized GM3 molecular species. C. S. and K. K.
analyzed the experiments in Figs. 3, 4, and 6. All authors analyzed theresults and approved the final version of the manuscript.
Acknowledgment—We are grateful to Dr. S. Anderson for English
editing of the manuscript.
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Biochimica et Biophysica Acta 1859 (2017) 2001–2011

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbamem

Identiﬁcation of a new B4GalNAcT1 (GM2/GD2/GA2 synthase) isoform,
and regulation of enzyme stability and intracellular transport byarginine-based motif
Fumi Shishido a,1, Satoshi Uemura b,c,⁎,1, Madoka Kashimura a, Jin-ichi Inokuchi a,⁎a
Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558,
Japanb
Division of Medical Biochemistry, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japanc
Division of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5258, Japana r t i c l e

i n f oArticle history:
Received 24 February 2017
Received in revised form 17 June 2017
Accepted 10 July 2017
Available online 12 July 2017
Keywords:
Glycosyltransferase
Retrograde transport
R-based motif
mRNA transcriptional varianta b s t r a c t
Glycosphingolipids (GSLs) are abundant in plasma membranes of mammalian cells, and their synthesis is strictly
regulated in the Golgi apparatus. Disruption of GSL homeostasis is the cause of numerous diseases. Hundreds ofmolecular species of GSLs exist, and the detailed mechanisms underlying their homeostasis remain unclear. We
investigated the physiological signiﬁcance of isoform production for β1,4-N-acetyl-galactosaminyl transferase 1/
B4GALNT1 (B4GN1), an enzyme involved in synthesis of ganglio-series GSLs GM2/GD2/GA2. We discovered a
new mRNA variant (termed variant 2) of B4GN1 through EST clone search. A new isoform, M1-B4GN1, which
has an NH2-terminal cytoplasmic tail longer than that of previously-known isoform M2-B4GN1, is translated
from variant 2. M1-B4GN1 has R-based motif (a retrograde transport signal) in the cytoplasmic tail. M1B4GN1 is partially localized in the endoplasmic reticulum (ER) depending on the R-based motif, whereas M2B4GN1 is localized in the Golgi. Stability of M1-B4GN1 is higher than that of M2-B4GN1 because of the Rbased motif. M2-B4GN1 forms a homodimer via disulﬁde bonding. When M1-B4GN1 and M2-B4GN1 were coexpressed in CHO-K1 cells, the two isoforms formed a heterodimer. The M1/M2-B4GN1 heterodimer was
more stable than the M2-B4GN1 homodimer, but the heterodimer was not transported from the Golgi to the
ER. Our ﬁndings indicate that stabilization of M1-B4GN1 homodimer and M1/M2-B4GN1 heterodimer by Rbased motif is related to prolongation of Golgi retention, but not to retrograde transport from the Golgi to the
ER. Coexistence of several B4GN1 isoforms having distinctive characteristics presumably helps maintain overall
enzyme stability and GSL homeostasis.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction
Glycosphingolipids (GSLs), complex carbohydrate-based molecules
comprised of hydrophilic and hydrophobic regions, are abundant in
the outer leaﬂet of plasma membranes of mammalian cells and are involved in a variety of important physiological processes, including cellproliferation, differentiation, and adhesion [1–5]. The hydrophilic headgroups of GSLs vary in terms of number and type of carbohydrate chains,resulting in the existence of several hundred molecular species of GSLs
[6,7]. Certain diseases result from disruption of GSL homeostasis; e.g.,
GM3 level is elevated in adipose tissue of Zucker fa/fa rats and ob/ob
mice, typical rodent models of obesity [8], and insulin sensitivity is en⁎ Corresponding authors.
E-mail addresses: s-uemura@tohoku-mpu.ac.jp (S. Uemura), jin@tohoku-mpu.ac.jp(J. Inokuchi).
1
These two authors contributed equally to the study.

http://dx.doi.org/10.1016/j.bbamem.2017.07.006
0005-2736/© 2017 Elsevier B.V. All rights reserved.

hanced in ST3 β-galactoside α2,3-sialyltransferase 5/ST3GAL5 (ST3G5)
knockout mice [9] (Fig. 1). In rodent obesity models, treatment with
GlcCer synthase inhibitors that signiﬁcantly reduce GM3 content improves insulin sensitivity and glucose homeostasis [10,11]. ST3G5
gene mutations were identiﬁed in patients with early-onset refractory
epilepsy or salt-and-pepper syndrome, characterized by severe intellectual disability, epilepsy, scoliosis, choreoathetosis, dysmorphic facial
features, and altered dermal pigmentation (Table 1) [12–16]. β1,4-Nacetyl-galactosaminyl transferase 1/B4GALNT1 (B4GN1), a key enzyme
in synthesis of complex gangliosides (GM1a, GD1a, GD1b, GT1b, GA1,
GM1b), also plays important roles in GSL homeostasis (Fig. 1). B4GN1
knockout mice, which lack all complex gangliosides in the central nervous system, display subtle neurological disorders at birth and subsequent nervous system neurodegeneration, indicating that complex
gangliosides are essential for maintenance of nervous system integrity[17–19]. In human native CD4+ T helper lymphocyte (Th), the expression level of GD2 synthesized by B4GN1 is increased by the stimulation

2002

F. Shishido et al. / Biochimica et Biophysica Acta 1859 (2017) 2001–2011

Fig. 1. Biosynthetic pathway of glycosphingolipids. Biosynthesis of ganglio-series GSLs
from LacCer. ST3G5: ST3 β-galactoside α2,3-sialyltransferase. ST8S1: ST8 α-N-acetylneuraminide α2,8-sialyltransferase 1. B4GN1: β1,4-N-acetyl-galactosaminyl transferase
1. B3GT4: UDP-Gal: βGlcNAc β1,3-galactosyltransferase, polypeptide 4. ST3G2: ST3 βgalactoside α2,3-sialyltransferase 2.

of anti-CD3 and anti-CD28 antibodies [20]. The GD2 is important for efﬁcient T cell receptor (TCR) clustering in the plasma membrane. In contrast, ST3G5 knockout mice, but not B4GN1 knockout mice, show a
reduction in the proliferation of Th cells when activated with anti-CD3
and anti-CD28 antibodies and the phenotype is recovered by the supplementation of GM3, GM1a, and GD1a [21]. Therefore, GM3 is necessary for the activation of Th cell in murine. Human B4GN1 mutations
have been identiﬁed in families from Kuwait, Italy, and Old Order
Amish who display an unusual neurodegenerative phenotype classiﬁed
as a complex form of hereditary spastic paraplegia (SPG26) (Table 1)[22–25].
We previously investigated the mechanisms whereby GSL biosynthesis is regulated by ST3G5, the primary glycosyltransferase responsible for synthesis of ganglio-series gangliosides. There are three ST3G5
isoforms, having NH2-terminal cytoplasmic tail lengths of 69 aminoacids (aa) (M1-ST3G5), 42 aa (M2-ST3G5), and 14 aa (M3-ST3G5)[26]. These isoforms are produced by leaky scanning from several
mRNA variants. M1-ST3G5 is localized primarily in the endoplasmic reticulum (ER) through an arginine (R)-based motif (a retrograde

Table 1
Distribution of gene mutations in human ST3GAL5 and B4GALNT1.
Genes

Mutationsa,bReference

ST3GAL5

c.584G N C (p.Cys195Ser)
c.601G N A (p.Gly201Arg)
c.862C N T (p.Arg288*)
c.862C N T (p.Arg288*)
c.862C N T (p.Arg288*)
c.1063G N A (p.Glu355Lys)
c.263dupG (p.Leu89Profs*13)
c.358C N T (p.Gln120*)c.395delC (p.Pro132Glnfs*7)
c.682C N T (p.Arg228*)
c.898C N T (p.Arg300Cys)
c.917_922dupTTACCG (p.Thr307_Val308dup)
c.1298A N C (p.Asp433Ala)c.1315_1317delTTC (p.Phe439del)c.852G N C (p.Lys284Asn)c.1458dupA (p.Leu487Thrfs*77)
c.1514G N A (p.Arg505His)c.838-2A N G (p.Gly280_Leu326del)c.1458dupA (p.Leu487Thrfs*77)[15]

B4GALNT1

[14]
[13]
[16]
[12]
[23]

[24]

[25]

a
The reference sequences of ST3GAL5 and B4GNT1 are NM_003896.3 and
NM_001478.4, respectively.
b
The mutations were described according to established mutation nomenclature(http://varnomen.hgvs.org/).

transport signal), located on its cytoplasmic tail. R-based motifs interact
with COP I subunits (β-COP and δ-COP) to function as signal sequences[27]. Binding afﬁnities to β-COP and δ-COP depend on not only R-based
motif sequence but also position in a lipid bilayer [28]. We demonstrated that ER targeting of M1-ST3G5 in vivo leads to suppression of its GM3
synthesis activity because this synthesis occurs in the Golgi. Both M2and M3-ST3G5 are localized in the Golgi, but the half-life of M2-ST3G5
is shorter than that of M3-ST3G5 [26].
In the present study, we investigated the biological signiﬁcance of
isoform production for glycosyltransferases, with focus on human andmouse B4GN1. EST clone search revealed a new isoform, M1-B4GN1,
produced from new mRNA variant 2. NH2-terminal lengths of humanand mouse M1-B4GN1 (hM1- and mM1-B4GN1) are respectively 81
and 40 aa, much longer than that of previously-known isoform M2B4GN1 (6 aa). hM1- and mM1-B4GN1 have R-based motif, a retrograde
transport signal, and are partially localized in the ER, whereas hM2- andmM2-B4GN1 are localized in the Golgi. Stability of hM1-B4GN1 washigher than that of hM2-B4GN1. Co-expression of hM1- and hM2B4GN1 resulted in enhanced stability of M2-B4GN1 through hM1-/
hM2-B4GN1 dimer formation via a disulﬁde bond. These ﬁndings indicate that overall stability of B4GN1 is increased by M1-B4GN1 isoform
production. Coexistence of several isoforms having distinctive membrane dynamics is presumably useful in maintenance of GSL homeostasis under varying physiological conditions.
2. Materials and methods
2.1. Plasmids
The plasmids used in this study are listed in Table 2. The open reading frame (ORF) of human M1-B4GalNT1 (hM1-B4GN1) was ampliﬁedusing cDNA prepared from human brain tissue, and primers 5′ATGCAGGAGAGGGCGAGAAGAAAG-3′ and 5′-TCACTGGGAGGTCATGC
ACTGCAG-3′. Resulting fragments were cloned into pGEM-T Easy Vector(Promega; Madison, WI) to generate pFS432. pFS444 encoding hM1B4GN1-FLAG was constructed using pFS432 and primers 5′AGGGAGACCCAAGCTTCACCATGCAGGAGAGGGCGAGAA-3′ and 5′CTTGTAATCGGGATCCTGGGAGGTCATGCACTGCAG-3′.
Resulting
Table 2
Plasmids used.
Plasmid name

Inserted ORF

VectorpFS 444
pFS 471
pFS 452
pKM 6
pKM 15
pKM 5
pFS 446
pFS 468
pFS 469
pFS 470
pFS 460
pFS 611
pFS 453
pFS 222
pFS 398
pFS 262
pFS 424
pFS 425
pFS 426
pFS 265
pKM 9
pFS 473
pFS 527
pFS 493
pFS 563
pFS 571
pFS 577
pFS 573hM1-B4GN1-FLAG
hM1-B4GN1-FLAG*
hM2-B4GN1-FLAG
mM1-B4GN1-FLAG
mM1-B4GN1-FLAG*mM2-B4GN1-FLAG
hM1-B4GN1(N)-GFP
hM1-B4GN1(N)-GFP R4S
hM1-B4GN1(N)-GFP R6S
hM1-B4GN1(N)-GFP R7S
hM1-B4GN1(N)-GFP R6/7S
hM1-B4GN1(N)-GFP R4/6/7S
hM2-B4GN1(N)-GFP
mM1-B4GN1(N)-GFP
mM1-B4GN1(N)-GFP R8S
mM1-B4GN1(N)-GFP R11S
mM1-B4GN1(N)-GFP R12S
mM1-B4GN1(N)-GFP R13S
mM1-B4GN1(N)-GFP R14S
mM1-B4GN1(N)-GFP R11/12/13/14S
mM2-B4GN1(N)-GFP
hM1-B4GN1-FLAG
hM1-B4GN1-FLAG R4/6/7S
hM2-B4GN1-FLAG
hM2-B4GN1-HA
mM1-B4GN1-FLAG
mM1-B4GN1-FLAG R11/12/13/14S
mM2-B4GN1-HA

pcDNA3-FLAG4
pcDNA3-FLAG4
pcDNA3-FLAG4
pcDNA3-FLAG4
pcDNA3-FLAG4
pcDNA3-FLAG4pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
pEGFP-N1
CSII-CMV-RfA
CSII-CMV-RfA
CSII-CMV-RfA
CSII-CMV-RfA
CSII-CMV-RfA
CSII-CMV-RfA
CSII-CMV-RfA

F. Shishido et al. / Biochimica et Biophysica Acta 1859 (2017) 2001–2011

2003

fragments were cloned into the HindIII-BamHI site of pcDNA3-FLAG4
vector using the In-Fusion cloning system (Takara Bio; Shiga, Japan) togenerate pFS444. Plasmids encoding hM2-B4GN1-FLAG, hM1-B4GN1FLAG*, and hM2-B4GN1-HA were created by similar procedures.
The ORF of mouse M1-B4GN1 was ampliﬁed using cDNA preparedfrom mouse brain tissue, and primers 5′-ATGGCTGGCCTTGGGTA
CCTGCGCGAGCCTCGG-3′ and 5′-TCACTCGGCGGTCATGCACTGTA-3′.
Resulting fragments were cloned into pGEM-T Easy Vector to generate
pKM2. To insert the Kozak sequence (GCCACC) before ATG(M1), M1B4GN1 was ampliﬁed using pKM2 and primers 5′-GGATCCGCCACCATGGCTGGCCTTGGGTACCTG-3′ and 5′- TGGATCCTCGGCGGTCATGC
ACTGTAGCC-3′. Resulting fragments were cloned into pGEM-T Easy
Vector to generate pKM4. The 1.7-kb BamHI fragment of pKM4 wasthen cloned into the BamHI site of pcDNA3-FLAG4 vector to generate
pKM6. Plasmids encoding mM2-B4GN1-FLAG and mM1-B4GN1-FLAG*
were created by similar procedures.
A pFS446 (hM1-B4GN1(N)-GFP) plasmid encoding fusion proteinsof the NH2-terminus of M1-B4GN1 and GFP was constructed using
pFS432 and primers 5′- TCTCGAGCTCAAGCTTCACCATGCAGGAGAG
GGC-3′ and 5′- GGCGACCGGTGGATCAAGAGGTAGCCGGAGGCCGG-3′.
Resulting fragments were cloned into the HindIII-BamHI site of pEGFPN1 vector using the In-Fusion system to generate pFS446. Plasmids
encoding hM2-B4GN1(N)-GFP, mM1-B4GN1(N)-GFP, and mM2B4GN1(N)-GFP were created by similar procedures.
Point mutants of hM1-B4GN1(N)-GFP, mM1-B4GN1(N)-GFP, hM1B4GN1-FLAG, and mM1-B4GN1-FLAG were created by site-directedmutagenesis using PrimeSTAR® Mutagenesis Basal Kit (Takara) according to the manufacturer's instructions.
For lentivirus production, a pFS473 (hM1-B4GN1-FLAG/CSII-CMVRfA) plasmid was constructed using pFS444 and primers 5′CACCATGCAGGAGAGGGCGA-3′ and 5′- TCAGATCTTATCGTCGTCATC
CTTGTAATC-3′. Resulting fragments were cloned into pENTR/D-TOPO
vector (Thermo Fisher; Waltham, MA) to generate pFS466. The CSIICMV-MCS plasmid was converted to a destination vector (CSII-CMVRfA) using the Gateway vector conversion system (Thermo Fisher).
The hM1-B4GN1-FLAG gene of pFS266 was inserted into lentiviral expression vector CSII-CMV-RfA, using the LR clonase reaction to generate
pFS473. Lentiviral expression plasmids encoding hM1-B4GN1-FLAG R4/
6/7S mutant, hM2-B4GN1-FLAG, hM2-B4GN1-HA, mM1-B4GN1-FLAG,
mM1-B4GN1-FLAG R11/12/13/14S mutant, and mM2-B4GN1-HA
were created by similar procedures.
For real-time PCR assay, hM1-B4GN1 sequence containing 5′-UTR was
ampliﬁed using cDNA prepared from human brain tissue, and primers 5′GCTTGCCGAGGCCACTCATCG-3′ and 5′- CCCGGGTGCTCGCGTACAGG-3′.
Resulting fragments were cloned into pGEM-T Easy Vector to generate
pFS635. The 18S rRNA sequence was ampliﬁed using cDNA preparedfrom human brain tissue, and primers 5′-TACCTGGTTGATCCTGCCAGTAG3′ and 5′-TAATGATCCTTCCGCAGGTTCACC-3′. Resulting fragments werecloned into pGEM-T Easy Vector to generate pFS636.

293 T cells (0.5 × 106) were seeded in a polylysine-coated 60-mm
dish 24 h before transfection, and then transfected with 3 μg CSIICMV-RfA cloning hM1- B4GN1-FLAG, hM2- B4GN1-FLAG, mM1B4GN1-FLAG, mM2-B4GN1-FLAG, and hM2-B4GN1-HA, 1.5 μg pCAGHIVgp, and 1.5 μg pCMV-VSV-G-RSV-Rev. plasmids, using Lipofectamine 2000. Cells were incubated 24 h at 37 °C, and medium was replaced with 4 mL high-glucose DMEM containing 10 μM forskolin. To
maintain virus stability, culture temperature was lowered from 37 to
32 °C ~ 32 h after transfection. Lentivirus-containing supernatant was
harvested 48 h after transfection, centrifuged at 200 ×g for 3 min to remove living cells, and either used immediately for experiments or snapfrozen in liquid nitrogen and stored at −80 °C for later experiments. For
lentiviral transduction of CHO-K1 cells, 4 mL lentiviral supernatant wasadded to 0.25 × 106 cells in a 60-mm dish, cells were cultured at 32 °C
for 24 h, culture temperature was raised from 32 to 37 °C, and incubation continued another 48 h.

2.2. Semi-quantitative reverse transcription PCR (RT-PCR)

2.6. Preparation of cell lysates

First-strand cDNA synthesis was performed using a First-Strand
cDNA Synthesis kit for RT-PCR (AMV; Roche Diagnostics; Basel, Switzerland) and total RNA isolated from mouse tissues or human brain tissue(Takara). We designed speciﬁc primers as follows to amplify B4GN1 (allvariants, or only variant 2): hB4GN1 all variants: 5′-GCTGGCAGTGCAAACCCTAGC-3′ and 5′-GTGAAAGCAGCCTCATGTCCCT-3′, hB4GN1
variant 2: 5′-ATGCAGGAGAGGGCGAGAAGA-3′ and 5′-GGCTGAACTTCCACACCCTGTAGG-3′, mB4GN1 all variants: 5′-CTAGTCTTGCTGCTCGC
CTG-3′ and 5′-GCCACGTCCCAGGTGCCTAG-3′, mB4GN1 variant 2: 5′CTCACGTTCTCTGAGAGCCTGG-3′ and 5′- GCCACGTCCCAGGTGCCTAG-3′.
PCR was performed using 50 ng cDNA, speciﬁc primers as above, and
KOD FX polymerase (Takara), according to the manufacturer's instructions. Cycling conditions were 94 °C for 2 min, followed by 34 cycles of
98 °C for 10 s and 68 °C for 40 s. Respective lengths of ampliﬁed fragments

Cells were washed twice with ice-cold phosphate-buffered saline(PBS), suspended in buffer A (50 mM Tris-HCl (pH 7.5), 150 mM NaCl,
10% glycerol, 4 M urea, and 1 × Complete protease inhibitor mixture(EDTA-free; Roche Diagnostics)), and lysed by sonication. Cell debris
was removed by centrifugation at 1000 ×g for 3 min at 4 °C, and supernatant (total cell lysates) was centrifuged at 100,000 ×g for 1 h at 4 °C.
Precipitate (integral membrane protein fraction) was suspended in 2×
SDS sample buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol,and trace amount of bromophenol blue) containing 5% βMe, and incubated 5 min at 37 °C. Integral membrane protein fraction was subjected
to PNGase F (New England Biolabs; Beverly, MA) digestion 1 h at 37 °C
according to the manufacturer's instructions. Protein concentrations of
total cell lysates were determined using a BCA protein kit (Pierce; Thermo Fisher).

of hB4GN1 all variants, hB4GN1 variant 2, mB4GN1 all variants, andmB4GN1 variant 2 were 599, 698, 539, and 618 bp.
2.3. Real-time PCRFirst-strand cDNA synthesis was performed using ReverTra Ace qPCR
RT Master Mix with gDNA Remover (Toyobo; Osaka, Japan) and total
RNA isolated from human tissues (Takara), or human cell lines. Primers
and probes speciﬁc for hB4GN1 all variants (Hs00155195_m1) were from
Applied Biosystems (Foster City, CA). hB4GN1 variant 2 (primers, 5′CATCCAAATGTCTTCAGCATTAAGAT-3′ and 5′-TCTCCCGGCCTCCTTTCTTCT3′; probe, 5′-TCGGGTGTGTGAGGAAACCGAGATG-3′) and 18S rRNA
(Hs·PT.39a.22214856.g) were from Integrated DNA Technologies (IDT;
Coralville, IA). Real-time PCR was performed with 2 × TaqMan Universal
Master Mix II (Applied Biosystems) using a Step One Plus real-time PCRsystem (Applied Biosystems). Standard curves were constructed using
pFS466 (hB4GN1 variant all), pFS635 (hB4GN1 variant 2), or pFS636(18S rRNA), and copy numbers of tissues or cells were calculated.
2.4. Cell culture
Chinese hamster ovary CHO-K1 (CHO) cells, human kidney 293T
cells, and human leukemia HAP1 cells [29] were cultured respectively
in Ham's F-12 medium (Wako; Osaka, Japan), high-glucose DMEM
(Nacalai Tesque; Kyoto, Japan), and low-glucose IMDM (Nacalai
Tesque), each supplemented with 10% fetal bovine serum, 100 U/mL
penicillin, and 100 μg/mL streptomycin. CHO-K1 cells were transiently
transfected using Lipofectamine 2000 (Thermo Fisher) according to
the manufacturer's instructions.
2.5. Preparation of lentiviral vectors

2004

F. Shishido et al. / Biochimica et Biophysica Acta 1859 (2017) 2001–2011

2.7. ImmunoblottingA
Human B4GalNT1 (GM2/GD2/GA2 synthase)

Integral membrane protein fraction as above was subjected to immunoblotting. Proteins were separated by SDS-PAGE and transferred
to an Immobilon PVDF membrane (Millipore; Billerica, MA). The membrane was incubated 1 h with 1:1000 dilution of anti-FLAG antibodies(Agilent Technologies; Santa Clara, CA), or anti-GFP (IgY fraction)(Aves Labs; Tigard, OR), washed, and incubated 1 h with 1:20,000 dilution of HRP-conjugated donkey anti-mouse IgG (GE Healthcare; Little
Chalfont, UK) or anti-chicken IgY (Aves Labs). Labeling was detectedusing an ECL Select kit (GE Healthcare).

Exon 11variant 1a
Exon 1b
variant 1b
Exon 1cvariant 1c
M2

2.8. Immunoﬂuorescence microscopy
Transfected cells were cultured on coverslips, ﬁxed 15 min with 3.7%
formaldehyde in PBS at room temperature, rinsed with PBS, permeabilized (if necessary) with 0.5% SDS or 0.5% Triton X-100 in PBS, treatedwith Image-iT FX Signal Enhancer (Thermo Fisher) for 30 min, incubated 1 h with anti-FLAG (monoclonal antibody: Agilent; polyclonal antibody: Sigma), anti-HA (InvivoGen; San Diego, CA), anti-KDEL(StressGen Bioreagents; Victoria, BC, Canada), or anti-GM130 (BD Bioscience; Franklin Lakes, NJ) diluted 1:100 with 1% BSA in PBS, washedthree times in PBS, and incubated 30 min with Alexa 488-conjugatedanti-rabbit IgG antibodies or Alexa 594-conjugated anti-mouse IgG antibodies (Thermo Fisher) diluted in 1% BSA in PBS to 5 μg/mL. Coverslips
were washed three times in PBS, mounted on glass slides using ProLong
Gold antifade reagent (Thermo Fisher), and analyzed by ﬂuorescencemicroscopy (model FV1000, Olympus; Tokyo, Japan).

Exon 2

Exon 1avariant 2
M1 M2

BMouse B4GalNT1 (GM2/GD2/GA2 synthase)
Exon 1

Exon 2

Exon 11variant 1
M2
variant 2
M1 M2

C
120
100

Ratio (%)

2.9. Lipid analysis
Lipids (GSLs) were analyzed as described previously [30].
2.10. Cycloheximide chase

80 v2 Allv2 Allv2 All60
40
20

CHO-K1 cells stably expressing hM1-B4GN1-FLAG, hM1-B4GN1FLAG R4/6/7S mutant, or hM2-B4GN1-FLAG, or co-expressing hM1B4GN1-FLAG and hM2-B4GN1-HA, were cultured in Ham's F-12 withor without 50 μg/mL cycloheximide for 5 h at 37 °C. Total lysates and integral membrane protein fraction were prepared as described above.

0

variant 2 All
Human brain

variant 2 All variant 2 All
Mouse brain
Mouse liverD
12

3.1. New mRNA variant of human and mouse B4GalNAcT1 (GM2/GD2/GA2
synthase) that produces an isoform with long cytoplasmic tail
Many glycosyltransferases are type II membrane proteins that have ashort cytoplasmic tail (2–17 aa) and a luminal catalytic domain. In contrast, mouse ST3G5 includes an M1-ST3G5 isoform having a long (69aa) cytoplasmic tail. M1-ST3G5 is localized mainly in the ER, and the cytoplasmic tail contains R-based motif, a retrograde transport signal [26]. To
clarify the biological signiﬁcance of isoform production for lipid-glycosyltransferases, we examined B4GN1, which catalyzes GalNAc transfer to
GM3, GD3, or LacCer, for possible production of isoforms having distinctive NH2-terminal cytoplasmic tail lengths. Isoforms of ST3G5 areproduced by leaky scanning from several transcriptional variants [26].
Human B4GN1 (hB4GN1) transcripts are classiﬁed into three variants(1a, 1b, 1c) according to the position of transcription initiation (Exon 1a,1b, 1c) (Fig. 2A) [31]. Because these three exons do not contain an initiation codon, hB4GN1 is translated from a common initiation codon (M2) of
Exon 2 in these transcripts, indicating that hB4GN1 expresses only one
isoform. We searched for an EST clone containing the sequence upstreamof M2, using the NCBI-BLAST program (http://blast.ncbi.nlm.nih.gov/
Blast.cgi). After eliminating clones that included the same sequence asvariants 1a, 1b, and 1c, we identiﬁed four EST clones (DC30908,
DC341828, DC400781, DC302562) that included Exon 2 extending to

B4GN1 18s rRNA

3. Results

v2

All

10
8
6
4
2
0
Brain Prostate Skeletal
muscle293T

HAP1Fig. 2. New transcriptional variant and quantitation of human and mouse B4GN1. (A andB) Structures of human (A) and mouse (B) B4GN1 genes and their mRNA variants. Grayboxes: exons. Horizontal lines: introns. M1 and M2: initiation codons. The accession
number of human and mouse B4GN1 variant 2 are LC273521 and LC273292,
respectively. (C) mRNA expression of B4GN1 variants in human and mouse tissues.
Variant 2 and total variants were ampliﬁed by semi-quantitative RT-PCR using brain(human, mouse) and liver (mouse) tissues. Expression levels of variant 2 are shown aspercentage of level of total variants (deﬁned as 100%). (D) Quantitative analyses of
hB4GN1 transcripts in various tissues and cell lines by real-time PCR. Expression levelsof variant 2 and total variant were normalized relative to 18S rRNA transcript.

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the 5′-upstream side (termed Exon 2′; Figs. 2A and S1). These clones startfrom a position between Exon 1a/1b and 1c, or between 1c and 2 (dots in
Fig. S1). We termed this new transcript variant 2 (Fig. 2A). Notably, theinitiation codon (M1) is present in the sequence upstream of M2 in
Exon 2′. These ﬁndings indicate that the new hB4GN1 isoform having along NH2-terminal cytoplasmic tail is translated from variant 2.
The gene structure of mouse B4GN1 (mB4GN1) was reported as variant 1 (Fig. 2B; M2 = initiation codon) [17,32]. Using the same method,
we searched for new mRNA variants of mB4GN1, and extracted 25 EST
clones similar to hB4GN1 variant 2 (Fig. S2). Many of these clones startin the region between Exons 1 and 2, suggesting that this region is not
spliced. Again, we termed the new transcript variant 2 (Fig. 2B). Variant
2 includes a new initiation codon (M1) upstream of M2; therefore, a
new isoform is presumably translated.
To determine expression levels of variant 2, we performed semiquantitative RT-PCR using total RNAs of brain (human and mouse)and liver (mouse) tissues expressing GM2 (Fig. 2C). Forward and reverse primers were designed to amplify all variants, or only variant 2.
Expression level of variant 2 as a percentage of the value for all variants
was 18 ± 5.0% for human brain, 9.4 ± 2.7% for mouse brain, and 11.2 ±
2.3% for mouse liver. Variant 2 was a minor but detectable transcript inbrain and liver tissues. Next, copy numbers of B4GN1 variant 2 and all
variants for human brain, prostate, skeletal muscle, 293 T cells, andHAP1 cells were determined by real-time PCR. 293 T (derived fromhuman embryonic kidney) and HAP1 cells [29] express GM2. In brain
tissues, copy numbers of B4GN1 variant 2 and all variants per 106 copies
of 18 s rRNA were 1.2 ± 0.2 and 8.9 ± 1.5, and the ratio of variant 2 to all
variants was 13.5 ± 0.44%, similar to the value obtained by semi-quantitative RT-PCR. In other tissues and cell lines, gene expression levels of
hB4GN1 were much lower than in brain tissue, and ratios of variant 2 toall variants were also low (prostate: 4.3 ± 0.5%, skeletal muscle: 3.2 ±
0.4%, 293 T: 1.0 ± 0.2%, Hap1: 1.1 ± 0.07%). We compared aa sequencesof the isoforms translated from variant 2 of human and mouse tissues,termed hM1-B4GN1 and mM1-B4GN1 (Fig. 3). NH2-terminal length ofhM1-B4GN1 was 41 aa longer than that of mM1-B4GN1. Each of these
isoforms had R-based motifs in the long cytoplasmic tail, similarly to
M1-ST3G5 (Fig. 3, underlining).
We performed 5′-RACE analysis using cDNA of human and mousebrain tissues in an attempt to clarify transcriptional start sites (TSSs)
of B4GN1 variant 2. Unfortunately, the sequence between Exons 1c
and 2 was very similar to other genome sequences, and 5′-terminal sequences of variant 2 were therefore not ampliﬁed speciﬁcally. Use of
total RNA of human cell lines 293 T and HAP1 as templates gave thesame results. We were thus unable to identify TSSs of human variant
2. However, we succeeded in identifying the TSSs of mB4GN1 variants(Fig. S2). The 5′-termini of all clones analyzed in this study started

from Exon 1, and there were numerous TSSs (Fig. S2, black triangles).
These ﬁndings indicate that variant 2 of mB4GN1 is a splicing variant
in which the intron between Exons 1 and 2 is not removed. The resultof 5′-RACE analysis of mB4GN1 transcripts suggest that hB4GN1 variant
2 is also a splicing variant in which the intron between Exons 1c and 2 is
not removed.
3.2. Analysis of leaky scanning in new B4GN1 mRNA variant 2
The ﬁrst AUG codon is usually recognized as an initiation codon by
the ribosome. However, in some cases (known as “leaky scanning systems”) the ﬁrst AUG codon is skipped, and a downstream AUG codon
is recognized as an initiation codon [33]. In mammals, the optimal context (motif) for recognition of an AUG start codon is GCCRCCAUGG.
Within this motif, the purine nucleotide (R = A or G) in position − 3is the most highly conserved and most functionally important. In
mST3G5 mRNA, leaky scanning occurs even though the sequence is
CCUAGCAUGC [26], and does not stop completely even when the sequence is changed to GCCACCAUGG. We investigated whether leaky
scanning occurs in B4GN1 mRNA variant 2. The sequences in hB4GN1
and mB4GN1 mRNA variant 2 were ACCGAGAUGT and GCCGAGAUGG(Fig. 4A). In both these variants, the nucleotide in position − 3 is G;
this is presumably sufﬁcient for recognition of AUG. To compare the molecular masses of M1-B4GN1 and M2-B4GN1, we constructed plasmids
in which a Kozak sequence (ks; CACC) was inserted before M1 or M2 of

A

B

M1

M2

ks-hM1-B4GN1-FLAG
ks-hM2-B4GN1-FLAG
hM1-B4GN1-FLAG*

CACC AUG C---TAGG AUG T-CACC AUG T-CGAG AUG C---TAGG AUG T--

ks-mM1-B4GN1-FLAG
ks-mM2-B4GN1-FLAG
mM1-B4GN1-FLAG*

CACC AUG G---CAGG AUG C-CACC AUG C-GGAG AUG G---CAGG AUG C--

kDa
70
57

C

-

-

-

-

kDa
70
57

Fig. 3. Comparison of aa sequence of NH2-terminal cytoplasmic regions of human andmouse B4GN1. Alignments were generated by the ClustalW program (DNA Data Bank ofJapan). Black boxes: identical residues. Gray boxes: aa similarities. Underlining: Arginine(R)-based motif. TM: transmembrane region. M1 and M2: initiation codons.

Fig. 4. Analyses of leaky scanning of human and mouse B4GN1. (A) Nucleotide sequencesof human and mouse B4GN1 (hB4GN1 and mB4GN1) used. hB4GN1 or mB4GN1 wasfused with FLAG tag at C-terminus. Kozak sequence (ks; CACC) was inserted before M1
or M2 (initiation codon) of B4GN1 (ks-hM1-B4GN1-FLAG, ks-hM2-B4GN1-FLAG, ksmM1-B4GN1-FLAG, and ks-mM2-B4GN1-FLAG). hM1- and mM1-B4GN1-FLAG*: thesequence just before M1 is the mRNA sequence of variant 2. (B and C) CHO-K1 cells
were transiently transfected with pcDNA3 vector (−) or a plasmid encoding hM1B4GN1-FLAG, hM2-B4GN1-FLAG, hM1-B4GN1-FLAG*, mM1-B4GN1-FLAG, mM2-B4GN1FLAG, or mM1-B4GN1-FLAG*. Integral membrane protein fractions were prepared,
treated with PNGase F, and analyzed by immunoblotting with anti-FLAG antibodies.

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B4GN1 (ks-hM1-B4GN1-FLAG, ks-hM2-B4GN1-FLAG, ks-mM1-B4GN1FLAG, ks-mM2-B4GN1-FLAG), and transiently transfected these plasmids into CHO-K1 cells. Integral membrane protein fractions were prepared and immunoblotted with anti-FLAG antibodies. Human andmouse B4GN1s have N-glycans [34]; we therefore treated integral fraction samples with peptide: N-glycosidase F (PNGase F) to remove Nglycans before immunoblotting. hM1-, hM2-, mM1-, and mM2B4GN1-FLAG proteins were detected respectively as 57.1, 49.6, 52.6,
and 51.0 kDa bands (Fig. 4B,C). We next replaced the Kozak sequencesof ks-hM1- and ks-mM1-B4GN1-FLAG with the original mRNA sequences (human: CGAG; mouse: GGAG), generated hM1- and mM1B4GN1-FLAG* (Fig. 4A), and transiently transfected the plasmids into
CHO-K1 cells. Integral membrane protein fractions were prepared,
treated with PNGase F, and immunoblotted with anti-FLAG antibodies.
hM1- and mM1-B4GN1-FLAG* bands migrated to the same positions
as did ks-hM1- and ks-mM1-B4GN1-FLAG bands (Fig. 4B,C). Taken together, these ﬁndings indicate that human and mouse M2-B4GN1 isoforms are not produced from B4GN1 mRNA variant 2 by leaky scanning.
3.3. Retrograde transport of M1-B4GN1(N)-GFP chimeric proteins by Rbased motif
Human and mouse B4GN1s have RR and RXR sequences (R-based
motifs) in the NH2-terminal cytoplasmic region (Fig. 3, underlining).
To function as retrograde transport signals, R-based motifs must be positioned distally from a lipid bilayer [28]. To assess the possible functionof the above sequences as R-based motifs, we transiently transfected
CHO-K1 cells with fusion proteins for the NH2-terminus of hM1-,
hM2-, mM1-, or mM2-B4GN1 containing the transmembrane region
and green ﬂuorescent protein (GFP) (hM1-B4GN1(N)-GFP, hM2B4GN1(N)-GFP, mM1-B4GN1(N)-GFP, mM2-B4GN1(N)-GFP) (Fig.
5A). Integral membrane protein fractions were prepared andimmunoblotted with anti-GFP antibodies. hM1-B4GN1(N)-GFP, hM2B4GN1(N)-GFP, mM1-B4GN1(N)-GFP, and mM2-B4GN1(N)-GFP chimeric proteins (the predicted molecular mass is 39.5 kDa, 31.5 kDa,
37.9 kDa and 33.6 kDa) were detected as 40 kDa, 28 kDa, 38 kDa, and30 kDa bands, respectively (Fig. 5B). Some extra bands were detectedunder the target bands and would be degradation products in lysosome.
These chimeric proteins were observed by ﬂuorescence microscopy(Figs. 5C, D, S3, S4, S5). Cells were ﬁxed, permeabilized, stained with
anti-GFP antibodies, and incubated with Alexa 488-conjugated antichicken IgY. hM1- and mM1-B4GN1(N)-GFP displayed a reticular immunoﬂuorescence pattern in the perinuclear and cytosolic regions.
This pattern was attributable to ER localization of hM1- and mM1B4GN1(N)-GFP because it could be superimposed on the ER patternproduced by an antibody against ER retention signal KDEL (Figs. S3,
S5). In contrast, hM2- and mM2-B4GN1(N)-GFP were localized in compact juxtanuclear reticulum. hM2- and mM2-B4GN1(N)-GFP were partially co-localized with GM130, a Golgi marker protein (Figs. S4, S5).
These ﬁndings suggested that R-based motifs of the M1-B4GN1 NH2terminus function as retrograde transport signals.
To further investigate this possibility, we generated hM1B4GN1(N)-GFP R4S, R6S, R7S, and R6/7S mutants by site-directed mutagenesis, and examined their subcellular localization (Figs. 5C, S3). Thesemutants were localized in both the Golgi and ER (“Golgi/ER” in Fig. 5C),or the Golgi only. We generated a hM1-B4GN1(N)-GFP R4/6/7S mutant
and observed its subcellular localization. It was localized entirely in the
Golgi, indicating that the 4R6R7R sequence is important for ER targetingin hM1-B4GN1(N)-GFP. Finally, we generated mM1-B4GN1(N)-GFP
R8S, R11S, R12S, R13S, R14S, and R11/12/13/14S mutants and examined
their subcellular localization (Figs. 5D, S5). The R11S, R12S, R13S, and
R14S mutants were localized mainly in the Golgi, the R8S mutant was
localized in the ER, and the R11/12/13/14S mutant was localized
completely in the Golgi. These ﬁndings indicate that the 11R12R13R14R
sequence is necessary for localization of mM1-B4GN1(N)-GFP in theER, and that the R-based motifs located on the NH2-terminal

cytoplasmic tail of hM1- and mM1-B4GN1(N)-GFP function collectively
as a retrograde transport signal.
3.4. Subcellular localization and enzyme activity of B4GN1 full-length
To examine the possibility that M1-B4GN1 full-length is also localized in the ER by R-based motifs, we used a lentivirus expression system
to express hM1-B4GN1-FLAG and hM2-B4GN1-HA in CHO-K1 cells, andapplied indirect immunoﬂuorescence microscopy with anti-FLAG andanti-HA antibodies to evaluate subcellular localization (Figs. 6A, S6A).
hM1-B4GN1-FLAG staining pattern was partially consistent with thatof the ER (KDEL) and the Golgi (GM130). Because hM1-B4GN1-FLAG
R4/6/7S mutant was localized completely in the Golgi, a part of hM1B4GN1-FLAG was localized in the ER by R-based motif. In contrast,
hM2-B4GN1-HA, which does not contain R-based motif in the cytoplasmic tail, was localized in the Golgi. M1-ST3G5, which has R-based motif,
is localized mainly in the ER [26], suggesting that retrograde transport ofhM1-B4GN1-FLAG does not occur efﬁciently. We next transfected cellswith mM1-B4GN1-FLAG and mM2-B4GN1-HA, and subjected them toanti-FLAG and anti-HA antibody staining (Figs. 6B, S6B). mM1-B4GN1FLAG was localized in both the ER and Golgi, whereas mM2-B4GN1HA and mM1-B4GN1-FLAG R11/12/13/14S mutant were localized onlyin the Golgi. These ﬁndings indicate that the R-based motif of M1B4GN1 full-length functions in part as a retrograde transport signal. In
addition, we also visualized the localization of non-tagged forms of
mM1-B4GN1 and mM2-B4GN1 using anti-B4GN1 antibody(9535Ex_ProteinA), which was raised against the luminal domain ofmouse B4GNT1 (504 aa). The localization patterns of the non-tagged
forms were quite similar to those of mM1-B4GN1-FLAG and mM2B4GN1-HA, indicating that the epitope tags do not affect the subcellular
localization of B4GN1 (Fig. S7).
We compared in vivo enzyme activity of hM1- vs. hM2-B4GN1 in
CHO-K1 cells, which express primarily GM3 ganglioside. Expression
levels of hM1- and hM2-B4GN1-FLAG were nearly identical (Fig. 7A).
To evaluate GSL composition, we separated acidic and neutral lipid fractions from total lipid extracts using DEAE-Sephadex A-25 column, andanalyzed the fractions by TLC. Presence of hM1- or hM2-B4GN1-FLAGin the cells resulted in enhanced expression of GM2 and GD1a in acidic
fraction, and reduced expression of LacCer and Gb4 in neutral fraction(Fig. 7B). No differences in lipid composition were observed, indicatingthat hM1- and hM2-B4GN1 display similar in vivo enzyme activities
when overexpressed.
3.5. Effects of hM1-/hM2-B4GN1 heterodimer formation on intracellular localization and stability
The observed expression level of mRNA variant 2 in human andmouse tissues (Fig. 2C, D) suggests that M1-B4GN1 is a minor isoform.
M1- and M2-B4GN1 isoforms presumably coexist in tissues and cells.
M2-B4GN1 isoform was reported to form a dimer via disulﬁde bond(80C and 82C) on the luminal side [35], suggesting that B4GN1 mayform not only M1/M1 or M2/M2 homodimers but also a M1/M2 heterodimer. To assess this possibility in our CHO-K1 cells, we co-expressed
hM1-B4GN1-FLAG (hM1-FLAG) (predicted molecular mass 68.2 kDa)
and hM2-B4GN1-HA (hM2-HA) (predicted molecular mass 60.2 kDa)
using a lentivirus expression system. Integral membrane protein fractions were prepared with or without β-mercaptoethanol (βMe), treatedwith PNGase F, and immunoblotted with anti-FLAG and anti-HA antibodies (Fig. 8A). In CHO-K1/hM1-FLAG and CHO-K1/hM2-HA cells,
M1/M1 and M2/M2 homodimers were detected respectively as~ 150 kDa and ~ 125 kDa bands, without βMe. In cells co-expressing
M1 and M2 isoforms, M1/M2 heterodimer was detected as an~130 kDa band. A faint hM1-FLAG band was detected in the same position as M1/M2 heterodimer under βMe(−) condition when cells
expressed only hM1-FLAG. hM1-FLAG was a single band underβMe(+) condition, indicating that the NH2-terminus of M1-FLAG was

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A

B
M1
hM1-B4GN1(N)-GFP

M2R

hM2-B4GN1(N)-GFP

TM

GFP

TM

GFP

kDa
43
36.5
28

kDa
43
36.5
28

*

mM1-B4GN1(N)-GFP

M1 M2
TM
R

GFP

mM2-B4GN1(N)-GFP

TM

GFP

C

*

*

-

*

-

D

hM1-B4GN1(N)-GFP

mM1-B4GN1(N)-GFP

WT

R4S

R6S

WT

ER

Golgi/ER

Golgi/ER

ER

ER

Golgi

R7S

R6/7S

R4/6/7S

R12S

R13S

R14S

Golgi/ER

Golgi/ER

Golgi

Golgi

Golgi

Golgi

R11/12/13/14S

mM2-B4GN1(N)-GFP

hM2-B4GN1(N)-GFP

R8S

WT

R11S

WT

Golgi
Golgi

Golgi

Golgi/ER

Golgi

ER

120

100

100
Ratio (%)

Ratio (%)

Golgi

120

80
60

60

WT

R11/12/13/14S

R14S

R13S

R12S

R11S

R8S

WT

WT

R4/6/7S

R6/7S

0

R7S

0

R6S

20
R4S

20
WT

40

hM2-B4GN1(N)-GFP

ER

80

40

hM1-B4GN1(N)-GFP

Golgi/ER

mM1-B4GN1(N)-GFP
mM2-B4GN1(N)-GFP

Fig. 5. ER targeting of M1-B4GN1(N)-GFP by R-based motif of cytoplasmic region. (A) Structure of cDNA constructs used. Each constructed plasmid encoded a fusion protein of the NH2terminus of hM1-B4GN1, hM2-B4GN1, mM1-B4GN1, or mM2-B4GN1 containing the transmembrane region, and GFP (hM1-B4GN1(N)-GFP, hM2-B4GN1(N)-GFP, mM1-B4GN1(N)-GFP,
and mM2-B4GN1(N)-GFP). TM: transmembrane region. M1 and M2: initiation codons. R: Arginine (R)-based motif. (B) CHO-K1 cells were transiently transfected with pcDNA3 vector (−), hM1-B4GN1(N)-GFP, hM2-B4GN1(N)-GFP, mM1-B4GN1(N)-GFP, or mM2-B4GN1(N)-GFP. Integral membrane protein fractions were prepared and analyzed by immunoblotting with
anti-GFP antibodies. The asterisk indicates the putative degradated forms of the respective proteins. (C and D) CHO-K1 cells were transiently transfected with M1-B4GN1(N)-GFP wild type(WT), M2-B4GN1(N)-GFP WT, or M1-B4GN1(N)-GFP mutants, in which the arginine (R) of the R-based motif was substituted with serine (S) (C: human; D: mouse). Cells were ﬁxed,
permeabilized with 0.5% Triton X-100 in PBS, stained for 1 h with anti-GFP antibodies, incubated with Alexa 488-conjugated anti-chicken IgY, and visualized by confocal laserscanning microscopy. For co-localization studies, cells were incubated with antibodies against GM130 (a cis Golgi marker) or KDEL (an ER marker) for 1 h, and then with Alexa 594conjugated anti-mouse IgG for 30 min. Localization patterns (Golgi, Golgi/ER, ER) were quantiﬁed by cell counting, and quantitative data were expressed as mean ± SD from three ormore independent counts. Bar: 10 μm.

partially uncleaved. We therefore speculated that a small amount of
M1/M1 homodimer was not fully denatured by SDS under βMe(−)
condition, and that apparent molecular weight was less than
~ 150 kDa. The amount of M1/M2 heterodimer was comparable tothose of M1/M1 or M2/M2 homodimers; i.e., efﬁciency of heterodimer

formation was similar to that of homodimer formation when isoform
expression levels were equal.
Subcellular localization of hM1-FLAG and hM2-HA was evaluated incells co-expressing these isoforms (Fig. 8B). hM1-FLAG was localized inboth the Golgi and ER, whereas hM2-HA was localized only in the Golgi.

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A

A
hM1-FLAG

hM1-FLAG R4/6/7S

hM2-HA

hB4GN1-FLAG-actinM1 M2

B
Neutral fraction

Acidic fraction

Ratio (%)

Golgi

120
100
80
60
40
20
0

Golgi/ER

ER

-

hM1

mM1-FLAG

hM1
R4/6/7S

hM2

mM1-FLAG
R11/12/13/14S

Golgi

Ratio (%)

Gb3
Gb4

GD1a

B

120
100
80
60
40
20
0

GlcCerLacCer

GM3
GM2

Golgi/ER

mM2-HAER

M1 M2
hB4GN1
-FLAG

-

M1

M2

hB4GN1-FLAG

Fig. 7. In vivo enzyme activities of hM1- and hM2-B4GN1. (A) Total lysates and integral
membrane protein fractions were prepared from CHO-K1 cells stably expressing hM1B4GN1-FLAG (M1) and hM2-B4GN1-FLAG (M2), treated with PNGase F, and analyzed
by immunoblotting with anti-FLAG antibodies (for integral membrane protein fractions)or anti-β-actin antibodies (for total lysates). (B) GSLs (acidic and neutral fractions)
extracted from CHO-K1 (−), CHO-K1/hM1-B4GN1 (M1), or CHO-K1/hM2-B4GN1 (M2)cells, corresponding to 2 mg protein, were separated on TLC plates and stained withorcinol sulfuric acid reagent.

co-expressed, expression levels of hM1-FLAG and hM2-HA in treatedcells were 51 ± 17% and 55 ± 6% of levels in untreated cells. These ﬁndings indicate that M2-HA stability is slightly enhanced by co-expression
with M1-FLAG. Because subcellular localization of M2-HA was unaffected by M1-FLAG co-expression, the increase in M2-HA stability was most
likely due to prolongation of Golgi retention.
4. Discussion

mM1

mM1
mM2
R11/12/13/14S

Fig. 6. Subcellular localization of M1-B4GN1 and M2-B4GN1 full-length. CHO-K1 cells
were stably transfected with hM1-B4GN1-FLAG (hM1-FLAG), hM1-B4GN1-FLAG R4/6/
7S mutant, hM2-B4GN1-HA (hM2-HA), mM1-B4GN1-FLAG (mM1-FLAG), mM1-B4GN1FLAG R11/12/13/14S mutant, or mM2-B4GN1-HA (mM2-HA) using a lentivirus
expression system (A: human; B: mouse). Cells were ﬁxed, permeabilized with 0.5% SDS
in PBS, stained with anti-FLAG or anti-HA antibodies and Alexa 488-conjugated antirabbit IgG, and visualized by confocal laser-scanning microscopy. For co-localization
studies, cells were incubated with anti-GM130 or anti-KDEL antibodies for 1 h, and thenwith Alexa 594-conjugated anti-mouse IgG for 30 min. Localization patterns (Golgi,
Golgi/ER, ER) were quantiﬁed by cell counting, and quantitative data were expressed asmean ± SD from three or more independent counts. Bar: 10 μm.

Similar localization patterns were observed for cells expressing only one
isoform (Fig. S8), suggesting that retrograde transport of M1/M2 heterodimer did not occur. Partial ER localization of M1/M1 homodimer
was not affected by appearance of M1/M2 heterodimer.
To investigate the effect of M1/M2 heterodimer formation on stability of M1- and M2-B4GN1 isoforms, cells expressing hM1-FLAG/hM2HA, hM1-FLAG, or hM2-HA were cultured for 5 h with or without cycloheximide (CHX), an inhibitor of protein synthesis. Integral membraneprotein fractions were prepared, treated with PNGase F, andimmunoblotted with anti-FLAG or anti-HA antibodies (Fig. 8C, D).
When a single isoform was expressed, expression levels of hM1-FLAGand hM2-HA in CHX-treated cells were respectively 58 ± 12% and 30± 11% of levels in untreated cells. In contrast, when the isoforms were

To elucidate the biological signiﬁcance of lipid-glycosyltransferase
isoform production, we searched for mRNA variants encoding new isoforms. An mRNA variant was found that encodes a new B4GN1 isoform(M1-B4GN1) that catalyzes synthesis of GM2, GD2, and GA2 in mice andhumans (Fig. 2). The new isoform has NH2-terminal length much longer
than that of previously known isoform M2-B4GN1 (Fig. 3). Human andmouse M1-B4GN1 isoforms (hM1-B4GN1, mM1-B4GN1) have respective lengths 81 and 40 aa, whereas hM2-B4GN1 and mM2-B4GN1
both have length 6 aa (Fig. 3). NH2-terminal aa sequences of hM1and mM1-B4GN1 have low similarity. On the other hand, both isoforms
have R-based motifs, and hM1- and mM1-B4GN1(N)-GFP chimeric proteins are localized in the ER. R-based motifs function as retrograde
transport signals when positioned distally from a lipid bilayer [28].
The consensus sequences of R-based motifs in mM1-ST3G5 are RR,
RXXXXR, and RXXXXXR. We use the term “functional distance” to describe the space essential for an R-based motif to function as retrograde
transport signal, and deﬁne this distance as the number of aa residues
from R to a lipid bilayer. Functional distances of RR, RXXXXR, andRXXXXXR in mM1-ST3G5(N)-GFP are 30, 45, and 53 aa [26]. The
R11S, R12S, R13S, and R14S single mutants in mM1-B4GN1(N)-GFP
are localized mainly in the Golgi, indicating that the four R residues(11R12R13R14R), but not RRR, RXR, or RR sequence, are essential for function as R-based motif (Figs. 5D, S4). The distance between the
11 12 13 14
R R R R sequence and a lipid bilayer is 27 aa; this is likely insufﬁcient for RRR, RXR, or RR sequence to function. The position of 51RX53R
sequence in hM1-B4GN1 is homologous to that in mM1-B4GN1(Fig. 3). Again, however, this sequence likely cannot function as a retrograde transport signal because of the short distance (29 aa). hM1-

F. Shishido et al. / Biochimica et Biophysica Acta 1859 (2017) 2001–2011

B4GN1 has 4RX6R7R sequence in addition to 51RX53R sequence, and theformer is located 75 aa from a lipid bilayer. Because the hM1B4GN1(N)-GFP R4/6/7S mutant is localized completely in the Golgi,
the 4RX6R7R sequence clearly functions as an ER targeting signal. The
hM1-B4GN1(N)-GFP R4S, R6S, and R7S mutants are localized in boththe ER and Golgi, indicating that the RR, RXR, and RXXR motifs function
in part as signal sequences (Figs. 5C, S3). The hM1-B4GN1(N)-GFP R6/
7S mutant is also localized in both the ER and Golgi (Figs. 5C, S3),

Me(-)

kDa
180
130

Me(+)

A

75

FLAG

kDa

HA

M1/M1
M1/M2 130

M1/M2
M2/M2

63

B
Merge

hM2-HA

hM1-FLAGC
hM1-FLAG
hM2-HAActin
CHX -Ratio of expression level

D

100

+

-

+CHX

80

*

**

60
40
20

Co-exphM2-HA
hM1-FLAG

hM2-HA

hM1-FLAG

0

+

-

+

2009

suggesting that only the 4R sequence functions as signal. A single R
has never been shown to be sufﬁcient to function as a signal sequence.
Another basic aa close to the R-based motif, such as 8K or 12R, mayalso function as a retrograde transport signal.
Expression levels of lipid-glycosyltransferases are extremely low,making it difﬁcult to detect endogenous proteins. We previously detected endogenous ST3G5 protein by immunoprecipitation and immunoblotting, using speciﬁc anti-ST3G5 polyclonal antibody and brain
tissues of St3gal5+/+ and St3gal5−/− mice [26]. Similarly, in an attempt
to detect endogenous B4GN1 protein, microsome fractions were prepared from liver or brain tissues of B4galnt1+/+ and B4galnt1−/− miceand treated with PNGase F. The fractions were analyzed by immunoblotting using anti-B4GN1 antibody (ab135902), which can recognize
overexpressed non-tagged B4GN1 proteins in CHO-K1 cells (Fig. S9A).
But, we were unable to observe the bands of endogenous B4GN1 protein(Fig. S9C, E). Moreover, we concentrated B4GN1 proteins including in
the microsome fraction of liver tissue by immunoprecipitation using
anti-B4GN1 antibody (9535Ex_ProteinA) (Fig. S9D). Nevertheless, endogenous B4GN1 protein could not be detected. In addition, the nontagged forms (mM1-B4GN1 and mM2-B4GN1) treated with PNGase F
were detected as two bands by immunoblotting using anti-B4GN1 antibodies (9535Ex_ProteinA or ab135902) (Fig. S9A), whereas tagged
forms of B4GN1 were detected as a single band by immunoblotting
using anti-FLAG or anti-HA antibody (Figs. 4, 7, and 8). Since the predicted molecular masses of mM1-B4GN1 and mM2-B4GN1 are
63.4 kDa and 59.2 kDa, respectively, the upper bands in Fig. S9A are obviously high molecular masses. This may suggest that the two bands are
due to the post-translational modiﬁcations, such as phosphorylations.
We examined subcellular localization, enzyme activity, and stabilityof overexpressed B4GN1 proteins. M1-B4GN1(N)-GFP chimeric protein
was completely localized in the ER, whereas M1-B4GN1 full-length waspartly localized in the Golgi. In view of the localization of M1-ST3G5 fulllength and M1-ST3G5(N)-GFP chimeric protein in the ER [26], the efﬁciency of retrograde transport in M1-B4GN1 full-length may be lower
than that in M1-ST3G5. COPI-coated vesicles including B-subcomplex(α-, β-, and ε-COP) and F-subcomplex (β-, δ-, γ-, and ζ-COP) are involved in retrograde transport from the Golgi to the ER [36]. R-based
motif interacts with acidic aa of the pocket formed by β-COP and δCOP in F-subcomplex [27]. Another retrograde transport signal,
K(X)KXX motif, interacts with α-COP or β’-COP in B-subcomplex [37,38]. Because F-subcomplex interacts with B-subcomplex [39], cargo
proteins having R-based motifs are presumably close to those having
K(X)KXX motifs when these proteins are packed into the same COPIcoated vesicle. It is therefore possible that the large luminal domainsof these cargo proteins are responsible for steric hindrance between
them. The molecular mass of GFPs, including M1-B4GN1(N)-GFP, isFig. 8. Homodimer and heterodimer formation of hB4GN1 isoforms. (A) Integral
membrane protein fractions were prepared from CHO-K1 cells stably expressing hM1B4GN1-FLAG (hM1-FLAG) or hM2-B4GN1-HA (hM2-HA), or stably co-expressing hM1B4GN1-FLAG and hM2-B4GN1-HA (CHO-K1/hM1-B4GN1-FLAG, CHO-K1/hM2-B4GN1HA, or CHO-K1/hM1-B4GN1-FLAG/hM2-B4GN1-HA), and treated with (+) or without(−) βMe. Samples were treated with PNGase F and analyzed by immunoblotting with
anti-FLAG (left panel) or anti-HA (right panel) antibodies. (B) CHO-K1/hM1-B4GN1FLAG, CHO-K1/hM2-B4GN1-HA, and CHO-K1/hM1-B4GN1-FLAG/hM2-B4GN1-HA cells
were ﬁxed, permeabilized with 0.5% SDS in PBS, stained with anti-FLAG (rabbit
polyclonal) and anti-HA (mouse monoclonal) antibodies, incubated with Alexa 488conjugated anti-rabbit IgG and Alexa 594-conjugated anti-mouse IgG, and visualized by
confocal laser-scanning microscopy. Right panel: merged images. Bar: 10 μm. (C and D)
CHO-K1/hM1-B4GN1-FLAG, CHO-K1/hM2-B4GN1-HA, and CHO-K1/hM1-B4GN1-FLAG/
hM2-B4GN1-HA cells were cultured with (+) or without (−) 50 μg/mL cycloheximide(CHX) for 5 h at 37 °C. C: Total lysates and integral membrane protein fractions wereprepared, treated with PNGase F, and analyzed by immunoblotting with anti-FLAG (forintegral membrane protein fractions), HA (for integral membrane protein fractions), or
anti-β-actin antibodies (for total lysates). D: Expression levels of CHX (−) and (+)
were quantiﬁed using the ImageJ software program. Expression levels of CHX (+) are
expressed as ratio relative to corresponding CHX (−) level. Quantitative data areexpressed as mean ± SD from three or more independent experiments. *P b 0.01; **P b
0.05. Co-exp.: co-expression with hM1-B4GN1-FLAG and hM2-B4GN1-HA.

2010

F. Shishido et al. / Biochimica et Biophysica Acta 1859 (2017) 2001–2011

Nomenclatureaaamino acid(s)B3GT4
UDP-Gal: βGlcNAc β1,3-galactosyltransferase polypeptide 4
B4GN1 B4GalNT1/β1,4 N-acetylgalactosaminyltransferase 1
Cerceramide

Lysosome

Degradation
Endosome

transRetrograde transport
between Golgi

Golgi retention= stabilization
medialGolgi

26.8 kDa. The molecular mass of native luminal domains of M1-B4GN1
is 56.5 kDa or for practical purposes 113 kDa, because B4GN1 forms
homodimers. Our ﬁndings suggest that cargo proteins having small luminal domains (e.g., GFP), in comparison with those having large luminal domains (e.g., B4GN1), are efﬁciently localized in the ER by R-based
motif. We observed reduced retrograde transport of M1-B4GN1(N)2GFP, which has two GFPs connected in tandem in the luminal domain(Fig. S10). The large luminal domain of B4GN1 may cause steric hindrance with other cargo proteins having K(X)KXX motif, resulting in reduced efﬁciency of retrograde transport.
M1-ST3G5 having R-based motif is localized mainly in the ER, leading to suppression of its GM3 synthesis activity in vivo [26]. M1B4GN1 is partially localized in the ER; however, enzyme activity of
hM1-B4GN1 in vivo was identical to that of hM2-B4GN1 (Figs. 6, 7B).
To investigate in vivo enzyme activities of hM1- and hM2-B4GN1, we
expressed these enzymes in CHO-K1 cells using a lentivirus expression
system. Such systems generally lead to excessive protein expression incomparison with endogenous protein expression. If our experimental
conditions resulted in saturation of hM1-B4GN1 in the Golgi, differencesin subcellular localization between hM1- and hM2-B4GN1 may nothave been reﬂected in their enzyme activity in vivo. In future study,
we will establish knock-out cells expressing only M1- or M2-B4GN1
using CRISPR/Cas9 system, and clarify the contribution of M1- or M2B4GN1 to ganglioside biosynthesis in the Golgi.
hM1-B4GN1 was stable relative to hM2-B4GN1 (Fig. 8C, D). hM1B4GN1 stability was reduced by R4/6/7S mutation (data not shown), indicating that R-based motif is necessary for such stability. hM1-B4GN1
was partially localized in the ER, whereas hM2-B4GN1 was localized
in the Golgi. hM1-B4GN1 stability can therefore be attributed simply
to ER localization. In contrast, when hM2-B4GN1 was co-expressedwith hM1-B4GN1, hM2-B4GN1 stability increased without ER localization (Fig. 8B, C, D). Under this condition, hM1-B4GN1 homodimer,
hM2-B4GN1 homodimer, and hM1-B4GN1/hM2-B4GN1 heterodimer
were all present, in a ratio of ~ 1:1:1 (Fig. 8A). Taken together, theseﬁndings suggest that hM1-B4GN1/hM2-B4GN1 heterodimer is nottargeted to the ER by retrograde transport, but is more stable thanhM2-B4GN1 homodimer (Fig. 9). ST3G5 and sphingomyelin synthase
2 are retained in the Golgi by retrograde transport between the Golgi
cisternae during Golgi maturation [40–42]. The R-based motif(4RX6R7R sequence) of hM1-B4GN1 may promote retrograde transport
between the Golgi cisternae or suppress export of heterodimer from theGolgi. Our ﬁndings indicate that Golgi retention, not ER localization, is a
key rate-limiting step in hB4GN1 degradation.
Expression of mRNA variant 2 accounted for only 10–20% of total
B4GN1 mRNA expression in human and mouse brain tissues (Fig. 2). Results of 5′-RACE analysis of mB4GN1 (Fig. 2S) suggested that hB4GN1
variant 2 may also be a splicing variant, and that expression level is regulated by a tissue-speciﬁc alternative splicing system. Because M1B4GN1 is produced only from mRNA variant 2, expression level of M1B4GN1 is substantially lower than that of M2-B4GN1. Most M1-B4GN1
therefore forms a heterodimer with M2-B4GN1, but not a homodimer
with itself, indicating that M1-B4GN1 expression level determines overall stability of B4GN1 through prolongation of Golgi retention by Rbased motif. mRNA variant 2 is more highly expressed in human brain
tissue than in other tissues. Since brain tissue contains not only a-series
but also b-series gangliosides, maintenance of B4GN1 protein level may
be necessary to control both synthesis pathways. Results of the present
and previous studies, taken together, indicate that coexistence of several B4GN1 isoforms, each with distinctive stability and subcellular localization, is essential for stable supplies and homeostasis of various GSLs.

R-based motif
cis
Retrograde transport
From Golgi to ER
ER

M1/M1

M1/M2

M2/M2

Fig. 9. Intracellular dynamics of B4GN1. M1/M1 homodimers are localized in the Golgi andpartially localized in the ER. M1-B4GN1 has R-based motif in its cytoplasmic tail, which is
involved in both retrograde transport from the Golgi to the ER and Golgi retention. M2/M2
homodimers without R-based motif are localized in the Golgi. M1/M2 heterodimers arealso localized in the Golgi even though they have R-based motif. M1/M2 heterodimers
are more stable than M2/M2 homodimers, but are not localized in the ER. This stability
is due to prolongation of Golgi retention by R-based motif, as explained in Discussion.

CHX
ER
GalCer
GalNAcGFP
GalT IGlcAT-P
GlcCer
Golgi
GSLks
LacCerO-FUT1
ST3G2
ST3G5
ST8S1

cycloheximide
endoplasmic reticulum
galactosylceramide
N-acetylgalactosamine
green ﬂuorescent protein
β1,4-galactosyltransferase I
glucuronyl transferase
glucosylceramide
Golgi apparatus
glycosphingolipid
Kozak sequence
lactosylceramide
o-fucosyltransferase
PNGase F peptide: N-glycosidase F
ST3 β-galactoside α2,3-sialyltransferase 2
ST3Gal5/ST3 β-galactoside α2,3-sialyltransferase 5
ST8 α-N-acetyl-neuraminide α2,8-sialyltransferase 1

Transparency document
The Transparency document, associated with this article can befound, in online version.

Conﬂict of interest
We do not have any conﬂict of interest.

F. Shishido et al. / Biochimica et Biophysica Acta 1859 (2017) 2001–2011

Acknowledgments
This study was supported by a Grant-in-Aid for Scientiﬁc Research (B)from MEXT (to J.I.) (16H04767), Mizutani Foundation for Glycoscience(J.I.), Naito Foundation (J.I.), ONO Medical Research Foundation (J.I.),
Uehara Memorial Foundation (J.I.), and the MEXT-Supported Program
for the Strategic Research Foundation at Private Universities(S1201031). The authors are grateful to Dr. S. Anderson for English editingof the manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbamem.2017.07.006.
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Glycoconj J
DOI 10.1007/s10719-017-9788-1

ORIGINAL ARTICLE

Identification of a new liver-specific c-type mRNA transcriptionalvariant for mouse ST3GAL5 (GM3/GM4 synthase)
Fumi Shishido 1 & Satoshi Uemura 2 & Takahiro Nitta 3 & Jin-ichi Inokuchi 1

Received: 22 June 2017 / Accepted: 26 July 2017
# Springer Science+Business Media, LLC 2017

Abstract GM3, a major lipid component of the plasma
membrane outer leaflet in mammalian cells, is synthesizedin the luminal side of Golgi by ST3GAL5 protein(ST3G5), a type II membrane protein. Two strains of
St3Gal5 knockout mice have been established for studies
of GM3 physiological function: St3Gal5-Ex5-KO (lacking exon 5, which contains the catalytic domain of
ST3G5), and St3Gal5-Ex3-KO (lacking exon 3, whichcontains the initiation codons). Results of the present
study demonstrate that GM3 synthesis is still present, at
a low level, in liver of St3Gal5-Ex3-KO mice. St3Gal5
has two mRNA transcriptional variants: a-type and b-type.
When exon 3 is deleted, ST3G5 is not translated from atype or b-type, as a result of initiation codon deletion orframe shift. Through NCBI database search and real-time
PCR analyses of various mouse tissues, we identified aliver-specific St3Gal5 transcriptional variant (c-type)
Electronic supplementary material The online version of this article(doi:10.1007/s10719-017-9788-1) contains supplementary material,
which is available to authorized users.
* Satoshi Uemuras-uemura@tohoku-mpu.ac.jp* Jin-ichi Inokuchi
jin@tohoku-mpu.ac.jp
1

Division of Glycopathology, Institute of Molecular Biomembrane
and Glycobiology, Tohoku Medical and Pharmaceutical University,
4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan

2

Division of Medical Biochemistry, Faculty of Medicine, Tohoku
Medical and Pharmaceutical University, 4-4-1, Komatsushima,
Aoba-ku, Sendai, Miyagi 981-8558, Japan

3

Division of Pathophysiology, Faculty of Pharmacy, Tohoku Medical
and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku,
Sendai, Miyagi 981-8558, Japan

capable of producing artificial ST3G5 (M*-ST3G5) having GM3 synthase activity in the absence of exon 3.
St3Gal5-Ex3-KO mice expressed c-type mRNA without
exon 3 (c-type−/−) in liver. The transmembrane and catalytic domains of M*-ST3G5 translated from c-type −/−were identical to those from wild-type, although the cytoplasmic regions differed. Expression of M*-ST3G5 in embryonic fibroblasts derived from St3Gal5-Ex3-KO miceled to GM3 synthesis; M*-ST3G5 thus displayed enzyme
activity in vivo. Taken together, our findings indicate that
expression of liver-specific c-type variant accounts for the
residual GM3 synthase activity observed in liver of
St3Gal5-Ex3-KO mice.
Keywords Sialyltransferase . mRNA transcriptional variant .
Ganglioside GM3

Abbreviations
ER
Endoplasmic reticulum
Ex
Exon
Golgi
Golgi apparatus
GSL
Glycosphingolipid
H3K4me1 Mono-methylation of residue Lys4 in histone H3
H3K4me3 Tri-methylation of residue Lys4 in histone H3
LacCerLactosylceramide
MEF
Mouse embryonic fibroblast
Neo
Neomycin resistance gene
ORF
Open reading frame
St3Gal5ST3 β-galactoside α2,3-sialyltransferase 5
ST3G5
ST3GAL5 protein
TLC
Thin-layer chromatography
TSSs
Transcriptional start sites

Glycoconj J

Introduction
GM3, one of the major gangliosides (sialic acid-containing
glycosphingolipids [GSLs]) in mammalian cells, is abundantin the outer leaflet of the plasma membrane. GM3 synthesis iscatalyzed by a sialyltransferase, ST3GAL5 protein (ST3G5),in the luminal side of Golgi apparatus [1]. ST3G5 possesses
sialylmotifs L, S, and VS, which are conserved among
sialyltransferases and are thought to function as a catalytic
domain [1].
Two transcriptional variants (termed a-type and b-type)of ST3G5-encoding mRNAs have been reported in mice
[2, 3]. Using a leaky scanning system, we demonstrated
translation of three isoforms (M1-, M2-, and M3-ST3G5)
from a-type and two isoforms (M2- and M3-ST3G5) fromb-type [4]. These isoforms differ greatly in terms of stability and subcellular localization. They are all transported
from endoplasmic reticulum (ER) to Golgi by an arginine(R)/lysine (K)-based motif located just above the transmembrane domain [5]. Only M1 isoform also has an Rbased motif, which functions as a retrograde transport signal and is positioned distally from a lipid bilayer.
Numerous M1 isoforms are localized in ER [4]. Both M2
and M3 isoforms are localized in Golgi, and the half-life of
M3 isoform is longer than that of M2 isoform [4]. Based on
result of our 2015 study, we proposed a mechanism inwhich R/K-based motif is involved in Golgi retention of
M3 isoform [5]. Multiple isoforms are involved in GM3
synthesis, but the biological significance of coexistence ofmore than one isoform in a single cell remains unclear.
We have previously investigated expression patterns of atype and b-type in various mouse tissues. The transcription
initiation point in a-type is close to that in b-type, suggestingthat transcriptional regulation is similar in the two variants.
Expression patterns of a-type and b-type in various tissues
were nearly identical, and both were highly expressed in brain,heart, and testis [4]. Northern blotting analysis for detection of
total St3Gal5 mRNA variants also revealed similar patterns inseveral tissues, except for liver [2]. Expression level of total
mRNA variants in mouse liver was high, whereas those of atype and b-type were low. These previous findings, taken together, indicate the presence in liver of other mRNA variants
besides a-type and b-type.
In the present study, we investigated a St3Gal5 knockout
mouse model (St3Gal5-Ex3-KO) in which exon 3, whichcontains M2 and M3 initiation codons, is replaced by neomycin resistance gene (Neo). These mice displayed GM3 synthase activity in liver, but not in inner ear or lymphocytic cells.
We identified a new St3Gal5 mRNA variant (c1-type),
expressed specifically in liver. The St3Gal5-Ex3-KO miceexpress artificial splicing isoforms (a-type−/−, b-type−/−, andc1-type−/−) in which the Neo region is absent. ST3G5 is nottranslated from a-type−/− and b-type−/− because of initiation

codon deletion and frame shift. On the other hand, liverspecific c1-type−/− can produce artificial ST3G5 that displays
enzyme activity without exon 3.

Materials and methods
Mice
St3Gal5 knockout mice (termed St3Gal5-Ex3-KO) were
produced as described previously [6]. After a sufficient number of backcross generations, mutant mice were maintained on
the KK/TaJcl background.
mRNA variant-specific amplification
First-strand cDNA synthesis was performed using a FirstStrand cDNA Synthesis kit for RT-PCR (AMV; Roche
Diagnostics; Penzberg, Germany). Total RNA was isolated
from tissues of KK/TaJcl and St3Gal5−/− mice. Specificprimers as follows were designed to amplify ST3G5 transcriptional variants (a-type, b-type, c1-type): a-type (F1-primer: 5′TCCCTAGCATGCACACAGAGG-3′; R4-primer: 5′-TTAT
CCGGTCAGGGTCCACATAGTGC-3′), b-type (F2-primer:
5′-AGCCTGCGCAGGTGTGGAC-3′; R4-primer: 5′-TTAT
CCGGTCAGGGTCCACATAGTGC-3′), c1-type (FX-primer: 5′-GATTTCTCGACACCTCTACTCACACAG-3′; R4primer: 5′-TTATCCGGTCAGGGTCCACATAGTGC-3′).
PCR was performed using 50 ng cDNA, specific primers,
and KOD FX polymerase (Takara Bio; Shiga, Japan), as per
the manufacturer’s instructions.
Plasmids
The open reading frame (ORF) of mouse M*- ST3GAL5(mM*-ST3G5) was amplified using cDNA prepared from liver of St3Gal5-Ex3-KO mice, and primers 5′-ATGT
CCTTGGCTGGACACGTTC-3′ and 5′-GGTT
TGCCGTGTTCCGAGTTC-3′. Resulting fragments werecloned into pGEM-T Easy Vector (Promega; Madison, WI)
to generate pFS615. pFS617-encoding M*-ST3G5 plasmid
was constructed using pFS615 and primers 5′-CACC
ATGTCCTTGGCTGGACACGTTC-3′ and 5′-GGTT
TGCCGTGTTCCGAGTTC-3′. Resulting fragments werecloned into pENTR/D-TOPO vector (Life Technologies;
Carlsbad, CA) to generate pFS617. CSII-CMV-MCS plasmid
was converted to Gateway® destination vector (CSII-CMVRfA) using the Gateway® vector conversion system (LifeTechnologies). For lentivirus production, M*-ST3G5 of
pFS617 was inserted into lentiviral expression vector CSIICMV-RfA, using the LR clonase reaction to generate
pFS620. pFS311 (M3-ST3G5/CSII-CMV-RfA) plasmid wascreated by similar procedures [7].

Glycoconj J

Real-time PCRFirst-strand cDNA synthesis was performed as describedabove, and total RNA was isolated from tissues of C57BL/6
mice. Primers and probes specific for mouse St3Gal5 c1-type(primers, 5′-ACTCAGAGGCTGTTTGAAACAGTCT-3′
and 5′-GTGAGTAGAGGTGTCGAGAAATCCAT-3′; probe,
5′-ATTTAAGCCCTCATGGACGCCGCTC-3′) and 18SrRNA (Hs99999901-s1) were from Applied Biosystems(Foster City, CA). Real-time PCR was performed with
2 × TaqMan universal master mix using a 7500 Real-Time
PCR system (Applied Biosystems). Tissue copy numbers
were calculated using appropriate standard curves.
5′-RACE analysis
5′-RACE was performed using a SMARTer RACE 5′/3′ Kit(Takara) per the manufacturer’s instructions with some modification. SMARTer first-strand cDNA, incorporating specificsequences at both ends, was synthesized from total RNA isolated from mouse liver tissue. 5′-RACE fragments were generated using a set of universal primer and gene-specific primer: 5′-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3′ and 5′-GATTACGCCAAGCTTGCATTGCTGTGTGAGTAGAGGTGTCGAGAAATCC3′. The resulting fragment was cloned into linearized pRACE
vector using an In-Fusion HD Cloning Kit (Takara). Isolated
plasmid DNAs were sequenced using universal primers:
M13F, 5′-GTTTTCCCAGTCACGACGTT-3′; M13R, 5′GGAAACAGCTATGACCATGA-3′.
Cell culture
Mouse embryonic fibroblasts derived from St3Gal5-Ex3-KO
mice (MEF−/−) [4] were cultured in low-glucose DMEM
(Nacalai Tesque; Kyoto, Japan) supplemented with 10% fetal
bovine serum, 100 U/mL penicillin, and 100 μg/mL
streptomycin.
Preparation of lentiviral vectors
293 T cells (0.5 × 106) were seeded in a polylysine-coated 60mm dish 24 h before transfection, transfected with 3 μg
pFS311 (mM3-ST3G5/CSII-CMV-RfA) or pFS620 (mM*ST3G5/CSII-CMV-RfA), 1.5 μg pCAG-HIVgp, and 1.5 μg
pCMV-VSV-G-RSV-Rev. plasmids using Lipofectamine
2000, and incubated 24 h at 37 °C. medium was then replacedby 4 mL high-glucose DMEM containing 10 μM forskolin.
Culture temperature was lowered from 37 to 32 °C ~ 32 h after
transfection to maintain virus stability. Lentivirus-containing
supernatant was harvested 48 h after transfection, centrifuged
at 200 × g for 3 min to remove living cells, and either used
immediately for experiments or snap-frozen in liquid nitrogenand stored at −80 °C for later applications. For lentiviral transduction of MEF−/− cells, 2.5 × 105 cells in a 60-mm dish wereadded with 4 mL lentiviral supernatant, cultured at 32 °C for
24 h, temperature lowered from 32 to 37 °C, and cells incubated 48 h more.
Preparation of cell lysates
Cells were washed twice with ice-cold phosphate-buffered saline (PBS), suspended in buffer A (50 mM Tris-HCl (pH 7.5),
150 mM NaCl, 10% glycerol, 4 M urea, and 1X cOmplete
Protease Inhibitor Cocktail (EDTA-free; Roche Diagnostics)),
and lysed by sonication. Cell debris was removed by centrifugation at 1000 × g for 3 min at 4 °C, and supernatant (total celllysates) was centrifuged at 100,000 × g for 1 h at 4 °C.
Precipitate (integral membrane protein fraction) was suspended
in 2X SDS sample buffer (125 mM Tris-HCl (pH 6.8), 4% SDS,
20% glycerol, and trace amount of bromophenol blue) containing 5% βMe, and incubated 5 min at 37 °C. Integral membraneprotein fraction was subjected to peptide: N-glycosidase F(PNGase F) (New England Biolabs; Beverly, MA) digestion
for 1 h at 37 °C as per the manufacturer’s instructions. Protein
concentrations of total cell lysates were determined using a
BCA protein kit (Pierce/ Thermo Fisher; Waltham, MA).
Immunoblotting
Integral membrane protein fractions as above were subjected toimmunoblotting. Proteins were separated by SDS-PAGE andtransferred to an Immobilon PVDF membrane (Millipore;
Billerica, MA), which was then incubated 1 h with antiST3G5 [8] or anti-β-actin antibodies (1:1000 dilution)(Sigma), washed, and incubated 1 h with HRP-conjugated donkey anti-rabbit IgG (1:20,000 dilution) or anti-mouse IgG (GEHealthcare Bio-Sciences; Piscataway, NJ). Labeling was detected using an ECL Select™ kit (GE Healthcare).
Immunofluorescence microscopy
Transfected cells were cultured on coverslips, fixed for 15 minwith 3.7% formaldehyde in PBS at room temperature, rinsed
with PBS, permeabilized with 0.5% SDS in PBS, treated with
Image-iT FX Signal Enhancer (Thermo Fisher) for 30 min,incubated 1 h with anti-ST3G5 [4] or anti-GM130 (BDBiosciences; Franklin Lakes, NJ) diluted 1:100 with 1%
BSA in PBS, washed three times in PBS, and incubated
30 min with Alexa 488-conjugated anti-rabbit IgG or Alexa
594-conjugated anti-mouse IgG antibodies (Thermo Fisher)
diluted in 1% BSA in PBS to 5 μg/mL. Coverslips were
washed three times in PBS, mounted on glass slides using
ProLong Gold antifade reagent (Thermo Fisher), and analyzed
by fluorescence microscopy (model FV1000; Olympus;
Tokyo, Japan).

Glycoconj J

Lipid analysis
Lipid (GSL) analysis was performed as described previously [5].

Results
GSL composition of liver and brain tissues
in St3Gal5-Ex3-KO mice
Proia’s group originally produced St3Gal5 knockout
mice termed St3Gal5-Ex5-KO by replacing exon 5(which contains sialylmotif L) with neomycin resistancegene (Neo) (see Fig. 1), and observed loss of GM3 synthesis in brain, skeletal muscle, and adipose tissues inthese mice [9]. Kozaki’s group subsequently produced
St3Gal5 knockout mice termed St3Gal5-Ex3-KO byreplacing exon 3 (which contains two initiation codons,
M2 and M3) [4, 6] with Neo (Fig. 1), and we found that
TLC analysis did not detect GM3 in inner ear cells, thymocytes, or CD4+/CD8+ T cells [10, 11]. However, GM3
synthesis was still present at low level in liver tissue ofthese mice (Fig. 2). In the present study, we separated
neutral and acidic GSL fractions from total lipid extractsof liver and brain tissues of wild-type (WT) and St3Gal5Ex3-KO mice using a DEAE-Sephadex A-25 column, andanalyzed each fraction by TLC. In WT mice, GM2 wasmainly detected in the liver. Liver of St3Gal5-Ex3-KO
mice showed much lower GM2 content (~50%) than thatof WT mice, and correspondingly higher LacCer accumulation and GA2 synthesis. TLC analysis revealed two

bands above GM3 in St3Gal5-Ex3-KO mice (Fig. 2a,left panel). These bands were identified by liquidc h r o m a t o g r a p hy - m a s s sp e c t r o m et r y ( L C - M S ) a s
sulfoglycolipids SM3 and SM2a (data not shown). Thesefindings indicate that GM3 synthase activity is reduced
but still present in liver of St3Gal5-Ex3-KO mice.
Brain tissues of WT mice express a-, b-, and c-series gangliosides (GM1, GD1a, GD1b, GT1b). Deletion of exon 3
results in a shift of ganglioside synthesis to the o-series ganglioside (GM1b, GD1α) pathway (Fig. 2b). Lipid patternchanges in St3Gal5-Ex3-KO mice are generally similar to
those in St3Gal5-Ex5-KO mice [9]. However, St3Gal5Ex3-KO brain tissue showed two faint bands unrelated tomigration of GM3 and GM2 (Fig. 2b, left panel). The upperband was identified by LC-MS as sulfoglycolipids SM1a (datanot shown), but we have not yet been able to identify lower
band (Fig. 2b, left panel, black dot). Our findings indicate that
deletion of exon 3 abolishes GM3 synthase activity in brain.

A

Liver
Acidic fraction

Neutral fraction
SM3
SM2a
GM3
GM2

LacCerGA2

Ori

WT
Exons 1 2
M1

34
M2 M3

5

67

8

B

+/+ +/+ -/- -/-+/+ +/+ -/- -/-St3Gal5

St3Gal5

Brain
Acidic fraction

Neutral fraction

Sialylmotif L TGA

St3Gal5-Ex5-KO
Neo
St3Gal5-Ex3-KO

GM1

Fig. 1 Gene structures of St3Gal5 knockout (St3Gal5-Ex5-KO andSt3Gal5-Ex3-KO) mice. The St3Gal5 gene is composed of eight exons.
St3Gal5-Ex5-KO mice were produced by replacing exon 5 (whichcontains sialylmotif L) with neomycin resistance gene (Neo). St3Gal5Ex3-KO mice were produced by replacing exon 3 (which includes
initiation codons M2 and M3) with Neo. Black boxes: exons.
Horizontal lines: introns

GlcCer
GalCerLacCer

GM1b

*
Neo

SM4
SM3
SM1a

+/+ +/+ -/- -/St3Gal5

GD1
Ori

Ori

GA2
+/+ +/+ -/- -/St3Gal5

GA1
Ori

Fig. 2 GSL composition of liver and brain tissues of St3Gal5-Ex3-KO
mice. Acidic and neutral GSL fractions were prepared from liver (a) andbrain (b) tissues of WT (+/+) and St3Gal5-KO mice (−/−), and separated
on TLC plates (amounts corresponding to 2 mg protein). GSLs were
stained by orcinol/sulfuric acid. Asterisk: GD1a, GD1b, and GT1bbands. Black dot: unidentified GSL (see text)

Glycoconj J

Expression of a-type and b-type mRNA variantsin St3Gal5-Ex3-KO miceMice express two transcriptional variants of St3Gal5 mRNA:a-type and b-type [2, 3]. Transcription of a-type starts fromexon 1; exon 2 is then removed by splicing, and exon 1 isconnected to exon 3 (Fig. 3a). a-Type expression was detected
as a 321-bp band by PCR using primers (F1/R4 primers) designed for exons 1 and 4. In contrast, transcription of b-typestarts from exon 2, and exon 2 is then connected to exon 3. bType expression was detected as a 265-bp band by PCR using
primers (F2/R4 primers) designed for exons 2 and 4. Because
exon 3 is replaced by Neo in St3Gal5-Ex3-KO mice, it ispossible that the Neo region is spliced, and exon 1 or exon 2 isdirectly connected to exon 4, generating a-type−/− or b-type−/−
as a mature mRNA (Fig. 3a). If so, 197-bp (a-type−/−) and141-bp (b-type−/−) bands should be amplified by PCR using
F1/R4 primers and F2/R4 primers, respectively.
We examined mRNA expression of a-type and b-type inbrain and liver tissues of WT (+/+) and St3Gal5-Ex3-KO(−/−) mice using PCR primers (Fig. 3b). In WT, both atype and b-type bands were detected in brain, whereas only
a-type band was detected in liver. Expression level of atype in liver was much lower than in brain. These findings
are consistent with real-time PCR data in our 2009 study[4]. In St3Gal5-Ex3-KO, we observed a-type−/− and/or btype−/− bands in brain and liver. Expression levels and patterns of these mRNA variants in St3Gal5-Ex3-KO weregenerally similar to those in WT. St3Gal5 mRNAs in
St3Gal5-Ex3-KO were present as spliced forms a-type−/−and b-type−/−. However, ST3G5 protein is not translated
from a-type−/− or b-type−/− as a result of deletion of initiation codons (M2 and M3) or frame shift. Residual GM3
synthesis activity in St3Gal5-Ex3-KO therefore cannot
explained by production of a-type−/− or b-type−/−.
Specific expression of a novel St3Gal5 mRNA variant
in liverTo account for observed GM3 synthesis in St3Gal5-Ex3-KO
mice, we investigated the possible presence of novel mRNAvariants that ST3G5 ORF even without exon 3. In the NCBI
database (ncbi.nlm.nih.gov), nine transcriptional and splicing
variants (including a-type and b-type) are presented as
St3Gal5 mRNA variants (Fig. 4). X1 and X2 indicate newexons of St3Gal5. We classified these variants into four types(a-, b-, c-, and d-types). Transcriptions of a-type (a1-a4), btype (b1, b2), and c-type (c1, c2) start respectively from exons
1, 2, and X2. Transcription of d-type starts before exon 1, andcontains exons 1, 2, and X1. Splicing variants without exon 4(a3-, a4-, b2-, and c2-types) encode the catalytic domain involved in GM3/GM4 synthesis, but do not encode the transmembrane domain. The soluble form of ST3G5, which cannot

A
Exons 1
WT

2

3

8
//

R4-primer

F1-primera-type

4

1

3

M1

M2,M3

1 3 4

a-type-/- 1 44

321 bp197 bpF2-primer

2

R4-primer

3

4

M2,M3
b-type2 3 4b-type-/- 2 4

B

265 bp141 bp

bp
500
400
300
200
100

321 bp265 bp197 bp141 bp

St3Gal5 +/+ -/- +/+ -/- M +/+ -/- +/+ -/a
b
a
b
Brain
Liver
Fig. 3 Expression of mRNA transcriptional variants a-type and b-type in
WT and St3Gal5-Ex3-KO mice. a. PCR products of a-type, a-type−/−, btype, and b-type−/−. a-type−/− and b-type−/− are transcripts in which exon 3
is removed by splicing. Numbers on gray background: sizes of PCR
products using F1/R4-primers or F2/R4-primers. Black boxes: exons.
Horizontal lines: introns. M1, M2, M3: initiation codons. b. Transcripts
were amplified by PCR using F1/R4-primers or F2/R4-primers, andcDNAs prepared from mouse brain and liver tissues. PCR products
were separated by agarose electrophoresis and stained with ethidium
bromideproduce GM3 in the luminal side of Golgi, is therefore translated from the splicing variant. Consequently, the mRNA variants that encode GM3 synthase are a1-, a2-, b1-, c1-, and dtypes. We presume that, among these variants, only c1-type
encodes an ORF of GM3 synthase when exon 3 is removed. In
WT, M2 and M3 isoforms are most likely translated as GM3
synthases from c1-type because the initiation codons (M2 andM3) are present in exon 3 of c1-type [4]. Deletion of exon 3 in
St3Gal5-Ex3-KO leads to generation of a novel initiation
codon (M*) in exon X2 of c1-type−/− (Fig. 5a). M*-ST3G5
isoform, translated from M*, has a catalytic domain for GM3
synthesis, and amino acid sequence of the NH2-terminal cytoplasmic region of this isoform is different from that of M2ST3G5 isoform (Fig. 5b).

Glycoconj J

Exon

a1-type(a-type)

1

2 X1

X2

3 4

5

A
Exons 1
WT

M1

WT

M2,M3

c1-typea3-type (X7)

X2 3

M2,M3
4

137 bp

B

M2
WT
-/-

M4

c2-type (X6)

M3

WT 16 SRTSLQWYTRTQHKMRRPSLLIKDICKC
TM-/- 31 PLLPAVLPSQRRWRGVRWISRHLYSHRC

M4

C

Fig. 4 Structures of mouse St3Gal5 gene and its mRNA variants.
St3Gal5 mRNA variants (a-type, b-type, X1 through X7) in the NCBI
database are shown. Transcripts are classified into four groups (a-, b-, c-,d-type) based on structure. Black boxes: exons in mouse St3Gal5. Exons
X1 and X2 are not present in known a1-type and b1-type variants. M1,
M2, M3, M4: initiation codons

1
MPSEFTSAKLRSDC
1 MSLAGHVHSSHVCSLRGCLKQSAVFKPSWT
M*

M2,M3

M2,M3

R4-primer4b2-type (X5)M1

261 bpM*
c1-type-/- X2 4

M4d-type (X1)

4

3

St3Gal5-Ex3-KO X2

a4-type (X4)8

R4-primer

X2

FX-primer

M4

c1-type (X2)

4

FX-primer

M2,M3

M2,M3

3

//

a2-type (X3)b1-type(b-type)

X2

2

bp
500
400
300
200
100

261 bp137 bpM +/+ -/- St3Gal5
Liver (c1-type)−/−

To determine whether c1-type and c1-type are present in liver of WT and St3Gal5-Ex3-KO, we performed
PCR using FX/R4-primers (Fig. 5a, c). 261-bp and 137bp bands were detected in WT and St3Gal5-Ex3-KO,
respectively. This finding indicates that mouse liver expresses c1-type, and that deletion of exon 3 in St3Gal5Ex3-KO results in production of c1-type−/− that encodes
M*-ST3G5. We evaluated c1-type expression in various
mouse tissues by real-time PCR, and found that it was
highest in liver (Fig. 6).
To identify transcriptional start sites (TSSs) of c1type, we performed 5′-RACE analysis using total RNAof mouse liver. Thirty-four clones containing 5′- end of
c1-type were obtained, and their sequences were determined (Fig. 7). In this figure, black dots indicate TSSs,
and numbers above the dots indicate number of clones.
Only one clone starts before M* (ATG), indicating that
although c1-type expression in mouse liver is high, theamount of c1-type capable of translating M*-ST3G5 isquite limited.

Fig. 5 Expression of c1-type in WT and St3Gal5-Ex3-KO mice. a. PCR
products of c1-type and c1-type−/−. c1-type−/− is a transcript in which
exon 3 is removed by splicing. Numbers on gray background: sizes of
PCR products using FX/R4-primers. Black boxes: exons. Horizontal
lines: introns. b. Amino acid sequences of NH2-terminal cytoplasmic
regions of WT and St3Gal5-Ex3-KO (−/−) mice. Alignments weregenerated using the ClustalW program (DNA Data Bank of Japan(DDBJ)). Black boxes: identical residues. Gray boxes: aa similarities.
M2, M3, M*: initiation codons. TM: transmembrane region. c.
Transcripts were amplified by PCR using FX/R4-primers, and cDNAs
were prepared from mouse liver tissue. PCR products were separated byagarose electrophoresis and stained with ethidium bromide

Subcellular localization and in vivo enzyme activityof artificial M*-ST3G5 isoform
To examine GM3 synthase activity of M*-ST3G5 in vivo, we
stably expressed mouse embryonic fibroblasts derived from
St3Gal5-Ex3-KO mice (MEF−/−) with M3- and M*-ST3G5,
using a lentiviral expression system. Integral membrane protein fractions were prepared and subjected to immunoblottingwith anti-ST3G5 antibodies. Expression level of M*-ST3G5

A

100
80

ST3G5

60

-actin40- M3 M*

B

20
0

kDa
43
36.5

Neutral fraction

GlcCerFig. 6 St3Gal5 c1-type transcripts from mouse tissues. Expression
levels of St3Gal5 c1-type transcripts from various tissues of C57BL/6
mice were analyzed by real-time PCR assay. Expression levels of c1-typewere normalized relative to 18S rRNA transcript

was slightly lower than that of M3-ST3G5 (Fig. 8a). Acidic
and neutral lipid fractions of cells were prepared and analyzed
by TLC. MEF−/− cells expressed only GM1b (o-series) as
acidic GSL, and glucosylceramide (GlcCer), LacCer, andglobosides (Gb3 and Gb4) as neutral GSLs. In MEF−/−/M3ST3G5 and M*-ST3G5 cells, GM3, GM2, GM1, and GD1a
were detected in acidic fractions, while LacCer and globosidelevels in neutral fractions were lower than those in MEF−/−.
Amounts of gangliosides in MEF−/−/M*-ST3G5 cells weresimilar to those in MEF−/−/M3-ST3G5 cells. These findings
indicate that in vivo enzyme activities of M3- and M*-ST3G5
are nearly identical.
Our 2009 study showed that the cytoplasmic region of
ST3G5 affects subcellular localization [4]. Amino acidsequences of the cytoplasmic region in M*-ST3G5 arequite different from those in M3-ST3G5. We used indirect
immunofluorescence assay to examine subcellular localization of M*-ST3G5 in MEF−/− cells (Fig. 8c). We confirmed that the fluorescence from anti-ST3G5 antibody

43

Acidic fraction

brain
lung
heart
colon
thymusspleenstomachtestispancreaskidneyliver
small intestine
adiposeduodenum

St3Gal5 mRNA/18S rRNA

Glycoconj J

GM1b

GM3 LacCer
GM2
Gb3
GM1
GD1a Gb4

OriginOrigin- M3 M**- M3 M*C
No expression

GM130

Merge

M3-ST3G5

GM130

Merge

M*-ST3G5

GM130

Merge

Exon X2

61

1
M*
CCCACAGATTTTATCCCCATGTCCTTGGCT
1 29
GGACACGTTCACTCCAGTCATGTTTGCTCA
21
CTCAGAGGCTGTTTGAAACAGTCTGCAGTA91

TTTAAGCCCTCATGGACGCCGCTCCTGCCT

120

121 GCTGTCCTTCCCTCGCAGCGGAGGTGGAGA

150

151 GGGGTGAGATGGATTTCTCGACACCTCTAC

180

1
31

30
60
90

181 TCACACAG

Fig. 7 Transcriptional start sites (TSSs) of mouse St3Gal5 c1-type. The
nucleotide sequence shown is that of exon X2 in mouse St3Gal5. Black
dots: TSSs. Numbers above dots: number of clones obtained by 5′-RACE
analysis. White letters on black background: artificial initiation codon(M*)

Fig. 8 In vivo enzyme activities and subcellular localization of M3ST3G5 and M*-ST3G5. MEF cells derived from St3Gal5-Ex3-KO
mice were stably expressed with M3-ST3G5 (M3) or M*-ST3G5 (M*)
using a lentiviral expression system. Minus sign (−): no transfection. a.
Total cell lysates and integral membrane protein fractions were prepared,
treated with PNGase F, and analyzed by immunoblotting with anti-βactin and anti-ST3G5 antibodies, respectively. b. Acidic and neutral
GSL fractions were extracted from cells, separated on TLC plates(amounts corresponding to 2 mg total protein), and GSLs were stainedby orcinol/sulfuric acid. c. Cells were fixed, permeabilized with 0.5%
SDS in PBS, stained with anti-ST3G5 antibody and Alexa 488conjugated anti-rabbit IgG, and visualized by confocal laser-scanningmicroscopy. For co-localization studies, cells were incubated with
antibody vs. GM130 (a cis-Golgi marker) for 1 h, and then with
Alexa594-conjugated anti-mouse IgG for 30 min. Bar: 10 μm

Glycoconj J

was absent in MEF−/− cells (no expression). Both M3- andM*-ST3G5 were partially colocalized with cis-Golgi
marker GM130, indicating that M*-ST3G5 is transported
from ER to Golgi as with M3-ST3G5.

Discussion
Two strains of St3Gal5 knockout mice (St3Gal5-Ex5-KO andSt3Gal5-Ex3-KO) have been established as models for studying physiological functions of ganglioside GM3. Strain
St3Gal5-Ex5-KO, produced by deletion of exon 5 (which
encodes sialylmotif L), has no GM3 synthesis activity in brain,
skeletal muscle, or adipose tissues [9]. Insulin sensitivity isenhanced in this strain because insulin receptor phosphorylation is up-regulated in skeletal muscle. This finding supports
the concept of type II diabetes therapy based on inhibition of
ganglioside synthesis [12, 13]. Strain St3Gal5-Ex3-KO, which
we produced by replacing exon 3 with Neo, has no GM3 synthesis in inner ear cells, thymocytes, or CD4+/CD8+ T-cells.
The knockout mice (St3Gal5-Ex3-KO) display selective degeneration of the organ of Corti, and inhibition of CD4+ T-cell
activation by treatment with anti-CD3 and anti-CD28 antibodies [10, 11]. The above findings indicate that either exon 5 orexon 3 deletion is sufficient to abolish GM3 synthase activity.
The present study showed that acidic GSL composition ofliver in St3Gal5-Ex3-KO is similar to that in WT (Fig. 2),
indicating that GM3 synthase activity is still present at low
level in liver. GM3 and GM2 are not expressed in liver of
St3Gal5-Ex5-KO (data not shown), indicating that ST3G5
is the sole GM3 synthase. We identified a liver-specificmRNA transcriptional variant (c1-type), and explained theresidual synthase activity by production of artificial c1-type(c1-type−/−) that lacks exon 3 (Figs. 5 and 6). In liver of
St3Gal5-Ex3-KO, M*-ST3G5, which has GM3 synthesis
activity, is presumably translated from c1-type−/−.
Sulfoglycolipids (SM3, SM2a, and SM1a) were detected inlivers or brains of St3Gal5-Ex3-KO (Fig. 2a and b) andSt3Gal5-Ex5-KO (data not shown). The unknown band inof St3Gal5-Ex3-KO brain (Fig. 2b, black dot) is most likely
also SB1a, but identification remains to be completed. LacCer
level is strikingly increased in St3Gal5-Ex3-KO liver (Fig.
2a), consistent with induction of SM3 and SM2a synthesis.
GAL3ST1/galactose-3-O-sulfotransferase 1 (CST) transcript
encoding SM3 synthase was previously detected by PCR in
WT liver, although expression level was low [14]. We detected CST transcripts by real-time PCR in WT and St3Gal5Ex3-KO livers, with similar expression levels (Fig. S1). The
presence of sulfoglycolipids (classified as a subset of acidic
glycolipids) thus may compensate for a lack of sialic acidcontaining gangliosides.
Expression of St3Gal5 c1-type was restricted to liver.
Modifications (acetylation or methylation) of histone H2A-,

H2B-, H3-, and H4-forming chromatins generally play important roles in initiation of transcription [15, 16]. One such modification known as an initiation point of transcription is trimethylation of residue Lys4 in histone H3 (H3K4me3). Data
for H3K4me3 states enrolled in ENCODE (https://www.
encodeproject.org), analyzed by Chip-seq using mouse tissues
from embryonic day 14.5, adult week 8, and adult week 24
were viewed by USCS genome browser (https://genome.ucsc.
edu) (Fig. S2). H3K4me3 peaks were consistently detected inclose proximity to exons 1 and 2. Interestingly, the peaks werealso detected in proximity to exon X2 in liver and bone marrowin adult week 8, and to brown adipose tissue in adult week 24.
These findings are in agreement with real-time PCR data (Fig.
6). The observed H3K4me3 states suggest that transcription of
c1-type does not occur in embryonic liver. Switching from a1and/or b1-type to c1-type most likely occurs at some point,
because St3Gal5 mRNAs were detected on embryonic day
15 [17]. We are currently examining possible expression of
c1-type in bone marrow and brown adipose tissue by realtime PCR analysis.
Tissue-specific gene expression is controlled by enhancers
located far away from TSSs. We estimated locations of enhancer regions through mono-methylation of residue Lys4 inhistone H3 (H3K4me1). Data for H3K4me1 states enrolled in
ENCODE were viewed by USCS genome browser similarlyas H3K4me3 (Fig. S3). H3K4me1 of liver at E14.5 was observed around exons 1 and 2, which are predicted transcriptional start positions. In contrast, H3K4me1 of liver at 8w wasobserved around exon X2 in addition to exons 1 and 2. Thesefindings suggest that, during development, regulatory regionsof transcription are altered by switching of transcriptional start
positions. The roles of enhancers in liver-specific expressionof c1-type will be clarified in future experimental studies.
Binding sites of transcription factors in 200-bp 5′-upstream
region from TSSs were searched and predicted using the
TFBIND program (tfbind.hgc.jp) (Fig. S4). Among these transcription factors, CCAAT/enhancer-binding protein (C/EBP)
regulates expression of multiple hepatocyte-specific genes [18]and is involved in liver differentiation and regeneration [19–21].
We also found the sequence (5′-(A/G)TTA(C/T)GTAA(C/T)-3′), similar to the binding site of D-site of albumin promoter (DBP)
expressed in adult mouse liver, although this sequence is notincluded in the TFBIND program list. Our ongoing studies arefocused on identification of regions with promoter activity, andof transcription factors involved in c1-type expression.

Acknowledgements
This study was supported by a Grant-in-Aid for Scientific
Research (B) from MEXT (to J.I.), Mizutani Foundation for
Glycoscience (J.I.), Naito Foundation (J.I.), ONO Medical
Research Foundation (J.I.), Uehara Memorial Foundation

Glycoconj J(J.I.), and the MEXT-Supported Program for the Strategic
Research Foundation at Private Universities. The authors are
grateful to Dr. S. Anderson for English editing of the manuscript, Dr. A. Suzuki (Tohoku Medical and Pharmaceutical
University), Dr. T. Moriguchi (Tohoku Medical andPharmaceutical University), K. Toshima (Tohoku Medical
and Pharmaceutical University), and Dr. A. Ishii for experimental support and useful discussion.

9.

10.

Compliance with ethical standards
Conflict of interest Authors declare no conflicts of interest.
Ethical approval This article does not contain any studies with humanparticipants or animals performed by any of the authors.

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〃ぜ.ゲザヂ...ʻ寺.毎.デー子 .ʻ争串やザザザ.ｄ.･やｻ.刈寸ﾅ公一ﾅﾅ".̅P､ﾅ令、

特: 集糖: 鎖関連遺伝子から眺める疾 謎、、

卜.,､錫.­"....今.."....., ず...』ʻ.ʻ〜...』ʻ.ʻ "...ｊ…→』.ぷ JJ

ガングリオシドGM３合成酵素欠損症からみる
スフィンゴ糖脂質機能
郷慎司１，２，井ノロ仁一ʼ

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ガングリオシドはその糖鎖構造中にシアル酸を含むスフィンゴ糖脂質の総称であり．脊椎動物の多ﾎlll胞社会構築と各細胞の機能（増殖分化など）制御に重要な分子群と考えられてきた．一方で．これまで各種ガングリオシド生合成酵素欠損マウスが作製されてきたが，その症状は予想よりはるかに軽度であり驚きをもって解析が続けられてきた．近年，ガングリオシド合成酵素の変異による欠損症が多数報告された.GM3合成酵素欠損症とGM3合成酵素欠損マウスで見いだされた症状は共通した部分だけでなく大きく違う部分もあった．
本稿では.GM3合成酵素欠損症からみえてきた聴覚におけるガングリオシド機能に関して概説するとともに．マウスとヒトでみられたGM3合成酵素欠損時の症状の違いを代謝の観点から新知見を交えて考察した

ス作用"と．同じ細胞膜上に発現する分子と作用する“シス1．序説

スフインゴ糖脂質は「セラミド」と「糖鎖」部分からなる両親媒性の分子であり．疎水性の強いセラミド部分を細胞膜の脂質二重層外層にアンカーし．親水性の糖鎖部位を外側に突出した形で発現しているスフィンゴ糖脂質の糖鎖部分は100種を超える構造多様性が存在し、その発現は細胞種ごとに厳密に制御されている．また．セラミド部分もスフィンゴシン塩基およびアシル鎖の炭素数．不飽和度の違い，水酸基の付加により多様な榊造が存在する!)．
スフィンゴ糖脂質は．さまざまな分子と特異的に相互作用し，生物学的機能を発揮している．糖鎖部分での相互作
用には．細胞外から相手の分子が接近し作用する“トラン'東北医科薬科大学・分子生体膜研究所・機能病態分子学(〒981-8558宮城県仙台市青葉区小松島4-4-1)
ﾕ川崎医科大学・病態代謝学教室（〒701=0192岡山県倉倣市
松島577）InSightSoi･GIycoSphingolipidSlimciionli･omGM３syntllaSedeliC腿ｎＣＶ
●

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SlliIljiGo'･2"dJin-ichilIlokuchi!('II1slilulcofMolecularBiomembraneandGlycobiology,Divisio11ofOlycopalllology,Tblloku
MedicalandPllarmaccuticalUIliverSily9４-４-IKomatsuSllima,Aoba･
ku,Scndai,Miyagi981-8558,Japall,2KawasakiMedicalSclloo1,5７７
Malsushima,Kurashiki,Okayama701-0192,Japan)
DOI:10.14952/SEIKAGAKU.2017.890644⑨2017公益社団法人日本生化学会

作用"の２通りが想定される．特にシス作用では細胞膜上で発現する各種受容体などの膜タンパク質に直接的に相互作用しその機能．局在安定性を制御するほか，間接的・直接的相互作用によって多様な分子とともに細胞膜上に"機能性微小膜領域"を形成することで細胞機能を制御していると考えられている．セラミド部分の構造もまた．他の細胞膜構成脂質およびタンパク質との相互作用や．細胞膜の物理化学的性質に関与すると考えられる．このようにス
フィンゴ糖脂質は多様な作用方法によって複雑に生物機能を制御していると考えられる2斗)

特にシアル酸を含有するスフインゴ糖脂質をガング
リオシドと総称する．その生合成は,GM3合成酵素(ST3GAL5)によってラクトシルセラミドにシアル酸が転移されることから始まる．その後順次糖が付加されてさまざまな構造のガングリオシドが生合成される（図1)，)．これまで，ガングリオシドは各種成長因子の受容体などのタンパク質と相互作用することによって．細胞の増殖，分化．移動などを制御していることが多数報告されてきている6．
)
本稿では，近年見いだされてきたヒトにおけるガングリ
オシド欠損症の知見とガングリオシド合成酵素欠損マウスの症状をまとめ．そこからみえてきた新しいガングリオシ
ドの機能を概説する．

生化学第８９巻第５号．pp.６４４-65I(2017)⑲645

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図１スフィンゴ糖脂質生合成経路
哺乳動物に発現する代表的なスフィンゴ糖脂質の生合成経路を示す．セラミド(Cer)に順次，糖が付加されることにより多様な糖脂質が生合成されるが，特にラクトシルセラミド(Laccer)に付加される糖の種類・結合様式によって多様性が形成される．

2．ガングリオシド欠損マウス

ガングリオシドはその糖鎖構造中にシアル酸を含む酸性スフインゴ糖脂質の総称である（図l).発見の経緯や，脳神経系に非常に多く存在していることから，長らく神経組織の形成や機能に必須であることが想定されてきた7.8)．

1990年代に入り．スフインゴ糖脂質．ガングリオシド

生合成に関わる酵素群のクローニングがなされ，各糖転移
酵素遺伝子欠損マウスが作製され解析された5･9･･13).ほぼすべて（ガラクトシルセラミドから合成される一部の糖脂質を除く）のスフィンゴ糖脂質を欠損するグルコシルセラ
ミド合成酵素'4'およびラクトシルセラミド合成酵素の欠損マウスは胎生中期で致死となるl5-17).これらの酵素を欠損した培斐細胞は生存・琳殖可能であることから．多様なスフィンゴ糖脂質櫛造は多細胞系の発達に明らかに必須である．
しかし驚くことに，神経系に多く存在し，重要な機能を担っていると考えられてきたガングリオシドを欠損させてもマウスは生まれてくる．我々のグループも含め.GM３合成酵素欠損マウスは一見明瞭な異常がなく，正常に成長し寿命にも大きな差はないことが報告されている!H･'り).ただし．のちに詳細に述べる聴覚障害に加え．行動異常など

種々の神経異常は確認されている１9･２(II.解析が進めば今後
さらに一見してわからない表現型が明らかにされると考えられるが，想像以上に垂篇な症状がないことに鷲かされるものであった.GD3合成酵素欠損マウスも軽度の神経異常を呈するが重篤ではなく．生存にも大きな影響はな
V'.GM2合成酵素欠損マウスも進行性の神経変性はみられるが，脳神経形成，生存に劇的な変化は確認されなかった２１)これらのマウスの詳細な解析結果は．各ガングリ

オシドが神経組織の恒常性維持や再生，各種臓器における機能維持に関与していることを示しているが，当初の予想
ほど神経組織形成や分化自体に影響がみられなかったことは筋きであった．これらの現象に関しては．特定の代謝経路の欠損により，代仙的に増加するガングリオシドもしくはスフィンゴ糖脂質分子種が，欠損したガングリオシド分
子種の機能をある程度代償をしているためと考えられる(図１,図４も参照)
これらの報告は，各ガングリオシド分子種固有の機能の存在を否定するものではない．いくつかのガングリオシドが有する非還元末端の共通構造が果たすことができる機
能厳密な特定糖鎖榊造でしか果たせない機能．付加されているシアル酸の数（電荷数）によって制御される機能など．ガングリオシドが制御しうる生物機能にはさまざまなﾉﾋ化学第８９巻鋪５･¥･(2017)

646

関与の形があることを示唆するものと考えられる．
これらのことから．ガングリオシド合成酵素ノックアウ

視

ながると考えられるそのためには，各スフインゴ糖脂質の代謝制御機構の解明糖脂質改変技術（糖転移酵素遺伝

子発現制御技術．合成酵素阻害剤開発)，糖脂質化学合成技術（可視化用ラベル糖脂質)．糖脂質検出技術（質量分析などの定性・定量法，抗体などの検出プローブ）などの技術開発各研究分野との連携も非常に重要である．
また．近年ではエクソーム解析により原因未知の病気の原因遺伝子変異も見つけやすい環境になってきた．実際
近年これらの技術を用いて，新たなスフィンゴ糖脂質代謝
関連遺伝子の変異が原因の疾患も見つかってきた．
3．ガングリオシド合成酵素欠損症

ヒトにおけるガングリオシド分解酵素の変異による「蓄積症」は1930年代のGM２ガングリオシドーシスの発見以降型)．多数報告されてきたものの型)．ガングリオシド「生合成酵素の欠損」による症例は報告がなかった．
そんな中．2004年．アメリカのアーミッシュの中で．
てんかん症状を示す子供のなかから,｢GM3合成酵素(ST3GAL5)欠損」の症例が初めて発見された24)(図2).
2013年にも新たにフランスのグループよりミトコンド
リア呼吸鎖複合体欠損症様症状患者の遺伝子解析より．
GM３合成酵素の変異が見つかり症例が報告された25).これらの症例ではST3GAL5のナンセンス変異が確認されている（図3)．これらの報告のGM3欠損患者の血清中ではGM3以降のガングリオシドが完全に検出されなくなり・

グロボ系，ネオラクト系の糖脂質が大幅に増加している．
他の組織で同様のスフィンゴ糖脂質の変化が起きているか
確認はとれていないが患者から採取し培誕した線維芽細胞でも同様にグロボ系スフィンゴ糖脂質の大幅な増加が確認されている．これらの患者ではてんかんの症状以外に．
発育遅延．視神経萎縮および脳皮質の異常による視覚異

排

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，

難聴唾==＝二能（消失したガングリオシドの機能）を真に反映しているのかという疑問が残ったままであった．現れた表現型は，
ノックアウトによってガングリオシドが消失したことに起デル生物）で発現する糖脂質の種類・量，表現型の詳細な解析．さまざまな種類の糖脂質改変モデル培養細胞株の作製・解析を行い．より多くの情報を集めることがますます必要となってくる．これらの情報を蓄輸・統合していくことで，鷲<ような新たなスフィンゴ糖脂質機能の発見につ莫

gこんかん発作

トマウスを用いた解析では，生体内のガングリオシド機因するのか．代償的に増加・出現したスフィンゴ糖脂質の影響か，あるいは他の脂質とのバランスの破綻によるもの
か，真の答に迫るのは困雌を要する問題である．
したがって単純にある特定のスフインゴ糖脂質の糖鎖柵造の合成を担う糖転移酵素のノックアウトマウスの解析だ
けでは．その糖脂質糖鎖柵造の重要性をダイレクトに示すことは雌しい、各スフィンゴ糖脂質欠損マウス（およびモ

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図２GM3合成酵素欠損患者の症状の推移（アーミッシユ中心に）GM3合成酵素欠損患者は，胎児期および生後まもなくまでは顕著な異常はみられない．生育に伴いさまざまな症状が発症す
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図３これまでに報告されたヒトにおけるガングリオシド生合
成酵素の変異(A)GM3合成酵素(ST3GAL5)アーミッシユで見つかったナンセンス変異(赤)に加え．近年シアリルモチーフ内に
ミスセンス変異(青)が報告されてきた.(B)GM2合成酵素(B4GALNTI).クウェーI､、イタリア．およびアーミッシュの患者からいくつかのミスセンス変異が報告されたドメイン
A:glycosyltral1sIbraseefhmily2domain,ドメインB:nucleotidediphospho-sugartransfbrasessuperfimilydomail1.

常精神発達遅滞，筋力低下．食欲不振，嘔吐などさまざ
まな症状およびミトコンドリアの呼吸鎖の異常が報告されている2+23)（図２，表1)
Boccutoらも2014年に「ソルト＆ペッパー症候群」患者のSNPアレイ解析，エクソーム解析などから．アクセ
プター基質，ドナー基質両方との結合に|卿わるST3GAL5のシアリルモチーフＳ内のミスセンス変異[c.994G>A
lranSition(p・E332K)]を同定した２9).患者由来の線維芽細胞ではガングリオ系スフインゴ糖脂質の消失，グロボ系
スフィンゴ糖脂質の大il偏な噌加が確認されている．また，
Boccutoらはこれらの線維芽ﾎl１l胞およびST3GAL5をノックダウンしたゼブラフィッシュにおいて．糖タンパク質Ⅳ結合型およびｏ結合型の変化を見いだしている．特に、複合型糖鎖の増加シアル酸修飾の増加傾向がみられる．これらがGM3欠損の代償機柵なのかは定かではないが，病態に何らかの影響を及ぼしているものと考えられる．
また．近年韓国でもST3GAL5の欠損症例が報告された30)．この症例では「レット症候群」様の症状を呈して生化学鋪８９巻第５号(2017)

647
表１ガングリオシド合成酵素欠損症の発見経緯・症状発見経緯・症状

変異
ST３GAL５

CI95S,G20IR
E322K
R288STOP

文献

レット症候群様症状

Leeら,2016

ソルト＆ペッパー症候群

Buccutoら,2014

infhntile-onselepilepsy症候群

Simpsonら,2004

筋低下
視覚異常

精神発達遅滞B４ＧＡＬＮＴＩ

K284N

ミトコンドリア呼吸鎖複合体欠損症

Fragakiら．Z013

色素異常

Wangら,2013

遺伝性痙性対麻庫Harlalkaら．２０１３

L487T
R505H

おり，生後まもなくの時期までは正常であるが，その後，

Ｇ
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ｃ
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！

精神遅滞やてんかん様症状などさまざまな症状が確認される．この報告の患者はST3GAL5のシアリルモチーフＬ“ ン / | ､ < " .

内の複合へテロ接合性変異(p.Gly201Argとp.Cysl95Ser)である．酵素活性測定は行われてはいないが，血清中のｌGM3,GD3は激減している．

また2013年には，遺伝性の神経変性疾患である遺伝

性痙性対麻庫のエクソーム解析から，クウェート，イ

タリア，およびアーミッシュにおいてGM2合成酵素(B4ALNTl)の変異も報告された31).患者由来線維芽細胞の糖脂質解析ではGM2の消失,GM3の増加が確認されている．

ST3GAL5内の変異の場所ひいては活性の残存率や地域性によって症状の違いはあるものの，多くの場合，

ST3GALSの変異によってGM3以降のガングリオシドが欠失した場合，神経系機能に大きな影響が見いだされており，またB4ALNTIの変異によりGM2以降のガングリオシ

ドが欠損した場合も神経変性を伴うことから，ヒトにおいて神経機能の維持にやはりガングリオシドが必須であることが示唆される．

ここで生じる大きな疑問は,GM3合成酵素欠損マウスとヒトのGM3合成酵素欠損症ではその重箆度が大きく異なる点である．ここにガングリオシド，スフィンゴ糖脂質の新たな機能示唆が隠れていると考えられる．これまで筆者らを含めてGM3合成酵素欠損マウスでは各臓器でGM3以降の消失．加えて代償的にアシアロ系糖脂質が発現す

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グロポ系蝕、質の畑鉋飢蓮■されても喝關

ガンケリオ纂ガングリオシドの輯■代償？

図４GM3合成酵素欠損マウスとGM3合成酵素欠損症患者のスフインゴ糖脂質代謝変化
GM3合成酵素欠損マウスでは大半の臓器でアシアロ系のガン
グリオシドGMlb｡GDIａの発現が確認される.GM3合成酵素欠損症患者ではグロボ系糖脂質の増加が確認されており，マウスとヒトでGM3合成酵素欠損時の糖脂質の流れが異なる可能性がある．代償的に発現・増加する糖脂質の違いが症状の違いに関連していると考えられる．

増加は報告されていない２４ʻ2932ʼ この代償して発現するス
フィンゴ糖脂質の違いがマウス， ヒト間の症状の違いに反映されている可能性が高い．

4.GM3合成酵素欠損マウスとGM3合成酵素欠損症の共通点ることを見いだしている!り'（図4)．GM3合成酵素欠損マ

代償発現するスフィンゴ糖脂質分子種の違いはあるが，マウスとヒトにおいてGM3合成酵素欠損時，共通してみたGMIbXaGDIQ,GDIcがある．これらはGM3合成酵素欠質である可能性が高いと考えた．

ウスが発現するアシアロ系糖脂質にはシアル酸を含有し損により消失したガングリオ系ガングリオシド(GMIA｡
GDla.GDlbなど）の異性体であり，これらが機能代償でられる症状があれば，消失したガングリオシドの機能の本
我々はGM3合成酵素欠損マウスが聴覚異常を有することを報告した'り１．その後の2013年にFlagakiらの報告ではきる生命現象が多数あるものと考えている．
これに対し，ヒトのGM3合成酵素欠損症の血清，線維フランス系のGM3合成酵素欠損症の患者で聴覚異常の記芽細胞ではガングリオ系の糖脂質の消失に加え，グロボ系

細は不明であった．そこで我々はアメリカペンシルバニア

糖脂質の増加が認められており．アシアロ系の糖脂質の載がなされた26).ただし,.･hearinglosgの記戦のみで，詳

州ランカスターのアーミッシュの人々で運営される病院で生化学第８９巻鋪うり。（2017）

648

あるSpeciaIClinicfbrchildrenとの共同研究で.ST3GAL5
c.694C>T変異を持つ複数名のGM3合成酵素欠損患者の聴A(）覚機能を聴性脳幹反応試験(ABR)で詳細に評価したところ．すべてのケースにおいて内耳蝸牛有毛細胞の機能異常が示唆された33I.内耳の詳細な形態的解析は不可能であるため．GM3合成酵素欠損症の患者でどのような異常が内耳に起きているかは不明であるが，マウス，ヒト共通
してGM3合成欠損の場合には聴覚機能に影響が出ることが明らかになった(Boccutoらの報告では聴覚にl則して明

制
刑

ＳＡ
Ｈ

確な記戦はない.Leeらの報告では鎮静状態の維持が雌しくABR試験ができなかったため．聴覚機能評価ができておらず．これらの患者の詳細な情報が期待される)．
これらのことからガングリオシドが聴覚器官においてどのような細胞に発現しているか,GM3欠損時にどこに障害が出るかを解析し、聴覚におけるガングリオシドの機能を明らかにすることで．ガングリオシド，特にGM３の機能の本質がみえると考えた
音波は外耳．中耳を経て．内耳の「蝸牛」へと伝えられる．蝸牛内の「コルチ器」が音波を神経信号に変換するコルチ器は感覚細胞である１列の「内有毛細胞(innerhaircell:IHC)」および3〜4列の「外有毛細胞(outerhaircell:OHC)とそれを支える支持細胞(supportingcell)からなる．有毛細胞はその上部に「聴毛（不動毛stereocilia)」と呼ばれる規則正しく配列した微細な繊毛構造を有している．蝸牛内に伝わった音波によって．有毛細胞の聴毛との間で力学的ひずみが生じ．不動毛に発現するイオン
チャネルが開きイオンが流入する．流入したイオンによって有毛細胞は脱分極を起こし神経伝達物質を放出する．このシグナルを蝸牛神経が受容し、電気信号として最終的に脳の聴覚野へと伝達され音として認識されるマウスは生後，蝸牛内の細胞の形態的・機能的成熟を経て生後約１４日目前後から聴覚が機能し始める蝸牛の主
要なガングリオシドはGM３である．生後まもなくのマウス蝸牛内のGM３の発現は微量であるが，生後１４日目前後にかけての成熟期間に大幅にその量を増加させる．加えて複雑なガングリオシドも発現してくる．このことは，生後の蝸牛の機能的・形態的成熟および維持にガングリオシドが重要な役割をしていることを示唆する．

特にGM３はコルチ器の内・外有毛細胞において細胞頂端側，聴毛において発現が強い（図5).GM3合成酵素欠損マウスでは．生後まもなく内有毛ﾎlll胞および外有毛ﾎlll胞の聴毛の変性が確認された．内有毛細胞では．いったん正常な細胞が形成されるが，生後１０日目前後から聴毛どうしの融合が確認された．この変性した聴毛では聴毛内の雌
聴原因過伝子産物のタンパク質の局在の変化・異常が確認されている．また．外有毛細胞の脱落も確認された．
内耳においてGM３とGD3のみを発現するGM2合成酵
素欠損マウスでは生後１４日前後でこのような聴覚異常
有毛細胞の変性はまったくみられないことから，細胞の極性形成．聴毛局在分子の輸送・局在決定にガングリオ

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図５マウス蝸牛内スフインゴ糖脂質の生後の発現変化とGM３,
GMI発現部位(A)聴覚器の構造とマウス蝸牛内スフインゴ糖脂質の生後の発現変化．蝸牛ではGM３が主要なガングリオシドである．生後発達とともにガングリオシド量が増加し，複雑なガングリオシドも発現してくる(B)蝸牛内コルチ器の構造とGM3,GMIの発現部位．抗ガングリオシド抗体を用いた染色結果をまとめた＋は染色性あり．一は染色性なしを示す.GM３は特に有毛細胞聴毛に強く発現するGM3合成酵素欠損マウスは有毛細胞聴毛の変性を示すが,GM３以降のガングリオシドを消失す
るGM2合成酵素欠損マウスは聴覚異常を示さない．このこと
はGM３が特に聴覚機能にとって重要なガングリオシドである
ことを示唆する．

シドGM３が重要な役割を担っていることが強く示唆される33..4)

ヒトGM3合成酵素欠損症の電気生理学的聴覚機能評

価でも内外有毛ﾎlll胞の異常が示唆されていることから，
GM3合成酵素欠損時，マウスでみられた変化がヒトでも起きている可能性が高い．ヒトGM3合成酵素欠損症の症状,GM3合成酵素欠損マウス,GM2合成酵素欠損マウスを用いた病態解析，スフィンゴ糖脂質解析，組織形態解析

で．内耳におけるGM３の機能にある程度迫ることはでき

たが．より詳細な分子機構・機能解明のため．今後モデル細胞などを用いた解析が必須になってくる．

解析手法や症例数の制限もあり.GM3合成酵素欠損マ

ウスとGM3欠損症患者でみられた症状に類似した点は現生化学第８９巻第５･i;･(2017)

649

在のところ少なく，不明な点が多い．共通して聴覚異常は判明したものの．やはりそれ以外にGM3合成酵素欠損マ
ウスでは患者でみられた重篤な症状はみられていない．今(A)

後ヒト．マウスのGM3合成酵素欠損時，代償的に発現するスフインゴ糖脂質の種類・量を精査し、症状との関連性を見いだしていく必要があるまた．我々はこれまで
GM３がインスリンシグナルの制御に関与していることや，
GM3合成酵素欠損マウスにおけるCD4+T細胞の機能異常を報告しており3,-洲)．現在も詳細な解析からGM3合成
酵素欠損マウスにおいて新たな表現型を見いだしている．
GM3欠損症患者でこれらの症状がみられるかを含め，今後多角的な検証を行いGM3機能の本質に迫りたい．

ＧＭ３
GM２
GM１
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GM3合成酵素欠損患者では筋力の低下がみられ，車い

重

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維持にGM３が重要であるとも考えられる

筆者らは最近､C2C12細胞を用いた筋分化モデルにおいて,GM３の新たな生物機能の可能性見いだした３9).
C2C12細胞はマウスサテライト細胞由来の筋芽細胞であ

­

(B)

＝

すでの生活を余儀なくされる．この筋力低下は発育不全，
栄養吸収の問題神経機能異常などさまざまな要因が絡んだ複合的な要因であることが想定されるが，筋細胞の機能

­

０ １ ２

へ

５．筋分化過程におけるGM3分子種の発現変化

一Ｏ

１. l,｡'１｡["̲ｖＥｕｓＡｒ別ｍ０５ＧＥＮｍ軌ｃ別借蝉ｃｃ計“l Ａｒ計r■ ＧＥＮｇ也凱ご可t踵Ｃぐ
１６:０Ｉ“》Ｚ４:Ｕ２４:ＵＺ４:ｌＺ４:１璽:０Ｚ２;０

糖脂質であるGM３の量的・質的変化を見いだした（図6)
GM3量は分化に伴い大幅に増加するが，それに加えシア

図６マウスC2C12細胞の分化過程におけるガングリオシドの分子種変化
マウスサテライト細胞由来筋芽細胞株C2C12を低血清条件下
で筋管へと分化誘導した.(A)TLCによるガングリオシド解
析．分化前(DayO)では2本（黒および灰色の矢頭）が主要なGM３であるが，分化に伴い移動度が低い（極性が高い）バ
ンドへとシフトした（灰色．白抜きの矢頭).(B)GM3分子
種のLC-MS/MS解析シアル酸分子種(Neu5Ac,Neu5Gc)とセラミドアシル鎖椛造を示す．分化前にはシアル酸構造としてNell5Ac,アシル鎖として24:0もしくは24:ｌの構造を持ったGM３が主要であるが，分化後にはNeu5Gc,1６:0を持つGM３が主要となる．

GM３のセラミドのアシル鎖構造は分化前には炭素数24のせ．類似点，相違点を見つめなおし検証していく古くて新る.１0%ウシ胎仔血清を含有する培地中では増殖するが．

低血清（2％ウマ血清）にすることによって分化が誘導され，細胞融合し筋管を形成する

C2C12細胞の分化過程のスフインゴ糖脂質をTLC,
HPLC,LC-MS/MSを用いて詳細に解析したところ．主要な

ル酸分子種がNeu5AcからNeu5Gcへと変化した．加えて．

ものが主要であったのに対し．分化に伴い炭素数１６のア
シル鎖を持ったGM３が増加し，筋管形成後は炭素数１６のアシル鎖を持ったNe5Gc-GM3が主要なものとなったつ
まり．筋分化過程においてGM３の量的増加と微細櫛造の変化とが同時並行して起こり．同じGM３であっても分化に伴い極性が高い分子種へと変化してゆくこのGM３の量的・質的変化は筋分化および分化後の筋肉の質の制御に重要である可能性を示す結果を得ている３，)．
現在のところ.GM3合成酵素欠損マウスはほぼ正常に生まれて生育し、体重にも大きな差はないことから．筋肉の発生に関してはおおむね行われていると考えている．現在，筋損傷時の再生の面や機能的な面に関して詳細に解析を行っている．
こういった．培養モデル細胞系を用いたガングリオシドの生化学的な解析知見，分子レベルでの機能解析知見は．
さまざまな報告が蓄祇されているガングリオシド代謝遺伝子改変マウスや新たに報告されてきたヒトにおける欠損症の知見が集まり始めた現在．これらの知見を照らし合わ

しい研究を続けることで．ガングリオシド機能の本質がみえてくるものと思われる．

このC2C12の系では，分化に伴ってGM３のシアル酸部

分．セラミドのアシル鎖部分の微細柵造変化が同時に起こるユニークな現象を見いだした．筋ジストロフイーのモ
デルマウスにおいて．ヒト(Neu5Ac)とマウス(Neu5Gc)のシアル酸構造の違いによって，症状の重症度が異なることも示唆されている4(1).また，近年．ガングリオシドのセラミド部分のアシル釧柵造の多様性に注目が集まりつつあり，我々のグループもこのC2C12の系以外にメタボリッ

クシンドロームの病態にこのアシル鎖構造が重要な鍵になっていることを示唆している41).

シアル酸構造．アシル鎖柵造を含めたガングリオシド詳

細櫛造とその機能に注目することでスフィンゴ糖脂質の新
たなフィールドの開拓が進むかもしれない．

生化学第８９巻第５号(2017)

650

は吉川弥里先生（現・就実大学薬学部)．筋分化研究に関6．スフィンゴ糖脂質機能解明に向けて上述のように，近年のシークエンス技術の進歩によって，これまで見つからなかったスフィンゴ糖脂質の生合成
酵素の変異による疾患が新たに見つかってきた．もちろん，その疾患の発見，症状の解析だけですべてがわかるわ

しては郷詩織さん（現・名古屋大学大学院生命農学研究科
博士後期課程）が中心になって行った仕事です．また，名古屋大学生物機能開発利用研究センター北島健先生にご指導．ご助言を賜りました．皆様に感謝申し上げます．
文

けではない.GM3合成酵素欠損マウスおよびヒト欠損症

で見いだされた聴覚機能障害はGM３の新たな機能を明示するものであるが，その詳細な分子機構は未知のままである．ヒト疾患やある一つの遺伝子改変マウスの単純な表現
型解析だけでは，ガングリオシドの本質を見いだすことはできない．ガングリオシド合成酵素遺伝子変異によって，どのガングリオシドが消失し，どのスフインゴ糖脂質が出現・増加したか，やはり詳細な脂質解析が重要な鍵である．どのようなガングリオシド（スフィンゴ糖脂質）の組成の場合にどこで何が起こるか，という地道な知識の積み重ねが必要になってくると考える．ヒトにおけるスフイン
ゴ糖脂質の生合成酵素の変異の症例に加え，これまで蓄積されたスフィンゴ糖脂質生合成酵素欠損マウスの知見，各種培養細胞モデルを用いた膨大な知見を統合していくこと
で，新たなスフィンゴ糖脂質の機能の本質が見いだされると思われる．

各スフインゴ糖脂質の機能解明には細胞膜機能性微小領域（ラフト）の実態解明が切り離せない問題である．近年，高感度のイメージング用プローブおよび技術の開発も進み，生細胞上でスフインゴ糖脂質が膜タンパク質と相互作用する様子を観察することも可能になってきた４２43'・従来の手法を用いた知見と新たな技術による知見とを組み合わせ，これまでのラフト像もブラッシュアップしていく必要がある．また．近年注目されるエキソソームをはじめとするマイクロベシクル上のスフィンゴ糖脂質を介した細胞間情報交換も想定されてきている．また，筋分化過程で見いだされたGM３のシアル酸分子種およびセラミドのアシ

ル鎖分子種の構造変化は，同一スフィンゴ糖脂質分子種の微細構造変化による生物機能制御を示唆する新たな知見である．こういった点に注目した研究の進展によって予想もしなかったスフィンゴ糖脂質機能の本質がみえてくると思われる．そのためには今まで以上に農学，理学，医学，工学，薬学などさまざまな分野との交流と技術．情報交換が必須である．これまでの知見を大事にし，新たな視点．技術を用いてチャレンジを続け，新たなスフィンゴ糖脂質研究が展開されることを期待する．

謝辞

本稿作成に記載されている我々の研究内容は，私立大学

戦略的研究基盤形成支援事業「生体膜糖鎖異常に起因する

生活習慣病発症機序の解明と臨床への応用｣，新学術領域

研究「統合的神経機能の制御を標的とした糖鎖の作動原理解明」の支援を受けて，主に東北医科薬科大学分子生体膜研究所で行われたものです．記載した聴覚に関する研究

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著者寸描●郷慎司（ごうしんじ）川崎医科大学病態代謝学講師．博士（農学)．
■略歴岐阜県生まれ．名古屋大学農学部卒業．同大学院生命
腿学研究科博士課程修了東北薬科大学（現・東北医科薬科大学）分子生体膜研究所学術フロンティア研究員助教を経て．
2017年より現職■研究テーマと抱負糖鎖の代謝制御機櫛と機能の解明特に
糖ヌクレオチドとシアル酸代謝に注目した研究■ウェブサイトllttps:"www.kawasaki-m.acjp/mcd/study/inib.
Php?di =２１１■趣味HPLC.

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K.,Wall9,CM.,Val･ki,A.,&.1ansscn,P.M.(2013)GI)'co6jo/m)',
23,833-843.
41)Veillol1,L.,G０,S.,Matsuyama,W､,Suzuki,A.,Nagasaki,M.,
Yatomi,Y.,&lllokllchi,J.(2015)PLoSO"e,10,e0129645.
42)Suzuki,K.G,Kasai,R.S.,FUiiwara,T.K.,&KuSumi,A・(2013)
Ａ伽ﾙofiyC'g〃βゎ１．117,373-390.
43)Suzuki,K.G.,KaSili,R.S.,Hirosawa.K.M.,Ncmolo,Y.L.,Islli.
bashi,M..Miwa,Y.,FMjiwara,T.K.,&KusumioA.(2012)M７/.
でﾙe"J､ＢｉO１８７. ７４-７８3)くちじんいち）● 井 ノ ロ 仁 一 (いのくち

東北医科薬科大学分子生体膜研究所所長．薬学部機能病態分子学教室教授薬学博士．
■略歴福岡県に生まれる．福岡大学薬学部を卒業．同大学院，生化学教室助手
を経て．1985年ミシガン大学医学部神経化学研究所博士研究員９１年生化学工業
東京研究所主任研究員を経て．９８年北海
道大学大学院薬学研究科助教授.2006年

より現職■研究テーマと抱負生体恒常性維持機構におけるスフィンゴ
糖脂質の分子種特異的な作動原理を解明し、生理活性糖脂質としての位置付けを明確にすることが．使命であると考えている．
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Biochemical and Biophysical Research Communications 485 (2017) 427e431

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

N-glycan-dependent cell-surface expression of the P2Y2 receptor andN-glycan-independent distribution to lipid rafts
Tetsuto Nakagawa, Chihiro Takahashi, Hitomi Matsuzaki, Shohei Takeyama, Shinpei Sato,
Ayaka Sato, Yoshiyuki Kuroda, Hideyoshi Higashi*
Division of Glyco-Signal Research, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Miyagi
981-8558, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 3 February 2017
Accepted 10 February 2017
Available online 14 February 2017

P2Y2 receptor (P2Y2R) is a G-protein-coupled receptor (GPCR) that couples with Gaq/11 and is stimulated
by ATP and UTP. P2Y2R is involved in pain, proinﬂammatory changes, and blood pressure control. Some
GPCRs are localized in lipid rafts for interaction with other signaling molecules. In this study, we prepared N-glycan-deﬁcient mutants by mutating the two consensus Asn residues for N-glycosylation to Gln
to examine intracellular localization and association with lipid rafts. Western blotting of the wild type
(WT) protein and mutants (N9Q, N13Q, N9Q/N13Q) in COS-7 cells showed that both Asn residues were
glycosylated in the WT. Fluorescent microscopy analysis showed that WT, N9Q and N13Q were expressedin the endoplasmic reticulum (ER), Golgi body, and cell membrane, but N9Q/N13Q was only found in the
ER. WT, N9Q and N13Q moved from the cell surface to endosomes within 15 min after UTP stimulation.
WT and the N9Q/N13Q glycosylation-deﬁcient mutant appeared in the detergent insoluble membrane
fraction, lipid raft. These ﬁndings suggest that P2Y2R is localized in lipid rafts in the ER during biosynthesis, and that N-glycosylation is required for subsequent expression in the cell membrane. In thepresence of epoxomicin, a proteasome inhibitor, there was a signiﬁcant increase in the level of N9Q/
N13Q, which suggests that N-glycan-deﬁcient P2Y2R undergoes proteasomal degradation.
© 2017 Elsevier Inc. All rights reserved.

Keywords:
Cell surface expression
ER-associated protein degradation
Lipid raft
N-linked glycosylation
P2Y2 receptor

1. Introduction
The P2Y receptor family consists of G-protein-coupled receptors(GPCRs) that are activated by nucleotides such as adenosine
triphosphate (ATP) and uridine triphosphate (UTP) and havevarious physiological functions. P2Y2 receptor (P2Y2R) is a Gaq/11coupled receptor that has ATP and UTP as ligands and is expressedin mammalian tissues as a mediator of pain and inﬂammation.
P2Y2R is associated with chloride secretion upon damage to respiratory tract epithelial cells [1], neuronal protection upon development of inﬂammation [2], phagocytosis of apoptotic cells bymacrophages [3,4], and axonal elongation and differentiation ofneurocytes [5]. There are an increasing number of reports on crosstalk of signals via ATP and bradykinin upon development ofinﬂammation [6e9], and we recently suggested that P2Y2R may be
associated with the bradykinin receptor, B2R [10]. We also showedthat sugar chains of B2R in Western blotting have a wide range ofsizes, while the sizes of the sugar chains of P2Y2R are uniformly
small.
Glycosylation promotes expression of proteins in the cell
membrane [11] and some GPCRs are localized in membrane
microdomains such as rafts for interaction with other signaling
molecules and regulation of their activity [12e14]. In this study, thecellular localization and distribution to lipid rafts of WT P2Y2R and
its mutants with loss of N-glycosylation sites were examined. The
results showed that most of WT P2Y2R localized in lipid rafts andwas expressed on the cell surface. Besides, a glycosylation-deﬁcient
P2Y2R mutant remained localized in lipid rafts, was not transferredbeyond the ER, and was degraded by proteasomes. These results
show that P2Y2R requires N-glycosylation for expression on the cellsurface.
2. Materials and methods* Corresponding author.
E-mail address: hhigashi@tohoku-mpu.ac.jp (H. Higashi).
http://dx.doi.org/10.1016/j.bbrc.2017.02.061
0006-291X/© 2017 Elsevier Inc. All rights reserved.

Preparation of Plasmids. Human P2Y2R genes with a C-terminal
c-Myc epitope tag (EQKLISEEDL) were prepared by PCR and428

T. Nakagawa et al. / Biochemical and Biophysical Research Communications 485 (2017) 427e431

inserted into the pIRES2-EGFP plasmid (Clontech) with EcoRI and
BamHI sites. The IRES and EGFP gene sequences were removed
using BamHI and NotI, followed by blunting and ligation. Phusion
High-Fidelity DNA Polymerase, restriction enzymes and a Quick
Ligation kit were purchased from New England Biolabs. A DNA
blunting kit was obtained from Takara Bio.
Cell culture. COS-7 cells were grown in D-MEM supplemented
with 10% fetal calf serum and kept in a humidiﬁed 5% CO2 atmosphere at 37 ! C. COS-7 cells were transiently transfected using
Viafect transfection reagent (Promega). In some experiments,
epoxomicin (Peptide Institute Inc.) and baﬁlomycin A1 (LC Labs)
were added at ﬁnal concentrations of 1 and 0.1 mM, respectively,
12 h before harvesting.
Preparation of membrane fractions. COS-7 cells cultured on a 6well plate were washed once with PBS and harvested using a cell
scraper. After centrifugation at 3000 rpm for 5 min at 4 ! C,
precipitated cells were suspended in ice-cold TBS and homogenized in 500 mL of TBS containing Protease Inhibitor Cocktail(Nacalai Tesque) with 15 strokes using a teﬂon homogenizer on ice.
The homogenate was centrifuged at 700 g for 5 min at 4 ! C, andsupernatant was centrifuged at 21,900 g for 90 min at 4 ! C. This
pellet was used as the total membrane fraction. In some experiment, the pellet was ﬁrst treated with Peptide-N-Glycosidase F(PNGase F, New England Biolabs). The pellet was resuspended in
100 mL of ice-cold TBS containing 0.1% Triton X-100, and the tube
was inverted using a rotator for 1 h at 4 ! C. The supernatant andpellet from centrifugation at 21,900 g for 90 min at 4 ! C were usedas the detergent-soluble and detergent-insoluble membrane fractions, respectively.
Chloroform-methanol precipitation of protein. Methanol (400 mL)
was added to 100 mL of detergent soluble membrane fraction, followed by 100 mL of CHCl3, and then 300 mL of water. The mixture
was vortexed well after each addition. After centrifugation at
17,400 g for 1 min at room temperature, the upper layer was
removed while leaving the intermediate protein layer. To eliminate
layer separation, 400 mL of methanol was added and the tube wasinverted a few times to mix gently. After centrifugation at 17,400 g
for 2 min, supernatant was removed and the dried pellet was used
as a protein sample for SDS-PAGE.
SDS-PAGE and Western blotting. SDS-PAGE was performed usinga Novex NuPAGE SDS-Gel system with 4e12% Bis-Tris gels (Thermo
Fisher Scientiﬁc). Proteins were electrophoretically transferred to
an Immobilon PVDF membrane (Merck Millipore) using a semi-dry

blotting system. After blocking with 5% skim milk in PBS, P2Y2R was
detected using anti-Myc mouse mAb (9B11; Cell Signaling Technology) followed by Horseradish Peroxidase conjugated antimouse IgG goat pAb (Jackson ImmunoResearch Lab.), and a Luminata Forte Western HRP Substrate (Merck Millipore).
Immunocytochemistry. COS-7 cells were cultured on Millicell EZ
8 well slides (Merck Millipore) for 1 day, stimulated with 0.1 mM
UTP 15 min before ﬁxation, and ﬁxed by 3% paraformaldehyde after
removal of the medium. After blocking with 5% skim milk in PBS,
P2Y2R was detected using anti-Myc monoclonal antibodies (9B11mouse mAb and 71D10 rabbit mAb; Cell Signaling Technology)
followed by Alexa Fluor 546 conjugated secondary antibodies(Thermo Fisher Scientiﬁc), and observed using an Olympus FV1000
confocal laser scanning microscope. The following antibodies wereused to detect organelles: anti-KDEL mouse mAb (10C3; Enzo Life
Science) for the ER, anti-RCAS1 rabbit mAb (D2B6N; Cell Signaling
Technology) for Golgi bodies, anti-Naþ-Kþ ATPase rabbit mAb(D2B6N; abcam) for plasma membrane, anti-hTransferin receptor
mouse mAb (H68.4; Thermo Fisher Scientiﬁc) for recycling endosomes, anti-EEA1 mouse mAb (4/EEA1; BD Biosciences) for earlyendosomes, and Alexa Fluor 546 conjugated secondary antibodies(Thermo Fisher Scientiﬁc).
3. Results
3.1. Preparation of N-glycan-deﬁcient mutants of P2Y2 receptor
P2Y2R has N-glycosylation consensus sequences (Asn-X-Ser/Thr) at three sites, including two (Asn9 and Asn13) in the N-terminal extracellular region and a third (Asn66) located in an intracellular region. For deletion of N-glycans, N9Q and N13Q point
mutants and the N9Q/N13Q double mutant were made by replacing
Asn9, Asn13 or both with Gln residues (Fig. S1). Each P2Y2R
construct had a c-Myc epitope tag attached to the C-terminal region
for detection in Western blotting and immunostaining. After
expressing WT or glycosylation-deﬁcient P2Y2R in COS-7 cells,
Western blotting was performed with anti-Myc antibody. Bands
were detected at 46 kDa for the WT protein, 42 kDa for N9Q and
N13Q, and 38 kDa for the glycosylation-deﬁcient N9Q/N13Q protein, indicating that both Asn9 and Asn13 are glycosylated in P2Y2R(Fig. 1A). The apparent absence of glycosylation in WT, N9Q and
N13Q P2Y2R (38 kDa bands) may have been due to transient
expression. Membrane fractions prepared from COS-7 cells

Fig. 1. Conﬁrmation of N-linked glycosylation of P2Y2R. (A) Membrane fractions of COS-7 cells expressing P2Y2R wild-type (WT) and mutants (N9Q, N13Q, N9Q/N13Q) analyzed byWestern blotting using anti-Myc antibody. (B) Membrane fractions treated with PNGase F and examined by Western blotting.

T. Nakagawa et al. / Biochemical and Biophysical Research Communications 485 (2017) 427e431

expressing WT or mutant P2Y2R were treated with PNGase F andthen subjected to Western blotting. The bands at 46 kDa for WT and
42 kDa for N9Q and N13Q were eliminated by PNGase F and only
the 38 kDa band remained, thus conﬁrming that both Asn9 and
Asn13 in P2Y2R undergo N-glycosylation (Fig. 1B).
3.2. Subcellular localization of N-glycan deﬁcient P2Y2 receptor
WT and mutant P2Y2R were expressed in COS-7 cells to observe
the distribution using confocal ﬂuorescent microscopy. Except
glycosylation-deﬁcient mutant N9Q/N13Q, P2Y2R appeared to
distribute on cell membrane and the distribution disappeared by
UTP stimulation (Fig. 2A). Furthermore, WT and N9Q/N13Q wereexpressed in COS-7 cells for ﬂuorescent double staining of marker
proteins for the ER, Golgi body, late endosome, and recycling endosome, and observation by confocal microscopy. The distribution

429

of WT P2Y2R was consistent with staining of the ER, Golgi body, andcell membrane (Fig. 2B). When the cells expressed WT P2Y2R were
stimulated with UTP, distribution to the cell membrane decreased,
and a granulated distribution overlapping early endosomes andrecycling endosomes was observed (Fig. 2B). N9Q/N13Q P2Y2R was
distributed only in the ER, and not in the cell membrane or Golgi
body (Fig. 2B), and showed no changes in distribution upon stimulation with UTP (Fig. 2A). We examined localization of P2Y2R inlipid rafts, based on resistance to detergent. Almost all N9Q/N13Q
proteins were found in the detergent insoluble membrane fraction,
lipid raft (Fig. 3A). While N9Q/N13Q P2Y2R was not transferred to
the Golgi body due to the absence of N-glycan, this mutant
remained in the detergent insoluble membrane fraction. These
results show that P2Y2R is distributed to lipid rafts in the ER
membrane prior to N-glycosylation in ER.

Fig. 2. Localization of P2Y2R WT and N9Q/N13Q mutant. (A) Intracellular localization in COS-7 cells observed by confocal laser scanning microscopy. In some experiments, cells
were treated with 0.1 mM UTP (UTP (þ)). (B) Colocalization of P2Y2R (red) and organelles (green) examined by double staining. Scale bars indicate 10 mm.

430

T. Nakagawa et al. / Biochemical and Biophysical Research Communications 485 (2017) 427e431

Fig. 3. Distribution to lipid rafts and proteolysis of P2Y2R mutants. (A) Detergent insoluble (Insol) and soluble (Solu) membrane fractions analyzed by Western blotting using antiMyc antibody. To ensure separation of lipid raft and non-raft proteins, the distribution of transferrin receptor (TfR) in the same sample was examined. (B) COS-7 cells expressing
P2Y2R WT or N9Q/N13Q were treated with the proteasome inhibitor epoxomicin (EXM) or the lysosome inhibitor baﬁlomycin A1 (BMA) 12 h before harvesting, and total membrane
fractions were analyzed by Western blotting.

3.3. ER-associated degradation of N-glycan-deﬁcient P2Y2 receptor
Epoxomicin, a proteasome inhibitor, or baﬁlomycin A1, alysosome-autophagy inhibitor, was added 12 h after transfection of
WT or N9Q/N13Q P2Y2R in COS-7 cells. Culture was then performed
for an additional 12 h. Western blotting and immunostaining with
anti-Myc antibody showed signiﬁcantly increased levels of WT and
N9Q/N13Q P2Y2R in cells treated with epoxomicin (Fig. 3B). This
result suggests that P2Y2R (38 kDa) without glycosylation isdegraded by proteasomes. Glycosylated WT P2Y2R (46 kDa) alsosigniﬁcantly increased because of degradation of proteins that were
folded or glycosylated incorrectly due to excess expression. The
level of WT P2Y2R was also increased by baﬁlomycin A1 (Fig. 3B).
There was also an increased level of receptors without glycosylation, which were not transferred to the cell membrane, and such
receptors may be degraded by autophagy. Collectively, these ﬁndings show that N-glycan modiﬁcation at either of the twoconsensus sequences in the N-terminal domain of P2Y2R is required
for transfer of the receptor to the cell membrane, and that nonglycosylated P2Y2R is degraded by proteasomes.

4. Discussion
Many GPCRs have N-glycosylation consensus sequences in theextracellular region, and most membrane proteins are likely to beglycosylated. Most of the roles of N-glycosylation in GPCRs areunclear, but glycosylation is known to be required for expression ofangiotensin II receptor (AT1R) and follicle-stimulating hormone
receptor (FSHR) in cell membranes [15e17], and for maintaining ahigher level of b2-adrenaline receptor in the cell membrane,
although glycosylation is not essential for this receptor [18]. In
contrast, there are many receptors, including a2-adrenaline receptor, H2 histamine receptor, and muscarinic receptor, for whichloss of N-glycosylation has almost no impact on cell surface
expression [18e20].
Expression of membrane proteins at a designated site requires
formation of the native fold, and misfolded proteins are excluded

from transport vesicles by a quality control mechanism in the ER[21,22]. In this mechanism, non-native folds are detected to faciliate
refolding or degradation. The mechanism involves molecularchaperones and lectin-like chaperones, which recognize the hydrophobic surface of non-native proteins, unbridged Cys residues,
and immature N-glycosylation. Calnexin, calreticulin, and BiP aremolecular chaperones related to GPCRs [23e28]. Calnexin andcalreticulin are homologous lectin-like molecular chaperones thatfacilitate maturation of glycoproteins by recognizing N-glycosylation in an early phase of modiﬁcation of membrane proteins andsecreted proteins during folding [29]. BiP binds to the hydrophobic
region on the surface of a protein during folding or to a misfolded
protein, and is involved in retrograde trafﬁcking to the ER or
membrane transport to the cytoplasm in ER-associated degradation(ERAD) [30,31].
The results of this study show that both N-glycosylationconsensus sequences in the extracellular region of P2Y2R are
modiﬁed, and that the N9Q/N13Q double mutant with complete
loss of glycosylation was not transferred to the cell surface and had
an extremely low expression level. Since N9Q/N13Q P2Y2R cannot
remain in the ER with lectin-like chaperones such as calnexin andcalreticulin, molecular chaperones such as BiP that recognize hydrophobic surfaces may be involved. The expression level of N9Q/
N13Q P2Y2R was low because the absence of N-glycosylation may
prevent proper folding and quality control by calnexin and calreticulin, causing lower folding efﬁciency. Thus, the expressed protein
is degraded by proteasomes as a misfolded protein, rather thanremaining in the ER.
Lipid rafts mature in the Golgi body through synthesis ofglycolipid and sphingomyelin structural components, and signaling
proteins associate with the rafts during the course of maturation.
However, the N-glycan-deﬁcient P2Y2R existed in the detergent
insoluble membrane fraction when the lipid raft was fractionated,while P2Y2R remained in the ER. Since the detergent insoluble
membrane fraction is product in the experiment, these results may
be an artifact, but it is possible that P2Y2R may associate with lipid
rafts in the ER because glucosylceramide synthases are also present

T. Nakagawa et al. / Biochemical and Biophysical Research Communications 485 (2017) 427e431

in the ER, and retrograde trafﬁc of vesiles can be observed from thecis Golgi. Prevously, we demonstrated that B2 bradykinin receptor
is closely associated with P2Y2R and has heterogeneous glycans
differ from P2Y2R [10]. If localization of P2Y2R in lipid rafts occurs
before glycosylation, this may lead to uniform and short sugar
chains. P2Y2R forms hetero-oligomers with various GPCRs[10,32,33], and P2Y2R localized in lipid rafts may recruit interacting
GPCRs.
This study shows that N-glycosylation of P2Y2R is involved inreceptor cell surface expression, and that P2Y2R is distributed tolipid rafts before N-glycosylation. P2Y2R lacking N-glycosylation at
one site was transferred to the cell surface but the effect of partial
loss of N-glycosylation on the receptor function of P2Y2R and association with other GPCRs are unclear.
Acknowledgements
Funding: This study was supported by the Strategic Study Base
Formation Support Project for Private Universities, Japan.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2017.02.061.
Transparency document
Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.bbrc.2017.02.061.
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expression, targeting, afﬁnity and quality control of the human AT1 receptor, Biochem. J. 390 (2005) 367e376.
[28] A. Anukanth, H.G. Khorana, Structure and function in rhodopsin. Requirements of a speciﬁc structure for the intradiscal domain, J. Biol. Chem. 269(1994) 19738e19744.
[29] D.B. Williams, Beyond lectins: the calnexin/calreticulin chaperone system of
the endoplasmic reticulum, J. Cell. Sci. 119 (2006) 615e623.
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barrier of the ER membrane by sealing the lumenal end of the translocon pore
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[31] B. Meusser, C. Hirsch, E. Jarosch, T. Sommer, ERAD: the long road todestruction, Nat. Cell Biol. 7 (2005) 766e772.
[32] K. Yoshioka, O. Saitoh, H. Nakata, Heteromeric association creates a P2Y-likeadenosine receptor, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 7617e7622.
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Biochem. Biophys. Res. Commun. 351 (2006) 559e565.

RESEARCH ARTICLE

crossmQinglei Hang,a Tomoya Isaji,a Sicong Hou,a Yuqin Wang,a,b Tomohiko Fukuda,aJianguo Gua,bDivision of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical
and Pharmaceutical University, Sendai, Miyagi, Japana; Department of Pharmacology, Pharmacy College,
Nantong University, Nantong, Jiangsu, Chinab

The N-glycosylation of integrin !5"1 is thought to control many fundamental aspects of cell behavior, including cell adhesion and migration. However, themechanism of how N-glycans function remains largely obscure. Here, we used aloss-of-function approach. Wild-type (WT) integrin !5 and N-glycosylation mutant
S3-5 (sites 3 to 5) integrin !5, which contains fewer N-glycans, were stably reconstituted in !5 knockout cancer cells. We found that the migration ability of S3-5 cells
was decreased in comparison with that of the WT. Interestingly, the levels of phosphorylated focal adhesion kinase and actin stress fiber formation were greatly enhanced in the S3-5 mutant. In a mechanistic manner, the internalization of active
but not total integrin !5"1 was inhibited in S3-5 cells, which is a process that is related to the enhanced expression of active integrin !5"1 on the cell surface. Importantly, restoration of N-glycosylation on the "-propeller domain of !5 reinstated thecell migration ability, active !5"1 expression, and internalization. Moreover, theseN-glycans are critical for !5–syndecan-4 complex formation. These findings indicatethat N-glycosylation on the "-propeller domain functions as a molecular switch tocontrol the dynamics of !5"1 on the cell surface that in turn is required for optimum adhesion for cell migration.
ABSTRACT

Received 11 October 2016 Returned formodification 3 November 2016 Accepted 29
January 2017
Accepted manuscript posted online 6
February 2017
Citation Hang Q, Isaji T, Hou S, Wang Y, Fukuda
T, Gu J. 2017. A key regulator of cell adhesion:
identification and characterization of importantN-glycosylation sites on integrin α5 for cell
migration. Mol Cell Biol 37:e00558-16. https://doi.org/10.1128/MCB.00558-16.
Copyright © 2017 American Society for
Microbiology. All Rights Reserved.
Address correspondence to Jianguo Gu,
jgu@tohoku-mpu.ac.jp.

KEYWORDS N-glycan, activation, complex formation, internalization

C

ell migration is essential not only for normal physiological processes such asembryonic development and wound healing but also for pathological changes
such as inflammatory disease and cancer (1). Integrin, the !" heterodimeric transmembrane receptor, plays a central role in cell migration by connecting an extracellularmatrix (ECM) to the cytoskeleton as well as functioning as a bidirectional signaling
molecule that transmits information across the cell membrane (2). Due to its importance, integrin has been highly implicated in the development of tumor malignancy.
Therefore, a detailed molecular understanding of the mechanisms involved in integrinmediated tumor cell migration is very important for our ability to intervene in theprocession of cancer.
In mammals, different ! and " subunit combinations make up a total of 24 integrins.
Integrins are found in either active or inactive conformations with respect to their highor low affinity for their ECM ligands (3), and an allosteric change that favors high affinity
can be induced by either cytoplasmic events (“inside-out” activation) or extracellularfactors (“outside-in” activation) (4), which triggers integrin clustering, cytoskeletal
remodeling, and the assembly of cell adhesion complexes, including focal adhesion(FA) and fibrillary adhesion (5). Indeed, cell migration is a highly dynamic process thatMay 2017 Volume 37 Issue 9 e00558-16

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is regulated by FA turnover in a coordinated manner, which requires the upregulationof cell matrix attachment at the leading edge of the cell and downregulation at its
trailing edge. An appropriate balance in the adhesion and de-adhesion of the integrins
is important for cell migration. The disassembly of FA requires the internalization of
integrins that are either then recycled back to the cell membrane at the leading edgeof migrating cells or degraded in the lysosome (6, 7). Thus, elucidating the precise
mechanisms that control the internalization of integrins is fundamental to understanding coordinated cell migration.
Current insight into this regulation is derived largely from studies focused on the
inner membrane, particularly cytoplasmic tails. For example, NPxY motif-binding proteins such as talin and kindlin (8), different small GTPases such as Rab and Rho families
and their regulators, as well as serine/threonine kinases have been implicated in theinternalization of integrins (9). However, little is known about the function of the outermembrane of integrins in this process.
Of note, integrins are known to be major glycan-carrying proteins. In fact, thefunctions of integrins are also dependent on their complex N-glycosylation modifications (10). Among the different types of integrins, !5"1, a major fibronectin (FN)
receptor, is believed to be a relatively well-characterized example, and N-glycosylation
is important for its mediated many biological functions such as cell adhesion andmigration (11, 12). Although the importance of the N-glycosylation of !5"1 for cell
migration has been highlighted, most studies have examined only total changes by
displaying or masking specific glycan epitopes via the overexpression and knockdown,
or knockout, of glycogenes. For instance, alterations in the oligosaccharide portion of
integrin !5"1 from the enhanced expression of some glycosyltransferase genes such as
N-acetylglucosaminyltransferase V (GnT-V), GnT-III, or !2,6-galactoside sialyltransferase
1 (ST6GAL1) can be used to regulate cell spreading and migration onto FN (10, 13, 14).
However, the use of these approaches makes it quite difficult to clarify the details of theunderlying mechanisms involved in the importance of N-glycosylation on !5"1.
To resolve these issues using the N-glycosylation mutants of integrin !5, we first
identified the individual N-glycosylation of !5 as a key regulator for cell migration. We
found that the N-glycosylation of the "-propeller domain on !5 was essential for theinternalization of active integrin !5"1 and membrane complex formation (e.g.,
syndecan-4), which in turn was important for its expression on the cell surface, which
may result in a form of cell adhesion that is more appropriate for migration.
RESULTS
N-Glycosylation on integrin !5 is required for !5"1-mediated cell migration.
Given the importance of !5 for cell migration, we hypothesized the involvement ofN-glycosylation. To test this idea, we chose MDA-MB-231, HeLa, and, U-251MG cells as
the cell models, which relatively express higher levels of endogenous integrins on thecell surface, including integrins !3, !5, !6, !V, "1, and "4, than do 293T cells (see Fig.
S1A in the supplemental material). First, we transfected either green fluorescent protein(GFP)-tagged wild-type (WT) or S3-5 mutant integrin !5 into !5 knockout (!5-KO)MDA-MB-231, !5-KO-HeLa, and !5-KO-U-251MG cells, which was established previously (15), because N-glycosylation on sites 3 to 5 (Fig. 1A) from among the 14 potential
N-glycosylation sites in integrin !5 is necessary for its dimer formation with "1 and total
cell surface expression in CHO-B2 cells (12). Consistently, in immunoprecipitation (IP)and flow cytometric analyses, WT-MDA-MB-231 and S3-5-MDA-MB-231 cells exhibitedcomparable !5"1 heterodimeric formation abilities (Fig. 1B) and the same expression
levels of !5"1 (Fig. 1C) or !5 (Fig. S1B) on the cell surface, as did HeLa and U-251MG
cells (Fig. S1C and D). It is noteworthy that due to the heterogeneity of glycans, WT !5
on SDS-PAGE gels usually shows a diffuse band, as shown in Fig. 1B, which seems tohave a relatively higher intensity than that of the S3-5 mutant. However, after theremoval of heterogeneity by treatment with the peptide:N-glycosidase F (PNGase F) for
deglycosylation, WT cells exhibited an expression level of !5 similar to that of S3-5
mutant cells (Fig. S1E). Together, these results suggested that S3-5 mutant !5 can also
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FIG 1 Comparison of FN-mediated cell migration in cell lines expressed with WT or S3-5 mutant !5. (A) Schematic diagram of potential
N-glycosylation sites on the WT and S3-5 integrin !5 subunits. Putative N-glycosylation sites (N84Q, N182Q, N297Q, N307Q, N316Q,
N524Q, N530Q, N593Q, N609Q, N675Q, N712Q, N724Q, N773Q, and N868Q) and point mutations are indicated. TM, transmembrane.
(B and C) WT- and S3-5-MDA-MB-231 cells exhibit comparable !5"1 heterodimer formation abilities (B) and the same expression levelsof total integrin !5"1 on the cell surface (C). (B, top) The stable cell lines were established as described in Materials and Methods. The
indicated cell extracts were immunoprecipitated (IP) with anti-GFP–agarose, followed by anti-"1 and !5 antibodies for Western
blotting. (Bottom) Whole-cell extracts were also subjected to Western blotting (as an input). (C) The expression levels of !5"1 wereanalyzed by flow cytometry. IgG was used as a control. (D to G) Individual migration tracks, persistence values, and mean speeds of
GFP-, WT-, and S3-5-MDA-MB-231 cells migrating on FN. (E) Movements of the indicated cells were observed by time-lapse video
microscopy (n ! 12, from triplicate experiments). (E to G) The directionality (E), displacement (F), and speed (G) of migration wereextracted from the track plots. (H and I) Cell wound closure (H) and abilities of migration toward FN (I) were determined by using
wound healing and Transwell assays. Representative images of MDA-MB-231 cells appear at the top. The migrated distances relative
to 0 h (H) and the numbers of migrated cells (I) of the MDA-MB-231, HeLa, and U-251MG cell lines were measured and statisticallyanalyzed, respectively (bottom, n ! 3 individual experiments). All values are reported as the means " SE (error bars), as determinedby Student’s t test. *, P # 0.05; **, P # 0.01; ***, P # 0.001. Bars, 250 #m (H) and 350 #m (I).

serve as a successful loss-of-function model to identify the roles of the remaining
N-glycosylation on !5 in MDA-MB-231, HeLa, and U-251MG cells.
Next, we compared cell movement on FN between GFP-, WT-, and S3-5-MDA-MB231 cells via time-lapse imaging for 12 h (Fig. 1D). As shown in Fig. 1D to G, GFP control
cells still exhibited some cell motilities, which might be due to the function of other FN
receptors such as integrin !V"1, but these motilities were much weaker than those of
WT cells, as reflected by the directionality (Fig. 1E), displacement (Fig. 1F), and mean
migration speed (Fig. 1G). Interestingly, although the cell motility of S3-5 cells was
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FIG 2 Changes in cell spreading, phosphorylation of FAK, and actin filament formation in S3-5 cells. (A) MDA-MB-231 cells weredetached, blocked, and then replated onto FN-coated plates. After incubation for 20 min, cells were fixed, and representative images
were taken. The percentages of rounded and spread cells were statistically analyzed (right) (n ! 9, from 3 individual experiments). (B)
MDA-MB-231 cells were detached, suspended in assay medium for 40 min, and then replated onto an FN-coated plate for theindicated times. Western blotting was performed with the indicated antibodies. (C, left) After culture on FN-coated dishes for 2 days,
the indicated MDA-MB-231 and HeLa cells were lysed and immunoblotted with the indicated antibodies. (Right) Relative ratios (p-FAK
versus FAK) (n ! 3 individual experiments); the relative ratio was 1.0 for S3-5 mutant cells. (D) Immunofluorescence labeling andconfocal microscopy of p-FAK (top) and actin stress fibers (bottom) in GFP, WT, and S3-5 mutant cells. MDA-MB-231 and HeLa cells
were cultured on FN-coated coverslips, and cells were fixed, permeabilized, and then visualized with p-FAK and phalloidin-Alexa Fluor
549 (actin), respectively. The relative fluorescence intensities of p-FAK and phalloidin were quantified by using ImageJ software (n !
6, from 3 individual experiments); relative fluorescence intensity was 1.0 for S3-5 mutant cells. All values are reported as the means "SE (error bars), as determined by Student’s t test. n.s, not significant (P # 0.05); *, P $ 0.05; ***, P $ 0.001. Bars, 120 "m (A) and 20"m (D).

stronger than that of control GFP cells, it was significantly weaker than that of WT cells(Fig. 1D to G). These phenomena were also confirmed via wound healing (Fig. 1H) andTranswell (Fig. 1I) assays. In addition, decreased wound closure (Fig. 1H, bottom) andmigration abilities (Fig. 1I, bottom) were also observed for both S3-5-HeLa and S3-5U-251MG cells. Of note, we previously showed that N-glycosylation on the calf domain
of !5 exhibited an inhibitory effect on cell proliferation via complex formation withepidermal growth factor receptor (EGFR) (15). Taken together, these results strongly
suggest that the remaining N-glycosylation, other than that on sites 3 to 5 of !5, plays
a crucial role in the mediation of cell migration.
Deletion of N-glycosylation on integrin !5 increases cell matrix adhesion. In
order to explore the underlying mechanisms involved in the N-glycosylation of !5mediated cell migration, we compared the cell spreading abilities of WT and S3-5 cells.
As shown in Fig. 2A, both WT- and S3-5-MDA-MB-231 cells exhibited comparable
abilities for cell spreading on FN. A similar result was also observed for HeLa or
U-251MG cells (data not shown), indicating that the remaining N-glycosylation on
integrin !5 had no significant effect on cell spreading.
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It is well known that integrin !5"1 facilitates cell migration, which in turn requires
a dynamic turnover of cell matrix associations, during which the activation of focal
adhesion kinase (FAK) is an important step (16). To determine whether N-glycosylation
on !5 affected the FN-mediated activation of FAK, serum-starved cells were replated
onto FN-coated plates and then harvested at the indicated times. The phosphorylationlevels of FAK (p-FAK) in the cell lysates were detected. As expected, Western blot (WB)
analysis revealed that the levels of p-FAK upon FN stimulation at each time point in
GFP-MDA-MB-231 cells were lower than those in WT cells (Fig. 2B). However, intriguingly, S3-5-MDA-MB-231 cells exhibited increased expression levels of p-FAK comparedwith the WT ones (Fig. 2B). In addition, when cells were grown on FN-coated dishes for
2 days, p-FAK levels were still significantly higher in S3-5 cells than those in WT cells,in both MDA-MB-231 and HeLa cells (Fig. 2C). Immunofluorescence staining clearly
showed that p-FAK containing cell matrix adhesion in S3-5 cells was consistently
more prominent in both size and staining intensity than that in WT-MDA-MB-231
cells (Fig. 2D, top). Furthermore, the actin stress fibers detected by phalloidin weremore abundant in S3-5-HeLa cells than in WT cells (Fig. 2D, bottom). Considering thedecreased cell migration ability of S3-5 cells, these findings indicate that integrin !5
N-glycosylation can regulate a modest level of cell-FN adhesion for migration.
Removal of N-glycosylation on integrin !5 increases the active form of "1 on
the cell surface. Next, we investigated the cause of increased cell-FN adhesion in S3-5
cells. Considering that the efficiency of cell adhesion on the ECM is generally thought
to be proportional to the amount of either active or total (i.e., active and inactive)
integrin on the cell surface (17), we compared the expression levels of both active andtotal integrin "1 in WT and S3-5 cells. The active form of "1 was detected with theantibody HUTS-4-recognizing epitopes in the region spanning positions 355 to 425(hybrid domain) of the common "1 subunit, the expressions of which are reported toparallel the activity of integrin "1 (18), and the level of active integrin "1 recognized bythis antibody but not total "1 was clearly increased upon Mn2! stimulation during
Western blot analysis (see Fig. S1F in the supplemental material). Flow cytometricanalysis clearly showed that approximately 10% of cell surface integrin "1 is in theactive conformation (Fig. 3A), which is consistent with data from a previous report (19),and that levels of the active form of "1 but not the total form on the cell surface weresignificantly increased in S3-5-MDA-MB-231, S3-5-HeLa, and S3-5-U-251MG cells compared with those in WT cells (Fig. 3A).
Given the increased expression levels of active "1 on the cell surface of S3-5 mutant
cells, we wondered whether the expression of total active "1 was also increased.
Interestingly, as shown in Fig. 3B (middle), both WT and S3-5 mutant cells exhibited
expression levels of both active "1 and total "1 similar to those in the whole-celllysates. However, increased expression levels of active "1 on the cell surface in S3-5
cells, as described above, were observed in the biotinylation experiment (Fig. 3B, top).
Furthermore, the increased cell surface expression of active integrin "1 (Fig. 3C,
middle), but not that of the total version (Fig. 3C, top), in mutant MDA-MB-231 cells wasconsistently confirmed by immunostaining. Taken together, these results indicate that
the N-glycosylation of !5 can regulate the localization of the active form of "1 on thecell surface, which might explain the enhancement of cell-FN adhesion in S3-5 mutant
cells.
N-Glycosylation on integrin !5 plays important roles in the internalization ofactive !5"1. Because the loss of N-glycosylation on !5 increased the expression levelsof active "1 on the cell surface, we sought to investigate the underlying mechanism.
Given that the endocytic trafficking of integrins, which involves their internalizationfrom the cell surface and recycling back to the plasma membrane, is important for cell
migration (20) and expression levels on the cell surface (21), we wondered whether
N-glycosylation on !5 regulates its internalization. To address this, biotin pulse-chaseexperiments were performed. After biotin labeling of cell surface proteins at 4°C, cells
were incubated at 37°C for different times to induce internalization, and the biotin
remaining in the form of cell surface proteins was then stripped at 4°C, leaving only
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FIG 3 Increased expression levels of active integrin "1 in S3-5 mutant cells. (A and B) The expression levels of active and total integrin"1 on the cell surface (CS) or in total cell lysates (TL) were analyzed by flow cytometry (A) or biotinylation (B), and immunoblotting
was performed with the indicated antibodies. The relative ratio (active "1 versus total "1) is shown at the bottom (n ! 3 individual
experiments), which was 1.0 for S3-5 mutant cells. (C) Comparison of the localization patterns of active integrin "1 in WT- andS3-5-MDA-MB-231 cells. Cells were cultured on FN-coated coverslips and then subjected to immunostaining analyses. The images weremerged with total (top) or active (middle) integrin "1 (red) and To-Pro-3 staining (blue). The relative fluorescence intensities of total "1 andactive "1 on cell surface were quantified by using ImageJ software (n ! 6, from 3 individual experiments); relative fluorescence intensity
was 1.0 for S3-5 mutant cells (bottom). All values are reported as the means " SE (error bars), as determined by Student’s t test. ***, P #0.001. Bars, 20 #m (C).

internalized proteins biotinylated. Internalization of integrin !5"1 was quantified by
the immunoprecipitation of streptavidin-agarose, followed by Western blot analysis. As
shown in Fig. 4A and B, the internalization of active "1 in WT cells was increased within
5 min and then decreased at 10 min, while it continued to increase even at 15 min
in S3-5 mutant cells, indicating delayed internalization in mutant cells. However,there were no significant differences in the internalizations of total !5 or "1 subunits
between WT and mutant cells. Consistently, the delayed internalization of active "1 inS3-5 mutant cells was also confirmed by a biotinylation-based capture enzyme-linked
immunosorbent assay (ELISA) (Fig. 4C). Taken together, these data indicate thatN-glycosylation serves as a switch to control the internalization of active, but notinactive, integrin !5"1.
Site 1 and site 2 N-glycosylation of the !-propeller domain on "5 mediates theregulation of cell migration. The data described above led us to investigate which
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FIG 4 Delayed internalization of active integrin "1 in S3-5 mutant MDA-MB-231 cells. (A) A biochemicalinternalization assay of active integrin "1, total integrin !5, and total integrin "1 was performed. The
indicated internalized integrins at the indicated times were immunoprecipitated by avidin-agarose andthen subjected to Western blotting for detection. The cell lysates were used as controls to show similar
expression levels of the indicated integrins between WT and S3-5 cells. (B) The percentages of internalized integrins were statistically calculated from the signal intensity of MesNa-resistant integrin at eachtime point relative to the value for the control groups (without MesNa), which was the total integrin on
the cell surface (n ! 3 individual experiments). (C) The proportions of internalized integrins of WT and
S3-5 mutant cells during the internalization period were also determined by a capture ELISA using
microtiter wells coated with anti-active integrin "1, anti-integrin !5, or anti-integrin "1 antibodies, as
described in Materials and Methods. The proportions of internalized integrins were statistically calculated(n ! 3 individual experiments). All values are reported as the means " SE (error bars), as determined byStudent’s t test. *, P # 0.05; **, P # 0.01; ***, P # 0.001.

N-glycosylation site(s) of integrin !5 was essential for regulation. We recently demonstrated that N-glycosylation of the calf domain on !5 (sites 10 to 14) (Fig. 5A) was
essential for its association with EGFR (15). This represented the first restoration of
N-glycosylation in both the calf and the remaining domains in S3-5 mutant cells.
However, as shown in Fig. 5B and C, restoration of N-glycosylation on the calf domain(sites 3 to 5 and 10 to 14) affected neither cell migration nor the expression of active"1 on the cell surface. Furthermore, deletion of the N-glycosylation sites on the calf
domain (sites 1 to 9) enhanced cell migration (Fig. 5B) and decreased the expression
level of active "1 on the cell surface (Fig. 5C), which was similar to the reaction in WT
cells and highlighted the importance of N-glycosylation on the "-propeller and thighdomains (sites 1 to 9) of integrin !5. Furthermore, the restoration of N-glycosylation on
the "-propeller domain (sites 1 to 5), but not that on the thigh domain (sites 3 to 9),
largely rescued the cell migration ability (Fig. 5B). Consistently, the increased expression
levels of active "1 on the cell surface of S3-5 cells was almost normalized in S1-5 mutant
cells compared with that in WT or S1-9 mutant cells (Fig. 5C). Moreover, importantly, thedelayed internalization of active "1 (Fig. 5D) and the enhanced expression levels of
p-FAK in S3-5 cells were also reversed in S1-5 but not S3-9 cells (Fig. 5E), indicating thatsite 1 and site 2 N-glycosylation on !5 was most important for active "1-mediatedappropriate cell adhesion for migration. Of note, as shown in Fig. S1B in the supplemental material, all the mutant cells exhibited almost the same expression levels of
integrin !5 on the cell surface compared with the WT ones, further suggesting that the
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FIG 5 Importance of N-glycosylation on the "-propeller domain of !5 for cell migration and !5–syndcan-4 complex formation. (A)Schematic diagram of potential N-glycosylation sites on the WT and mutational !5 subunits (S3-5; S1-9; S3-5,10-14; S1-5; and S3-9). (B)Comparison of the cell migration abilities among WT and mutant cells with restored S1-9; S3-5,10-14; S1-5; or S3-9 !5. The stable cell lines
were established as described in Materials and Methods. The cell migration abilities were analyzed (n ! 3 individual experiments). (C) The
expression levels of active and total "1 were analyzed by flow cytometry. (D) The internalization of active integrin "1 in WT, S3-5, S1-5,
and S3-9 mutant cells was determined by a capture ELISA. The proportions of internalized active "1 were statistically calculated (n ! 3
individual experiments). (E, top) After culture on FN-coated dishes for 2 days, the indicated MDA-MB-231 cells were lysed and immunoblottedwith the indicated antibodies. (Bottom) Relative ratios (p-FAK versus FAK) (n ! 3 individual experiments); the relative ratio was 1.0 for S3-5 mutant
cells. (F) The surface expression levels of syndecan-4 on WT and S3-5, S1-5, and S3-9 mutant cells were analyzed by flow cytometry. (G, top) The
indicated MDA-MB-231 cell extracts were immunoprecipitated with anti-GFP–agarose followed by anti-syndecan-4 (Syn-4), active "1, total "1,
and !5 antibodies for Western blotting. (Bottom) Whole-cell extracts were also immunoblotted with the indicated antibodies. All values arereported as the means " SE (error bars), as determined by Student’s t test; *, P # 0.05; ***, P # 0.001. Bar, 350 #m (B).

remaining N-glycosylation sites, with the noted exception of sites 3 to 5 of integrin !5,
had no effect on its expression.
Next, we explored the underlying mechanisms of the N-glycosylation of integrin!5-mediated activation and migration. Considering that integrin-mediated cellular
events are thought to be regulated not only by a conformational change caused bytheir activations but also by their cross talk with other membrane proteins (22), we
further investigated !5"1-mediated complex formation. Here, we chose one representative membrane protein, syndecan-4, a transmembrane heparan sulfate proteoglycan
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Downloaded from http://mcb.asm.org/ on April 16, 2017 by Tohoku Medical and Pharmaceutical Universitythat can interact with integrin !5"1 (23) and has been reported to be a control protein
for !5"1 trafficking (24). As shown in Fig. 5F, WT and S3-5, S1-5, and S3-9 mutant cells
exhibited same expression levels of syndecan-4 on the cell surface as determined byflow cytometry, suggesting that N-glycosylation on !5 had no effect on the expressionof syndecan-4. However, immunoprecipitates with anti-GFP–agarose showed that theinteraction between syndecan-4 and !5 was significantly decreased in S3-5 cells (Fig.
5G). Importantly, the decreased association between !5 and syndecan-4 could bereversed in S1-5 but not S3-9 cells (Fig. 5G), which indicated that site 1 and site 2
N-glycosylation on !5 was required for its cooperation with syndecan-4. In addition,these four cell lines exhibited almost the same association abilities between !5 andactive or total "1 (Fig. 5G), which further suggested that N-glycosylation on !5 had no
significant effect on the heterodimerization of !5"1. It is notable that the expression
levels of active "1 on the cell surface were significantly different among these mutants(Fig. 5C), but the total expression levels of active "1 were almost the same, as shown in Fig.
3B and 5G. In fact, based on data from previous reports, the active "1 integrins werepredominantly cytoplasmic (19, 25), as also shown in Fig. 3C. Taken together, these results
clearly showed that site 1 and site 2 N-glycosylation on the "-propeller domain of !5 plays
a crucial role in regulating appropriate cell adhesion for migration by controlling theinternalization of its active form and complex formation (e.g., syndecan-4), which
demonstrates a novel mechanism for integrin !5-mediated cell migration.
DISCUSSION
Previously, our group identified specific N-glycosylation on integrin !5 that plays
crucial roles in several biological functions. In detail, we first underscored the importance of three N-glycosylation sites (sites 3 to 5 and particularly site 5) on the"-propeller domain of !5 in its heterodimeric formation with "1 (12). Also, we recently
identified that N-glycosylation on the calf domain, at sites 10 to 14, was essential for the!5-mediated inhibitory effect on EGFR signaling and cell proliferation (15). Here, we
expanded our understanding of the N-glycosylation of !5 and found that site 1 and site
2 N-glycosylation on the "-propeller domain plays a key role in driving integrin !5"1
dynamics and cell migration, which is a central role for integrin. Mechanistically, site 1
and site 2 N-glycosylation regulates complex formation with syndecan-4, which probably enhances active integrin !5"1 internalization and promotes FA disassembly toprovide modest cell-substrate adhesiveness for cell migration (Fig. 6). Taken together,
these findings support the idea that individual integrin !5 N-glycosylation differentiallyfunctions as a molecular switch to regulate the biological functions of !5"1.
The N-glycosylation of !5 is widely believed to play important roles in cell migration
through several experimental methods, including the remodeling of N-glycans bytreatment with N-glycanase F or an N-linked oligosaccharide-processing inhibitor andmanipulation of the expression levels of some important glycosyltransferases (14).
Apparently, the use of these models to comprehensively explore the functions of !5
N-glycosylation and its underlying mechanisms is insufficient, because those studies
lacked strict control. In the present study, we clearly demonstrated that the removal ofthe majority of !5 N-glycosylation, except for that on sites 3 to 5, affected neither !5"1
heterodimer formation nor cell surface expression or cell spreading, but it dramaticallysuppressed cell migration in several cancer cell lines, including MDA-MB-231, HeLa, andU-251MG cells. These data suggest that !5 N-glycosylation, with the exception of sites
3 to 5, is important for cell migration. These results seem contradictory compared tothose of our previous study, in which CHO-B2-S3-5 mutant cells exhibited cell spreading
and migration abilities on FN comparable to those of the WT (12). These differences
could be explained at least in part by the lack of !2,6-sialylation expression in the
CHO-B2 cell line (26), since !2,6-sialylation plays very important roles in integrin!5"1-mediated cell migration and other functions (27).
FAK is a key molecule in integrin-mediated cell adhesion and migration, and the
autophosphorylation of FAK at tyrosine 397 plays important roles not only in intracellular signaling but also in cell motility (28). Unexpectedly, the phosphorylated levels of
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FIG 6 Proposed model for the regulation of integrin !5"1-mediated cell migration by N-glycosylation. (Left)
Integrin !5"1 may form a complex with several receptors (e.g., syndecan-4), which may trigger active !5"1
internalization and the turnover of FA to provide the appropriate cell-substrate adhesiveness for efficient cell
migration. (Right) However, in the case of the !5 mutant lacking N-glycosylation on sites 1 and 2, the cells exhibit
increased active !5"1 expression on the cell surface and enhanced cell adhesion signaling as a result of its
decreased ability to associate with related proteins, which results in the suppression of cell migration. The present
study provides new insights into the concept that modest cell adhesion is best for cell migration (40). The solid
arrows indicate a normal signal pathway in integrin !5"1-mediated cell migration, while the dotted arrows indicatethat these events are aberrant.

FAK upon FN stimulation in S3-5 mutant cells were higher than those in WT cells.
Furthermore, actin stress fiber formation was also greatly enhanced in the mutant cells.
These data further support the notion that more cell-substrate adhesiveness, less cell
migration, and a modest amount of cell adhesion are important for cell migration (29).
In fact, cell migration is a strictly controlled dynamic process, which requires regulated
cycles of polarized cell-substrate attachment to form focal contact at the leading edgeof the cell with corresponding detachment at its trailing edge (30). Therefore, animbalance of attachment and detachment in this process may result in abnormal
adhesion (31). Such a cell adhesion phenotype is compatible with data from a previous
study in which a loss of the protein annexin A2 inhibited intestinal epithelial cell
migration while promoting enhanced cell matrix adhesion (21). Given that the conformational activation of integrins on the cell surface supports cell adhesion and spreading, whereas the transition of integrins toward an inactive bent conformation causes
cell detachment and rounding (32, 33), it is possible that the increased cell adhesion
ability is due to an aberrant activation of integrin. In agreement with this hypothesis,
our flow cytometric and biotinylation analyses showed that S3-5 mutant cells highly
expressed the active form of !5"1 on the cell surface. Similarly, Park et al. previously
reported that aberrant activation of integrin !4"7 suppressed lymphocyte migration
and underscored the importance of a proper balance in the adhesion and de-adhesionof integrin (34). The enhanced expression of active "1 on the cell surface might havebeen due to different trafficking events, since the internalization of the active form of!5"1 but not the total version was significantly delayed in S3-5 cells compared withthat in WT cells. These results suggested that !5 N-glycosylation may have a significant
effect on the expression and internalization of active !5"1. Our finding is analogous
to those of a previous report indicating that the downregulation of the protein
neuropilin-1 (Nrp1) resulted in a delay in active integrin !5"1 internalization but notthat of the total version (35). Therefore, it is reasonable to consider that the internalization of active and inactive integrins may occur through distinct molecular mechanisms. Actually, inactive and active integrins !5"1 undergo different trafficking pathMay 2017 Volume 37 Issue 9 e00558-16

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ways: the Rab4-dependent short loop for inactive integrin !1 (19) and the Rab11dependent long loop used by active "5!1 (20). Thus, further studies elucidating thefunctions of "5 N-glycosylation in regulating its degradation and recycling are required
for a comprehensive understanding of how N-glycosylation coordinates the trafficking
of integrin "5!1 during cell migration.
Integrin "5!1 internalization is the first step in its trafficking. It is required for thedisassembly regulation of FA and plays a key role in efficient cell migration (34). However,the current mechanism for integrin "5!1 internalization remains largely unclear, since moststudies focus mainly on intracellular trafficking regulation without extracellular information(7, 9). Here, we found the site 1 and site 2 N-glycosylation on "5 could regulate active "5!1
internalization, which may broaden our understanding of the functions of the extracellulardomains in "5!1 trafficking. In addition, the internalization of "5!1 is also related to theassistance of other transmembrane proteins such as the FN receptor syndecan-4 (24, 36),
myelin-associated glycoprotein (37), EGFR (38), and Nrp1 (35). As a model, here we focusedon syndecan-4 and found that its associative ability with "5!1 was dependent on "5
N-glycosylation. In particular, site 1 and site 2 N-glycosylation of "5 was most importantfor interactions and cell migration. Of course, we could not exclude the effects of cell
migration on the remaining N-glycosylation sites. In fact, the restoration of site 1 andsite 2 N-glycosylation in the S3-5 mutant efficiently rescued the expression of the active
form of !1 to a level similar to that in WT cells, whereas the cell migration ability was
only partially rescued. This is a possible reason why N-glycosylation on the calf domain(sites 10 to 14) of "5 is necessary for EGFR-"5 complex formation, as previously
reported (15), which may also play important roles in "5!1 trafficking and cell migration, as discussed above. Moreover, it is of note that the internalization ratio of active!1 in S3-9 cells was slightly higher than that in S3-5 cells to some extent but was not
significant (Fig. 5E), and also, there was a weak interaction between "5 and syndecan-4
in S3-9 mutant cells (Fig. 5G), which may indicate that N-glycosylation on the thighdomain of "5 also has some effects on the internalization of active !1. Considering theroles of the bulky glycocalyx of the ECM in integrin-mediated tumor phenotypes (39),
it is tempting to speculate that individual N-glycosylation on integrin may exhibit asuitable physical or specific structure that functions as a nexus for integrin-mediatedcomplex formation to integrate both the ECM and cytokines in the microenvironment
surrounding tumors.
In summary, we have delineated the mechanism for N-glycosylation on the !-propeller
domain of "5 as a controller that coordinates cell-FN adhesion and internalization
dynamics of "5!1 for cell migration. These findings will improve our understanding of
how cell-substrate adhesiveness is regulated and the importance of a modest level ofcell adhesion for cell migration.
MATERIALS AND METHODS
Antibodies and reagents. The experiments were performed by using the following antibodies:monoclonal antibodies (MAbs) against integrin "5, integrin !1, integrin "6, p-FAK, and FAK (BDBiosciences, San Jose, CA); MAbs against human "5!1 (HA5), active integrin !1 (HUTS-4), integrin "3, andintegrin !4 and rabbit polyclonal antibody against integrin "5 (Millipore, Billerica, MA); mouse MAb to
syndecan-4 (Santa Cruz Biotechnology, Santa Cruz, CA); antibody against integrin "V (BioLegend, San
Diego, CA); MAb against the human integrin !1 subunit (P5D2) (Developmental Studies Hybridoma Bank,
University of Iowa, IA); MAb against "-tubulin (Sigma-Aldrich, St. Louis, MO); and Alexa Fluor 647– or
546 – goat anti-mouse IgG and Alexa Fluor 546-phalloidin (Thermo Fisher Scientific, Waltham, MA). The
peroxidase-conjugated goat antibodies against mouse and rabbit IgG were obtained from Chemicon
and Cell Signaling Technology (Danvers, MA), respectively. 2-Mercaptoethanesulfonic acid sodium salt
(MesNa), iodoacetamide, o-Phenylenediamine dihydrochloride (OPD), and FN were obtained from Sigma;
control mouse IgG1 was obtained from Tonbo Biosciences (San Diego, CA); and sulfo-NHS-SS biotin(sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate), streptavidin-conjugated horseradish peroxidase, and To-Pro-3 were obtained from Thermo Fisher Scientific. PNGase F, agarose-conjugatedanti-GFP antibody (RQ2), streptavidin-conjugated agarose, and Ab-Capture Extra beads were obtained
from New England BioLabs, Medical & Biological Laboratories Co. Ltd. (Nagoya, Japan), Millipore, andProteNova Co. Ltd. (Kagawa, Japan), respectively.
Cell lines and cell culture. The 293T and HeLa cell lines were provided by the RIKEN cell bank
(Tokyo, Japan). The MDA-MB-231 cell lines were purchased from the American Type Culture Collection(Manassas, VA). The U-251MG cell line was a gift from Jun Nakayama (Shinshu University Graduate School
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Downloaded from http://mcb.asm.org/ on April 16, 2017 by Tohoku Medical and Pharmaceutical Universityof Medicine, Japan). Integrin !5-KO-MDA-MB-231, !5-KO-HeLa, and !5-KO-U-251MG cells were previously established in our laboratory (15). The stable cell lines used in this study were established as
mentioned below. All cell lines were maintained at 37°C in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) under a humidified atmosphere containing 5% CO2,
except for virus production.
Integrin !5 expression vectors. The pENTR-D-Topo vectors of GFP and GFP-tagged integrin !5 withaltered N-glycosylation sites (the WT and the S3-5; S1-9; S3-5,10-14; and S1-5 mutants) were previously
established in our laboratory (12, 15). The mutation vector S3-9 was constructed by using an in-fusion kit(TaKaRa Bio) according to the manufacturer’s instructions and then cloned into the CSII-EF-Rfa vector byusing a Gateway cloning system kit (Thermo Fisher Scientific) to acquire all lentivirus expression vectors.
Virus production, infection, and stable cell line construction. Virus production and infection were
performed as described previously (15). In brief, CSII-EF-Rfa-based lentivirus vectors were cotransfectedwith pCAG-HIVgp and pCMV-VSV-G-RSV-Rev into 293T cells. After transfection for 48 h, the lentivirus
supernatants were collected. The indicated integrin !5 KO cells were infected with the resultant viralsupernatant for 72 h, and GFP-positive cells were then sorted 3 times by using a FACSAria II instrument.
Stable cell lines were used in subsequent studies.
Cell spreading and cell adhesion response assays. Six-well plates were coated with FN (10 "g/ml)
in phosphate-buffered saline (PBS) overnight at 4°C and then blocked with 1% bovine serum albumin(BSA) in DMEM for 1 h at 37°C. The indicated cells were detached and suspended in assay medium (50mM HEPES and 0.1% BSA in serum-free DMEM [pH 7.4]) for 40 min at 3 ! 104 cells/ml. After a 20-min
incubation, nonadherent cells were gently removed by washing with PBS, attached cells were fixed with
4% paraformaldehyde (PFA) in PBS, and photos were then taken by phase-contrast microscopy.
To assay the cell adhesion response upon FN treatment, the indicated cells were also detached andsuspended in the assay medium for 40 min at 1 ! 106 cells/ml. After replating onto FN-coated 6-wellplates for 0, 15, 30, and 45 min, nonadherent cells were gently removed by washing with PBS, and theattached cells were collected, lysed, and then analyzed by WB, as described below.
Video microscope. A glass-bottom dish (Asahi Techno Glass, Japan) was precoated with 10 "g/ml
FN at 4°C overnight and then blocked with 1% BSA in DMEM for 1 h at 37°C. A 2-ml aliquot of the cellsuspension (2.5 ! 104 cells/ml) in serum-free DMEM was added to each glass-bottom dish. Afterincubation for 1 h to allow cells to adhere to FN, the serum-free medium was gently replaced with 2 ml
growth medium, followed by mounting with 1 ml glycerol. The cells were then monitored, and theimages and videos were acquired by using inverted microscopes (Axio Observer.D1; Carl Zeiss) every 10
min at 37°C with 5% CO2 in a heated chamber with temperature and CO2 control (Onpu-4 & CO2; Air
Brown, Japan). Cell motility was analyzed in roughly 12 cells by measuring the x and y coordinates oftracked cells over 12 h. The directionality (ratio of the Euclidean distance versus accumulated distance,
0 # values # 1) parameters, displacement (micrometers), and the manual tracking plug-in velocity(micrometers per minute) were analyzed by using Chemotaxis and Migration Tool software (version 2.0).
Wound healing assay. A confluent layer of cells cultured on an FN-coated (10 "g/ml) 6-well plate
was starved in serum-free medium for 12 h and then scraped by using a p200 pipette tip. Serum-free
medium was replaced with 3% FBS-containing DMEM. Wounded areas were photographed under a lightmicroscope at 0 and 24 h for MDA-MB-231 cells, at 36 h for HeLa cells, and at 48 h for U-251MG cells.
Wound closure was measured by observing the distances between the sides of the wound at theindicated times.
Cell migration assay. Each Transwell (BD BioCoat control 8.0-"m-pore-size inserts) was coated only
on the bottom side with 10 "g/ml FN at 4°C overnight. Cells were prestarved in serum-free medium for
12 h, trypsinized, and suspended in complete DMEM. The suspended cells were centrifuged, and thesupernatants were removed. The resultant cell pellets were resuspended with assay medium (0.1% BSA
in DMEM containing 3% FBS) and diluted to 1 ! 105 cells/ml, and the cell viabilities were confirmed bytrypan blue staining. Aliquots of 500 "l of the cell suspension were added to each FN-coated Transwell,followed by incubation at 37°C for 5 h for HeLa cells and for 4 h for MDA-MB-231 or U-251MG cells. Afterincubation, cells on the upper side were gently removed by scraping with a cotton swab. The membranes
in the Transwells were fixed with 4% PFA for 30 min, followed by staining with 0.5% crystal violet for 4
h. Cells that had migrated to the lower side were counted by using a phase-contrast microscope.
Western blot and immunoprecipitation analyses. For WB, the indicated cells were washed with
ice-cold PBS and then lysed in the cell lysate (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100)with protease and phosphatase inhibitors (Nacalai Tesque, Kyoto, Japan) for 30 min. After centrifugation,
the supernatant was collected, and protein concentrations were determined by using a bicinchoninicacid (BCA) protein assay kit (Pierce, Rockford, IL). The protein samples were resolved by nonreducing
SDS-PAGE for !5 and active $1 or reducing SDS-PAGE for other proteins. After electrophoresis, theproteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore) and detectedwith the indicated primary and secondary antibodies by using the Immobilon Western chemiluminescenthorseradish peroxidase (HRP) substrate (Millipore), according to the manufacturer’s instructions. For IP,
cells were lysed in 0.1% Triton–Tris-buffered saline (TBS) buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl)with a protease inhibitor and then centrifuged. The supernatants were immunoprecipitated with
anti-GFP–agarose for 1 h at 4°C with rotation, and the immunoprecipitates were subjected to SDS-PAGE.
PNGase F treatment. The indicated cell lysates were treated with PNGase F at 37°C for 1 h according
to the manufacturer’s instructions or not treated and then subjected to SDS-PAGE with the indicatedantibodies.
Flow cytometric analysis. The indicated semiconfluent cells were detached from 10-cm culture
dishes and subsequently stained with either mouse IgG or primary antibodies for 1 h on ice, followed by
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incubation with Alexa Fluor 647– goat anti-mouse IgG for 1 h. During incubation, the cells were mixed
gently every 10 min by flicking. After incubation, cells were washed and then analyzed by using a
FACSCalibur flow cytometer (BD Biosciences).
Cell surface biotinylation. Cells were gently washed twice with PBS and then incubated with
ice-cold PBS containing 0.2 mg/ml sulfo-NHS-SS biotin for 1 h at 4°C. After incubation, cells were washed
3 times with ice-cold PBS, and the cells were harvested by using lysis buffer. A total of 200 !g of thebiotinylated protein was precipitated with streptavidin-conjugated agarose (20 !l) for another 1 h at 4°C
with rotation and then subjected to SDS-PAGE as described above.
Immunofluorescence. Cells were plated onto FN-coated glass coverslips (MatTek Corporation,
Ashland, MA) for 48 h, washed with PBS, fixed with 4% PFA, and blocked with PBS– 0.1% Triton
X-100 –10% BSA. Antibodies against p-FAK, active "1, or P5D2 were used, followed by incubation with
anti-mouse Alexa Fluor 546-conjugated secondary antibodies and Alexa Fluor 546-phalloidin. Finally, thecells were incubated with To-Pro-3 for 30 min. The confocal images were also acquired by using a
60!/1.35-numerical-aperture (NA) oil immersion objective lens (FV1000; Olympus, Tokyo, Japan). The
fluorescence intensities were quantified by using ImageJ software.
Capture ELISA. Corning (Corning, NY) 96-well enzyme immunoassay (EIA)/radioimmunoassay (RIA)
plates were coated with 5 !g/ml the appropriate anti-integrin antibodies in 0.05 M Na2CO3 (pH 9.6) at
4°C overnight and then blocked in PBS containing 0.05% Tween 20 (PBS-T) with 5% BSA for 1 h at room
temperature. The indicated integrins were captured by incubation of 50 !l (about 80 !g) of the cell lysate
overnight at 4°C. Unbound material was removed by extensive washing with PBS-T, and wells wereincubated with streptavidin-conjugated horseradish peroxidase in PBS-T containing 1% BSA for 1 h at
4°C. Following further washing, biotinylated integrins were detected by a chromogenic reaction with
OPD for 20 min, and the absorbances at 450 nm were measured.
Biotinylation-based integrin internalization assay. Cells grown on FN-coated 10-cm dishes wereserum starved for 1 h prior to the assay and washed with ice-cold PBS, and surface proteins were
biotinylated with 0.2 mg/ml sulfo-NHS-SS biotin in PBS for 1 h, followed by washing in TBS andplacement in a cold room. For internalization, cells were then incubated in prewarmed complete DMEM
at 37°C for 0, 2.5, 5, 10, and 15 min, whereas the control groups (without MesNa) remained on ice. Surface
biotin was then stripped from the cells with 10 min of incubation in 50 mM MesNa–TBS (pH 8.6) twice,followed by washing and quenching of MesNa with 20 mM iodoacetamide in TBS for 10 min. Cells of thecontrol group were not subjected to surface reduction (without MesNa) in order to obtain total
surface-labeled integrin. After quenching, the cells were lysed, precipitated with streptavidin-conjugatedagarose, and subjected to WB or a capture ELISA.
Statistical analysis. Results are reported as the means " standard errors (SE). Statistical analyses
were performed by using Student’s t test and GraphPad Prism version 5. Statistical significance wasdefined as a P value of #0.05.

SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/
MCB.00558-16.
SUPPLEMENTAL FILE 1, PDF file, 0.8 MB.
ACKNOWLEDGMENTS
Q.H. performed all the experiments with the help of T.I., S.H., Y.W., and T.F. Q.H. and
T.I. constructed the virus expression and the #5 knockout vectors. Q.H., T.F., and S.H. did
the cell sorting experiments and constructed the related stable cell lines. J.G. designed
the experiment. Q.H. and J.G. analyzed the data, prepared the figures, and wrote themanuscript. All authors discussed the results and commented on the manuscript.
We declare no competing financial interests.
This work was partially supported by grants-in-aid for scientific research (15H04354
to J.G. and 24570169 to T.I.) and for Challenging Exploratory Research (15K14408 to J.G.)
from the Japan Society for the Promotion of Science, a grant from the National Natural
Science Foundation of China (no. 31670807), and a Strategic Research Foundation
grant-aided project for private universities from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan.

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Molecular and Cellular Biology

Biochimica et Biophysica Acta 1861 (2017) 1036–1045

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen

Alteration of N-glycan expression proﬁle and glycan pattern ofglycoproteins in human hepatoma cells after HCV infection
Tian Xiang a, Ganglong Yang b, Xiaoyu Liu a, Yidan Zhou c, Zhongxiao Fu a, Fangfang Lu a, Jianguo Gu d,
Naoyuki Taniguchi e, Zengqi Tan b, Xi Chen f, Yan Xie a, Feng Guan b,⁎, Xiao-Lian Zhang a,⁎aState Key Laboratory of Virology. Hubei province Key Laboratory of Allergy and Immune-related diseases, Medical Research Institute, Department of Immunology of Wuhan University School of
Basic Medical Sciences, Wuhan 430071, Chinab
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Chinac
University of Illinois at Urbana-Champaign, School of Molecular and Cellular Biology, Department of Microbiology, IL 61801, USAdDivision of Regulatory Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aobaku, Sendai, Miyagi 981-8558, Japane
Systems Glycobiology Group, Global Research Cluster, RIKEN and RIKEN-Max Planck Joint Research Center, 2-1 Hirosawa, Wako, Saitama 351-0198, Japanf
Wuhan Institute of Biotechnology, Medical Research Institute of Wuhan University, Wuhan 430071, Chinaa r t i c l e

i n f oArticle history:
Received 10 November 2016
Received in revised form 9 February 2017
Accepted 11 February 2017
Available online 14 February 2017
Keywords:
Glycosyltransferase expression analysis
Hepatitis C virus (HCV)
Lectin microarray
Mass spectrometry
N-glycanα1,6-fucosyltransferase 8 (FUT8)

a b s t r a c t
Background: Hepatitis C virus (HCV) infection causes chronic liver diseases, liver ﬁbrosis and even hepatocellular
carcinoma (HCC). However little is known about any information of N-glycan pattern in human liver cell after
HCV infection.
Methods: The altered proﬁles of N-glycans in HCV-infected Huh7.5.1 cell were analyzed by using mass spectrometry. Then, lectin microarray, lectin pull-down assay, reverse transcription-quantitative real time PCR (RT-qPCR)
and western-blotting were used to identify the altered N-glycosylated proteins and glycosyltransferases.
Results: Compared to uninfected cells, signiﬁcantly elevated levels of fucosylated, sialylated and complex N-glycans were found in HCV infected cells. Furthermore, Lens culinaris agglutinin (LCA)-binding glycoconjugateswere increased most. Then, the LCA-agarose was used to precipitate the speciﬁc glycosylated proteins and identify that fucosylated modiﬁed annexin A2 (ANXA2) and heat shock protein 90 beta family member 1 (HSP90B1)
was greatly increased in HCV-infected cells. However, the total ANXA2 and HSP90B1 protein levels remained unchanged. Additionally, we screened the mRNA expressions of 47 types of different glycosyltransferases and foundthat α1,6-fucosyltransferase 8 (FUT8) was the most up-regulated and contributed to strengthen the LCA binding
capability to fucosylated modiﬁed ANXA2 and HSP90B1 after HCV infection.
Conclusions: HCV infection caused the altered N-glycans proﬁles, increased expressions of FUT8, fucosylated
ANXA2 and HSP90B1 as well as enhanced LCA binding to Huh7.5.1.
General signiﬁcance: Our results may lay the foundation for clarifying the role of N-glycans and facilitate the development of novel diagnostic biomarkers and therapeutic targets based on the increased FUT8, fucosylated
ANXA2 and HSP90B1 after HCV infection.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction
Hepatitis C virus (HCV) is an enveloped, positive-sense RNA virusthat is a major cause of chronic liver disease and infects nearly 185 million people worldwide. Approximately 350,000 to 500,000 people die
Abbreviations: ACA, Amaranthus caudatus agglutinin; AFP, alpha-fetoprotein; ANXA2,
annexin A2; ECA, Erythrina cristagalli agglutinin; FUT8, α1,6-fucosyltransferase 8; HCC,
hepatocellular carcinoma; HCV, Hepatitis C virus; HCVcc, HCV cell culture; HSP90B1,
heat shock protein 90 beta family member 1; LCA, Lens culinaris agglutinin; NS3,
nonstructural protein 3; PFN-1, Proﬁlin-1; POFUT1, Protein O-fucosyltransferase1.
⁎ Corresponding authors at: Department of Immunology, Wuhan University School of
Medicine, 115 Donghu Road, Wuhan, 430071, China.
E-mail addresses: fengguan@jiangnan.edu.cn (F. Guan), zhangxiaolian@whu.edu.cn(X.-L. Zhang).

http://dx.doi.org/10.1016/j.bbagen.2017.02.014
0304-4165/© 2017 Elsevier B.V. All rights reserved.

each year from hepatitis C-related liver diseases, according to the2014 WHO report [1,2]. In the case of HCV infection, pathological mechanisms have generally been well characterized from a biochemical andbiophysical perspective. However, little is known about the N-glycan
expression proﬁle and glycan pattern of glycoproteins of host cell proteins after HCV infection [3–6].
N-linked glycosylation is one of the most common types of posttranslational protein modiﬁcation [7]. The sugar composition of oligosaccharides and the number and size of branches in the sugar tree
vary widely [8,9]. As of yet, alterations of protein N-linked glycosylation
have been identiﬁed as biomarkers for the development of many diseases, including cancer [10–14]. Several studies have shown that changes in the β-1, 6-branched and bisected N-glycan expression wereassociated with replicative immortality, tissue invasion and metastasis

T. Xiang et al. / Biochimica et Biophysica Acta 1861 (2017) 1036–1045

of cancer cells [15,16]. The enzyme activities of glycosyltransferases andtheir gene expressions showed distinct differences between normal
cells and cancer cells, and certain glycosyltransferases were involved
in signal transduction processes related to epithelial–mesenchymaltransition (EMT), metastasis and therapeutics in cancer cells [17,18].
Sialyl lewis X epitope with α1,3 Fuc was markedly increased in advanced breast cancer patients [19]. Furthermore, increased α-1-3fucosylation of α-1-acid glycoprotein (AGP) and fucosylated haptoglobin were reported as novel biomarkers for pancreatic cancer [20,21].
In many cases of viral infected host cells, N-linked glycans are viral
receptors and/or co-receptors thought to be involved in cellular surface
attachment and entry of viruses [22]. The surface proteins of manyenveloped as well as non-enveloped human viruses recognize terminal
sugar chain moieties of glycan and select glycans for binding to speciﬁc
tissues and hosts [23–25]. For example, human inﬂuenza A virus infection is restricted at the level of virus entry in Lec1 cells, which lack complex N-linked glycosylation by a mutation in the Nacetylglucosaminyltransferase I (GnT1) gene, and thus cannot initiate
infection [26]. All viruses studied to date use host (speciﬁc cellular subtype)-encoded glycosyltransferases and glycosidases to add and remove sugar residues from their glycoproteins cotranslationally or
shortly after translation of the protein [27–29]. Thus, detection of theglycopattern of host cells and the proﬁling of N-linked glycans from isolated glycoproteins can reﬂect the progression of viral infection and are
beneﬁcial for the rapid identiﬁcation of abnormal protein glycosylation[26,30,31].
Human hepatoma cell line Huh7.5.1 is the only available cell line for
HCV infection in vitro currently. In this study, the alteration of N-linked
glycans after HCV infection of Huh7.5.1 was examined by MatrixAssisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF-MS). The altered glycoproteins were analyzed by lectin
microarrays and lectin pull-down analysis, which were further conﬁrmed by confocal microscopy and western blotting. The mRNA levelsof glycan-related glycosyltransferase genes were analyzed using multiple reverse transcription-quantitative real time PCR (RT-qPCR). The integrated strategy described here may provides valuable information toreveal the molecular mechanisms of HCV infection and facilitate the discovery of new diagnostic biomarkers and therapeutic targets.
2. Materials and methods
2.1. Cell culture and viral infection
Huh7.5.1 cells were cultured in complete Dulbecco's modiﬁed
Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) (HyClone
Laboratories, Logan, UT, USA). The HCV cell culture (HCVcc) infection
was performed according to our previous publications [32,33]. In brief,
Huh7.5.1 cells in 24-well plates were infected with HCVcc (MOI of ∼1;
0.8 × 106 copies/ml) at 37 °C for 4 h. The supernatants were discarded,
and the infected cells were washed twice with phosphate buffered saline (PBS) and incubated in DMEM containing 10% FBS for 72 h.
2.2. Total cellular protein extraction
Huh7.5.1 cells and HCVcc infected Huh7.5.1 cells (2 × 107, respectively) were lysed with Tissue Protein Extraction Reagent (T-PER)(Thermo Scientiﬁc; San Jose, CA) according to the manufacturer's instructions. The solution was centrifuged at 12000 g for 20 min. The supernatant of the total protein was stored at −80 °C. The concentrationof the protein was measured by a BCA kit (Beyotime, Shanghai, China).
2.3. N-linked glycans separation and derivation
The total proteins from Huh7.5.1 and HCV-infected cells were concentrated and desalted using a size-exclusion spin ultraﬁltration unit
(Amicon Ultra-0.5 10 KD, Millipore; Billerica, MA). The total proteins1037

were then denatured with 8 M urea, 10 mM DTT, and 10 mM IAM andcentrifuged at 14,000 g for 15 min. The denatured protein was modiﬁedwith sialic acid as previously described [18,34]. In brief, the denatured
proteins were desalted with water for three times and reconstituted
with 100 μl of 1 M acetohydrazide, 20 μl of 1 N HCl, and 20 μl of 2 M
EDC and incubated at room temperature (RT) for 4 h (every sialic acidreside will obtain additional 56.0676 mass). Next, the proteins were incubated with PNGase F (New England BioLabs; Ipswich, MA) at 37 °C
overnight to release N-glycans. The efﬂuents were collected andlyophilized.
2.4. Desalting of N-glycans
Desalting was performed as previously described using Sepharose
4B (Sigma-Aldrich) [35]. Sepharose 4B was washed with MW (methanol/H2O (1:1 v/v)) and BMW (1-butanol/methanol/H2O (5:1:1 v/v/v))
under centrifugation. Glycans were dissolved in 500 μl of BMW andadded to the microtube. After incubation for 45 min, N-glycans were
washed three times with BMW, eluted with MW and lyophilized.
2.5. Mass spectrometry
Alteration of N-glycans in total proteins was characterized by
MALDI-TOF/TOF-MS (UltraﬂeXtreme, Bruker Daltonics; Bremen, Germany). Lyophilized N-glycans were dissolved in 10 μl of MW, spotted
onto a MTP AnchorChip sample target and air-dried. Then 1 μl of
20 mg/ml DHB in MW was added to recrystallize the glycans. Samples
were evaluated on mass spectrometry (MS) in positive reﬂectronmode (ion source 1 voltage: 25 kV; ion source 2 voltage: 22.5 kV; lens
voltage: 7.5 kV; reﬂector voltage: 26.4 kV; reﬂector 2 voltage: 13.4 kV)
across the m/z range of 700 to 3500, with gating of ions below m/z
400. For these studies, each spot was measured using 5000 laser shotsin groups of 500 shots per sampling area of the spot. MS was calibrated
before being run, using a Peptide Calibration Standard II (external standard) from Bruker Daltonics (Part NO. 222570). The m/z data were analyzed and annotated using GlycoWorkbench software [36].
2.6. Lectin microarray analysis
Lectin microarray analysis was performed as previously described[17,18]. In brief, 40 commercial lectins from Vector Laboratories (Burlingame, CA) and Sigma-Aldrich were immobilized onto a microplate.
Total proteins (2 mg) from Huh7.5.1 cells and HCVcc infected
Huh7.5.1 cells were labelled with ﬂuorescent dye Cy3 (GE Healthcare;
Bucking-hamshire, UK) and about 10–15 μg protein was spotted foreach individual spot on the lectin microplate. The Mean Fluorescence
Intensity (MFI) at 570 nm was determined on a SpectraMax® i3x microplate reader (Molecular Devices, Sunnyvale, CA), and average backgrounds were removed. The lectin microarray data were normalized
as described previously [37], and the change of protein glycosylation
during HCV infection was evaluated by comparison of the data from
HCV infected cells versus Huh7.5.1 cells and then presented by using
MeV software (Version 4.6.0) [38].
2.7. Confocal microscopy
Huh7.5.1 cells were plated in confocal dishes (NEST Biological Technology Co., Ltd., Shanghai, China) at a density of 5 × 104 cells/ml. After
24 h, HCVcc (MOI of 1) was added to the Huh7.5.1 cells and incubatedat 37 °C for 4 h. The supernatants were discarded, and the cells were
washed twice with PBS and incubated in DMEM containing 10% FBS
for 72 h. Cells were then ﬁxed with Intracellular (IC) Fixation Buffer(eBioscience) for 30 min at 4 °C and then permeabilized with Permeabilization Buffer (1×, eBioscience) for 30 min at RT (25 °C). Biotin-labelled LCA, Erythrina cristagalli agglutinin (ECA) and Amaranthus
caudatus agglutinin (ACA) were incubated with permeabilized cells for1038

T. Xiang et al. / Biochimica et Biophysica Acta 1861 (2017) 1036–1045

1 h at RT. FITC-streptavidin was then used to detect the binding ability ofbiotin-lectin with cells and nuclei were stained with DAPI (100 ×,
Ribobio, Guangzhou, China) in 1 × PBS for 10 min at RT. The stained
cells were observed and analyzed using a Leica-LCS-SP8-STED confocal
microscope (Heidelberg, Germany).
2.8. Lectin pull-down assay
Lectin pull-down assays were performed according to previous publications [39,40]. Total cell lysates (2 mg of protein) were incubatedwith LCA-agarose (Vector Laboratories, Burlingame, CA) (40 μl) at 4 °C
for 16 h. The precipitated samples were washed with Glycoprotein Eluting Solution (Vector Laboratories, Burlingame, CA) and analyzed with
SDS-PAGE. The bands of proteins that were differently expressed between Huh7.5.1 cells and HCV-infected cells were extracted from the
SDS gel and prepared for identiﬁcation with LC-MS (TripleTOF5600+,SCIEX, USA).
2.9. In-gel digestion and LC-MS/MS analysis
Gel processing and in-gel digestion were performed as described in
the previous study [41]. In brief, gel slices were destained, then DTT(10 mM DTT/50 mM NH4HCO3), iodoacetamide and trypsin (0.025 μg/
μl trypsin/10 mM NH4HCO3) solution were used to reduce, alkylate
and digest respectively. After digestion, peptides were extracted fromthe gel cubes with 150 μl extraction buffer (60% acetonitrile/5% formicacid) and incubated for 15 min at RT in a shaker. Supernatant was transferred to a new tube and gel pieces were further extracted with 100 μl
extraction buffer. Prior to LC-MS, extracts were concentrated in a vacuum centrifuge and desalted with Millipore ZipTip C18.
Peptides were detected by RPLC-ESI-MS/MS which was carried out
on a hybrid quadrupole-TOF LC/MS/MS mass spectrometer (TripleTOF
5600 +, AB Sciex) equipped with a nanospray source. Peptides wereﬁrst loaded onto a C18 trap column (5 μm, 5 × 0.3 mm, Agilent Technologies) and then eluted into a C18 analytical column (75 μm × 150 mm,
3 μm particle size, 100 Å pore size, Eksigent). Mobile phase A (3% DMSO,
97% H2O, 0.1% formic acid) and mobile phase B (3% DMSO, 97% ACN,
0.1% formic acid) were used to establish a 30-min gradient, which comprised of: 0 min in 5% B, 15 min of 5–35% B, 1 min of 35–80% B, the gradient was maintained in 80% B for 5 min, followed by 0.1 min of 80–5%
B, and a ﬁnal step in 5% B for 8.9 min. A constant ﬂow rate was set at
300 nl/min. MS scans were conducted from 350 to 1500 amu, with a
250 ms time span. For MS/MS analysis, each scan cycle consisted of
one full-scan mass spectrum (with m/z ranging from 350 to 1500 andcharge states from 2 to 5) followed by 40 MS/MS events. The thresholdcount was set to 120 to activate MS/MS accumulation and former target
ion exclusion was set for 18 s.
Raw data from TripleTOF 5600 + were analyzed with ProteinPilot
Software. Data were searched against the UniProt human reference proteome database using the following parameters: Sample Type, Identiﬁcation; Cys Alkylation, Iodoacetamide; Digestion, Trypsin. Search Effort
was set to Rapid ID.

2.11. Transfection
The siRNA (small interfering RNA) oligonucleotides against FUT8(GenBank accession no: NM_178155.2) were designed by Invitrogen
BLOCK-iT™ RNAi Designer and synthesized by Invitrogen (Shanghai,
China). The siRNA sequence of FUT8 (si-FUT8) was 5″GGACUGCACAAUCGAUACATT-3″ (target position: 2606–2624), and siNC (5″-CAGCCTAACGTGTTGTCCATTCGCT-3″) was used as a negative
control. Huh7.5.1 (5 × 104) cells were plated in six-well plates overnight
and then transiently transfected with siRNA after the cells reached 70–
90% conﬂuence using INTERFERin® in vitro siRNA transfection(Invitrogen, CA, USA) according to the manufacturer's protocol.
2.12. Flow cytometry (FCM)
Huh7.5.1 (5 × 104) cells were plated in six-well plates overnight andthen transiently transfected with siRNA. After 24 h, HCVcc (MOI of 1)
was added to the Huh7.5.1 cells and incubated at 37 °C for 4 h. The supernatants were discarded, and the infected cells were washed twicewith PBS and incubated in DMEM containing 10% FBS for 72 h. Cells
were then ﬁxed with Intracellular (IC) Fixation Buffer (eBioscience)for 30 min at 4 °C and then permeabilized with Permeabilization Buffer(1×, eBioscience) for 30 min at RT (25 °C). Biotin-labelled LCA was incubated with permeabilized cells for 1 h at RT. FITC-streptavidin (1:500;
Abcam; UK) was then used to detect the binding ability of lectin withcells and analyzed on a BD Accuri C6 Flow cytometer (BD Biosciences).
2.13. Western blotting analysis
HCVcc-infected cells and Huh7.5.1 cells (2 × 107, respectively) werepre-transfected with or without FUT8-siRNA. Total proteins were extracted with T-PER Regent and then pulled down by LCA-agarose. Protein concentrations were quantiﬁed using the BCA Protein Assay Kit(Beyotime, China), after which 30 μg of each of the protein lysates was
loaded onto the SDS-PAGE gel. The electrophoresed proteins weretransferred to PVDF membranes (Millipore, USA). The membranes
were blocked in 5% nonfat milk and incubated with diluted nonstructural protein 3 (NS3), ANXA2, HSP90B1 and PFN-1 antibodies (1:3000;
Abcam; UK), followed by incubation with a horseradish peroxidase(HRP)-conjugated secondary antibody (1:5000; Santa Cruz; USA). βactin was used as a control (1:10,000; Abcam; UK).
2.14. Statistical analysis
Data are presented as the mean ± SD and were analyzed byGraphPad Prism V.5.00 software (GraphPad Software, San Diego CA,
USA). Differences between groups were tested by one-way ANOVA
followed by the Newman-Keuls post hoc test. Two-sided p-values
under 0.05 were considered statistically signiﬁcant (*, p b 0.05, **,p b 0.01, ***, p b 0.001).
3. Results
3.1. Altered N-glycan proﬁle in HCV infected Huh7.5.1 cells

2.10. Quantiﬁcation of HCV and glycosyltransferase mRNA expressions byreverse transcription-quantitative real time PCR (RT-qPCR)
The total RNA from HCV-infected cells and Huh7.5.1 cells was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA). First-strand
cDNA was synthesized from the total RNA using ReverTra Ace-α Firststrand cDNA Synthesis Kit (Toyobo, Osaka, Japan). The primers of HCV
and 47 glycosyltransferases genes are listed in Supplementary Table
S1. The relative mRNA expression of HCV and each of the 47 glycosyltransferases was quantiﬁed using real-time RT-PCR with the SYBRGreen real-time PCR Master Mix kit (Toyobo, Osaka, Japan). GAPDH
was used as the endogenous control [33].

QRT-PCR and western blotting were used to detect HCV RNA andNS3 protein levels in Huh7.5.1 cells (Fig. 1A). MALDI-TOF/TOF-MS proﬁles of total N-glycans in HCVcc-infected Huh7.5.1 cells and Huh7.5.1
cells were then compared to reveal abnormal N-glycosylation. Representative MALDI-TOF-MS spectra of N-glycans with signal-to-noise ratios N5 from total glycoproteins were annotated using
GlycoWorkbench software. MS proﬁles of N-glycans and proposed
structures are shown in Fig. 1B and C (Magniﬁed ﬁgures for Fig. 1B
and C are presented in supplementary materials as Figs. S2 and S3 respectively). In total, 23 N-glycans structures were proposed from theHCVcc-infected Huh7.5.1 cells and Huh7.5.1 cells, of which 21 N-glycans

T. Xiang et al. / Biochimica et Biophysica Acta 1861 (2017) 1036–1045

1039

Fig. 1. MALDI-TOF-MS spectra of N-glycans. (A) qRT-PCR and western blotting analysis were used to detect the relative HCV RNA expression and NS3 protein level post 72 h of HCV
infection in Huh7.5.1 cells. The N-glycans from the control Huh7.5.1 cells (B) and HCVcc-infected Huh7.5.1 (C) samples were separated, as described in M&M. The lyophilized Nglycans were resuspended in MW, and the mixture was spotted on a MTP Anchor Chip sample target and air-dried. MALDI-TOF-MS was performed in positive-ion mode. Peaks(signal-to-noise ratio N 5) were selected for relative proportion analysis, and m/z data were analyzed and annotated using GlycoWorkbench software. All analyses were performed atleast three times.

and 14 N-glycans were derived from HCVcc-infected Huh7.5.1 cells andHuh7.5.1 cells, respectively (Table 1). There were 12 common N-glycan
structures in both uninfected Huh7.5.1 cells and HCVcc-infected cells,
but they displayed different intensities. Two structures (Nos. 6 and 8)
were found only in Huh7.5.1 control cells, and 9 structures (Nos. 3, 9,
12, 14, 17, 18, 20, 21, and 23) were unique to HCVcc-infected cells(Table 1). The structures of identiﬁed glycans were annotated by the
MS/MS date and partial results are shown in the supplementary information (Fig. S1). Changes in the relative variation of N-glycans in HCV
infection are summarized in Table 2. The proportions of fucosylated(47.6% vs. 28.6%) and sialylated (57.1% vs. 42.9%) and complex glycan
type (71.4% vs. 42.9%) were higher in HCVcc-infected cells than in the
Huh7.5.1 control (Table 2). However, the proportions of oligomannose
and hybrid-type N-glycans were down-regulated in HCVcc-infected
Huh7.5.1 cells (Table 2).
3.2. Glycan patterns of glycoproteins in HCVcc-infected cells
Lectins are carbohydrate-binding proteins that recognize speciﬁc
glycans based on subtle differences in structure. In our study, the glycan

1040

T. Xiang et al. / Biochimica et Biophysica Acta 1861 (2017) 1036–1045

Table 1
Proposed structures and their molecular ions in MALDI spectra of N-glycans from Huh7.5.1 and HCVcc infected cell.a
Relative intensity
Experimental m/zNo.

Huh 7.5.1

Huh 7.5.1
HCVcc-Huh7.5.1

Calculated m/z

Glycan structure

HCVcc-Huh7.5.1

AverageCV%

AverageCV%11257.488

1257.529

1257.423

0.052

18

0.125

7

2

1419.543

1419.593

1419.476

0.122

3

0.148

9

3

1485.662

1485.534

ND

ND

0.024

9

4

1581.596

1581.652

1581.528

0.158

5

0.273

13

5

1743.646

1743.715

1743.581

0.490

17

0.892

16

6

1825.743

1825.634

0.028

54

ND

ND

7

1905.707

1905.634

1.000

0

1.000

0

8

1987.836

1987.687

0.024

38

ND

ND

2012.719

ND

ND

0.046

31

9

1905.771

2012.820

102067.776

2067.850

2067.687

0.040

6

0.111

5

11

2156.957

2156.971

2156.802

0.023

10

0.089

46

2174.942

2174.772

ND

ND

0.025

34

2358.024

2357.907

0.072

29

0.041

26

2376.036

2375.877

ND

ND

0.036

11

12
13

2358.09514152504.1172504.173

2503.965

0.128

26

0.088

8

16

2522.071

2522.1302521.935

0.009

22

0.073

26

17

2723.154

2723.040

ND

ND

0.023

13

18

2741.254

2741.009

ND

ND

0.015

42

2869.346

2869.098

0.006

25

0.071

15

2887.289

2887.067

ND

ND

0.045

39

3070.355

3070.203

ND

ND

0.009

30

3216.524

3216.261

0.003

4

0.042

11

3234.463

3234.230

ND

ND

0.019

33

19

2869.32920
21
22

3216.492

23aCV, coefﬁcient of variation. ND, not detected in the samples.

pattern of glycoproteins in HCVcc-infected Huh7.5.1 cells was further
conﬁrmed using lectin microarray, which consisted of 40 types of different lectins. Hierarchical clustering analysis (HCA) of all 40 lectins was
performed by MeV 4.6.0 (Fig. 2A). According to the results, 5 of 40
lectins (Table 3, shown in blue) in HCVcc-infected cells showed a statistically signiﬁcant MFI exceeding 1.5 times over control Huh7.5.1 cells.
Among these, Fucα1-6GlcNAc, Galβ1-4GlcNAc and Galβ1-3GalNAcαSer/Thr (T) were binding ligands for LCA, ECA and ACA, respectively,
with N1.6 times stronger binding signals than the control MFI (Fig. 2A,
B). In accordance with the results from MALDI-TOF/TOF-MS/MS analysis, lectin microarrays also identiﬁed that fucosylated glycans (LCA
binding) were elevated in HCVcc-infected Huh7.5.1 cells. We further
conﬁrmed the glycan proﬁles using FITC-LCA, -ECA and -ACA lectinbinding assays by confocal microscopy (Fig. 2C). These ﬁndings showedthat HCVcc-infected Huh7.5.1 cells had greatly increased ﬂuorescence
Table 2Different types of N-glycans in Huh7.5.1 and HCV infected Huh7.5.1 cells.
N-glycan types

Huh7.5.1

HCVcc-Huh7.5.1

Oligomannose-type
Hybrid-type
Complex-type
Biantennary
Tri- and tetra-antennary
Fucosylated
Sialylated

42.9%
14.2%
42.9%
28.5%
21.4%
28.6%
42.9%

28.6%
0%
71.4%
23.8%
52.3%
47.6%
57.1%

signal intensities of these three lectins and were in line with the resultsof lectin microarray analysis (Figs. 2A, B).
3.3. Validation of glycoproteins immunoprecipitated with LCA
Since fucosylated glycans were the most elevated in HCVcc-infected
Huh7.5.1 cells, we performed fucosylated glycan-binding lectin LCAagarose pull-down and extracted LCA-binding proteins for SDS-PAGE
analysis as indicated in Fig. 3A. Four differently expressed protein
bands between Huh7.5.1 cells and HCVcc-infected cells (Fig. 3A, indicated by the red arrow) underwent LC-MS/MS analysis. The data of identiﬁcation of each band by LC-MS/MS analysis are shown in the
Supplementary Table S2. In this Table S2, numbers 1 to 3 are
HSP90B1, ANXA2 and Proﬁlin-1 (PFN-1), respectively. These three proteins (HSP90B1, ANXA2 and PFN-1) have high protein score according
to LC-MS/MS analysis and their molecular weights (MWs) are also similar to the sizes of the corresponding bands which we cut and analyzed
from Fig. 3A. In addition, HSP90B1, ANXA2 and PFN-1 are pivotal oncogenic proteins involved in HBV/HCV infection and the progression of
HCC, thus having the potential to be used as diagnostic or prognostic
biomarkers [42–45]. Therefore, these three proteins were chosen toevaluate the LC-MS/MS results by western blotting. We observed thatthe total levels of these three proteins were not increased in HCVcc-infected cells compared to Huh7.5.1 cells (Fig. 3B), however the LCA-precipitated protein levels of HSP90B1 and ANXA2, but not PFN-1, wereincreased in HCVcc-infected cells (Fig. 3B). Thus, our results further

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Fig. 2. Variation in glycans recognized by lectin microarrays in Huh7.5.1 and HCVcc-infected Huh7.5.1 cells. (A) Variations in the expression of glycans was recognized by 40 different
lectins presented as a heatmap. (B) Representative fold changed histogram of glycans recognized by LCA, ECA and ACA between HCVcc-infected Huh7.5.1 cells and Huh7.5.1. Data arepresented as the mean ± SD. (C) Analysis of the altered expression of glycans evaluated by lectins using confocal microscopy. Signals from a merge image of FITC-conjugated lectins(green) and DAPI staining of nucleus (blue) in control samples (left) and HCVcc-infected samples (right) are shown (objective magniﬁcation 40×). All analyses were performed atleast three times.

support the notion that HCV infection enhances α1,6 fucosylation observed in the speciﬁc glycoproteins.
3.4. Variation of glycosyltransferase expression patterns in HCV-infected
Huh7.5.1 cells
To further identify which glycosyltransferases were changed after
HCV infection in Huh7.5.1 cells, the differences in the expression of glycosyltransferases between Huh7.5.1 control cells and HCVcc-infected
cells were investigated using RT-qPCR. 47 glycosyltransferases gene
(Table S1) belonging to 4 different families, including sialyltransferases(STs), galactosyltransferases (GalTs), N-acetylglucosaminyltransferases(GnTs) and fucosyltransferases (FUTs) were studied [46–48]. The
fucosylation-related gene Fut8 in HCVcc-infected cells reached thehighest level expression in comparison to the other 46 glycosyltransferase genes, and was 4 times higher than that of control cells (the data in
Fig. 3C represents the FUT glycosyltransferase families). FUT8 is the only
enzyme responsible for the α1,6-fucosylation of N-glycans, catalyzing
the transfer of fucose from GDP-L-fucose to the asparagine-linked Nacetylglucosamine [49]. Signiﬁcant changes of the FUT8 mRNA expression by RT-qPCR were consistent with results from mass spectrometry(Table 2), lectin microarray analysis (Fig. 2A and B), confocal microscopy (Fig. 2C), western blotting analysis (Fig. 3D) and FCM (Fig. 3E), whichindicated that the α1,6-fucosylation of N-glycans was enhanced in
Huh7.5.1 cells after HCV infection.

Next, we evaluated whether fucosylation modiﬁcations of ANXA2and HSP90B1 were mediated by FUT8. We determined that FUT8
siRNA signiﬁcantly suppressed α1,6 fucosylation in HCVcc-infected
cells compared with control siRNA from 90.6% to 72.4%, as evaluatedby LCA staining shown in Fig. 3E. Western blotting also showed that
FUT8 siRNA signiﬁcantly decreased LCA-precipitated protein levels of
ANXA2 and HSP90B1 in HCVcc-infected cells (Fig. 3F). These results
conﬁrmed that the elevated FUT8 expression in HCVcc-infected cells
led to increased fucosylation of ANXA2 and HSP90B1, which changed
the glycoconjugate and glycan pattern of liver cells during HCV
infection.
4. Discussion
In the present study, we analyzed the changes of N-linked glycan expression proﬁles after HCV infection in Huh7.5.1 cells using MALDI-TOF/
TOF-MS. We found that the levels of fucosylated, sialylated and complex
N-glycans were greatly enhanced (Table 2). Combined with the increased fucosylated glycans (LCA binding) of lectin array, our present
data suggest that increased fucosylated N-glycans might be, at least,
one of important factors involved in HCV infection of liver cells. In
Table 3, Galβ-GlcNAc/GalNAc-reactive lectin (ECA, MAL-I, ACA) signals
were also enhanced signiﬁcantly. The increase of these lectin signals
means the increase of asialo-glycans [50]. Elevated levels of both sialoand asialo-glycans suggest that branched N-glycans might be increased

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T. Xiang et al. / Biochimica et Biophysica Acta 1861 (2017) 1036–1045

Table 3Differences in glycan patterns between control and HCVcc-infected cells by lectin microarray analysis.
LectinAbbreviation

Carbohydrate speciﬁcity

Fold changed between HCVcc-Huh7.5.1 and Huh7.5.1

Lens culinaris agglutinin
Amaranthus caudatus agglutinin
Erythrina cristagalli agglutinin
Maackia amurensis I
Aleuria aurantia
Lotus tetragonolobus agglutinin
Phaseolus vulgaris
Arachis hypogaea
Ricinus communis
Elderberry
Ulex europaeus agglutinin I
Galanthus nivalis
Triticum aestivum
Solanum tuberosum
Phaseolus vulgaris
Narcissus pseudonarcissus
Bauhinia purpurea
Vicia villosa
Pisum sativum
Hippeastrum hybrid
Maackia amurensis II
Maclura pomifera
Eunoymus europaeus
Urtica dioica
Psophocarpus tetragonolobus
Wisteria ﬂoribunda
Phytolacca americanca
Artocarpus integliforia
G. simplicifolia
Soybean agglutinin
Dolichos biﬂorus
Griffonia simplicifolia
Griffonia simplicifolia
Hippeastrum hybrid amaryllis
Datura stramonium
Griffonia simplicifolia
Lycipersicon esculentum
Agaricus bisporus
Helix pomatia
Sophpra japonica

LCA
ACA
ECA
MAL-I
AAL
LTA
PHA-E
PNA
RCA 120
EBL
UEA-I
GSL-I
WGA
STL
PHA-L
NPA
BPL
VVA
PSA
HHL
MAL-II
MPL
EEL
UDA
PTL
WFA
PWM
ALA
GSL-I-B4
SBA
DBA
GSL-II
GSL-I-A4
HHA
DSA
GS-I
LEA
ABA
HPA
SJA

Fucα1-6GlcNAc, α-D-Man
Galβ1-3GalNAc
Galβ1-4GlcNAc
Galβ1-4GlcNAc
Fucα1-6GlcNAc, Fucα1-3(Galβ1–4)GlcNAc
Fucα1-3(Galβ1-4)GlcNAc, Fucα1-2 Galβ1-4GIcNAc
complex-type N-glycan
Galβ1-4 GalNAc
Galβ1-4GlcNAc, Gal
Neu5Acα6Gal/GalNAc
Fucα1-2 Galβ1-4 GlcNAc
αMan
Chitin oligomers, NeuAc
GlcNAcβ1-4 GlcNAc, Mixture Man5 to Man9
Tri/tetra-antennary complex-type N-glycan
Non-substituted α-1,6Man
Galβ1-3 GalNAc, GalNAc
GalNAcαMan, αGlc
High-Man, Mana1-3Man, Mana1-6Man
Neu5Acα(2-3)
Galβ3GalNAc
Galα3Gal
GlcNAcGalNAc, Gal
GalNAcβ1-4 GlcNAc, Galβ1-3(-6)GalNAc
Branched (LacNAc)nGalβ1-3GalNAc, GalNAc
α-Linked Gal
α-or β-terminal terminal GalNAc, GalNAcα1-3GalNAc
Blood group A antigen, GalNAcα1-3 GalNAc
Agalactosylated Tri/tetra-antennary glycans
α-Linked GalNAc
α-1,6Man(GlcNAcβ1-4)n, Galβ1-4GlcNAc
Melibio, α-D-Galactose
GlcNAc trimmers/tetramers
Galβ1-4 GalNAc
α-Linked terminal GalNAc
βGalNAc

1.861⁎⁎
1.697⁎⁎
1.670⁎⁎
1.615⁎
1.503⁎
1.364
1.250
1.249
1.221
1.216
1.201
1.196
1.178
1.174
1.150
1.133
1.057
1.023
1.018
0.992
0.986
0.985
0.969
0.958
0.938
0.932
0.915
0.907
0.906
0.891
0.859
0.838
0.811
0.808
0.746
0.745
0.743
0.675
0.649
0.624

⁎ p b 0.05.
⁎⁎ p b 0.01.

after HCV infection, which actually was supported by mass spectrometry analysis (increased ratios of Tri- and tetra-antennary N-glycans inTable 2). Roles of those branching N-glycans in HCV pathology remain
further study.
It has been reported that α1,6 fucosylation plays an important role in
TGF-β, EGF, HGF and T-cell receptor-mediated signaling in many physiological and pathological functions, including cancers and inﬂammatory bowel disease [51–53]. In addition, an enhanced degree of α1,6fucosylation has also been observed in many viral infections, including
hepatitis B virus (HBV) [41], herpes simplex virus (HSV) [54] andhuman T-cell leukemia virus [55]. For example, high FUT8 expression
was reported to be a good indicator of poor prognosis in HBV-related
HCC [41]. However, the relationship between FUT8 and HCV has notbeen reported. In our study, we demonstrated that FUT8 mRNA exhibited the highest induced expression among 47 types of different glycosyltransferase mRNAs after HCV infection. Knockdown of FUT8
signiﬁcantly decreased the enrichment of ANXA2 and HSP90B1 in
LCA-precipitated proteins (Fig. 3F). FUT8 expression is elevated in HCC
and is also critical for the regeneration of the liver by increasing thephosphorylation levels of AKT in response to epidermal growth factor(EGF) and hepatocyte growth factor (HGF) [56,57]. The interactions between FUT8 and ANXA2 or HSP90B1 might play important roles in HCVpromoted HCC progression.
ANXA2 and HSP90B1 were previously reported to be involved in
HBV-related HCC and may serve as biomarkers to predict HCC metastasis [42,58–60]. In particular, core fucosylation of ANXA2 has been

observed in a study on hepatic cancer cells and was observed to be related to the metastatic potential of hepatic cancer [61]. Combining the detection of serum ANXA2 and alpha-fetoprotein (AFP) substantially
improved the diagnostic efﬁciency (96.52%) and the negative predictive
value (96.61%) for HCC [58,59]. ANXA2 can form a tetrameric complex
with the S100A10 subunit and, in this manner, regulates the cell membrane cytoarchitecture, dynamics of endosomal membranes and structural organization in physiological conditions [62,63]. During HIV-1
infection, ANXA2 served as a cellular cofactor that supports viral infection of macrophage by binding with phosphatidylserine in the humancell membrane [64], or enhanced cytomegalovirus plaque formation inhuman foreskin ﬁbroblast [65]. Furthermore, the knockdown of
ANXA2 or treatment with ANXA2 antibodies signiﬁcantly increased
drug sensitivity and decreased proliferation and migration of cancer
cells by inhibiting the EGFR-dependent PI3K-AKT downstream pathways [66–68]. HCV NS5A protein activated PI3K-AKT signaling by enhancing sterol regulatory element-binding protein (SREBPs)
expression and facilitating the entry and replication of HCV in liver
cells [69–72]. Thus, we assume that HCV-induced FUT8 expression
might activate AKT signaling and enhance ANXA2 fucosylation to promote HCC progression or mediate drug-resistance. These will be needed
to further conﬁrm in our future study.
HSP90B1 is an endoplasmic reticulum (ER) heat shock protein 90
paralog that acts as a protein chaperone and plays an important role inmaintaining liver development and hepatocyte function [43]. Increased
expression of HSP90B1 by HBx-induced NF-κB activation feedback

T. Xiang et al. / Biochimica et Biophysica Acta 1861 (2017) 1036–1045

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Fig. 3. FUT8 modiﬁed ANXA2 and HSP90B1 in HCVcc-infected Huh7.5.1 cells. (A) LCA–agarose pull-down and SDS-PAGE analysis. Four protein bands differently expressed in LCAprecipitated samples (indicated by red arrow) were cut and extracted for MS analysis. (B) The protein levels of ANXA2, HSP90B1 and PFN-1 in total cells or LCA-precipitated proteins
were analyzed with western blotting. β-actin was used as endogenous control. (C) The mRNA expression of FUT glycosyltransferases families was analyzed with RT-qPCR in control
cells or HCVcc-infected cells. Data are presented as the mean ± SD. **, p b 0.01, ***, p b 0.001. (D) Huh7.5.1 cells and HCVcc-infected cells were pre-transfected with or without FUT8siRNAs. The protein expression of FUT8 was evaluated by western blotting analysis. (E) The relative glycosylated activity of FUT8 of the cells was evaluated by FITC-LCA staining andanalyzed by FCM. (F) Huh7.5.1 cells were pre-transfected with FUT8-siRNA and then either were or were not infected with HCVcc. The total proteins were extracted from control or
HCVcc-infected cells and then incubated with or without LCA-conjugated agarose. The protein levels of ANXA2 and HSP90B1 in total cells or LCA-precipitated proteins were analyzed
by western blotting. All analyses were performed at least three times.

enhances HBV production in HBV-transfected cells and a mouse model[60]. Moreover, the up-regulated HSP90B1 expression was observed in
HBV-infected patients and is associated with poor outcomes of HBV-related HCC [42,60]. In this study, we found that HCV infection induced an increase of fucosylated ANXA2 and HSP90B1, as demonstrated by LCApulldown and western blot analysis (Figs. 3A-E), but it did not have an effect on total protein expression of ANXA2 and HSP90B1. Kim et al. reported that HCV E2 protein increased surface expression of HSP90B1 in liver
cells, which resulted in inhibition of apoptosis and a maintenance of
prolonged infection states [73]. This phenomenon likely occurred because
various components of HCV virus are involved in regulating host cell function at different levels. However, we still need to further study which
component of HCV regulates FUT8 expression. Nonetheless, these results
indicate that in HCV infection, ANXA2 and HSP90B1 are fucosylated andthus are promising glycoprotein biomarkers that may predict HCV infection and HCV-associated HCC. In addition, increased binding ability of LCA
to HCVcc-infected HCC cells (Fig. 3E) could possibly be used to monitor
HCV infection in HCC.
Our results in Fig. 3C showed that protein O-fucosyltransferase 1(POFUT1) was also increased after HCV infection. POFUT1 can boost
the activation of Notch signaling through adding O-fucose to conserved
threonine or serine residues in the epidermal growth factor-like repeatsof Notch receptors and ligands [74]. POFUT1 overexpression in HCC wascorrelated with aggressive tumor behaviors and associated with poor
prognosis. It promoted cell proliferation, migration and invasion in hepatoma cells [75] and oral squamous cell carcinoma (OSCC)-derived celllines [76]. These indicate that POFUT1 may serve as a diagnostic marker
and a therapeutic target for some tumor. But the relationship between
POFUT1 and viral infection has not been reported, and molecular details
underlying this HCV-infection induced POFUT1 expression change andthe roles that this regulatory mechanism plays in HCV pathology needfurther research.

In summary, the information of N-glycan pattern and their glycosyltransferases which were simultaneously obtained in our study provide
insight into the relation between N-glycans and their glycoproteins in
the context of HCV infection. Furthermore, we identiﬁed that HCV enhances FUT8 expression and thus promotes the fucosylation of ANXA2and HSP90B1 in Huh7.5.1 cells. This observation may provide valuable
information for the discovery of new diagnostic biomarkers and therapeutic targets based on the increased FUT8, fucosylated ANXA2 and
HSP90B1 after HCV infection, and may lay the foundation for clarifying
the role of N-glycans in HCV infection and hepatitis C-related liverdiseases.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbagen.2017.02.014.

Conﬂict of interest
None.

Author contributions
Conceived and designed the experiments: XLZ. Performed the experiments: TX, GLY, XYL, YDZ, ZXF, FFL. Analyzed the data: TX, FG,
XLZ. Wrote the manuscript: TX, XLZ. Revised the manuscript: XLZ, TX,
JGG, NT, ZQT, XC, YX.
Transparency document
The Transparency document associated with this article can befound, in online version.

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T. Xiang et al. / Biochimica et Biophysica Acta 1861 (2017) 1036–1045

Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (21572173, 31370197 and 31221061), National Outstanding Youth Foundation of China (81025008), the Hubei
Province's Outstanding Medical Academic Leader Program (523276003), and Hubei Province Major Knowledge Innovation Project(Natural Science Foundation) (2016CFA062, 2016ACA150).
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Carbohydrate Polymers 170 (2017) 241–246

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Chitosan oligosaccharides inhibit epithelial cell migration throughblockade of N-acetylglucosaminyltransferase V and branched GlcNAc
structure
Qingsong Xu a,c,∗ , Wenjing Wang b , Chen Qu a , Jianguo Gu c , Heng Yin d , Zhihao Jia a ,Linsheng Song a , Yuguang Du e,∗∗aCollege of Fisheries and Life Science, Dalian Ocean University, Dalian 116023, China
Dalian Elite Analytical Instruments Company Limited, Dalian 116023, Chinac
Tohoku Medical and Pharmaceutical University, Sendai, Miyagi, 981-8558, Japand
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, ChinaeInstitute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, Chinab

a r t i c l e

i n f oArticle history:
Received 9 November 2016
Received in revised form 10 March 2017
Accepted 24 April 2017
Available online 25 April 2017
Keywords:
Chitosan oligosaccharides
N-Acetylglucosaminyltransferase V
Branched N-glycan
Cell migrationa b s t r a c t
Chitosan oligosaccharides (COS) have been shown to regulate various cellular and biological functions.
The aim of this study was to investigate the antimetastatic potency of COS and the underlying mechanism. Here, we established a stably N-acetylglucosaminyltransferase V (GnT-V)-overexpressed MCF10A
cell line. As expected, GnT-V overexpression greatly promoted cell migration in the transfectants byusing wound healing assay. However, the induction in the cell migration was significantly suppressedby an addition of COS. Curiously, COS inhibited the protein expression of GnT-V in a dose dependent
manner. Consistent with that, the reactivities with datura stramonium (DSA) and leuko-agglutinating
phytohemagglutinin (L4-PHA) lectins, which specifically recognize branched N-acetylglucosamine (GlcNAc) structure, were also suppressed by COS. Taken together, these results demonstrated COS inhibitedbreast epithelial cell migration through down-regulation of GnT-V and its products, branched N-glycans,indicating that COS may serve as a potential novel therapeutic candidate for the treatment of metastatic
breast cancer.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Chitin, a linear biopolymer comprising N-acetylglucosamine
residues, is the second most abundant polysaccharide in nature.
Nevertheless, its high molecular weight and low solubility seriously limited its practical application (Sayari et al., 2016). As theAbbreviations:COS,
chitosanoligosaccharides;
GnT-V,
NV;
DSA,
daturastramonium;
L4-PHA,
acetylglucosaminyltransferase
leuko-agglutinating phytohemagglutinin; GlcNAc, N-acetylglucosamine; GalT,
galactosyltransferase; GlcNAcT, N-acetylglucosaminyltransferase; DP, degree ofpolymerization; ATCC, American Tissue Culture Collection; SDS-PAGE, sodium
dodecyl sulfate polyacrylamide gel electrophoresis; FN, fibronetin; BSA, bovine
serum albumin; LLC, Lewis lung carcinoma; MMP-2, matrix metalloproteinase2; MMP-9, matrix metalloproteinase-9; MT1-MMP, membrane-type matrix
metalloproteinase-1.
∗ Corresponding author at: College of Fisheries and Life Science, Dalian Ocean
University, 52. Heishijiao Street, Shahekou District, Dalian 116023, China.
∗∗ Corresponding author.
E-mail addresses: xuqingsong@dlou.edu.cn (Q. Xu), ygdu@ipe.ac.cn (Y. Du).
http://dx.doi.org/10.1016/j.carbpol.2017.04.075
0144-8617/© 2017 Elsevier Ltd. All rights reserved.

degradation and deacetylation product of chitin, chitosan oligosaccharides (COS) have attracted more and more attentions recently,
because the latter are not only easily soluble in water (generally, themolecular weight of COS is 10 kDa or less) and free amino groups ind-glucosamine units, but also readily absorbed through the intestine, quickly getting into the bloodstream (Chae, Jang, & Nah, 2005).
Therefore, COS have many important biological activities, such as
immuno-stimulation, antimicrobial effect and antitumor activity(Zou et al., 2016). The antitumor activity of COS has been known
since 1986. Suzuki et al. demonstrated hexa-N-acetylchitohexaose
and chitohexaose had antitumor activity against sarcoma 180 solid
tumor (Suzuki et al., 1986). Zou et al. found that the antitumor
effects on S180 tumor-bearing mice could be observed in bothoral and intraperitoneal administration of COS (Zou et al., 2016),
which strongly suggest COS might be considered promising candidate as antitumor functional food or pharmaceutical adjuvant
in oncotherapy. Also, our previous studies showed that COS could
induce apoptosis of human hepatocellular carcinoma cells via upregulation of Bax (Xu et al., 2008), and inhibit breast cancer cellular

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Q. Xu et al. / Carbohydrate Polymers 170 (2017) 241–246

transplantation tumor angiogenesis through interference with thenitric oxide signaling pathway (Wu, Yao, Bai, Du, & Ma, 2010).
Recently, tumor metastasis and invasion has become a hot
research field attracting much attention. Metastasis is a sequential
process whereby cancer cells penetrate the basement membrane, invade blood/lymphatic vessels, survive in the vasculature
before extravasation to next sites, and finally adapt to newhost environments to proliferate and form metastases (Langley& Fidler, 2007). Alterations in glycan structures are associatedwith many physiological and pathological processes, including cell
migration, differentiation and tumor invasion (Zhao et al., 2008). NAcetylglucosaminyltransferase V (GnT-V) catalyzes the attachmentof a UDP-GlcNAc to a mannose of N-glycan by a !1,6-linkage to forma branched GlcNAc structure. !1,6 GlcNAc-branched N-glycans
can be subsequently modified by !1,4 galactosyltransferase (GalT)
and !1,3 N-Acetylglucosaminyltransferase (GlcNAcT) to form polyN-acetyllacotosamine for the elongation of N-glycans, which arefurther processed to form other sugar motifs such as sialyl Lewis
X, which contributes to increasing cancer invasion and metastasis(Taniguchi & Korekane, 2011). It has also been reported that GnT-V
activity and !1,6 GlcNAc branched N-glycan levels are increasedin highly metastatic tumor cell lines (Pochec et al., 2013). Consistently, cancer metastasis is greatly suppressed in GnT-V knockout
mice (Granovsky et al., 2000). Meanwhile, elevated expression
and activity of GnT-V in hepatocellular carcinoma is a common
early event involved in tumor invasion during hepatocarcinogenesis (Liu et al., 2013). These results strongly demonstrated thatglycosyltransferase-modified N-glycan structures could modulate
cell migration and thereby cancer metastasis as well.
As a non-toxic natural product, COS has very important researchsignificances on physiological and pathological conditions, especially on the antitumor activity. However, the effect of COS oncancer metastasis still remains elusive. In this study, we investigated the effect of COS on cell migration induced by GnT-V,
and found COS could suppress GnT-V expression and its products,
which may give a hint for developing a novel therapeutic candidate
for some metastatic cancers.
2. Materials and methods
2.1. Preparation of the COS
COS were prepared by the enzymatic hydrolysis of chitosan
according to the method we reported previously (Li, Liu, Xu, Du,& Xu, 2014). The products of the enzymolysis were a mixture ofseveral oligosaccharides with a degree of deacetylation over 95%
and an average molecular weight ≤1000 Da. The percentage composition of COS was 3.7%, 16.1%, 28.8%, 37.2% and 14.2%, with adegree of polymerization (DP) of 2–6.
2.2. Cell line and cell culture
Human immortalized MCF-10A breast epithelial cell line, sharing many features of mesenchymal cancer cell lines (Sarrio et al.,2008), was obtained from American Tissue Culture Collection(ATCC) and cultured according to ATCC recommendations. Briefly,
MCF10A was cultured in DMEM/F12 medium, supplemented with
5% horse serum, 20 ng/ml EGF, 100 ng/ml cholera toxin, 10 "g/ml
insulin, and 0.5 "g/ml hydrocortisone, under a humidified atmosphere of 5% CO2 and 95% O2 at 37 ◦ C.
2.3. GnT-V overexpression and retroviral infection
The cDNA of human GnT-V was amplified using pENTR-d-Topofor the Gateway Conversion System (Invitrogen), according to therecommended protocol. The cloned genes were integrated into thevirus expression vector, pBABE-puro (Addgene, Inc. Cambridge,MA), and inserted into the Gateway Conversion System utilizing
an LR Clonase reaction. The GnT-V construct was transfected into
Phoenix-Ampho cells with Lipofectamine 2000 (Invitrogen) for the
preparation of viral supernatants. After virus infection, the infected
cells were selected with 5 "g/ml puromycin to get the GnT-V stablytransfected MCF10A cells (GnT-V transfectants). For mock treatment, the same protocol was performed with the empty virus
expression vector.
2.4. RT-PCR for GnT-V mRNA expression analysis
GnT-V transfectants were incubated with COS (0, 50, 100
and 200 "g/ml) for 24 h, then cellular total RNA was extractedusing cold TRIZOL reagent (Takara, Dalian, China) according to
the manufacturer’s protocol. RNA samples (2.0 "g) were used as
templates to perform the RT-PCR assay. PCR primers were as follows: GnT-V: forward sequence: GACCTGCAGTTCCTTCTTCG andreverse sequence: CCATGGCAGAAGTCCTGTTT; and ˇ-actin: forward sequence: GGGTCAGAAGGATTCCTATG and reverse sequence:
GGTCTCAAACATGATCTGGG. The ˇ-actin mRNA was used as a control in PCR runs and the reaction products were visualized byelectrophoresis in 1.5% agarose gels containing ethidium bromide.
2.5. Western blot and lectin blot analysis
GnT-V transfectants were incubated with COS (0, 50, 100 and
200 "g/ml) for 72 h, then equal amounts of proteins were separated
by 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF)
membranes. The membranes were blocked with 5% skim milk in
PBS containing 0.1% Tween 20 (PBST) at room temperature for 2 h.
After incubation, the membranes were probed with the appropriate
antibodies, or with biotinylated DSA and biotinylated L4-PHA lectin(Seikagaku kogyo Inc., Japan). Immunoreactive bands were visualized utilizing a Vectastain ABC kit (Vector Laboratories, CA) andan ECL kit (Amersham, UK). Antibody against GnT-V (24B11) was
obtained from FUJIREBIO Inc. (Tokyo, Japan), and the anti-#-tubulin
antibody was from Sigma.
2.6. An in vitro assay of 2-D cell migration across an artificial gap
An in vitro cell migration assay was performed to investigate the ability of cell motility as previously described (Xuet al., 2011). Briefly, the GnT-V transfectants were incubated with
COS (200 "g/ml) for 48 h, then a confluent layer of cells was
scraped/wounded using a yellow tip. The open gap was thenobserved microscopically over time when the cells moved in andfilled the damaged area. Micrographs were taken at 0, 12 and 24 hafter wounding under an inverted microscope (Leica, German). The
pictures showed the cells as they began to migrate toward the center of the wound. Wound closure was measured by showing the
distances between the sides of the wound at the indicated times.
2.7. Cell adhesion evaluation
Cell adhesion assay was performed as previously described (Xuet al., 2011). Briefly, the GnT-V transfectants were incubated with
COS (100 and 200 "g/ml) for 72 h, then 3 "g/ml of fibronectin (FN)
were coated in 96-well plates (Corning Inc., PA) at 37 ◦ C for 1 h, andthen blocked with 1% bovine serum albumin (BSA) in DMEM/F12
for 1 h. The cells in suspension were added to each well (5 × 104
per well) and the plates were incubated at 37 ◦ C for 30 min. Afterincubation, attached cells were fixed with 25% glutaraldehyde, andstained with 0.5% crystal violet. The absorbance at 590 nm was

Q. Xu et al. / Carbohydrate Polymers 170 (2017) 241–246

243

Fig. 1. Effects of GnT-V overexpression on N-glycan expression and cell morphology in MCF10A cells. Stable GnT-V-expressed cells were harvested for RT-PCR (A) andimmunoblotting analysis (B) to confirm GnT-V expression. Equal amounts of protein (20 !g) were separated on 7.5% SDS-PAGE under reducing conditions, and the membranes
were probed with antibody against GnT-V (B), L4-PHA lectin (C), and DSA lectin (D). "-tubulin was used as a loading control. Square brackets indicated the L4-PHA and DSA
specific reactive bands, and asterisks indicated nonspecific staining of L4-PHA and DSA, since those bands did not disappear after treatment with 100 mM acetic acid. The
morphological changes of the GnT-V transfectants were photographed under a phase-contrast microscope (E). Photographs were taken of living cells using a 20× objective.
Insets, representative amplified cell morphology. The arrow indicates aggregated cells, while the arrowhead shows scattering cell with a spindle shape.

detected on an automated micro-titer plate spectrometer (Biotech
Instruments Inc., USA).
2.8. Expression of the branched N-glycans were analyzed by flow
cytometry
To examine the branched N-glycans on the cell surface, a flow
cytometry analysis was performed as previously described (Xuet al., 2011). Briefly, the GnT-V transfectants were incubated with
COS (200 !g/ml) for 72 h, then the cells were suspended and incubated with biotinylated DSA or L4-PHA lectin for 30 min, followed
by streptavidin Alexa Fluor 488 conjugate (Invitrogen). Negative
controls were performed the same treatment without biotinylated
lectins. A FACSCalibur instrument (BD Biosciences), equipped with
CELL QuestPro software was used to analyze the fluorescence intensity of cells.

L4-PHA lectin, which specifically recognizes branched GlcNAc, wasalso up-regulated in the GnT-V transfectants (Fig. 1C/D). The wild
type MCF10A cells formed a typical compact cobblestone epithelial morphology indicated by arrow, while the GnT-V transfectants
exhibited obvious mesenchymal morphological changes including
loss of cell–cell contact and spindle shape indicated by arrowhead(Fig. 1E), which indicates that cells have more migratory ability.
3.2. COS suppressed cell migration and cell adhesion induced by
GnT-V

3. Results

Previously, Gu et al. reported that GnT-V influenced cell migration and FN-mediated cell adhesion (Gu & Taniguchi, 2008). In thisstudy, an in vitro assay of cell migration and FN-dependent celladhesion was performed. Comparing with control cells, the GnT-V
transfectants efficiently moved in and filled the open gap at 12 and24 h after wounding. However, the increased wound closure wasgreatly suppressed in the presence of COS at 200 !g/ml final concentration (Fig. 2A/B). Cell adhesion on fibronectin was significantly
increased in the GnT-V transfectants compared with control cells.
Similarly, COS significantly suppressed FN-dependent cell adhesion
induced in the GnT-V transfectants (Fig. 2C). These results demonstrate that COS could functionally affect cell behavior.

3.1. Overexpression of GnT-V increased expression levels of
branched N-glycans and cell migration

3.3. COS inhibited GnT-V protein expression

GnT-V, a glycosyltransferase encoded by the Mgat5 gene whichcatalyzes the formation of #1,6 GlcNAc branches on N-glycans,is believed to be associated with cancer growth and metastasis(Miyoshi, Terao, & Kamada, 2012). To investigate effects of COS
on breast epithelial cell migration, we established a GnT-V stable
expression cell line. The expression levels of GnT-V were verified
by RT-PCR and immunoblot analysis, and both gene and protein
levels of GnT-V were dramatically increased in GnT-V-transfected
MCF10A cells (Fig. 1A/B). In addition, the reactivity with DSA or

To further verify the nature of COS as a potential antagonistof cancer metastasis, we evaluated the gene and protein expression of GnT-V, which was critically responsible for aforementioned
cell behavior. As shown in Fig. 3A, the gene expression levels of
GnT-V in the transfectants were around 10-fold higher than thosein the control or mock cells, and COS did not obviously affect its
expression. However, although the underlying mechanisms remain
unclear, COS significantly inhibited the protein expression of GnT-V
at 100 and 200 !g/ml concentrations (Fig. 3B). These results sug-

2.9. Statistical analysis
Student’s t-test was executed for comparison of two groups.
P < 0.05 was defined as statistically significant.

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Q. Xu et al. / Carbohydrate Polymers 170 (2017) 241–246

Fig. 2. Effects of COS on cell migration and cell adhesion induced by GnT-V. (A) The scraped gap was detected using a microscope, and the distances between the two sides
were recorded at the indicated times. (B) Quantitative data of the cell migration distance were obtained from three independent experiments. The distance between the twosides immediately after scraping (0 h) was set as 100. The relative migratory distance was defined as the width of the wound relative to the distance at 0 h. (C) Different
cells were spread on FN-coating plates in the presence or absence of COS for 30 min. The attached cells were detected as described in “Materials and methods”. # P < 0.05
compared to the mock control group, * P < 0.05, ** P < 0.01 compared to the GnT-V transfectant group without COS treatment.

by COS in a dose dependent manner (Fig. 4A/B). It is well known
that branched GlcNAc N-glycans, which enhance the formationof complexes on the cell surface such as integrins, galectins andgrowth factor receptors, lead to an increase in cell motility and cellular signaling (Dennis, Lau, Demetriou, & Nabi, 2009; Kariya & Gu,2011). The flow cytometry analysis indicated that branched GlcNAc N-glycans expressed on the cell surface were also significantly
suppressed by COS in GnT-V transfected cells (Fig. 4C/D).
4. Discussion

Fig. 3. Effects of COS on GnT-V expression. (A) The gene expression of GnT-V wasanalyzed by RT-PCR, and ˇ-actin was used as a loading control. (B) The protein
expression of GnT-V was analyzed by Western blot, and !-tubulin was used as aloading control. The arrow indicated the protein expression of GnT-V.

gest COS may be related with protein translation, post-translational
processing and stability of GnT-V.
3.4. COS suppressed branched GlcNAc structures
The branched N-glycans modified by GnT-V were evaluated bylectin blot analysis. As shown in Fig. 1, the reactivities with DSA or
L4-PHA lectin, which specifically recognizes branched N-glycans
were clearly increased in the GnT-V transfectants compared to thecontrol cells. Interestingly, these enhancements were suppressed

More and more attentions have been given to the development of chitosan or its derivatives as antitumor agents in thepast decades (Mattaveewong et al., 2016). Although the antitumor
activity of COS has been studied in vivo and in vitro (Park, Chung,Choi, & Park, 2011), the molecular mechanisms of the antitumor
activity are still obscure. Our previous studies indicated that COS
could induce hepatoma cell apoptosis (Xu et al., 2008) and inhibit
tumor angiogenesis (Wu et al., 2010). In this study, we focused on
the antimetastatic potency of COS and the underlying mechanism.
In a GnT-V stable expression cell line, COS successfully inhibited
GnT-V induced cell migration and FN-mediated cell adhesion. Inline with these results, Shen et al. found COS administered significantly inhibited lung metastasis in Lewis lung carcinoma (LLC)
bearing mice. A roughly 80% reduction in metastatic lung tumors
was observed in LLC-bearing mice treated with 500 mg/kg of COS(Shen, Chen, Chan, Jeng, & Wang, 2009). Nam et al. investigated theantimetastatic property of COS by evaluating motility and invasion in MDA-MB-231 human breast carcinoma cells. COS led toa concentration-dependent decrease in cell migration and inhibited the invasion of MDA-MB-231 cells through a matrigel-coatedmembrane (Nam & Shon, 2009). Meanwhile, our new study demonstrated that COS (200 "g/ml) could significantly suppressed the
EGF-induced mouse epithelial GE11 cells motility (Xu, Wang, Yang,
Du, & Song, 2017). In this study, our results indicated that antimetastatic potency of COS was involved in alterations of GnT-V
expression and branched GlcNAc N-glycans.
GnT-V is one of the important glycosyltransferases involvedin tumor metastasis and carcinogenesis. Three different pathways

Q. Xu et al. / Carbohydrate Polymers 170 (2017) 241–246

245

Fig. 4. Effects of COS on the expression levels of branched GlcNAc N-glycans. Equal amounts of protein (20 !g) were used for lectin blot analysis. After separation on 7.5%
SDS-PAGE and the proteins were probed with DSA lectin (A) and L4-PHA lectin (B), "-tubulin was used as a loading control. Square brackets indicated the DSA and L4PHA specific reactive bands, and asterisks indicated nonspecific staining of DSA and L4-PHA, since those bands did not disappear after treatment with 100 mM acetic acid.
Expression levels of branched GlcNAc N-glycans on the cell surface were detected by FACS analysis. Prior to detection, cells were incubated with biotinylated DSA lectin (C)and L4-PHA lectin (D), followed by treatment with streptavidin Alexa Fluor 488 conjugate.

were identified to be related to the facilitation of cancer metastasis by GnT-V. The first pathway was the promotion of epidermal
growth factor receptor signaling by lattice formation between polylactosamine on #1,6 GlcNAc branches and galectin-3, resulting
in the blockage of receptor endocytosis (Partridge et al., 2004).
The second pathway involved in the up-regulation metastasis byrepressing protease degradation through the addition of #1,6 GlcNAc branches (Ihara et al., 2002). In the third pathway, GnT-V
inhibited E-cadherin/#-catenin complex function, which led to therelease of cancer cells from the original tissue (Pinho et al., 2013).
Therefore, suppression of GnT-V could be a promising anti-cancer
way. In this study, we clearly showed COS inhibited GnT-V expression and its products to block cell migration. Similar to our findings,
Sato et al. demonstrated staurosporine could suppress the expression of GnT-V by 30–40% and alter the N-glycosylation of cellsurface proteins to affect the metastasis of colorectal adenocarcinoma cells (Sato, Takahashi, Kawado, Takayama, & Furukawa,
2005). GnT-V is present in the Golgi apparatus in a membranebound form and is released from cells after cleavage by certain
proteases. Secretase is an important protease for GnT-V secretion
processing, which can increase the secretion rate of endogenous
GnT-V (Nakahara et al., 2006). There have been some studies on
secretase inhibition activities of COS and its derivatives. An investigation of #-secretase inhibitory activity of COS was performed by
Byun et al. They demonstrated that the inhibition pattern of COS
was a noncompetitive by Dixon plot, and the inhibition constant(Ki) of COS was 3.87–6.47 !M (Byun, Kim, Park, Lin, & Kim, 2005).
Phenolic acid conjugated COS also exhibited significant #-secretaseinhibitory activity (Eom et al., 2013). Thus, we proposed that theeffects of COS on GnT-V expression might be related with secretaseactivity.
Furthermore, Ko’s group reported that tissue inhibitor of
metalloproteinase-1 (MMP-1) was aberrantly glycosylated by GnTV showing a weaker inhibition with respect to both MMP-2 and
MMP-9, and this aberrancy was closely associated with cancer cell

invasion and metastasis in vivo as well as in vitro (Kim et al., 2008).
Furthermore, GnT-V modulated the transcriptional activation of
membrane-type matrix metalloproteinase-1 (MT1-MMP) in cancer
cells. It not only promoted proteolytic activity for cancer cells, butalso led to the activation of MMP-2. Consequently, the activation of
the two MMPs triggered by GnT-V intensified the cancer invasive
potential (Lee et al., 2013). On the other hand, Kim et al. reported
that COS suppressed activation and expression of MMP-2 in humandermal fibroblasts (Kim & Kim, 2006). Nam et al. proved that COS
inhibited the metastasis of MDA-MB-231 human breast carcinoma
cells by blockage of MMP-9 (Nam & Shon, 2009). Based on thoseobservations, we speculated that COS regulate GnT-V expression to
influence metalloproteinase activities, and finally affect cell migration.
5. Conclusions
In summary, this study demonstrated that COS could be a potential therapeutic candidate for the metastatic breast cancer. The
antimetastatic potency of COS involves in the suppression of GnT-V
expression and branched N-glycans. Further research and development of COS might provide an effective, inexpensive and safe
antitumor agent.
Conflict of interest
The authors declared that they have no conflict of interest.
Acknowledgement
This work was supported by grants from the National Natural Science Foundation of China (31372347), and National Natural
Science Foundation of China (31670807).

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Q. Xu et al. / Carbohydrate Polymers 170 (2017) 241–246

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Biochemical and Biophysical Research Communications 486 (2017) 1090e1096

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Differential expression of ST6GAL1 in the tumor progression ofcolorectal cancer
Sen Zhang a, 1, Jishun Lu b, c, 1, Zhijue Xu b, Xia Zou a, b, Xue Sun d, Yingjiao Xu b,
Aidong Shan b, Jiaoyang Lu a, Xialin Yan a, Yalu Cui b, Wei Yan b, Yuguo Du d, Jianguo Gu c, e,
Minhua Zheng a, Bo Feng a, **, Yan Zhang b, *a

Department of General Surgery, Shanghai Minimally Invasive Surgery Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, 197, Ruijin
Er Road, Shanghai 200025, China
Key Laboratory of Systems Biomedicine (Ministry of Education), and Collaborative Innovation Center of Systems Biomedicine, Shanghai Center for Systems
Biomedicine (SCSB), Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, Chinac
Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1
Komatsushima, Aobaku, Sendai 981-8558, Miyagi, Japand
State Key Lab of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing
100085, Chinae
Department of Pharmacology, Pharmacy College, Nantong University, Nantong 226001, Jiangsu, Chinab

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 16 March 2017
Accepted 31 March 2017
Available online 1 April 2017

Elevated expression of b-galactoside a2,6-sialyltranferase 1 (ST6GAL1) has been observed in colorectal
cancer (CRC) and demonstrated to be important for its tumorigenesis. Here, we found that ST6GAL1
expression was signiﬁcantly higher in non-metastatic tumors (stage I and II) than that in metastatic
tumors (stage III and IV) using 62 pair-matched tumor/normal tissues. To elucidate the molecularmechanisms of how ST6GAL1 affected the CRC progression, we performed a global identiﬁcation of thesubstrates of ST6GAL1 in the colon adenocarcinoma cell line SW480. A total of 318 membrane proteins
were identiﬁed differentially affected by ST6GAL1 overexpression using metabolic labeling and proteomic analysis. Subsequent bioinformatic analysis revealed a list of potential substrates that might mediate
the different functions of ST6GAL1 in CRC including cell movement, cell death and survival. Taken
together, these results indicate a dynamic change in the expression of ST6GAL1 during the CRC progression and provide a list of sialylated proteins potentially relevant to the different functions of ST6GAL1
in CRC.
© 2017 Elsevier Inc. All rights reserved.

Keywords:
Metabolic labeling
Sialylation
ST6GAL1
Colorectal cancer

1. Introduction
Sialic acid is the most abundant terminal monosaccharide ofglycoconjugates on the eukaryotic cell surface. It is known to belinked via an a2,3 or a2,6 bond to Gal/GalNAc, or a2,8 bond to sialic
acid in proteins. Given the relatively strong electronegative charge

Abbreviations: CRC, colorectal cancer; EGFR, EGF receptor; OE, overexpression;
qPCR, quantitative RT-PCR; ST6GAL1, b-galactoside a2,6-sialyltranferase 1; WT,
wild-type.
* Corresponding author.
** Corresponding author.
E-mail addresses: yanzhang2006@sjtu.edu.cn (Y. Zhang), fengbo2022@163.com(B. Feng).
1
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bbrc.2017.03.167
0006-291X/© 2017 Elsevier Inc. All rights reserved.

of sialic acids and their location at the outmost reaches of the cellsurface, sialic acids may regulate numerous cell-cell and cell-matrix
interactions. Not surprisingly, altered sialylation has been longassociated with tumorigenesis and tumor progression and it isusually driven by the dysregulated expression of certain sialyltransferases and sialidases responsible for the addition andremoval of sugars [1e3].
Among these, b-galactoside a2,6-sialyltranferase 1 (ST6GAL1),
which primarily produces a2,6 linked sialic acids on N-glycans, hasreceived much attention in recent years as elevated expression ofthis sialyltransferase was observed in multiple types of carcinomas
including colorectal, ovarian, breast, prostate, ovarian, andpancreatic carcinomas [4]. The selective enrichment of ST6GAL1mediated a2,6 sialic acids on tumor cells is signiﬁcant in that it
may elicit various biologic outcomes. It has been also reportedly

S. Zhang et al. / Biochemical and Biophysical Research Communications 486 (2017) 1090e1096

involved in tumorigenesis, epithelial mesenchymal transition, celladhesion, migration and invasion, apoptosis, drug resistance, immune evasion and maintenance of cancer stem cell property[4e10]. In contrast to these important roles, there is limited information regarding the molecular details of how ST6GAL1-catalyzed
a2,6 sialylated proteins mediate these functions [11]. ST6GAL1
could regulate the cell migration and invasion partly by increasing
the a2,6 sialylation of b1 integrin [12e14]. Sialylation of the Fas
death receptor by ST6GAL1 provides protection against Fasmediated apoptosis in colon carcinoma cells [15]. In addition,
there are also several other reported proteins whose activities are
regulated by sialylation which includes CD45, EGFR, and PECAM[12e19]. Clearly, these limited proteins could not provide thecomprehensive explanation for its diverse roles in tumors. To
obtain a better understanding of the mechanistic roles of this sialyltransferase, identiﬁcation of its additional function-related sialylated proteins is of critical importance.
The up-regulated expression of ST6GAL1 was ﬁrst described incolorectal cancer (CRC) [20]. Increasing evidence has indicated that
ST6GAL1 may serve as a major inhibitor of several cell deathpathways and contribute to the colorectal tumorigenesis [6,15,21].
However, the mechanistic roles of ST6GAL1 in the tumor progression are still obscure. In the current study, interestingly, we foundthat ST6GAL1 was signiﬁcantly induced in tumors at stage I and II
but not in those at stage III and IV when compared with their paired
normal tissues. Subsequent metabolic labeling and proteomics
analysis in the colon cancer cell line SW480 revealed a list of proteins that may mediate the effects of ST6GAL1 on tumor development and progression. These results indicate a dynamic change in
ST6GAL1 expression in human CRC progression and suggest thedistinct roles of ST6GAL1 at different stages.
2. Materials and methods
Tissue Specimens d From May 2009 to Jun 2012, a total of 62
CRC cases with pathological stage I (n ¼ 19), II (n ¼ 20), III (n ¼ 17)
and IV (n ¼ 6) were recruited from Ruijin Hospital (Shanghai,
China) in accordance with the guidelines set by the Ethical Committee of Ruijin Hospital. All participants gave the written,
informed consent and no participants had received any medication
prior to sample collection. Staging of CRC was performed according
to the Union Internationale Contre Le Cancer (UICC). Tumor tissues
and paired normal colonic tissues, located approximately 10 cm
from the distal edge of the tumor were snap frozen in liquid nitrogen 30 min after vessel ligation and stored in the liquid nitrogenprior to analysis. The inclusion and exclusion criteria of patients,
and protein extraction were performed as described in Ref. [22].
Cell Lines and Cell Culture d The HEK 293T and SW480 celllines were purchased from ATCC (Manassas, VA, USA). The HEK
293T cells were cultured as described previously [10]. SW480 and
ST6GAL1-overexpressing cells were maintained in Leibovitz's L15
Medium (HyClone, Logan, Utah, USA) with 10% FBS under a humidiﬁed atmosphere at 37 " C without CO2 addition. The ST6GAL1overexpressing cells were established by virus transfection andcell-sorting using FACS Aria II (BD Bioscience, Bedford, Massachusetts, USA).
Western Blot Analysis and Immunoprecipitation d Western
blot analysis and immunoprecipitation were performed as
described previously [10]. For details, please refer to our
supplementary materials.
Flow Cytometry Analysis of Cells d Cell cytometry was performed as reported in Ref. [23]. Cells were grown to about 90%
conﬂuency and detached using trypsin containing 1 mM EDTA at
37 " C, and washed three times with cold PBS. Then, cells were
stained with 1 mg/ml Alkyne 555 (Invitrogen, Eugene, Oregon, USA)1091

for 30 min at RT temperature or with 10 mg/ml SNA-FITC (Vector,
Burlingame, California, USA) or 10 mg/ml MAA-FITC (EY Laboratories, San Mateo, California, USA) for 60 min on ice. Finally, cells
were washed three times with PBS and analyzed by ﬂow cytometry(BD Biosciences, San Jose, California, USA).
qPCR for mRNA Expression Analysis d The qPCR analysis was
performed as described previously [24]. The sequences of primers
used for quantitative RT-PCR (qPCR) analysis are as follows: GAPDH(50 -TTCAACAGCAACTCCCACTCTT-30 and 50 -TGGTCCAGGGTTTCTTACTCC-30 ), EGFR (50 -ACCTGCGTGAAGAAGTGTCC-30 and 50 ABCB1(50 -GCCAAAGCCAAAACGTCTTCCTCCATCTCATAGC-30 ),TATCAGC-30 and 50 -TTCCAATGTGTTCGGCAT-30 ).
Cell Migration Assay d Cell migratory ability was examinedwith Transwell (BD BioCoat™ control inserts, 8.0-mm inserts; BD
Biosciences, Bedford, Massachusetts, USA) as described previously[23]. Transwells were coated only on the bottom side with 10 mg/ml
ﬁbronectin or collagen I at 4 " C overnight.
Metabolite Labeling and Fluorescence Detection d cells(4.0 # 106) were cultured with 100 mM of the Ac4ManNAz or
ManNAc for 60 h. After metabolic labeling, the cells were incubatedwith Alkyne 555, catalyst (CuSO4, NaVc, TBTA) for click chemistry[25]. The obtained cells were then detected with a laser-scanningconfocal microscope (Nikon, Tokyo, Japan) or ﬂow cytometry (BDBiosciences, San Jose, California, USA) for the check of the labeling
efﬁciency. For details, please refer to [26].
Protein Enrichment and Mass Spectrometry Analysis d Afterwashing with PBS, 1 # 107 metabolic labeled cells were used toperform the subsequent click reaction with biotinylated alkyne andglycoprotein enrichment by streptavidin-agarose resin as described
previously [25]. The beads were pelleted by centrifugation andwashed, reduced and alkylated followed by on bead trypsin
digestion as shown in Ref. [27]. The released peptides wereanalyzed on a Q Exactive Plus mass spectrometer equipped with an
Easy-nLC 1000 (Thermo Fisher Scientiﬁc, Odense, Denmark).
Spectra was searched against a human proteins database (Uniprot
Complete Proteome e taxonomy Homo sapiens, release 2016.08)
using the Andromeda module of MaxQuant software v. 1.5.5.1. For
details, please refer to our supplementary materials.
Bioinformatic Annotation and Analysis d N- and O-glycoprotein prediction analysis using the NetNGlyc 1.0 and the NetOGlyc 4.0 servers was performed. Gene ontology (GO) enrichment
analysis was carried out using DAVID Bioinformatics Resources 6.8.
Functional analysis was generated using the Ingenuity Pathways
Analysis (IPA) software v7.1 (Ingenuity Systems).
3. Results
To explore the roles of ST6GAL1 in the CRC progression, we ﬁrst
evaluated its expression in the tissue specimens at different stages
by western blot analysis (Fig. 1A and S1). 62 patients wererandomly selected based only on the criteria of matched age andgender (Table 1). Interestingly, the expression of ST6GAL1 wassigniﬁcantly increased in tumors at the stage I and II as comparedwith their pair-matched normal tissues, while no statistical difference was observed when the tumors were developed to the stage III
and IV (Fig. 1B). Furthermore, the expression of ST6GAL1 in tumors
at stage I and II was signiﬁcantly higher than that at stage III and IV(Fig. 1C). These results clearly indicate a dynamic change in
ST6GAL1 expression in the colorectal tumors at different stages,
suggesting important roles of ST6GAL1 in CRC progression.
Next, to explore molecular mechanisms of how ST6GAL1
affected the CRC progression, we ﬁrst overexpressed this sialyltransferase in SW480, a human colon adenocarcinoma cell linewith the low expression of ST6GAL1. Western blot, lectin blot, cell
cytometry and qPCR results demonstrated the high expression of

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S. Zhang et al. / Biochemical and Biophysical Research Communications 486 (2017) 1090e1096

B

Stage I
#43

#51

#72

#88

#116

T

T

T

T

T

N

N

N

N

N

ST6GAL1GAPDH
Stage II
#36

#66

#67

#77

#80

#84

T

T

T

T

T

T

N

N

N

N

N

Stage III+IV(n=39)(n=23)

1

#17

#39

#55

#62

#68

#81

T

T

T

T

T

T

N

N

N

N

Relative changes (tumor/normal)

C

Stage IIIN

ST6GAL1GAPDH
Stage IV
#44
T N

Stage I+II

2

ST6GAL1

N

n.s.

0

N

GAPDH

#53
T N

#61
T N

#94
T N

#103
T N

Tumor
Normal*3

Relative expression

A

#2-8
T N

ST6GAL1
GAPDH

*
5
4
3
2
1
0

Stage I

Stage II

Stage III

Stage IV

(n=19)(n=20)(n=17)(n=6)

Fig. 1. ST6GAL1 was signiﬁcantly higher in colorectal tumors at stage I and II but not in those at stage III and IV compared with their pair-matched normal tissues. A) Representative
images of the immunoblot for ST6GAL1 in primary colorectal tumors at different stages. For the rest of our immunoblot images, please refer to our supplementary materials. B)
ST6GAL1 bands detected from the immunoblot were quantiﬁed by densitometric scanning and protein intensity values were normalized by GAPDH. The normalized ST6GAL1
intensity in tumors was compared with that in its pair-matched normal tissues. Error bars indicate standard derivation. C) The ratio (tumor/normal) for each sample pair was
calculated. Middle lines indicate Median values (n.s.: no signiﬁcance; *, p < 0.05 by paired t-test or Mann-Whitney U test).

Table 1
Clinical features of samples used in this study.
UICC Stage

Stage I

Stage II

Stage III

Stage IV

NumberAge (year)
median/range
GenderMale
FemaleLocationa
AC
TC
DC
SC
RC
CEA (ng/ml)
median/range
CA19-9 (ng/ml)
median/range
CA125 (ng/ml)
median/range
AFP (ng/ml)
median/range

19

20

17

6

59/47-76

60/50-78

59/40-77

61/35-74

11
8

13
7

10
7

3
3

4
1
2
4
8

4
e
4
2
10

3
e
e
1
13

e
e
2
1
3

2.62/1.36e8.90

4.19/0.94e376.43

2.86/0.7e891.16

10.43/4.43e303.24

6.53/2.00e42.70

11.42/0.80e91.70

18.9/2.00e1554.6

185.35/51.60e3770.46

10.00/3.9e32.6

14.80/7.40e34.90

13.60/7.20e62.30

84.55/14.80e238.80

2.96/1.48e4.39

3.15/1.57e6.14

3.19/1.89e8.40

5.42/4.88e5.88

aAC, ascending colon; TC, transverse colon; DC, descending colon; SC, sigmoid colon; RC, rectum.

ST6GAL1 in the ST6GAL1-overexpressing SW480 cells (Fig. 2AeD).
Then, to identify the sialylated proteins differentially affected by
ST6GAL1, SW480 and ST6GAL1-overexpressing cells were separately labeled with Ac4ManNAz and ManNAc followed by theenrichment and identiﬁcation of the sialylated membrane proteins.
Fig. 3A showed the schematic representation of our strategy.

Ac4ManNAz is intracellularly deacetylated and used to incorporate
azidos to tag the sialic acid linked glycans and the sialylated proteins [26]. The azido-tagged proteins on the cell surface were thenreacted with biotin-alkyne and enriched with streptavidin-beads.
The sialylated proteins were efﬁciently and speciﬁcally labeled byazido sugars, as shown by the strong signals in the glycoproteins

1093

S. Zhang et al. / Biochemical and Biophysical Research Communications 486 (2017) 1090e1096

ST6GAL1
GAPDH

B

D
Blot:

SNA
MAA
(kDa) WT OE (kDa) WT OE
180
130

180
130

95

95

72

72

55

55

43

43

34

34

Fold changes in the mRNAlevel of ST6GAL1

300
200
5

Negative control
SNA
MAA

SW480

OE

ST6GAL1 OE

C
WTCell counts

A

4
3
2
1
0

WT

OE

GAPDH

Reactivity against
lectinsFig. 2. ST6GAL1 were efﬁciently overexpressed in colon cancer cells SW480. A-D) The efﬁciency of the ST6GAL1 overexpression in SW480 was conﬁrmed by the analyses of western
blot, qPCR, lectin blot and cell cytometry. GAPDH was used as an internal control.

Fig. 3. Sialylated proteins in SW480 and ST6GAL1-overexpressing cells were efﬁciently labeled and enriched. A) Schematic representation of the cell surface sialylated proteinlabeling, enrichment and identiﬁcation. B-C) The labeling efﬁciency of cell surface sialylated proteins with Ac4ManNAz was checked by the analyses of immunostaining andcytometry. D-E) The selectivity and efﬁciency of the enrichment of these proteins were examined by the analyses of silver staining and western blot with streptavidin 680.

from the cells incubated with Ac4ManNAz but not from those
pretreated with ManNAc according to the ﬂuorescence-staining
and FACS analyses (Fig. 3B and C). We also checked the efﬁciencyof sialylated protein enrichment by silver staining and western blot
analyses. The amount of proteins as well as the signals against

streptavidin 680 in the elute fraction of Ac4ManNAz treated cells
were markedly higher as compared with those incubated with
ManNAc (Fig. 3D and E), indicating the efﬁcient and selectiveenrichment of the azido sugar modiﬁed proteins.
The MS analysis of the elute fractions collected from the cells

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S. Zhang et al. / Biochemical and Biophysical Research Communications 486 (2017) 1090e1096

cultured with Ac4ManNAz led to the identiﬁcation of a total of 709
membrane proteins by searching the data against the Uniprot
Complete Proteome Human Database and GO analysis (Cellular
Component). Prediction analysis of these proteins indicated that567 of them could be N-glycosylated and 625 of them could be Oglycosylated (Fig. 4A). Considering sialic acids mainly exist as theterminal sugars on the N-glycans, O-glycans and glycolipids, theseresults suggest that most of the labeled glycoproteins arising fromthe Ac4ManNAz treatment might be sialylated.
Next, we examined the differential regulation of glycoproteins
upon ST6GAL1 overexpression in SW480 cells based on our identiﬁed set of 709 membrane proteins. Proteins were considered as
differentially expressed between the wild-type SW480 and
ST6GAL1-overexpressing cells if their fold change in relative
expression!2 (47e49). Based on these selection criteria, we found a
total of 318 glycoproteins were differentially affected (up-regulatedor down-regulated or speciﬁcally expressed in either cells) after

A

C
32

52

-Log (p-value)
35

515

ST6GAL1 overexpression in SW480. Volcano plot-log10 (intensityof the protein in ST6GAL1 overexpression) vs. log2 (fold change of
ST6GAL1 overexpression/wild-type) was constructed to graphicallydisplay the quantitative data using the overlapped proteins between control and ST6GAL1-overexpressing cells (Fig. 4B). To
evaluate the potential roles of ST6GAL1 in CRC, these proteins wereused for the functional analysis by the IPA software. Fig. 4C showed
the top 15 most signiﬁcantly perturbed functions upon the overexpression of ST6GAL1 in SW480. The ﬁrst three were cell movement, cell death and survival, and cell growth and proliferation,
which are in good agreement with numerous reports on thefunctions of ST6GAL1 under pathological conditions [4]. Table S1
listed the proteins that are related with these functions.
To experimentally conﬁrm these changes, two proteins ABCB1(~10 folds up) and EGFR (~2 folds up), which showed big differencein the extent of their changes, were selected to perform the qPCR,
immunoprecipitation and western blot analyses (Fig. 4DeF). ABCB1

30

25

20

15

10

5

0

Cellular Movement

110

Cell Death and Survival
Cellular Growth and Proliferation
Cellular Function and MaintenanceTotal proteins
Predicted N-glycosylated proteins
Predicted O-glycosylated proteins

Organismal Survival
Cell Morphology
Cell-To-Cell Signaling and Interaction

B
Ratios of ST6GAL1 OE/WT (Log2)

Cellular Assembly and Organization
Infectious Diseases
Immune Cell Trafficking
Hematological System Development
Cardiovascular System Development
7

8

9

10

11

D

E
10
9
2
1
0

EGFR ABCB1

Tissue Development

F
Relative changes ofmRNA level (OE/WT)

Relative changes basedon MS (OE/WT)

Organismal Development

12

Intensity in ST6GAL1 OE (Log10)IP:7cell lysate
SNA
WT OE WT OE

6

MAA
WT OE

EGFR

2

ABCB1

1

GAPDH
0

EGFR ABCB1

Fig. 4. Identiﬁcation of the sialylated proteins and functional analysis by IPA. A) Comparative analysis of N-glycoprotein and O-glycoprotein predictions using the NetNGlyc 1.0 and
NetOGlyc 4.0 servers. B) Volcano plot illustrates differentially regulated membrane proteins. The x-axis represented the log10 (intensity in ST6GAL1-overexpressing cells). The y-axis
represented the log2 (ratio) where ‘ratio’ was the ratio of the averages of LFQ Intensities of ST6GAL1-overexpression over control. The non-axial horizontal lines denoted ± 2-foldchange. The colored dots showed proteins considered as up- (red) or down- (blue) regulated, while the non-regulated proteins were displayed as gray dots. C) ST6GAL1 overexpression signiﬁcantly perturbed many functional pathways including cellular movement, cell death and survival. IPA analysis of the differentially regulated proteins on the plasma
membrane (!2-fold) revealed the top 15 most signiﬁcantly perturbed functions upon the overexpression of ST6GAL1 in SW480. The functional pathways included in this analysis
were shown along the y-axis of the bar chart. The x-axis indicates the statistical signiﬁcance on the left. Calculated using the right-tailed Fisher exact test, the p-value indicatedwhich biological annotations were signiﬁcantly associated with the input molecules relative to all functionally characterized mammalian molecules. D) The intensity ratio (ST6GAL1
overexpression/WT) of ABCB1 or EGFR was shown based on the MS analysis. E) The transcriptional levels of EGFR and ABCB1 in WT and ST6GAL1 overexpressing cells were checked
by qPCR analysis. F) The sialylation pattern of EGFR and ABCB1 after ST6GAL1 overexpression was investigated by immunoprecipitation analysis with SNA and MAA beads and its
protein level in whole cell lysate was analyzed by western blot. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of thisarticle.)S. Zhang et al. / Biochemical and Biophysical Research Communications 486 (2017) 1090e1096

were signiﬁcantly upregulated at both mRNA and protein levels,
indicating the different expression of this protein observed in our
MS data is mainly due to its increased mRNA level. In contrast, there
was little change in either mRNA or protein level of EGFR after
ST6GAL1 overexpression. Since immunoprecipitation analysis
showed a signiﬁcant increase in the a2,6 sialylation level, it ishighly likely that ~2 folds upregulation of EGFR observed in our MS
data is attributed to its enhanced sialylation.

4. Discussion
Elevated expression of ST6GAL1 has been observed in multiple
types of carcinomas, among which colorectal cancer was the ﬁrst
reported [20]. Although it has been well documented that ST6GAL1
expression contributes to the colorectal tumorigenesis [6], themechanistic roles of this protein in tumor progression are still
obscure. Interestingly, we found that as compared with their pairmatched normal tissues, the expression of ST6GAL1 was signiﬁcantly increased in tumors at stage I and II (non-metastatic), but not
at stage III and IV (metastatic). Meanwhile, ST6GAL1 expression in
the tumors at stage I and II was statistically higher than that at stage
III and IV, suggesting a dynamic change of ST6GAL1 expression in
CRC progression. Metabolic labeling revealed 318 sialylated membrane proteins differentially affected by ST6GAL1 overexpression.
Considering the distinct roles of different linkages of sialylation incell biology, these results suggest important roles of the dynamicchange in ST6GAL1 expression during the CRC progression.
We showed that high expression of ST6GAL1 was decreased at
the advanced stage of CRC. Consistent with our observation, there isevidence that the mRNA level of ST6GAL1 is signiﬁcantly increased
in colorectal carcinoma specimens, but not in highly invasive tumors and tumors exhibiting distant metastases when comparedwith their pair-matched normal tissues [28]. These results suggestthat high expression of this enzyme may not beneﬁt the CRC progression to the advanced/metastatic stage. Supporting this hypothesis, a very recent work showed that knockdown of ST6GAL1
dramatically enhances the migration and invasion of the coloncancer cell line SW620, and also promotes its liver metastasis
in vivo [29]. Furthermore, our MS data showed that ST6GAL1
overexpression increased the expression of ICAM-1, a liver metastasis suppressor of colon cancer (data not shown). It is well-known
that sialyl Lewis structure serves as a key mechanism for directing
tumor cell adhesion to the endothelium and the a2,3 linked sialic
acid on sialyl Lewis structure is critical for this function. A previous
report showed that a2,3 sialylated type 2 chain structures is predominantly expressed in colorectal tissues associated with themalignant transformation, in particular, with the lymphatic spreadof distal colorectal adenocarcinoma [30]. Therefore, it is possiblethat decreased expression of ST6GAL1 and a2,6 sialylation may
beneﬁt the tumor metastasis by increasing the a2,3 sialylated
structures on cell surface because a2,3 and a2,6 sialyltranferases
share the common glycan substrates.
Our IPA analysis revealed a list of novel glycoproteins that could
mediate these functions above. Among the list, we found thatoverexpression of ST6GAL1 resulted in an increased transcription of
ABCB1 by more than ~6 folds. Consistent with this, it has beenpreviously reported that ST6GAL1 expression could induce theexpression of ABCB1 in human leukemia cells [31]. In addition, a
recent study showed that KAI1 is signiﬁcantly decreased after
ST6GAL1 knockdown in SW620 cells. This protein could also befound in our list, which was increased by ~2-fold after ST6GAL1
overexpression. These results partially suggest the reliability of thelist we obtained, which may contribute to the identiﬁcation of
additional molecules linking the functions of ST6GAL1 in tumor.

1095

5. Declaration
We declare that this manuscript is original, has not been published before and is not currently being considered for publicationelsewhere. There is no conﬂict of interest concerning thismanuscript.
Acknowledgement
This work was supported by The National Natural Science
Foundation of China (31370806, 31170771, 31600643, 31670807),
The National Basic Research Program of China (2012CB822103,
2011CB910603), Shanghai Municipal Commission of Health andFamily Planning Program (201640030), Shanghai Jiao Tong University Interdiscipline with Medicine Program (YG2013MS26),
China Postdoctoral Science Foundation (2016M600310).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2017.03.167.
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Glycoconj J (2017) 34:219–227
DOI 10.1007/s10719-016-9754-3

ORIGINAL ARTICLE

Specific N-glycan alterations are coupled in EMT induced
by different density cultivation of MCF 10A epithelial cells
Qingsong Xu 1,2 & Xueming Niu 1 & Wenjing Wang 3 & Wen Yang 1 &Yuguang Du 4 & Jianguo Gu 2 & Linsheng Song 1

Received: 11 August 2016 / Revised: 22 November 2016 / Accepted: 28 November 2016 / Published online: 29 December 2016
# Springer Science+Business Media New York 2016

Abstract Epithelial-mesenchymal transition (EMT) is a process in tumor progression during which cancer cells undergo
dramatic changes acquiring highly invasive properties. In awidespread adoption of TGF-β-induced EMT model, we havepreviously observed that expression of bisecting GlcNAc on
N-glycans was dramatically decreased. Herein, we performedin vitro studies with the MCF10A cell line. In response to low
cell density, MCF10A cells suffered spontaneously morphologic and phenotypic EMT-like changes, including elongatedspindle shape, extended out from edge of the cell sheet, cytoskeleton reorganization, vimentin and fibronectin up-regulation, catenins redistribution, and cadherin switching.
Moreover, these phenotypic changes were associated withspecific N-glycan alterations. Interestingly, the amounts ofbisecting GlcNAc structure were declined, by contrast, theformation of β1-6 GlcNAc branches were obviously upregulated during the EMT induced by sparse cell conditions.
We further investigated N-glycans on the β1-integrin, which
is a good target of some glycosyltransferases. The reactivity
with E4-PHA lectin decreased, whereas the staining for L4-* Qingsong Xu
xuqingsong@dlou.edu.cn* Jianguo Gu
jgu@tohoku-mpu.ac.jp
1

College of Fisheries and Life Science, Dalian Ocean University, 52.
Heishijiao Street, Shahekou District, Dalian 116023, China

2

Tohoku Medical and Pharmaceutical University,
Sendai, Miyagi 981-8558, Japan

3

Dalian Elite Analytical Instruments Company Limited,
Dalian 116023, China

4

Institute of Process Engineering, Chinese Academy of Sciences,
Beijing 100190, China

PHA lectin, which recognizes branched GlcNAc, increased insparse cell conditions compared with dense cell conditions.
Taken together, these results demonstrated that specific N-glycan alterations are coupled in EMT process and promoted
cells migration at a low cell density.
Keywords Epithelial-mesenchymal transition . Cell density .
N-glycan . Migration . Tumor

Introduction
Metastasis is a sequential process whereby cancer cells penetrate the basement membrane, invade blood/lymphatic vessels,
survive in the vasculature before extravasating to secondary
sites, and finally adapt to new host environments to proliferate
and form metastases [1]. This process is mediated by theepithelial-mesenchymal transition (EMT) whereby tightly connected epithelial cells convert to disorganized motile mesenchymal cells with defined morphology, gene characteristic andprotein expression. Therefore, EMT is regarded as a differentiated or morphogenetic process, which contributes to the pathogenesis of diseases, such as cancer metastasis and tissue fibrosis [2]. A growing body of evidences indicates that EMT isstimulated by signals from the tumor microenvironment, including a variety of growth factors and cytokines (such astransforming growth factor-β and epidermal growth factor)[3]. In addition, EMT is regulated by a series of intracellularsignaling networks, including ERK1/2, PI3K/Akt, Smads,
RhoB, β-catenin, and so forth [4]. These factors alter the epithelial profile by repressing genes encoding epithelial junction
complexes and cytokeratins, and induce the expression of theirmesenchymal counterparts, N-cadherin and vimentin. A defined feature of EMT is a reduction in E-cadherin levels anda concomitant induction of N-cadherin [5]. However, the220significance of EMT during cancer progression and even its
relevance in human tumor remain a matter for debate.
It is widely known that the tumor microenvironment has a
profound impact on determining the gene expression patterns[6]. Kim and his collaborators suggested that the gene expression patterns of cancer cells could vary significantly according
to the conditions under which they were cultured. Culture media
formulations and cell density could modulate the cancer celltranscriptome [7]. Indeed, some researchers found that lowered
cell density, conditions under which cell contact was reduced,
lead to reducing expression of genes associated with mammary
epithelial cell differentiation and increasing expression of genes
associated with breast cancer [8]. Other researchers proposed that
limiting cell spreading, either by increasing cell density or byculturing cells on precisely defined micro-patterned substrata,
blocked expression of characteristic markers of EMT in cells[9]. MCF10A mammary epithelial cells have been used to investigate EMT pathways in premalignant cells, because they readily
undergo EMT when exposed to some stimulus, such astransforming growth factor β (TGF-β) [10]. Moreover,
MCF10A cells have been found to exhibit many EMT-likechanges when grown at different density: cells grown undersparse conditions express higher levels of mesenchymal markers,
while cells grown under confluent conditions express higher
levels of epithelial markers [11]. However, the extent to what
density-dependent alterations in MCF10A cells fully recapitulate
the EMT program and how this process occurs remain unknown.
Glycans, for instance glycosphingolipids (GSLs), N-glycans,and O-glycans are related to numerous of biological phenomenathat include virus/bacteria-host interactions, inflammation, morphogenesis, cell-cell interactions, and cancer progression [12].
Some research data indicated that aberrant formation of glycans
was detected in different EMT models. Guan et al. [13] reported
the function of GSLs in phenotypic alteration of cell growth,motility, and adhesion during EMT, and the expression ofGM2 and Gg4 were found to be dramatically decreased or depleted after TGF-β treatment. Overexpression of thefucosyltransferase 8, which is related to enhance corefucosylation of E-cadherin, decreased Src phosphorylation andsuppressed cell migration in giant lung carcinoma cell line 95C,
but low core fucosylation of E-cadherin activated Src andpromoted an EMT-like phenomenon [14]. The expression of
β-galactoside α 2, 6-sialyltranferase 1 (ST6GAL1), which
modifies N-glycans with terminal α 2, 6-sialylation, was upregulated in different carcinomas and in an EMT model induced by TGF-β [15]. Our previous results showed that expression of N-acetylglucosaminyltransferase III (GnT-III) andbisecting GlcNAc on N-glycans were obviously decreased in
an EMT model induced by TGF-β. GnT-III prolonged Ecadherin turnover and prevented β-catenin translocation into
the cell nucleus from E-cadherin/catenin complex [10]. Nacetylgalactosaminyltransferase 6 (GALNT6), which participates the primary step of O-glycosylation, could stabilize O-

Glycoconj J (2017) 34:219–227

glycosylated fibronectin, promote the acinar disruption, andfurther stimulate cellular alteration similar to those of EMT innormal mammary epithelial cells [16]. Above-mentioned results strongly indicated that remodeling of glycan structuresby glycosyltransferases could modulate cell migration andthereby tumor metastasis as well. It is therefore very important
to clarify the mechanisms for the alteration of glycans underphysiological and pathological conditions.
In this study, we performed new experiments in which we
assessed the effect of differential density on cellular morphology and phenotype in human MCF10A mammary epithelial
cells. Interestingly, the alterations of bisecting GlcNAc andβ1-6 GlcNAc branched N-glycan were coupled with cells
spontaneous EMT-like changes at a sparse condition, whichmight be contributed to diagnosis and therapy of breast cancer.

Materials and methods
Cell line and cell culture
MCF10A, a human non-tumorigenic immortalized mammary
epithelial cell line, was cultured in DMEM/F12 medium, supplemented with 5% horse serum, 20 ng/ml EGF, 10 μg/ml
insulin, 0.5 μg/ml hydrocortisone, and 100 ng/ml cholera toxin, under a humidified atmosphere containing 5% CO2 at
37 °C. Cells were plated at 5 × 106, 1 × 106, 5 × 105 and1 × 105 on 15-cm dishes for density gradient culture, followed
by incubation for 3 days. Cells at 5 × 106 and 1 × 105 were
defined as a dense culture and a sparse culture, respectively.
PCR for mRNA expression analysis
Total RNA was prepared with Trizol (Invitrogen), and 2.0 μgof total RNA was reverse transcribed using the Superscript III
RNase H Reverse Transcriptase kit (Invitrogen) according to
the manufacturer’s instructions. The sequences of the primers
used for the PCR amplification were shown in Table 1. The
GAPDH mRNA was used as a control in PCR runs and thereaction products obtained were submitted to electrophoresis
in 1.5% agarose gels containing ethidium bromide.
Western blot and lectin blot analyses
Cells cultured under different conditions, as indicated, were
washed with PBS and then lysed with lysis buffer (10 mM
Tris-HCl, 1% Triton-X100, 150 mM NaCl, aprotinin, leupeptin
and 1 mM phenylmethylsulfonyl fluoride). Insoluble materials
were removed by centrifugation at 15, 000 rpm for 10 min at
4 °C. Equal amounts of protein were separated using 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), transferred to nitrocellulose and probed with the appropriate antibodies, as indicated, or with biotinylated erythro-Glycoconj J (2017) 34:219–227

221

Table 1

Primers used for RT-PCR experiments

Gene

Primer sequence (5′-3′)E-cadherin

ForwardACGCATTGCCACATACA

P-cadherin

ReverseForwardCGTTAGCCTCGTTCTCA
GGTGGTTCTCCGCAATG

ReverseGCCGCCTTCAGGTTCTC

N-cadherin

ForwardGAAAGACCCATCCACG

VimentinReverseForward

CCTGCTCACCACCACTAATGGCTCGTCACCTTCG

GnT-III

ReverseForward

AGTTTCGTTGATAACCTGTCCGCCGCGTCATCAACGCCATCAA

GnT-V

ReverseForwardCAGGTAGTCGTCGGCGATCCAGACCTGCAGTTCCTTCTTCG

GAPDH

ReverseForwardCCATGGCAGAAGTCCTGTTTAGCCACATCGCTCAGACA

ReverseTGGACTCCACGACGTACT

agglutinating phytohemagulutinin (E4-PHA) lectin, biotinylated
leuko-agglutinating phytohemagglutinin (L4-PHA), and biotinylated Datura stramonium (DSA) lectin (Seikagaku kogyo
Inc., Japan). Immunoreactive bands were visualized using a
Vectastain ABC kit (Vector Laboratories, CA) and an ECL kit(Amersham, UK). Monoclonal antibodies against E-cadherin, Pcadherin N-cadherin, β-catenin, p120-catenin, γ-catenin, and αcatenin were purchased from BD Biosciences, and the anti-αtubulin antibody was from Sigma. Monoclonal antibodies
against Fibronectin and HRP-labeled anti-mouse IgG were obtained from Cell Signaling.

biotinylated L4-PHA lectin, followed by streptavidin Alexa
Fluor 488 conjugate (Invitrogen). Negative controls underwent
the same procedure without biotinylated lectin. The analyses
were performed using a FACS Calibur instrument (BDBiosciences), equipped with CELL QuestPro software.
Immunoprecipitation
For immunoprecipitation, the supernatant (2 mg of protein) was
incubated with anti-β1 integrin (P5D2) antibody (3 μg/ml),
which was obtained from the Developmental Studies
Hybridoma Bank, University of Iowa, for 1 h at 4 °C. Protein
G beads (30 μl in 50% slurry) were then added, followed byincubation overnight at 4 °C with a rotator. After washing 3 times
with lysis buffer, the immunoprecipitates were subjected to 7.5%
SDS-PAGE, and the separated proteins were transferred to anitrocellulose membrane. The membrane was incubated with
E4-PHA or L4-PHA lectin for lectin blot analysis, or with anantibody for immunoblot analysis.
Statistical analysis
Statistical analysis was performed with SPSS 13.0 software.
Student’s t-test was performed for comparisons between two
groups. P < 0.05 was considered to be statistically significant.

Results
The MCF10A cells suffered spontaneous EMT-likephenotypic changes at low cell density

Microscopy and cell image
Cellular morphology was confirmed using phase-contrast observations. For immunofluorescence assay, cells were seeded on
glass-bottom dishes before fixation. After washing twice with
PBS, the cells were fixed for 30 min in 4% paraformaldehyde
solution at 37 °C. For permeabilization, the cells were treatedwith 0.2% (v/v) Triton X-100 in PBS. The fixed cells were
blocked with 2% BSA in PBS for 1 h and then incubated withmonoclonal antibodies against E-cadherin, N-cadherin and βcatenin in blocking buffer for 1 h at room temperature.
Following three washes in PBS, the cells were incubated witha 1:500 dilution of Alexa Fluor® secondary antibody or with a
1:300 dilution of Alexa Fluor® 488 phalloidin for actin(Invitrogen) for 1 h at room temperature. After washing threetimes with PBS, the cells were observed using an Olympus fluorescence microscope (FV1000 system).

To determine how gradual differences in MCF10A cell density
affected patterns of phenotypic changes, cells were plated at
5 × 106, 1 × 106, 5 × 105 and 1 × 105 on 15-cm dishes for density
gradient culture, followed by incubation for 3 days. Cells at
5 × 106 and 1 × 105 were defined as a dense culture and a sparse
culture, respectively. As shown in Fig. 1, cells cultured undersparse conditions had adequate space available for cell spreading.
In contrast, cells cultured under dense conditions had not enoughspace for full spreading, resulting in a reduction in cell size.
Furthermore, image analysis of cell morphology for different
densities revealed that MCF10A suffered spontaneous morphologic changes depending on cell confluence, showing Bfibroblast
like^ spindle morphology in sparse culture conditions andepithelial-like compact morphology in dense cultures, suggestingthat they might be susceptible to EMT at a low cell density.

Flow cytometry

Effects of spontaneous EMT-like phenotypic changes
on expression of EMT marker molecules in MCF10A cells

Flow cytometry was performed as previously described [17].
Briefly, cells were detached by trypsinizing and incubated with

We next investigated whether the expression of some important
genes identified as EMT markers may correlate with these

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Glycoconj J (2017) 34:219–227

Fig. 1 MCF10A cells suffer spontaneous, morphologic- EMT-likechanges in response to different cell densities. Cells were plated at
5 × 106, 1 × 106, 5 × 105 and 1 × 105 on 15-cm dishes for density gradientculture, followed by incubation for 3 days. The cell densities were
photographed by phase-contrast microscopy at 200 × magnification

phenotypic changes. By RT-PCR, we observed significant decrease in E-cadherin and P-cadherin expression in sparse cultured cells, whereas the N-cadherin and vimentin genes wereincreased (Fig. 2a). To further confirm the molecular events associated with the plasticity of MCF10A cells, we characterized
the relative proteins modulated during the phenotypic changes byWestern blot. As expected, the total level of E-cadherin and Pcadherin expression were greatly down-regulated in sparse condition, while the mesenchymal N-cadherin and fibronectin were
obviously up-regulated. In addition, the downstream elements ofE-cadherin, the p120-catenin, β-catenin, γ-catenin and α-catenin, were evaluated in different cell densities. The expression
levels of β-catenin were nearly no changes, however, theexpression levels of p120-catenin, γ-catenin and α-catenin wereeffectively decreased in a density-dependent manner (Fig. 2b).
Furthermore, we found that cadherin switching also occurred
during the morphologic changes (Fig. 3). The immunofluorescence data indicated that E-cadherin expression in cell-cell junctions became jagged and faint in the sparse condition, whereas,
N-cadherin expression became enhanced at cell borders. βcatenin staining was decreased in cell-cell contacts and accumulated significantly in the cytosol and nuclei during sparse culture.
F-actin distribution also dramatically changed from cell-cell borders to the stress fiber at low cell density. These results suggested
that MCF10A cells suffered spontaneous, EMT-like phenotypicand intrinsic properties alteration in a sparse condition.

Fig. 2 Effects of cell density on
expression of EMT marker
molecules in MCF10A cells. a
RT-PCR was carried out toexamine the expression levels ofE-cadherin, P-cadherin, Ncadherin, and vimentin in
different density cells. The
expression level of GAPDH wasused as a load control. b Cell
lysates from different density cells
were immunoblotted (IB) with
anti-E-cadherin, P-cadherin, Ncadherin, fibronectin, p120catenin, β-catenin, γ-catenin and
α-catenin antibodies. α-tubulin
was used as a load control. Cells
inoculated at 5 × 106, 1 × 106,
5 × 105 and 1 × 105 on 15-cm
dishes were defined as 1, 2, 3, and4 density gradient cultureGlycoconj J (2017) 34:219–227

223

Fig. 3 The distributions of E-cadherin, N-cadherin and β-catenin were
evaluated by immunofluorescence assay. Dense and sparse cultured cells
were stained with monoclonal antibodies against E-cadherin, N-cadherin
and β-catenin respectively, followed by incubation with Alexa Fluorconjugated secondary antibody. Localization of F-actin was examinedby staining with Alexa Fluor 488 phalloidin. The small triangle showed
N-cadherin expression, and the arrow indicated β-catenin expression.
Cells inoculated at 5 × 106, and 1 × 105 on 15-cm dishes were definedas Dense and Sparse culture. Scale bar, 50 μm

Spontaneous EMT-like phenotypic changes induced
specific N-glycan alterations

were evaluated in a spontaneous EMT process. As shown in
Fig. 4a/b, the expression of GnT-III gene was greatly declined,
and the relative ratio was decreased 2.4 fold in sparse condition compared to dense condition. Meanwhile, the products of
GnT-III detected by E4-PHA lectin, which preferentially recognizes bisecting GlcNAc, were declined in a cellular density
dependent manner (Fig. 4c). Normally, introduction of thebisecting GlcNAc suppresses further processing and elongation of N-glycans catalyzed by other branching enzymes such

Aberrant expression of glycans has been observed in cancer
development and progression [18]. In a TGF-β-induced EMT
model, we have previously observed that expression of
bisecting GlcNAc was dramatically down-regulated. To confirm whether the phenomenon described above would be also
observed in another EMT model, MCF10A epithelial cells

Fig. 4 Effects of spontaneous, EMT-like phenotypic changes on GnT-III
and bisecting GlcNAc. a RT-PCR was carried out to examine theexpression levels of GnT-III gene, and the expression level of GAPDH
was used as a load control. b The relative ratio of Gnt III densities
normalized to GAPDH was shown. The quantitative data were obtained
from three independent experiments, and the p value was calculated using
Student’s two-tailed t test. Error bars indicate SD. c Equal amounts ofprotein (20 μg) were separated on 7.5% SDS-PAGE, and the membranes

were probed with E4-PHA lectin, and α-tubulin was used as a load
control. The asterisks indicated nonspecific staining of E4-PHA, sincethose bands did not disappear after treatment with 100 mM acetic acid.
Cells inoculated at 5 × 106, and 1 × 105 on 15-cm dishes were defined as
Dense and Sparse culture. Cells inoculated at 5 × 106, 1 × 106, 5 × 105 and1 × 105 on 15-cm dishes were defined as 1, 2, 3, and 4 density gradientculture224

Glycoconj J (2017) 34:219–227

as N-acetylglucosaminyltransferase V (GnT-V), which isstrongly associated with cancer metastasis, because neitherof them can utilize the bisected oligosaccharide as a substrate[19]. Indeed, the expression of GnT-III gene was reduced in
low cell density, while the expression of GnT-V gene wasincreased in sparse cultured cells (Fig. 5a). As the result of
up-regulated GnT-V, the cell surface β1, 6 GlcNAc branching
structure, which is recognized by L4-PHA lectin in a FACS
analysis, increased 1.3-fold in sparse culture compared todense culture (Fig. 5b/c). Meanwhile, the reactivity with
DSA and L4-PHA lectin (Fig. 5d/e), which preferentially recognizes whole cellular branched GlcNAc, were dramaticallyenhanced in low cell density culture. These results indicatedthat spontaneous EMT induced by low cell density specifically down-regulated bisecting GlcNAc and up-regulated
GlcNAc branched N-glycan.

glycosylation of integrins is related to tumor metastasis [17].
To examine whether the alteration of N-glycans on the cellsurface was correlated with special glycans changes on glycoprotein, we further investigated N-glycans on the β1 integrin,
which is a good target of some glycosyltransferases. As shownin Fig. 6 a/b/c, the reactivity with L4-PHA lectin, which recognizes β1-6 GlcNAc branched structures, increased 1.3-foldon β1 integrin subunit in sparse cells compared to dense cells.
In contrast, the reactivity with E4-PHA lectin, which recognizes bisecting GlcNAc, decreased obviously on β1 integrin
in low cell density culture. These results suggested that spontaneous, EMT-like phenotypic changes can influence composition of N-glycans on some important glycoproteins.

EMT influence composition of N-glycans on β1 integrin
proteinIntercellular communication is dependent on a dynamic andcomplex network of cytokines, growth factors, chemokines,
and matrix remodeling enzymes [20]. Cellular confluent condition has an important influence on cell communication, andthen affects the cell normal physiological function. EMT is

There is a certain set of glycoproteins on cell surface, such as
cadherin, EGFR, galectin and integrins family, and theFig. 5 Effects of spontaneous
EMT-like phenotypic changes on
GlcNAc-branched N-glycan. a The
expression of GnT-V gene wasdetermined by RT-PCR. b Cellsurface expression levels of Nglycans were examined by FACS
analysis. Prior to analysis, cells
were incubated with biotinylated
L4-PHA lectin, followed byincubation with streptavidin Alexa
Fluor 488 conjugate. c The
quantitative data were obtained
from three independent
experiments, and the p value wascalculated using Student’s
two-tailed t test. Error bars indicate
SD. Equal amounts of protein(20 μg) were separated on 7.5%
SDS-PAGE, and the membranes
were probed with DSA lectin d or
L4-PHA lectin e, and α-tubulin wasused as a load control. The asterisks
indicated nonspecific staining of
DSA and L4-PHA, since thosebands did not disappear after
treatment with 100 mM acetic acid.
Cells inoculated at 5 × 106, and1 × 105 on 15-cm dishes were
defined as Dense and Sparse
culture. Cells inoculated at 5 × 106,
1 × 106, 5 × 105 and 1 × 105 on
15-cm dishes were defined as 1, 2,
3, and 4 density gradient culture

Discussion

Glycoconj J (2017) 34:219–227

225

Fig. 6 Effects of EMT on composition of N-glycans on β1 integrin. a
Cell lysates from different density cells were immunoprecipitated (IP)using anti-β1 integrin antibody. Immunoprecipitates were run on a
7.5% SDS-polyacrylamide gel and probed with the biotinylated E4PHA and L4-PHA lectins. b Bar graphs, quantification of bisecting
GlcNAc structures (E4-PHA reactivity) that are modifying β1 integrin.
c Bar graphs, quantification of β1-6 GlcNAc-branched N-glycan
structures (L4-PHA reactivity) that are modifying β1 integrin. Amountsof N-glycan structures were determined from the ratios of densities of E4PHA or L4-PHA reactivity after normalization to β1 integrin. The
modification of N-glycan structures in sparse cultured cells wereexpressed as the fold decrease or increase, compared with the density
cultured cells, which was taken as 1. The p value was calculated using
Student’s two-tailed t test. Error bars indicate SD. Cells inoculated at
5 × 106, and 1 × 105 on 15-cm dishes were defined as Dense andSparse culture

known to be closely related to enhancement of cell motility. In
the EMT process, the phenotype conversion from less mobile
epithelial cells to fibroblastic mesenchymal cells with higher
motility is accompanied by declining expression of epithelial
cell molecules and enhancing expression of mesenchymal molecules. This process is implicated in the promotion of tumor
invasion and metastasis [21]. A growing body of evidencesindicated that cellular microenvironments are closely linked to
EMT, such as growth factors [22], cytokines [23], cell density[11], and growth matrix [9] etc. However, the relationship between cellular microenvironments and EMT is a little bit complicated. In this study, we clearly observed spontaneous EMT
process in a low cell density condition. The E-cadherin expression in cell-cell contacts, or the total level, was greatly downregulated, meanwhile, the N-cadherin expression was distinctly
up-regulated in sparse culture. Cadherin switching usually refers to a switch from the expression of E-cadherin to that of Ncadherin. N-cadherin promotes motility when expressed in epithelial cells, and cells that express more E-cadherin remain lessmotile. Cadherin switching is an important event in EMT process. Previous studies implicated that EGF could induce EMT
in breast cancer cells, while EGFR activation disturbed cell-celladhesion by destabilizing the E-cadherin/catenin complex, promoted EMT and contributed to the acquisition of a motile phenotype [24]. The newly synthesized E-cadherin and catenincomplex move together to the cell surface. Our results indicatedthat the expression of p120-catenin and α-catenin were declined in a cellular density dependent manner, which would
destabilize the E-cadherin/catenin complex and facilitate EMT
process. The similar notion was proposed by David Sarrio andhis collaborators [11].
Cell surface glycan characteristics can vary during cell-cellinteractions, growth regulation and differentiation. Altered glycan signatures are also observed in tumor cells, and play a criticalrole in the escape of tumor cells from immune surveillance aswell as metastasis [19]. N-Acetylglucosaminyltransferase III(GnT-III) transfers N-acetylglucosamine (GlcNAc) from UDPGlcNAc to a β1,4 mannose in N-glycans to form a Bbisecting^
GlcNAc linkage. Bisecting GlcNAc alters not only the composition, but also the conformation of the N-glycan [25]. GnT-III isgenerally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways. Introduction of the bisecting GlcNAc suppresses further processing and elongation of N-glycans catalyzed
by N-acetylglucosaminyltransferase V (GnT-V) [26]. GnT-III has
been proposed as an antagonist of GnT-V, thereby contributing to
the suppression of cancer metastasis. In fact, overexpression of
GnT-III in highly metastatic melanoma cells reduced β1-6branching in cell-surface N-glycans and increased bisected Nglycans [27]. Furthermore, it is known that the β1-6 GlcNAcbranched N-glycans, which may enhance the complex formationsuch as integrins and galectins on cell surface, resulting in an
increase in cell motility and cell signaling [28, 29]. It has also
been reported that β1-6 GlcNAc-branched N-glycans levels are
increased in highly metastatic tumor cell lines [30]. Consistently,
cancer metastasis is greatly suppressed in GnT-V knockout mice[31]. Our recent research indicated that bisecting GlcNAc Nglycans structure catalyzed by GnT-III was dramatically declined
in TGF-β-induced EMT model [10]. In this study, it was aninteresting notion that bisecting GlcNAc N-glycans structure
was also down-regulated in cellular density-dependent EMT
model. Moreover, when epithelial cells converted into fibroblastic mesenchymal cells, the β1-6 GlcNAc-branched N-glycan
structure was obviously enhanced, which might increase cell
migration and contribute to EMT process. Based on the discussion above, we speculated that there are bidirectional regulation
mechanisms. Cell-cell contact can modulate N-glycan alteration,
whereas specific N-glycan alterations facilitated EMT process. A
detailed characterization of the bidirectional regulation

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Glycoconj J (2017) 34:219–227

mechanisms between specific N-glycan and EMT are required
for further studies.
A series of glycoproteins on cell surface were modified by
special glycans structure, such as cadherin, EGFR, galectin andintegrins family. Our FACS analysis results indicated that N-glycans expressed on the cell surface were altered in sparse condition, which might influence the function of some important glycoproteins and cell migration. It has been reported that β1-4bisecting GlcNAc on E-cadherin glycosylation is a mechanism
involved in TGF-β-induced EMT [32]. Janik et al. also proposedthat an increase in β1-6-GlcNAc sugar chains of the integrin
resulted in the stimulation of cell migration [33]. Our results
indicated that the β1-6 GlcNAc-branched N-glycan structure
were obviously increased on β1-integrin, which might be related
to the EMT. In this study, the mechanism of EMT induced bysparse cell culture could be ascribed to two aspects. One was thatsparse condition promoted the breakdown of cell-cell junctions,as described above; the other was that alteration of N-glycans
might influence some important surface glycoproteins stability
and function, which mediated cell migration, as reported previously [34, 35].
In conclusion, we successfully constructed an EMT modelutilizing different cellular density culture in MCF10A epithelial cells, and demonstrated that density-dependent specific Nglycan alterations were coupled in EMT process and promoted cells migration. Our results provided a new insight into themechanism of epithelial-mesenchymal transition.
Acknowledgements This work was supported by grants from the
National High Technology Research and Development Program ofChina (863 Program, No. 2014AA093604), The Technology
Foundation for Selected Overseas Chinese Scholar, Ministry of
Personnel of China (Q.S. Xu), and the Ocean Public Welfare Scientific
Research Project, State Oceanic Administration of China (Grant No.
201405003).
Compliance with ethical standards

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Conflicts of interest The authors declared that they have no conflict ofinterest.
Ethical approval This article does not contain any studies with humanparticipants or animals performed by any of the authors.

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mammary acinar morphogenesis through O-glycosylation offibronectin. Neoplasia. 13(4), 320–326 (2011). doi:10.1593/neo.101440
Xu, Q., Akama, R., Isaji, T., Lu, Y., Hashimoto, H., Kariya, Y.,
Fukuda, T., Du, Y., Gu, J.: Wnt/beta-catenin signaling downregulates N-acetylglucosaminyltransferase III expression: the implications of two mutually exclusive pathways for regulation. J.
Biol. Chem. 286(6), 4310–4318 (2011). doi:10.1074/jbc.
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Takahashi, M., Kizuka, Y., Ohtsubo, K., Gu, J., Taniguchi, N.:
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19. Takahashi, M., Kuroki, Y., Ohtsubo, K., Taniguchi, N.: Core fucose
and bisecting GlcNAc, the direct modifiers of the N-glycan core:
their functions and target proteins. Carbohydr. Res. 344(12), 1387–
1390 (2009). doi:10.1016/j.carres.2009.04.031
20. Balkwill, F.R., Capasso, M., Hagemann, T.: The tumor microenvironment at a glance. J. Cell Sci. 125(Pt 23), 5591–5596 (2012).
doi:10.1242/jcs.116392
21. Cai, Z., Wang, Q., Zhou, Y., Zheng, L., Chiu, J.F., He, Q.Y.:
Epidermal growth factor-induced epithelial-mesenchymal transition in human esophageal carcinoma cells–a model for the studyof metastasis. Cancer Lett. 296(1), 88–95 (2010). doi:10.1016/j.
canlet.2010.03.020
22. Buonato, J.M., Lan, I.S., Lazzara, M.J.: EGF augments TGFbetainduced epithelial-mesenchymal transition by promoting SHP2binding to GAB1. J. Cell Sci. 128(21), 3898–3909 (2015).
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23. Matthaei, M., Gillessen, J., Muether, P.S., Hoerster, R., Bachmann,
B.O., Hueber, A., Cursiefen, C., Heindl, L.M.: Epithelialmesenchymal transition (EMT)-related cytokines in the aqueous
humor of Phakic and Pseudophakic Fuchs' dystrophy eyes. Invest.
Ophthalmol. Vis. Sci. 56(4), 2749–2754 (2015). doi:10.1167/iovs.15-16395
24. Hardy, K.M., Booth, B.W., Hendrix, M.J., Salomon, D.S., Strizzi,
L.: ErbB/EGF signaling and EMT in mammary development andbreast cancer. J. Mammary Gland Biol. Neoplasia. 15(2), 191–199(2010). doi:10.1007/s10911-010-9172-2
25. Gu, J., Sato, Y., Kariya, Y., Isaji, T., Taniguchi, N., Fukuda, T.: A
mutual regulation between cell-cell adhesion and N-glycosylation:
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26. Pinho, S.S., Figueiredo, J., Cabral, J., Carvalho, S., Dourado, J.,
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Carneiro, F., Seruca, R., Taniguchi, N., Reis, C.A.: E-cadherin
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N.: Suppression of lung metastasis of B16 mouse melanoma by Nacetylglucosaminyltransferase III gene transfection. Proc. Natl.
Acad. Sci. U. S. A. 92(19), 8754–8758 (1995)
28. Kariya, Y., Gu, J.G.: N-glycosylation of beta 4 integrin controls theadhesion and motility of keratinocytes. PLoS One. 6(11), e27084(2011). doi:10.1371/journal.pone.0027084
29. Dennis, J.W., Lau, K.S., Demetriou, M., Nabi, I.R.: Adaptive regulation at the cell surface by N-glycosylation. Traffic. 10(11), 1569–
1578 (2009). doi:10.1111/j.1600-0854.2009.00981.x
30. Pochec, E., Litynska, A., Amoresano, A., Casbarra, A.:
Glycosylation profile of integrin alpha(3)beta(1) changes with melanoma progression. Biochim. Biophys. Acta. 1643(1–3), 113–123(2003). doi:10.1016/j.bbamcr.2003.10.004
31. Granovsky, M., Fata, J., Pawling, J., Muller, W.J., Khokha, R.,
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Gartner, F., Seruca, R., Reis, C.A., Oliveira, C.: Loss and recoveryof Mgat3 and GnT-III mediated E-cadherin N-glycosylation is amechanism involved in epithelial-mesenchymal-epithelial transitions. PLoS One. 7(3), e33191 (2012). doi:10.1371/journal.
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33. Janik, M.E., Litynska, A., Vereecken, P.: Cell migration-the role ofintegrin glycosylation. Biochim. Biophys. Acta. 1800(6), 545–555(2010). doi:10.1016/j.bbagen.2010.03.013
34. Isaji, T., Gu, J., Nishiuchi, R., Zhao, Y., Takahashi, M., Miyoshi, E.,
Honke, K., Sekiguchi, K., Taniguchi, N.: Introduction of bisecting
GlcNAc into integrin alpha5beta1 reduces ligand binding anddown-regulates cell adhesion and cell migration. J. Biol. Chem.
279(19), 19747–19754 (2004). doi:10.1074/jbc.M311627200
35. Zhao, Y., Nakagawa, T., Itoh, S., Inamori, K., Isaji, T., Kariya, Y.,
Kondo, A., Miyoshi, E., Miyazaki, K., Kawasaki, N., Taniguchi,N., Gu, J.: N-acetylglucosaminyltransferase III antagonizes the effect of N-acetylglucosaminyltransferase V on alpha3beta1 integrinmediated cell migration. J. Biol. Chem. 281(43), 32122–32130(2006). doi:10.1074/jbc.M607274200

ǤǤ Ȁ  



Received: 30 May 2017
Accepted: 31 August 2017
Published: xx xx xxxx

Inhibition of fucosylation by͸Ǧƪ 
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͸Ƭ  ͷ    ǤǡǦ ǡ͸ǦƪǦǦ ȋ͸ Ȍǡ 
 Ǧ ǡƤ  
  ͸   Ǥ ƪ 
 ͸    ǡ 
Ƥ ǤǦ 
Ƥ  ͸ Ǥ  
ƥ  ǡ͸ Ǧ ǡ  Ǥǡ͸    
    βͷǡ 
 Ǧ ǡǦǡǦǡǦ Ǥ  
͸      ǡ͸ ǤGlycosylation is by far the most prolific form of protein modification in mammalian cells. Accumulating data
have made it clear that glycan structures expressed on glycoproteins have essential roles in various biological processes such as inflammation, growth, differentiation, carcinogenesis, and cancer metastasis1, 2. Alteration during
glycosylation is now regarded as a feature event in the progression of cancer. Among all forms of carbohydrate
modification involved with the progression of cancer, fucosylation is considered one of the most important3, 4.
In regards to liver cancer, core fucosylation is a pre-eminent factor. Core fucosylation, also known as
α1,6-fucosylation, is catalyzed by α1,6-fucosyltransferase (Fut8) to transfer fucose residue from guanosine
5/-diphosphate (GDP)-fucose to the innermost asparagine-linked GlcNAc via an α1,6 link, which is a processthat has been implicated in the progression of liver cancer5. Early work by Breborowicz, J. et al. suggested thatcore fucosylated alpha-fetoprotein (AFP) was a reliable marker that could be used to distinguish hepatocellular
carcinoma (HCC) from chronic hepatitis and liver cirrhosis6.
Recent studies have shown that core fucosylation is up-regulated both in the serum and on the cell surface of liver cancer cells7, 8, although contradictory results were also reported whereby an increased level oftetra-antennary N-glycan, rather than core fucosylation, was associated with hepatocellular carcinoma9.
Consistently, Yamashita, F. et al. associated a high expression of core fucosylation with a poor prognosis 10.
Furthermore, our previous studies indicated that loss of Fut8 gene significantly suppressed liver regeneration11
and chemical-induced HCC12 via the down-regulation of several cell-signaling pathways. Taken together, these
Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical  ǡͺǦͺǦͷǡǦǡǡǡͿ;ͷǦ;ͻͻ;ǡ Ǥ͸
   ǡ      ȋ ȌǡͷǦͷǦͷǡǡ ǡ͹ͶͻǦ;ͻͼ;ǡ Ǥ Ǥ Ǥȋǣ̻ǦǤ ǤȌ
ͷ

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studies clearly show that core fucosylation plays crucial roles in liver cancer, which enabled us to predict that the
inhibition of core fucosylation could provide a novel strategy for liver cancer therapy9.
How to inhibit core fucosylation in HCC? As described above, biosynthesis of core fucosylated N-glycans
requires donor substrate GDP-fucose, N-glycans and Fut8. Fut8 is localized in the inner lumen of Golgi bodies,thus, conventional wisdom holds that an inhibitor of Fut8 cannot be efficiently delivered to a Golgi apparatus.
On the other hand, GDP-fucose in the cytoplasm is known to be synthesized by two distinct pathways13, 14. One
is called the de novo pathway, and this pathway converts GDP-mannose into GDP-fucose via enzymatic reactions catalyzed by GDP-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3, and 5-epimerase
4-reductase (FX)15–17. Blocking this pathway forces cells to make use of another pathway, which is called thesalvage pathway. This pathway uses fucose kinases to convert L-fucose into GDP-fucose18, 19. GDP-fucose is thendelivered into the Golgi apparatus via GDP-fucose transporters. Finally, GDP-fucose serves as a donor substrate
and is transferred into the oligosaccharides of protein to synthesize core fucose by the action of Fut820. Thus,
the inhibition of GDP-fucose production is desirable in order to block fucosylation. Previous efforts to deletecore fucosylation have focused mostly on the manipulation of Fut8 by knockout or knockdown of its gene.
Additionally, there have been efforts to knockout the key enzymes for GDP-fucose production such as GMD and
FX and impair the Golgi GDP-fucose transporter21–25. The methods described above, however, are not suitablefor pharmacological application.
A variety of glycosyltransferases inhibitors have been developed, and mainly based on donor or accept substrates mimics26. Several GDP-fucose analogs have been reported to be inhibitors of FUTs27, 28. However, thosecharged groups (GDP portion) prevent uptake into cells, which limits their use in biological systems. On the other
hand, a specific fluorinated analog of fucose, 2-fluoro-L-fucose (2FF), has been reported to easily enter cells via
passive diffusion wherein it is metabolized into a corresponding donor substrate analog of GDP-fucose, GDP-2FF,
via the salvage pathway29–31. Since GDP-2FF accumulates in cells, it will also lead to a shutdown of the de novo
pathway that synthesizes natural GDP-fucose29. In fact, the addition of 2FF has efficiently suppressed the endogenous production of GDP-fucose, which dramatically inhibited the formation of fucosylation in both cancer andplant cells31–33. Therefore, 2FF has been used to reduce cell-surface fucosylated glycans such as Lewis antigens for
E-selectin binding in colon carcinoma cells31, and has blocked core fucosylation in HL-60 cells29. However, theeffect that 2FF exerts on liver cancer cells remains unclear.
In this study, we examined the effects of 2FF in live cancer HepG2 cells and further clarified the underlying
molecular mechanisms. We found that treatment with 2FF greatly decreased core fucosylation levels and bothsuppressed downstream signaling and tumor formation, which suggested that 2FF might be a novel candidate forliver cancer therapy.



   ͸ Ǥ Several analogues of L-fucose have shown inhibitory
effects on fucosylation. One such analogue is 2FF, as shown in Fig. 1A. To investigate whether 2FF also inhibits
fucosylation levels in HepG2 hepatoma cells, we first carried out lectin blot testing to detect fucosylation levels
by probing with aleuria aurantia lectin (AAL), a specific lectin that preferentially binds to Fuc α1-6GlcNAc34.
The fucosylation levels were inhibited by 2FF in a dose-dependent manner. The inhibitory effects of 2FF wereobserved even at a final concentration at 10 µM, and remarkably appeared at 100 µM (Fig. 1B). In addition, timecourse studies have indicated that the effect of 2FF persisted for more than 7 days without further adding 2FF(Fig. 1C). After the removal of 2FF, its effect at least lasted for another 3 days (Fig. 1D). These alterations in thefucosylation expression on the cell surface were further confirmed by flow cytometric analysis using AAL lectin.
The reactive abilities of AAL lectin were persistently decreased in the HepG2 cells during 7-day culture in thepresence of 2FF, compared with the control cells cultured without 2FF (Fig. 1E). To explore whether the decreased
fucosylation was due to a suppression of fucosyltrasferase gene expression by 2FF, we performed semi-quantitative RT-PCR to detect typical fucosylation related genes such as α1,3-fucosyltransferase 4 (Fut4), α1,3-fucosyltransferase 7 (Fut7), and Fut8 because 2FF was known to inhibit not only the products of Fut8 but also the
products of Fut4 and Fut7 in HL-60 cells29. As shown in Fig. 1F, the treatment with 2FF did not significantly affectgene expression level. These data again indicated that 2FF strongly down-regulates fucosylation levels via aninterruption of intracellular GDP-fucose synthesis, and/or that GDP-2FF may inhibit fucosyltransferases, which
includes Fut8, in HepG2 cells.

͸Ǧ   ͸ ͸ Ǥ To further verify the effects

of 2FF in regulating carbohydrate structures, the N-glycan profiles of those cells were analyzed by mass spectrometry. MALDI-TOF mass spectrometry of N-glycans clearly supported the suppression of core fucosylation
by treating HepG2 cells with 2FF (Fig. 2A). Without 2FF, fucosylated complex N-glycans were accompanied bylarge signals from high mannose-type glycans (Fig. 2A upper). These fucosylated glycans, mostly core fucosylated
glycans, were considerably displaced by corresponding afucosylated glycans in 2FF-treated HepG2 cells (Fig. 2A
lower). The percentages of fucosylated complex glycans among all complex glycans in an m/z range of from 1,500
to 3,500 were reduced from 75 to 8% by treatment with 2FF (Fig. 2B). Curiously, sialylated complex glycans wereobserved in an m/z of more than 3,500 in both measurements of the MS spectra. Afucosylated sialylated glycans
were also significantly increased by treatment with 2FF. In fact, upon 2FF treatment, reactivity against both concanavalin A (ConA) and wheat germ agglutinin (WGA) showed no change. An SNA lectin blot showed a tendency
whereby sialylation was up-regulated upon 2FF treatment in a dose-dependent manner (Fig. 2C). That phenomenon could be interpreted as sialyation is up-regulated by a lack of terminal fucosylation, which was blocked by
2FF, since fucosyltransferase and sialytransferase can compete for the same terminal structure as an accepted
substrate29. These results clearly demonstrated that fucosylation, particularly core fucosylation, is down-regulated
via 2FF treatment to HepG2 cells.

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Figure 1. Treatment with 2FF suppressed fucosylation on glycoproteins. (A) The structure of 2FF as previously
described29. (B) HepG2 cells were cultured with 2FF for 3 days at different concentrations, as indicated. Cell
lysates were prepared, and then subjected to lectin blot analysis using AAL, which preferentially recognizes corefucose, as described in “Methods”. GAPDH was used as a loading control. Control refers to cells treated with thesame amount of DMSO. (C) HepG2 cells were cultured in the presence of 100 µM 2FF for different time points(0 h-7 days). Equal amounts of proteins were probed with AAL lectin as described above. (D) HepG2 cells
were cultured in the presence of 100 µM 2FF for 2 days, then the culture medium containing 2FF was removed
and change into normal culture medium for different time points (24 h, 2d, 3d, 5d, 7d). (E) HepG2 cells wereincubated with 100 µM 2FF at different time points, and then stained with (grey line) or without (dark line)
biotin-conjugated AAL followed by streptavidin-conjugate Alexa Fluor 647 and subjected to flow cytometry
analysis. (F) RT-PCR was carried out using total RNA extracted from HepG2 cells treated with or without 2FF
to detect the expression level of representative genes involved in fucosylation. The expression level of GAPDH
was examined as a loading control. The relative mRNA levels were normalized with the GAPDH control gene.

  Ǥ In consideration of theimportance of core fucosylation in physiological and pathological functions 11, 12, 35, we speculated a potential
role for 2FF in the regulation of cell proliferation. To test this hypothesis, we investigated the proliferation of
HepG2 cells, which were pre-treated with or without 2FF for 3 days followed by 24 h serum-starvation. As shownin Fig. 3A, cell proliferation was significantly suppressed following treatment with 2FF, compared with control
cells without 2FF treatment (Fig. 3A). There was no significant difference in cell viabilities between the control
and 2FF-treated cells (data not shown). This phenomenon was also observed in Hela cells (Fig. 3D). The ability
to grow in an anchorage-independent manner has always been regarded as one of the hallmarks of cancer, andthis ability is believed closely related to cell proliferation and cell-survival signals. Therefore, we performed aclonogenicity study using a soft agar assay. Colony formation abilities were significantly suppressed in the HepG2
cells treated with 2FF, compared with control cells (Fig. 3B). Each colony size treated with 2FF was much smaller
than the control without 2FF (Fig. 3C). In contrast, Rillahan, C.D. et al., reported that inhibition of fucosylationin HL-60, Ramos, or CHO-K1 cells did not significantly affect cell proliferation29. These contradictory data could
be speculated that the different phenotypes could be due to cell types. Since different cell types differentiallyexpress growth factor receptors to control cell proliferation, for example, HepG2 cells highly express EGFR andc-Met, while HL-60 cells and CHO-K1 cells do not express EGFR36, 37. In fact, HL-60 cells abundantly express
insulin-like growth factor I receptor (IGF-IR), which may be modified by different glycans compared with thoseon EGFR, to contribute to the cell growth38, 39. Furthermore, even same receptor may also be modified by different
glycans in different cells.
͸Ǧ Ǥ As described above, 2FF treatment had a major
impact on cell growth. Then, we used a transwell assay to examine the migratory ability of HepG2 cells following 2FF treatment. As shown in Fig. 4A, the integrin-mediated cell migration was significantly inhibited in͸

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Figure 2. Comparison of N-glycan profiles in HepG2 cells treated with and without 2FF. (A) MS spectrum of
the permethylated glycans from the cells treated with DMSO only (upper) and DMSO containing 2FF (lower).
(B) Percentages of fucosylated complex glycans in total complex glycans from HepG2 cells. The values were
calculated from peak areas in the range of m/z 1,500-3,500 of the MS spectra. (C) HepG2 cells were cultured
with 2FF for 3 days at different concentrations as indicated, and then equal amounts of cell lysates were probedwith Con A, which specifically recognizes alpha-linked mannose and glucose; WGA, which selectively binds to
N-Acetyl glucosamine (GlcNAc) and SNA, which specifically recognizes α 2, 6 sialylation. GAPDH was used asa loading control.
2FF-treated cells compared with control cells. In a similar manner, the migratory ability that was suppressed by
2FF was also observed in Hela cells (Fig. 4B). Collectively, these data strongly suggested that 2FF could effectively
inhibit cell proliferation and cell migration in vitro.

͸    βͷ ǦǤ N-Glycosylation is known to be essential for the functional expression of membrane glycoproteins suchas EGFR and integrins, and regulates several functions: cell-surface expression, dimerization, endocytosis andspecific ligand binding40, 41. Recently, we showed that a loss of core fucosylation via knockout of the Fut8 genedecreased the interaction between EGFR and its ligand, including downstream signaling, which in turn suppressed cell proliferation11, 12. In addition, the deletion of core fucosylation significantly suppressed integrin
α3β1-mediated cell migration42. Therefore, issues having to do with the down-regulation of cell proliferation andmigration were related in these molecules.
First, we performed a pull down assay using the biotinylated PhoSL, which preferentially recognizes core fucosylated N-glycans, to examine the effect of 2FF on the core fucosylation of EGFR and integrin β143. As shown in
Fig. 5A, core fucosylation was expressed on EGFR (upper panel) and integrin β1 (lower panel) in the control cells,
but not in the 2FF-treated HepG2 cells, suggesting that 2FF efficiently blocked core fucosylation in these membrane proteins. Then, we asked whether 2FF affected intracellular signaling. To address this point, we examinedthe expression levels of phospho-EGFR and its downstream signaling such as phospho-AKT and phospho-ERK,
as well as integrin’s downstream signal phospho-FAK. Interestingly, all of these signals were suppressed in the
2FF-treated HepG2 cells, compared with the control cells (Fig. 5B). Based on these observations, it is not difficult
to conclude that the decreased core fucosylation by 2FF might impair the biological functions of some important
membrane receptors such as EGFR and integrin β1, which subsequently inhibits intracellular signaling.

ơ ͸   in vivoǤ Based on the inhibitory
effects of 2FF on malignant phenotypes observed in vitro, we used a well-established xenograft tumor model tofurther examine the tumorigenicity of HepG2 cells in vivo. The same amounts of control and 2FF pre-treated
HepG2 cells (4 × 106) were injected subcutaneously into the left and right flanks of each mouse, respectively.
Based on the inhibitory effect of 2FF on fucosylation that lasted for 7 days at least (Fig. 1C); 100 µl of the DMEM
solution containing with 2FF at 100 µM were directly injected into each tumor tissue from three directions after
the inoculation for 7 days, and once a week for 3 weeks. All the mice were euthanized at 25 days after the first
injection of 2FF. Tumor tissues were isolated, photographed and then weighed. Interestingly, significant decreases
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Figure 3. Effects of 2FF treatment on cell proliferation. HepG2 and Hela cells were pre-treated with or without
100 µM 2FF for 3 days and then starved for 24 h and replaced with DMEM containing 3% FBS to assay cellproliferation. (A) The pre-treated cells were incubated in 2FF containing medium for another 4 days. The time
point at the replacement was deemed as 0 h. Cells at the same areas were photographed and counted every 24 h.
The results are shown as fold changes relative to the numbers of those at 0 h, and presented as the mean ± s.e.m.
(*p < 0.05 by t-test). (B) Soft agars were prepared as described in the “Methods”. Controls and 2FF-treated
HepG2 cells (5,000 per/well) were cultured on the soft agar for 3 weeks. The colonies that formed were stainedwith crystal violet. The stained colonies were counted (**p < 0.01 by t-test). Three independent experiments
were performed. (C) Representative images showed different sizes of colonies treated with or without 2FF. Scalebars indicate 500 µm. (D) Pre-treated Hela cells were incubated in 2FF containing medium for another 4 days.
The time point at the replacement was deemed as 0 h. Cells were photographed at the same areas and counted
every 24 h. The results are shown as fold changes relative to the numbers of those at 0 h, and presented as themean ± s.e.m. (*p < 0.05 by t-test, **p < 0.01 by t-test).

were noted in both the size (Fig. 6A,B) and weight (Fig. 6C) of tumor tissues in the 2FF-treated side, comparedwith the control side 25 days after inoculation. The time course of tumor growth also clearly showed the inhibitory effects of 2FF on tumor formation (Fig. 6D). Furthermore, AAL lectin blot showed that the levels of corefucosylation were apparently decreased in the tumor tissues that grew from the HepG2 cells treated with 2FF,
compared with the controls (Fig. 6E). Importantly, in accordance with the results in vitro (Fig. 5), the cellularsignals including p-EGFR, p-AKT, p-ERK and p-FAK in the 2FF-treated tumor tissues were decreased comparedwith those in the controls treated without 2FF (Fig. 6F). These results addressed the efficient effects of 2FF in vivo.

 

Liver cancer ranks as the second leading cause of death from cancer44. Effective therapy for liver cancer remains
one of the biggest challenges for public health45. Increased levels of core fucosylation, usually higher expressionsof either Fut8 or GDP-fucose, have lately been recognized as reliable indicators for liver cancer3. Therefore, regulation of the core fucosylation of N-glycans can serve as an essential strategy for liver cancer therapy. For example,
knockout of the Fut8 gene in mice strongly suppressed a chemical-induced hepatocellular carcinoma12. Also,
down regulation of the Fut8 expression by miRNAs inhibited the progression of liver cancer35. In addition, an
abrogation of ability to form or transport GDP-fucose was also worked9. Those findings prompted us to investigate whether there is a pharmacological approach that could block core fucosylation in liver cancer cells.
In this study, we used the easily handled 2FF, an analog of fucose, to inhibit GDP-fucose formation. Considereda universal inhibitor of fucosylation, 2FF is used to reduce cell-surface fucosylated glycans such as Lewis antigens
and core fucosylated N-glycans29. Our results, as determined by western blot and flow cytometry in liver cancer
cells, also confirmed this conclusion (Fig. 1B–E). The inhibitory effects on fucosylated glycans did not seem tobe due to an inhibition of the expression levels of fucosyltransferases, since there were no significant changes
between cells treated with or without 2FF in the mRNA levels of typical fucosylation-related genes such as Fut4,
Fut7 and Fut8, which produce Lewis X (LeX) or sialyl Lewis X (SLeX) and core fucosylated N-glycans, respectively(Fig. 1F). Surprisingly, changes in the N-glycan structures analyzed by mass spectrometry have shown that the
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Figure 4. Effects of 2FF treatment on integrin-mediated cell migration. (A) The migration abilities of HepG2
cells toward FN were detected by transwell assay as described in “Methods”. HepG2 cells pre-treated with andwithout 2FF for 3 days were seeded into the top chambers of a transwell. After incubation for 24 h, the migrated
cells were fixed and stained with 0.5% crystal violet. Representative images of the bottom surface were captured
using phase-contrast microscopy (upper). The numbers of migrated cells in nine random microscopic fields(×200) were counted and averaged. Data represent the mean ± s.e.m. of three independent experiments(lower) (**p < 0.01 by t-test). Scale bars indicate 50 µm. (B) The migration abilities of Hela cells toward FN
were detected. Hela cells pre-treated with or without 2FF for 3 days were seeded into the top chambers of thetranswell, and incubated for 4 h. The migrated cells were calculated as described above. Data represented themean ± s.e.m. of three independent experiments (**p < 0.01 by t-test).

majority of fucosylation in HepG2 cells is core fucosylation, while α1,2; α1,3 or α1,4 fucosylated N-glycans wereat undetectable levels (Fig. 2). However, unexpectedly, sialylation levels seemed to be enhanced in the 2FF-treatedcells (Fig. 2). It could be partly explained by that fucosyltransferases and sialyltransferases may compete for thesame terminal glycan structures as an accepted substrate29. In fact, Rillahan, et al. reported that a treatment withthe sialylation inhibitor resulted in a complete loss of sialic acids and a notable increase in overall fucosylation.
Conversely, knockout of Fut9, a key enzyme for the synthesis of LeX or SLeX, resulted in an increase in sialylation46. The detailed molecular mechanisms are required for further study. Thus, it is not difficult to speculate thatthe effects of 2FF in HepG2 cells share similar phenomena as observed in Fut8 deficient liver cells. As expected,
the treatment with 2FF significantly suppressed cell proliferation and colony formation (Fig. 3)11, 12. Alterationsof glycans can have a significant impact on cell proliferation, migration and invasion, all of which contribute to
the transformation progress and subsequent metastasis of malignancy47–49. In order to further understand thelink between 2FF-induced core fucosylation deficiency and cellular progression, we examined cell migration,and found that 2FF inhibits integrin-mediated cell migration (Fig. 4). These results clearly indicated that 2FF
effectively suppresses both cell proliferation and cell migration.
Previously, we reported that core fucosylation was required for the binding of EGF to a receptor and forthe regulation of EGFR-mediated intracellular signaling in embryonic fibroblast cells50. In addition, the attenuated EGFR-mediated signaling was confirmed in Fut8 knockout HepG2 cells12. Conversely, the incorporationof core fucose by Fut8 promoted EGFR dimerization and phosphorylation in lung cancer cells51. Furthermore,N-glycosylation is now known as a critical determinant of EGFR conformation, and specifically the orientation of
the EGFR ectodomain relative to the membrane52. In agreement with these observations, this study showed thattreatment with 2FF also decreased the levels of phospho-EGFR and downstream signaling such as phospho-ERK
and phospho-AKT (Fig. 5B). It is reasonable to conclude that the inhibitory effect of 2FF on cell proliferation
can at least be partially mediated via attenuated EGFR-mediated signaling. On the other hand, integrins are
known to associate with EGFR in order to cooperatively regulate cell proliferation, migration, differentiation andsurvival in fibroblasts and epithelial cells53. Also, with EGFR they share some of the elements in signal pathways
such as AKT and ERK54. Among the various types of integrins, integrin β1 plays crucial roles in cell migration
and adhesion55–57. A knockout of β1 has sufficiently inhibited cell migration and affected the EFGR-mediated cell
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Figure 5. Treatment with 2FF suppressed core fucosylation on EGFR and integrin β1, as well as down stream
signaling. HepG2 cells were cultured with and without (control) 2FF for 3 days, and harvested for lectin pull
down assay or immunoblotting as described in “Methods”. (A) The cell lysates were pooled and incubated with
10 µl of indicated bead-bound PhoSL, which specifically recognizes core fucose. The lectin precipitated samples
and equal amounts of cell lysates were then subjected to SDS-PAGE, and probed with anti-EGFR (upper panel)and anti-integrin β1 (lower panel) antibodies. GAPDH were used as a loading control. (B) Cell lysates were
immunoblotted with anti-phospho-EGFR, anti-EGFR, anti-phospho-AKT, anti-AKT, anti-phospho-ERK1/2,
anti-ERK1/2, anti-phospho-FAK, and anti-FAK antibodies, respectively. GAPDH was used as a loading control.

proliferation in a cell density-dependent manner in MDA-MB231 breast cancer cells58. In the present study, 2FF
suppressed cell migration and the attenuated integrin-mediated specific downstream signaling of phospho-FAK(Fig. 5B), which is responsible for integrin-mediated cell migration59, 60. In fact, we previously reported that deletion of Fut8 significantly suppressed integrin α3β1-mediated cell migration42. Nonetheless, we cannot exclude
the possibility that 2FF may influence the functions of other glycoproteins, since core fucose widely exists on thesurfaces of cells.
It was also worth noting that although we addressed the importance of core fucosylation in cell behaviors since
2FF mainly inhibited core fucosylation in HepG2 cells, we could not conclude that other fucosylations expressedat marginal levels were unimportant. Previous studies have shown that oral administration of 2FF inhibits neutrophil extravasation via the suppression of Lewis antigen expression and delays tumor growth in immune deficient
mice31. In order to further address the effects of 2FF in vivo, we subcutaneously inoculated HepG2 cells pretreatedwith 2FF (intratumoral administration before treatment) into nude mice. After tumors were formed, 2FF solutions were directly injected into each tumor tissue. Consistently, treatment with 2FF prevented tumorigenesis as
well as core fucosylation in vivo (Fig. 6). These data strongly suggested that the chemical compound 2FF is easy tohandle and exerts a potential inhibitory effect on tumor growth.
In addition, we also cannot exclude the potential effects on sialylation. As described above, sialylation andfucosylation can be regulated mutually. However, the phenomenon, an increase in sialylation in the 2FF-treatedcells, observed in the present study could not be simply explained by mutual regulation. The increased sialylation may associate with some unknown mechanisms, and could also influences cell biological functions, which
requires further study. In fact, an aberrant expression of sialylation has long been associated with metastatic cell
behaviors such as invasion and enhanced cell survival, and the increased α2,6 sialylation on N-glycans catalyzed
by β-galactoside α2,6 sialyltranferase 1 (ST6Gal I) is frequently observed in many types of the cancers61, 62.
In summary, our study provided evidence that 2FF inhibited core fucosylation in the HepG2 cells ofcell-surface glycoproteins, which in turn inhibited cellular signaling for cell proliferation and migration as well
as for tumor growth. We believe that 2FF shows promise as a therapeutic compound for hepatoma and othercancers.

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Figure 6. Effects of 2FF on tumor growth and fucosylation in vivo. HepG2 cells (4 × 106) were inoculated
into both the left (control cells, -) and right posterior flanks (2FF-treated cells, + ) of nude BALB/c-nu mice as
described in “Methods”. (A) Photographs illustrate the representative features of tumor growth 25 days after
injection (upper panel), and each corresponding pair of dissected tumor tissues (lower panel). Changes ineach of the corresponding pair of tumor volumes (B) and weights (C) were also measured. (D) Tumor growth
curves were measured every 5 days (n = 6, *p < 0.05 by t-test). (E) Tumor tissues were homogenized and lysates
were blotted with AAL lectin. GAPDH was used as a loading control. (F) The lysates from tumor tissues were
immunoblotted with anti-phospho-EGFR, anti-EGFR, anti-phospho-AKT, anti-AKT, anti-phospho-ERK1/2,
anti-ERK1/2, anti-phospho-FAK, and anti-FAK antibodies, respectively. GAPDH was used as a loading control.



Ǥ Experiments were performed using the following antibodies: Rabbit anti-

bodies against EGFR, p-EGFR, AKT, p-AKT, ERK1/2, and p-ERK1/2 were purchased from Cell Signaling
Technology (Danvers, MA, USA); Mouse antibodies against integrin β1, FAK and p-FAK were purchased fromBD Biosciences (Franklin Lakes, NJ, USA); Rabbit antibody against glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was purchased from Santa Cruze Biotechnology (Santa Cruz, CA, USA). Peroxidase-conjugated goat
antibodies against rabbit and mouse IgG were obtained from Cell Signaling Technology and Promega, respectively. Biotinylated aleuria aurantia lectin (AAL), concanavalin A (ConA) and wheat germ agglutinin (WGA) werepurchased from Seikagaku Crop (Tokyo, Japan). Biotinylated Pholiota Squarrosa lectin (PhoSL), which specifically recognizes core fucosylated N-glycans, was a generous gift from Dr. Yuka Kobayashi (J-oil Mills, Tokyo,
Japan). Alexa Fluor 647 goat anti-mouse secondary antibody and fetal bovine serum (FBS) were purchased from
Invitrogen (Life Technologies). 2FF was purchased from Synchem, Inc., IL, USA. Fibronectin (FN) and dimethyl
sulfoxide (DMSO) were obtained from sigma (St. Louis, MO, USA); and, Dulbecco’s modified Eagle’s medium(DMEM) was purchased from Gibco (Grand Island, NY, USA).

®

 Ǥ Human hepatoma HepG2 and Hela cell lines were purchased from the RIKEN

cell bank (Japan). Cells were cultured in DMEM containing 10% FBS and incubated at 37 °C in a humidified
atmosphere with 5% CO2. The cells were incubated with and without 2FF dissolved in DMSO for indicated times.
The final concentrations of 2FF were diluted with culture media at 0.5~500 µM. The same amounts of DMSO wereused as controls.

 Ǥ Cells cultured in the presence or absence of 2FF under different

conditions, as indicated, were harvested, washed by cold PBS and then scraped off culture plates with lysis buffer
containing 20 mM Tris-HCl (pH = 7.4), 1% Triton X-100, 150 mM NaCl, and protease and phosphatase inhibitors(Nacalai Tesque). After incubation for 15 min at 4 °C, cell debris were removed by centrifugation for 10 min at
13,000 g. Protein concentrations were measured using a BCA protein assay kit (Pierce). Equal amounts for eachsample were run on 6, 7.5 or 10% SDS-PAGE gels, as indicated.
For the pull-down assay, 500 µg of cell lysates were collected and incubated with 10 µL FG streptavidin beads(Tamagawa Seiki Co., Tokyo, Japan) and 2 µg biotinylated PhoSL for 2 h at 4 °C. The precipitates were then washed
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by cold lysis buffer and boiled in SDS loading buffer. Resolved proteins were then transferred onto a PVDF membrane (Millipore) for detection.
For Western blot assay, the membranes were blocked with 5% skim milk in TBS containing 0.1% Tween-20(TBST) for 1 h at room temperature (RT) and then probed with specific antibodies at 4 °C overnight. Detection
was achieved using secondary HRP-conjugated IgG against rabbit or mouse IgG and finally visualized using the
ECL system (Amersham).
For lectin blot assay, the membranes were blocked with 3% bovine serum albumin (BSA) in TBST followed by
specific lectins. The bands were finally visualized using a Vectastain ABC Kit (Vector Laboratories).

ǦǤ Total RNAs of HepG2 cells treated with and without 2FF were extracted by Trizol reagent(Invitrogen). 1 µg of total RNAs were reverse-transcribed using a Prime Script RT Reagent kit with gDNA
Eraser (Takara, Japan). The specific primers used for the PCR amplification were as follows: Fut4, forward,
5′-ACTACCACCAACTGAGCCAACATGTGA-3′, reverse, 5′-AAGGAGGTGATGTGGACAGCGTAG-3′;
Fut7, forward, 5′-AGTACCGCTTCTACCTGTCCTTTGAGAA-3′, reverse, 5′-AGCCTGTCACGCCAGG
CAAAGAA-3′; and, Fut8, forward, 5′-CACTTGGTACGAGATAATGAC-3′, reverse, 5′-CACATGATGGAGCTGACAGCC-3′. The GAPDH mRNA served as the control. The obtained reaction products were then placed into 2%
agarose gels containing ethidium bromide for electrophoresis.

 Ǥ Cells were harvested and adjusted to a concentration of 1-5 × 106 cells/mL incold PBS. Then cells were stained with biotinylated AAL for 1 h on ice, followed by streptavidin-conjugate Alexa
Fluor 647 for 1 h in the dark on ice. A negative control was prepared without AAL. After incubation, cells were
washed 3 times with ice-cold PBS, and then analyzed using a FACSCalibur flow cytometer (BD Biosciences).
 Ǥ Membrane fractions of cells were prepared as described previously63. Pellets were dis-solved in 100 µL of 0.1 M Tris-HCl (pH = 8.6), then 20 µL of 200 mM DTT in 1 M Tris-HCl (pH = 8.6) containing
1% SDS was added with heating to 80 °C for 10 min. After cooling to RT, 40 µL of 5% Nonidet P-40 and 15 µL of
distilled water were added to the mixture. Glycopeptidase F (16 mU, Takara Bio Inc.), an amidohydrolase thatremoves intact N-glycans from glycoproteins, was added to the solution, which was then incubated at 37 °C for
16 h. After the addition of 234 µL of ethanol to the solution, the mixtures were cooled to −20 °C and kept in afreezer for 20 min followed by centrifugation at 15,000 rpm for 20 min. Supernatants were transferred into new
microtubes and dried via a centrifuge evaporator. The residues were dissolved in 200 µL of water, and then loaded
onto a solid-phase extraction cartridge (Sep-Pak C18, 50 mg, Waters Corp) and washed with 1 mL water. The
flow-through and washings were combined and applied to a HyperSep Hypercarb SPE cartridge (25 mg). Afterwashing with 1 mL water and then with 1 mL 0.1% TFA, glycans were eluted with 1 mL of 50% acetonitrile containing 0.1% TFA. The eluted solution was dried in vacuo, and then permethylated as described previously63. Mass
spectrometry was performed using a MALDI-TOF mass spectrometer (Ultraflex). Ions were generated using
a 337-nm nitrogen laser and were accelerated to 20 kV. All spectra were obtained with a delayed extraction of
140 ns in a reflectron mode, and were the result of a signal averaging of 300 laser shots. For sample preparation, 2,5-dihydroxybenzoic acid was used as a matrix.

Ǥ HepG2 and Hela cells were pre-treated with or without 2FF at a final concentration of
100 µM for 3 days followed by 24 h serum-starvation. Then, 3 × 103 cells were then seeded into 6-cm dishes andcultured under 3% FBS media with or without 100 µM 2FF. Cells were photographed in the same area when theindicated times were reached. Cell numbers at each time point were counted and normalized to those at 0 h when
the cell culture media were replaced with 3% FBS.
Ǥ A total of 1 mL 0.5% base agar in complete culture medium was added into each 6-cm dishand set aside for 5 min to allow the agars to solidify. The pre-treated cells described above were then mixed with
the 0.33% agarose (5,000 cells/mL) and layered onto the base agar. Agar dishes were incubated at 37 °C and fed
with complete culture medium. Culture media were changed every 4–5 days. Three weeks later, agar dishes were
stained with 0.005% crystal violet overnight. The colony numbers were counted from three independent plates.
Representative photographs were taken.

 Ǥ Cell migration assays were performed using a transwell chamber (BD

BioCoatTM control inserts, 8.0-mm inserts; BD Biosciences) according to the manufacturer’s instructions. Briefly,
the bottom sides of transwells were coated with 10 µg/mL fibronectin (FN) and let stand at 4 °C overnight. Cells
were harvested by trypsin containing 1 mM EDTA and re-suspended in the serum-free DMEM medium at a concentration of 1 × 105 cells/mL for HepG2 and 4 × 104 cells/mL for Hela. Then, 500 µl of suspension was added tothe upper chamber and cells were allowed to migrate to the lower chamber containing 500 µL complete medium
at 37 °C. At the end of the cell migration, the filter side of the upper chamber was cleaned using cotton swabs
and the migrated cells located at the reverse side of the chamber were fixed in 4% paraformaldehyde. After being
washed with PBS, cells were stained with crystal violet and let stand overnight. Cells that migrated across thefilters were counted using a phase-contrast microscope, and the values were averaged.

Ǥ Four-week old male BALB/c-nu mice were obtained from Charles River Laboratories,

Japan, and acclimated for one week. Mice were raised in 22 ± 3 °C with saturated humidity and a standard light/
dark cycle. Plenty of food and water was guaranteed.
The same amounts of control and 2FF pre-treated HepG2 cells (4 × 106) were injected subcutaneously into theleft and right flanks of each mouse, respectively. Based on the inhibitory effect of 2FF on fucosylation lasted for
7 days at least (Fig. 1C), 100 µl of the DMEM solution containing with 2FF at 100 µM were directly injected into
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each tumor tissue from three directions after the inoculation for 7 days, and once a week for 3 weeks. All the mice
were euthanized at 25 days after the first injection of 2FF. Tumor tissues were isolated, photographed and thenweighed. And the same amounts of tumor tissues were homogenized for lectin blot with AAL and Western blot
with several indicated antibodies. Measurement of tumor sizes began on the fifth day, and sizes were monitored
every 5 days by measuring the tumor length and width with a vernier caliper. Tumor volumes were estimatedaccording to the following formula: volume (mm3) = (L × W2)/2, where L and W are the length and width of
the tumor tissues. All experiments were performed according to protocols approved by the Tohoku Medical andPharmaceutical University Research Ethics Board.

 Ǥ Statistical analyses were performed using a student’s t-test with GraphPad Prism5 soft-

ware. Results were presented as the mean ± s.e.m. Statistical significance was accepted at p < 0.05. All experiments
were repeated at least three times.

 

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 

This work was partially supported by a Grant-in-Aid for Scientific Research (15H04354 to JG), for Challenging
Exploratory Research (15K14408 to JG) from the Japan Society for the Promotion of Science, and by a Strategic
Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.



Y.Z. performed all the experiments with the help of T.F., Q.H., S.H. and T.I. A.K. performed the mass spectrometry
analysis. Y.Z. and T.F. performed the animal experiments. T.F., Q.H., S.H. and J.G. designed the experiment. Y.Z.,
T.F. and J.G. analyzed the data, prepared the figures and wrote the manuscript. All authors discussed the results
and commented on the manuscript.

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Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-11911-9.
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cancers
Review

Roles of Integrin ↵6 4 Glycosylation in Cancer

Yoshinobu Kariya 1, *, Yukiko Kariya 1 and Jianguo Gu 2, *1
2*

Department of Biochemistry, Fukushima Medical University School of Medicine, 1 Hikarigaoka,
Fukushima City, Fukushima 960-1295, Japan; ykk-kari@fmu.ac.jp
Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology,
Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai,
Miyagi 981-8558, Japan
Correspondence: kariya@fmu.ac.jp (Y.K.); jgu@tohoku-mpu.ac.jp (J.G.); Tel.: +81-24-547-1144 (Y.K.);+81-22-727-0216 (J.G.); Fax: +81-24-548-8641 (Y.K.); +81-22-727-0078 (J.G.)

Academic Editor: Helen M. Sheldrake
Received: 30 May 2017; Accepted: 30 June 2017; Published: 5 July 2017

Abstract: Malignant transformation is accompanied with aberrant glycosylation of proteins. Suchchanges in glycan structure also occur in the integrins, which are a large family of cell surface receptors
for the extracellular matrix and play key roles in tumor progression. There is now increasing evidence
that glycosylation of integrins affects cellular signaling and interaction with the extracellular matrix,
receptor tyrosine kinases, and galectins, thereby regulating cell adhesion, motility, growth, andsurvival. Integrin ↵6 4 is a receptor for laminin-332 and the increased expression level is correlatedwith malignant progression and poor survival in various types of cancers. Recent studies haverevealed that integrin ↵6 4 plays central roles in tumorigenesis and the metastatic process. In thisreview, we summarize our current understanding of the molecular mechanisms of tumor progression
driven by integrin ↵6 4 and also discuss the modification of glycans on integrin 4 subunit to address
the important roles of glycan in integrin-mediated tumor progression.
Keywords: integrin; glycosylation; cancer; N-acetylglucosaminyltransferase-V (GnT-V); epithelial tomesenchymal transition (EMT); galectin-3

1. Introduction
Integrins are a large family of heterodimeric transmembrane receptors comprising ↵ andsubunits. In mammals, 18↵ and 8 subunits have been characterized, and the combination ofthem forms 24 distinct integrins. Integrins bind to extracellular matrix proteins including collagen,fibronectin, laminin, osteopontin, and tenascin in the extracellular domain, which leads to the assemblyof signaling complexes including focal adhesion kinase, paxillin, and Src in the cytoplasmic domain
and the rearrangement of actin cytoskeleton. The transducing signals from integrin receptors tocytoskeletal and adhesive machinery regulate cell adhesion, migration, proliferation, differentiation,and tumor progression.
Of note, integrins are known to be major glycan-carrying proteins. In fact, the functions ofintegrins are also dependent on their complex N-glycosylation modifications [1]. Among the different
types of integrins, ↵5 1, a major fibronectin receptor, is believed to be a relatively well-characterized
example, and N-glycosylation is important for its mediated many biological functions such as celladhesion and migration [2,3]. Alterations in the oligosaccharide portion of integrin ↵5 1 from theenhanced expression of some glycosyltransferase genes—such as N-acetylglucosaminyltransferase-V(GnT-V), N-acetylglucosaminyltransferase-III (GnT-III), or ↵2,6-galactoside sialyltransferase 1—canbe used to regulate the cell spreading and migration onto fibronectin [1,4,5]. Furthermore, we
recently found that N-glycosylation on the calf domain of ↵5, putative sites 10–14, was essential

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2 of 10for the ↵5-mediated inhibitory effect on epidermal growth factor receptor (EGFR) signaling and cellproliferation [6], while N-glycosylation on sites 1–2 on the -propeller domain of ↵5 played a keyrole in driving integrin ↵5 1 dynamics and cell migration [7]. Taken together, these findings support
the idea that individual integrin ↵5 N-glycosylation differentially functions as a molecular switch toregulate the biological functions of ↵5 1. Similarly, recent studies have revealed that glycosylation of
the integrin 4 subunit is important for integrin ↵6 4 functions, the expression of which is associatedwith cancer progression. In this review, we summarize our current understanding of integrin ↵6 4 incancer and also discuss function of glycans on integrin 4 subunit.
2. Structure and Functions of Integrin ↵6 4
Integrin ↵6 4 is an essential component of the hemidesmosome that provides stable adhesionof basal epithelial cells to the underlying basement membrane [8–10]. The integrin 4 can form
heterodimer only with the ↵6 integrin. Patients with genetic mutations in either integrin ↵6 or 4
subunit suffer from the junctional epidermolysis bullosa with pyloric atresia (JEB-PA), which is an
autosomal-recessive disorder clinically characterized by mucocutaneous fragility and gastrointestinal
atresia [11–13]. The extracellular domain of integrin 4 associates with extracellular matrix,
laminin-332, which is a major component of the hemidesmosome [14,15] (Figure 1). The cytoplasmic
domain of integrin 4 is much longer (>1000 amino acid) than that of other integrin subunits (<50amino acid) [16], and the large cytoplasmic domain of integrin 4 interacts with other hemidesmosome
component, plectin, collagen XVII (BP180/BPAG2), and BP230 (BPAG1) [9,10] (Figure 1). The adhesion
complex consisting of those hemidesmosome proteins plays an important role in maintaining thehemidesmosome structure. Mice carrying a target deletion of the integrin 4 cytoplasmic domain
display extensive epidermal detachment at birth and die shortly thereafter from a syndrome resembling
the human JEB-PA [17]. The integrin 4 cytoplasmic domain contains several serine, threonine, andtyrosine phosphorylation sites (Figure 1), and the phosphorylation of integrin 4 cytoplasmic domain
is caused by activation of receptor tyrosine kinases (RTKs) [18], and directly by protein kinase [19].
3. Integrin ↵6 4 in Cancer
Integrin ↵6 4 was first discovered as a tumor-specific antigen [20,21]. Subsequent studies
demonstrated that increased expression level of integrin ↵6 4 was correlated with malignant
progression and poor survival in squamous cell carcinoma (SCC) of the skin [21,22], lung [23], headand neck [24], and cervix [25]. Further studies have reported that high expression levels of integrin↵6 4 were found in several types of cancer—including breast, bladder, colon, ovarian, pancreatic,
prostate, and thyroid—and linked to poor prognosis [26]. In a mouse model of active H-Ras andIB↵-driven human cutaneous SCC, integrin 4-negative keratinocytes (derived from JEB-PA patientswith null ITGB4 gene mutations) failed tumor formation but reintroduction of integrin 4 gene into thecells restored it [27], suggesting that integrin 4 plays an essential role in human SCC development.
Association of integrin ↵6 4 with laminin substrates significantly promotes cancer cell adhesion,
migration, invasion, proliferation, and tumorigenesis through the activation of Rac1, PKC, PI3K, andERK signaling pathways [10,14,26,28–32] (Figure 1). The PI3K activation response to integrin ↵6 4
ligation is involved in invasive potential of carcinoma cells, and Tyr1494 in the cytoplasmic domainof the integrin 4 is required for the activation [33]. Ligand binding to the extracellular domain of
integrin ↵6 4 induced phosphorylation at serine and tyrosine residues in integrin 4 cytoplasmic
domain, which were associated with a metastatic phenotype of cancer cells [34]. Phosphorylationof Tyr1494 and Tyr1526 in integrin 4 leads to recruitment of tyrosine phosphatase Shp2 and Shc to
the 4 cytoplasmic domain, respectively, followed by activation of Ras-MAP kinase pathways, andpromotes cell cycle progression [31,35–37] (Figure 1). However, crystallographic studies have beenshown that the structural environment of Tyr1494 and Tyr1526 are not compatible with binding to theSH2 and PTB binding domains of Shp2 and Shc, respectively [38]. Furthermore, both Tyr residues
are not well solvent-exposed. It is therefore questionable whether these residues are involved in theCancers 2017, 9, 79

3 of 10recruitment of SHP2 and Shc, and the subsequent coupling of the integrin ↵6 4 to the MAPK signaling
pathways [39].
During cancer progression, integrin ↵6 4 is released form hemidesmosomes and the numberof hemidesmosomes is decreased, which facilitates the cancer cell migration and invasion [26,39].
Serine phosphorylation of integrin 4 cytoplasmic domain by PKC induces relocation of integrin↵6 4 from hemidesmosomes to cell protrusions in cancer cells [40]. Compared with carcinoma insitu or normal tissue, increased phosphorylation at Ser1356 in the integrin 4 cytoplasmic domain was
found in around 60% of invasive cutaneous SCC. Triple mutation at Ser1356 , Ser1360 , and Ser1364 to
non-phosphorylatable alanines in the integrin 4 cytoplasmic domain stabilized hemidesmosome-likestructures and reduced cell migration in SCC cells [41]. Thus, the phosphorylation at specificsites in the integrin 4 cytoplasmic domain leads to the disruption of stable adhesion structure,
hemidesmosomes [19,37,42,43], thereby facilitating the migration of cancer cells (Figure 1). Integrin
4 is also phosphorylated by the associations with several RTKs—including EGFR, ErbB2, andMet [18,32]—which are often mutated or amplified in tumors. RTKs activate Src-family kinases,
and thereby phosphorylates integrin 4 cytoplasmic domain. Tyrosine phosphorylation of integrin 4
through Src family kinase, Fyn, which is activated by EGFR, causes disruption of hemidesmosomes,
thereby promoting squamous carcinoma invasion [44]. Conversely, integrin ↵6 4 regulates theexpression of ErbB2 and the subsequent Src-family kinase-dependent phosphorylation of RTKs andactivation of Ras, STAT-3, and c-Jun [45]. These findings suggest that cooperative signaling between
integrin 4 and RTKs promotes cancer progression.
Metastasis of cancer cells is a major cause of death in patients with cancer. A first step inmetastasis of cancer cells is to move from the primary site and invade into the stroma. In theprocess of metastasis, some cancer cells undergo epithelial to mesenchymal transition (EMT), which
is characterized by loss of epithelial phenotype with cell-cell adhesion and cell polarity, and gainof fibroblast-like morphology [46]. EMT induces cell motility, and stem cell-like properties, thereby
enhancing cancer invasion, metastasis, and chemoresistance [47]. A cDNA microarray analysis
using clinical samples of pancreatic ductal adenocarcinoma revealed that high levels of integrin 4
expression were significantly correlated with the hallmarks of EMT, with high tumor grade, and withthe presence of lymph node metastasis [48]. Overexpression of integrin 4 promoted cell motilityof pancreatic ductal adenocarcinoma cell lines in combination with down-regulation of E-cadherin
and up-regulation of vimentin expression [48]. Integrin ↵6 4 also promotes EMT in hepatocellular
carcinoma by upregulating the expression of transcription factor Slug that inhibits the transcriptionof E-cadherin gene [49]. A recent report has demonstrated that cells with an intermediate level ofintegrin 4 expression exhibited a hybrid epithelial/mesenchymal phenotype and contained cancer
stem cell-enriched populations in triple-negative breast cancer cells. Therefore, integrin 4 canbe a mechanistically driven prognostic biomarker for identifying the more aggressive subtypes ofmesenchymal carcinoma cells in triple-negative breast cancer cells [50]. A subpopulation of the PC-3
prostate cancer cell line, TEM4-18, displayed the hallmarks of EMT, including frank loss of E-cadherin
expression and upregulation of E-cadherin repressor ZEB1 compared to parent cells [51]. Surprisingly,
the ZEB1-mediated EMT in TEM4-18 cells repressed integrin 4 and laminin-332 expression by
the binding of ZEB1 to the promoter elements of integrin 4 and laminin 2 (one of the subunitof laminin-332) genes. The ZEB1 expression exhibited enhanced trans-endothelial migration but
decreased transwell migration and invasion of cancer cells [51]. These results suggest that integrin↵6 4 is associated with EMT, but the regulatory mechanism of EMT by integrin ↵6 4 might dependon cancer types.
Exosomes are cell-derived small membrane vesicles (30–100 nm) containing proteins, lipids, RNA,and DNA that can be horizontally transferred to recipient cells [52]. Recent evidence suggests that
exosomes play a critical role in the development of cancers, such as activation of fibroblasts, promoting
angiogenesis, enhancing invasiveness and chemoresistance [52]. Hoshino et al. have reported that
exosomes containing integrin ↵6 4 and ↵v 5 derived from tumor cells were associated with lungCancers 2017, 9, 79

4 of 10and liver metastasis, respectively [53]. Furthermore, exosomal integrin ↵6 4 uptake activated Src
and upregulated pro-migratory and pro-inflammatory S100 molecules in resident cells. These results
suggest that exosomal integrin ↵6 4 determines metastatic organotropism and could be a biomarker
for lung-specific metastasis.
4. Roles of Glycans in Integrin 4 Function
N-glycosylation is a common protein post-transcriptional modification occurring on asparagine
in the asparagine-X-serine/threonine motif, where X can be any amino acid except proline. Integrins↵6 and 4 have nine (Asn78 , Asn223 , Asn284 , Asn370 , Asn731 , Asn748 , Asn891 , Asn927 , Asn958 ) [54] andfive (Asn327 , Asn491 , Asn579 , Asn617 , and Asn695 ) N-glycosylation potential sites in each extracellulardomain, respectively [55] (Figure 1). Although the N-glycans on integrin 1 is required for theheterodimer formation with integrin ↵5 [56], the presence of N-glycans on integrin 4 is not essential
for integrin ↵6 4 heterodimer formation [55]. In contrast, a defect of N-glycosylation in integrin 4
decreases its function such as cell spreading, adhesion, and migration on its substrate, laminin-332, as
well as localization to lipid rafts [55].
Overexpression of 1,6-N-acetylglucosamine (GlcNAc)-branched N-glycans is often found intumor tissues, and the increase in 1,6-GlcNAc-branched N-glycans is directly associated withmalignancy and poor prognosis [57]. The addition of the 1,6-GlcNAc-branched N-glycans iscatalyzed by GnT-V, a member of the family glycosyltransferase [58] (Figure 2). GnT-V knockout
mice showed reduced 1,6-GlcNAc-branched N-glycans, resulting in suppression of mammary
tumor growth and metastasis induced by the polyomavirus middle T oncogene [59]. In vitro,
1,6-GlcNAc-branched N-glycans-modified integrins ↵3 1 and ↵5 1, and laminin-332 strongly
promoted cancer cell motility [60–62]. In contrast, introduction of bisecting GlcNAc by GnT-III
expression suppresses 1,6-GlcNAc branching formation catalyzed by GnT-V [58], resulting insuppression of cancer metastasis (Figure 2). These findings indicate that 1,6-GlcNAc-branchedN-glycans catalyzed by GnT-V play important roles in tumor malignancy and progression.
Galectins are a family of soluble lectins that bind -galactoside-containing glycans such as
N-acetyllactosamine (Gal 1,4-GlcNAc 1,3). The most studied member of the galectin family,
galectin-3 is known to be associated with cancer aggressiveness and metastasis [63,64]. The bindingof galectin-3 to -galactoside sugars on glycoproteins crosslinks between the glycoproteins andregulates diverse cellular functions in cancer cells. 1,6-GlcNAc-branched N-glycans catalyzed
by GnT-V can be elongated with N-acetyllactosamine repeats (polylactosamine), which acts as ahigh-affinity ligand for galectin-3 (Figure 2). Previously, we found the molecular complex consistingof integrin ↵6 4, EGFR, and galectin-3 in gastric cancer cell line MKN45 cells, which highly express
GnT-V [65,66]. The formation of integrin ↵6 4/EGFR/galectin-3 complex was inhibited by either thepresence of a competitive inhibitor of galectin-binding to -galactoside structure, -lactose or GnT-III
expression [66]. In addition, the breakdown of the tri-molecular complex by an anti-galectin-3 antibody
inhibited integrin ↵6 4 clustering and cell migration [66]. Similar effect was also observed on thelaminin-332/integrin ↵6 4 association. In GnT-III-overexpressing MKN45 cells, the modification oflaminin-332 increased bisecting GlcNAc, thereby decreasing 1,6-GlcNAc branched N-glycans, as wellas integrin ↵6 4 clustering and cell motility [61]. These findings indicate that galectin-3 cross-links
among integrin ↵6 4, EGFR, and laminin-332, thereby inducing efficient signaling and the following
cellular function.
Mucin type O-glycosylation (hereafter referred to as O-glycosylation) is one of the mostabundant forms of post-translational modification of secreted and membrane-bound proteins that
contains a range of N-acetylgalactosamine (GalNAc)-Serine/Threonine O-linked oligosaccharaides(O-glycans) [67]. Sialic acids occupy terminal positions of N-glycans and O-glycans in glycoproteins,and altered sialylation has long been associated with the cancer progression. Desialylation of O-glycans
on integrin 4 by sialidase NEU1 suppressed colon cancer cell adhesion to laminin-332, tyrosine
phosphorylation of integrin 4, and metastasis of human colon cancer cells [68]. In contrast, sialylation

Cancers 2017, 9, 79

5 of 10of integrin 4 was downregulated during EMT but then reverted and upregulated in the mesenchymal
state after EMT [69]. These results indicate that sialylation of integrin 4 is dynamically regulated
and contributes to cancer progression. Although there are some data using lectin suggesting that
Cancers 2017,9, 79 occur on the integrin
5 of 9 structure
O-glycosylationmay
4 [55,68], direct evidence for the O-glycan
Cancers 2017, 9, 79
5 of 9and O-glycosylation site in the molecule has not been presented. Further studies including mass
structure and O-glycosylation site in the molecule has not been presented. Further studies including
structure
and O-glycosylationsite
in thethe molecule
has not
been
presented. Further
studies
includingspectrometry
are analysis
requiredfor
study
O-glycosylationon
integrin
4.
massanalysis
spectrometryarerequired
for the about
study
about
O-glycosylationon thethe integrin
Ά4.
mass spectrometry analysis are required for the study about O-glycosylation on the integrin Ά4.

Figure 1. Structure and functions of integrin Ά4. Integrin Ά4 contains laminin-332 binding sites [14]Figure 1. Structure and functions of integrin
4. Integrin 4 contains laminin-332 binding sites [14]and five N-glycosylation sites (Asn327, Asn491, Asn579, Asn617, Asn695) in its extracellular domain [55],327 , Asn491 , Asn579 , Asn617 , Asn695 ) in its extracellular domain [55],and fiveFigure
N-glycosylation
sites
(Asn
and 1.
theStructure
binding sites
Plectin [70],BP180, BP230[71,72],Ά4
and
ErbB2 [45]in its cytoplasmic
and forfunctionsof integrin
Ά4. Integrin
contains
laminin-332
bindingdomain.
sites [14]1356, Ser1360
1364
1526,BP230
1736 induces579
and theandbindingsites for
Plectin[70],
BP180,[71,72],[45] disassemblyin
its cytoplasmic
,327
Ser
, 491
Tyr
and
Thr617
Phosphorylationof Ser
, Asn
, Asn
, Asn
, Asn695)and
inhemidesmosome
itsErbB2
extracellulardomain[55],
five N-glycosylationsites (Asn
1526
1360
1526
1736
[19,37,42,43].
ofpromotes
recruitment
of,ErbB2
Shc,
in itsturn
activateshemidesmosome
Ras,
Rafandthe binding Phosphorylationsites for[70],
BP180,[71,72],[45]
cytoplasmic
domain.
domain.
Phosphorylation
ofPlectin
Ser1356,Tyr
Ser,BP230
Ser1364, TyrandandwhichThrin
induces
is associatedwith
PI3K activation[33]. N-Glycosylation
ERK and Rac-JNK
signaling[31,37].
Tyr, 1494
1356, Ser1360
1736 induces
1526, Ser1364
Tyr
, and
Thr
hemidesmosome
disassembly
Phosphorylationof Serdisassembly[19,37,42,43].
Phosphorylationof1526Tyrpromotes
recruitmentof Shc,which in turn
sites
are
shown
by
flags.
Numbersandboxes indicate
the numberof amino
residueand thefour
1526[19,37,42,43]. Phosphorylation of Tyr promotes
recruitmentof Shc,whichacidin turn
activates
Ras,
Raf1494
activates Ras,
Raf-ERKand
Rac-JNKsignaling[31,37].
Tyrisassociated
with
PI3K
activation[33].
fibronectin type III repeats, respectively.
Star shape indicates phosphorylation site. TM,
1494
is associated with PI3K activation [33]. N-Glycosylation
ERK and Rac-JNK signaling [31,37]. Tyr
N-Glycosylationsites areshown
by flags. Numbers
and boxes
indicate
the number of amino acidtransmembraneregion.
HD, hemidesmosome.
EGFR, epidermal
growthfactor receptor.
sites are shown by flags. Numbers and boxes indicate the number of amino acid residue and the four
residuefibronectin
and the four
fibronectin
typerespectively.
III repeats, respectively.
Star shapeindicates phosphorylation
site.
type
III repeats,
Star shape indicatesphosphorylationsite. TM,
TM, transmembraneHD,hemidesmosome.
hemidesmosome.
EGFR,
epidermal
growthfactor receptor.
transmembrane region.
region. HD,
EGFR,
epidermal
growth factor
receptor.

Figure 2. Glycosylation reactions catalyzed by GnT-V. GnT-V catalyzes the formation of Ά1,6-GlcNAcbranched structures. Ά1,6-GlcNAc-branching can be elongated with N-acetyllactosamine repeats(polylactosamine), which acts as a high-affinity ligand for galectin-3. Enhanced expression of GnT-V
results in increased migration and metastasis of cancer cells. GnT-III adds GlcNAc to the core
Figure Figure2.
Glycosylation
reactionscatalyzed
byGnT-V
GnT-V.
GnT-V
catalyzes
the formation of
2. Glycosylation reactions
catalyzed
by GnT-V.
catalyzes
the formationof Ά1,6-GlcNAcmannose to form bisecting N-acetylglucosamine (GlcNAc) in N-glycans, which inhibit the Ά1,6branchedstructures.
Ά1,6-GlcNAc-branchingcanbeelongatedwithN-acetyllactosamine
1,6-GlcNAc-branchedstructures.
1,6-GlcNAc-branchingcanbeelongatedwithN-acetyllactosamine
GlcNAc branching formation catalyzed by GnT-V and the resultant increase in cancer migrationrepeats
and
which
acts asa high-affinity
ligand for
galectin-3.
Enhanced expressionof GnT-V
repeats(polylactosamine),(polylactosamine),
which
acts
as a high-affinity
ligandfor galectin-3.
Enhanced
expression ofmetastasis.

and metastasis of cancer cells. GnT-III adds GlcNAc to the core
GnT-V results
resultsininincreased
increasedmigration
migrationand metastasis of cancer cells. GnT-III adds GlcNAc to the coremannose to form bisecting N-acetylglucosamine (GlcNAc) in N-glycans, which inhibit the Ά1,6mannose to form bisecting N-acetylglucosamine (GlcNAc) in N-glycans, which inhibit the 1,6-GlcNAc
GlcNAc branching formation catalyzed by GnT-V and the resultant increase in cancer migration andbranchingformation catalyzed by GnT-V and the resultant increase in cancer migration and metastasis.
metastasis.

Cancers 2017, 9, 79

6 of 10

5. Conclusions and Perspective
In normal stratified and complex epithelial tissues, integrin ↵6 4 is an essential component of
the hemidesmosomes. However, integrin ↵6 4 also overexpresses in several types of cancers andthe expression level is correlated with malignant progression and poor survival in cancer patients.
Integrin ↵6 4 significantly promotes cancer cell adhesion, migration, invasion, proliferation, andtumorigenesis through the activation of Rac1, PKC, PI3K, and ERK signaling pathways, which are
induced by the interaction with other molecules including RTKs and laminin-332. Phosphorylationof the cytoplasmic domain in integrin 4 also contributes to the cancer progression by activationof Ras-MAP kinase pathways and hemidesmosome disassembly. In addition, the expression levels
of integrin 4 are closely correlated with the hallmarks of EMT, and also exosomal integrin ↵6 4
determines metastatic organotropism.
The biosynthesis of glycan is primarily determined by the glycosyltransferases, the expression
level of which is controlled at the level of gene transcription, and by enzymatic activity and chaperone.
Since the expression profile of glycolsyltransferases in cancer cells is quite different from that of normal
cells, the resultant glycan structure is aberrant and specific to cancer. Therefore, alteration of glycan
structures is one of the hallmarks of cancer. Recent studies have revealed that integrin ↵6 4 functions
are regulated by glycosylation of integrin 4. The formation of integrin ↵6 4/EGFR/galectin-3
complex through N-glycans induces integrin ↵6 4 clustering and cell migration. Specifically,
sialylation of integrin 4 seems to be associated with cancer progression. Therefore, glycosylation on
integrin 4 may be a useful biomarker and a novel therapeutic target for cancer.
Acknowledgments: This work was partially supported by the Japan Society for Promotion of Science KAKENHI
Grant Number JP25860243 to Yoshinobu Kariya; JP15H04354 to Jianguo Gu.
Conflicts of Interest: The authors declare no conflict of interest.

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Contribution of sialidase NEU1 to suppression of metastasis of human colon cancer cells throughdesialylation of integrin 4. Oncogene 2009, 28, 1218–1229. [CrossRef] [PubMed]
Du, J.; Hong, S.; Dong, L.; Cheng, B.; Lin, L.; Zhao, B.; Chen, Y.G.; Chen, X. Dynamic Sialylation inTransforming Growth Factor- (TGF- )-induced Epithelial to Mesenchymal Transition. J. Biol. Chem. 2015,
290, 12000–12013. [CrossRef] [PubMed]
Wilhelmsen, K.; Litjens, S.H.; Sonnenberg, A. Multiple functions of the integrin ↵6 4 in epidermal
homeostasis and tumorigenesis. Mol. Cell Biol. 2006, 26, 2877–2886. [CrossRef] [PubMed]
Koster, J.; Geerts, D.; Favre, B.; Borradori, L.; Sonnenberg, A. Analysis of the interactions between BP180,
BP230, plectin and the integrin ↵6 4 important for hemidesmosome assembly. J. Cell Sci. 2003, 116, 387–399.
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Steenbergen, R.D.; Snijders, P.J.; Sonnenberg, A. Hemidesmosome formation is initiated by the 4 integrin
subunit, requires complex formation of 4 and HD1/plectin, and involves a direct interaction between 4
and the bullous pemphigoid antigen 180. J. Cell Biol. 1998, 142, 271–284. [CrossRef] [PubMed]© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

ORIGINAL RESEARCH
published: 29 January 2018
doi: 10.3389/fimmu.2018.00078

Core Fucosylation of the T Cell
Receptor Is Required for T Cell
Activation
Wei Liang1†, Shanshan Mao1†, Shijie Sun1†, Ming Li1, Zhi Li 2, Rui Yu1, Tonghui Ma1,
Jianguo Gu 3, Jianing Zhang 4, Naoyuki Taniguchi 5 and Wenzhe Li1*
1
College of Basic Medical Sciences, Dalian Medical University, Dalian, China, 2 Clinical Laboratory, Dalian Municipal Central
Hospital, Dalian, China, 3 Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology,
Tohoku Medical and Pharmaceutical University, Sendai, Japan, 4 School of Life Science and Medicine, Dalian University ofTechnology, Panjin, China, 5 Systems Glycobiology Research Group, Advanced Science Institute, RIKEN, Saitama, Japan

Edited by:Michael Loran Dustin,
Harvard University, United States
Reviewed by:
Balbino Alarcon,
Consejo Superior de Investigaciones
Científicas (CSIC), Spain
Christoph Wülfing,
University of Bristol, United Kingdom*Correspondence:
Wenzhe Liliwenzhe@dlmedu.edu.cn†

These authors have contributed
equally to this work.
Specialty section:
This article was submitted
to T Cell Biology,
a section of the journal
Frontiers in Immunology
Received: 30 September 2017
Accepted: 11 January 2018
Published: 29 January 2018

Citation:
Liang W, Mao S, Sun S, Li M, Li Z,
Yu R, Ma T, Gu J, Zhang J,
Taniguchi N and Li W (2018) Core
Fucosylation of the T Cell Receptor
Is Required for T Cell Activation.
Front. Immunol. 9:78.
doi: 10.3389/fimmu.2018.00078

CD4+ T cell activation promotes the pathogenic process of systemic lupus erythematosus (SLE). T cell receptor (TCR) complex are highly core fucosylated glycoproteins,which play important roles in T cell activation. In this study, we found that the corefucosylation of CD4+ T cells was significantly increased in SLE patients. Loss of corefucosyltransferase (Fut8), the sole enzyme for catalyzing the core fucosylation of
N-glycan, significantly reduced CD4+ T cell activation and ameliorated the experimental autoimmune encephalomyelitis-induced syndrome in Fut8−/− mice. T cell activation
with OVA323–339 loaded major histocompatibility complex II (pMHC-II) on B cell was
dramatically attenuated in Fut8−/−OT-II CD4+ T cells compared with Fut8+/+OT-II
CD4+ T cells. Moreover, the phosphorylation of ZAP-70 was significantly reduced in
Fut8+/+OT-II CD4+ T cells by the treatment of fucosidase. Our results suggest that corefucosylation is required for efficient TCR–pMHC-II contacts in CD4+ T cell activation,and hyper core fucosylation may serve as a potential novel biomarker in the sera from
SLE patients.
Keywords: core fucosylation, T cell receptor, T cell activation, systemic lupus erythematosus, T–B cell interaction

INTRODUCTION
Systemic lupus erythematosus (SLE) is a severe autoimmune disease that characterized by the
production of autoantibodies and the subsequent inflammatory disorders (1). Although the pathogenesis is not completely understood, the activation of CD4+ T cells seems plays an essential rolein the onset and development of SLE (2). Appropriate CD4+ T cell activation is crucially important
for adaptive immune responses and autoimmunity, but hyper-activation of these cells results in
autoimmune diseases. T cell recognition of peptide-loaded major histocompatibility complex II(pMHC-II) on the antigen-presenting cells (APCs) by T cell receptors (TCRs) is the most important
checkpoint for CD4+ T cell activation (3). During antigen recognition, the CD4 coreceptor binds
to the non-polymorphic surfaces of the membrane-proximal domains of the same pMHCs, which
results in a marked increase in the sensitivity of T cells to pMHCs on APC. When adequate agonistic
TCR signaling creates a favorable microenvironment for binding, CD28-B7 molecules provide
costimulatory lower the thresholds for TCRs triggering and activation. The signaling throughTCR induces a conformational change in leukocyte function-associated antigen-1, which greatly

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increases its affinity for intercellular adhesion molecule 1 andcontributes to immune synapse formation T cells and APC (4, 5).
Glycosylation plays a regulatory and often pivotal role in
T cell activation (6–10). Several studies have reported thatglycosylation could contribute to higher activation thresholds
of T cells. For instance, β1,6N-acetylglucosaminyltransferase
V (Mgat5) deficiency mediates lower T lymphocyte activationthresholds, and subsequently improves T cell activity in vitro
and results in autoimmune disease in vivo (11, 12). Deletion of
sialyltransferase ST3Gal-1 increase the sensitivity of TCRs to
low-affinity ligands in CD8+ T cells (13). Fucosyltransferase 1transgenic mice show increased TCR signaling and apoptosisthat results in thymocyte maturation arrest (14). Notably,
reduced N-glycosylation of TCR chains can improve functional
avidity and recognition by T cells (15) suggesting that the glycosylation of TCR has a unique role in the regulation of T cell
activation.
T cell receptors are heavily core-fucosylated glycoproteins.
The core fucosylation of protein is catalyzed by core fucosyltransferase (Fut8), which transfers fucose residue from GDPfucose to the innermost N-acetylglucosamine (GlcNAc) residueof N-linked glycans via an α1,6 linkage in the Golgi apparatus
of mammalians (Figure S1 in Supplementary Material). Fut8mediated core fucosylation is an important post-translational
process (16), which regulates protein conformation, stability, andfunctional expression. Studies have shown that the N-glycans
at Asn70 [GlcNAc(α1,6Fuc)-β1,4GlcNAc: (A2G2F)], Asn185(A2G2F), and Asn203 in the α chain (Cα) and Asn236 in the β chain(Cβ) extend from the surface of TCR on Drosophila melanogaster
cells (6, 17). Interestingly, they found that the Cα and Cβ of TCR
were connected by the hydrogen bonds of the core fucose residue
from Asn185α (A2G2F) to side chains of Glu181β and Ser182β (6, 17),
suggesting a crucial role of core fucosylation on the conformation of TCR. However, to the best of our knowledge, none of theprevious studies had addressed the regulatory role of TCR corefucosylation on CD4+ T cell activation.
B cells play a role in evoking T cell responses by functioning as APCs, and the presentation of peptide by MHC-II on the
B cells initiates T cell activation (18). Therefore, it is reasonable
to anticipate that the core fucosylation has significant functional
implications in T–B cell interaction, and thus affect the CD4+
T cell activation. In this study, we provide the first confirmationthat SLE patients exhibited hyper core fucosylation on CD4+
T cells, which significantly enhanced the activation of their CD4+
T cells. Knockout of Fut8 gene resulted in attenuated T–B cellinteraction via TCR–pMHC and the consequential reduced CD4+
T cell activation. Our data suggest that the core fucosylation may
serve as a potential novel biomarker with promising clinical andtherapeutic implications in SLE patients.

Fut8+/− mice (C57BL/6). OT-II (Jackson Laboratory) is a
C57BL/6 TCR transgenic strain, expressing a receptor specificfor peptide OVA323–339. Fut8+/+OT-II mice and Fut8−/−OT-II mice
were generated by crossing heterozygous Fut8+/−OT-II mice.
Mice were maintained in the specific pathogen-free laboratory
animal facility of Dalian Medical University. All animal work
was approved by the Ethics Committee at the Dalian Medical
University.

Patients

Serum samples were collected from a total of 17 SLE patients(14 women, 3 men; mean age, 49 years; range, 18–67 years) withand healthy controls (12 women, 12 men; mean age, 18–48 years)(Table S1 in Supplementary Material). The diagnosis of underlying disease was made based on clinical manifestation, serology, imaging, and/or histopathology. These participants were
Chinese, recruited at Dalian municipal central hospital. The antinuclear antibodies (ANA) titers of AD patients were detectedwith using Anti-nuclear Antibodies IgG Kit (EUROIMMUN,
Germany). The Ethics Committee at the hospital approved thestudy protocol.

Antibodies

Anti-CD16/32 (2.4G2), anti-CD3(145-2c11), anti-CD28 (37.51),
FITC-anti-MHC II (M5/114.15.2), FITC-anti-CD69 (H1.2F3),
PE-labeled anti-CD4 (GK1.5), APC-labeled anti-CD8 (53-6.7),
biotin-labeled anti-TCRβ (H57-597), and PE-Cy5-labeled antiTCRβ (H57-597) were obtained from e-Bioscience; anti-GAPDH,
horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG
and HRP-conjugated donkey anti-human IgG were obtained
from proteintech; additional biotin-conjugated lens culinaris
agglutinin (LCA) were purchased from Vector; anti-TCRαβ(ab25336), anti-pZAP70 (ab194800), anti-ZAP70 (ab32410),
Natural streptavidin protein (FITC) (ab136201), and streptavidin(HRP) (ab7403) were purchased from Abcam.

Histological Analysis

Formalin-fixed tissue samples were paraffin-embedded andsections were analyzed by hematoxylin–eosin (H&E) staining.
The sections were stained with biotin-conjugated LCA. Briefly,
sections were deparaffinized three times in xylene and hydrated
through a 100, 90, 80, and 70% ethanol to phosphate-bufferedsaline (PBS). To quench the endogenous peroxidase activity,
slides were incubated with 3% H2O2 for 30 min. Then, the slides
were incubated with biotin-conjugated LCA, and washed threetimes with PBS. The slides probed with HRP–streptavidin for
30 min, and visualized with 3,3′-diaminobenzidine. The intensityof LCA-positive staining in the spleen was analyzed by integrated
optical density using Image-Pro® Plus software (version 6.0;
Media Cybernetics, USA).

Cell Lysate

MATERIALS AND METHODS

Cells were solubilized in lysis buffer [Tris–HCl (50 mM), 1%
Triton X-100, 10% glycerol, phenylmethylsulfonylfluoride(100 µM), leupeptin (5 µg/mL), aprotinin (1 µg/mL), NaF(100 mM), 150 mM NaCl, 2 mM EDTA, and sodium orthovanadate (1 mM)] for 15 min at 4°C. Cell lysate was centrifuged at

MiceFut8−/− mice were generated as previously described (19),and homozygous wild-type (Fut8+/+) and Fut8−/− mice on the
C57BL/6 background were obtained by crossing heterozygous

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Western Blot and Lectin Blot Analysis

20,000 × g for 10 min at 4°C, and the supernatant was subjected
to immunoprecipitation or Western blot, as indicated below.

Protein samples were electrophoresed on 10% polyacrylamidegels. After electrophoresis at 240 mA for 30 min, proteins weretransferred to PVDF membranes. Membranes were blocked in
5% BSA in TBS-T (10 mM Tris–HCl, 150 mM NaCl, and 0.1%
Tween 20) at room temperature for 1 h, and then incubatedwith the biotin-labeled LCA, which preferentially recognizes
the core fucose, or primary Abs in 1% BSA in TBS-T overnightat 4°C. After washing, the membranes were covered with the
HRP-conjugated streptavidin or HRP-labeled secondary Abs atroom temperature for 1 h, and visualized with an ECL system(Amersham).

Fut8 Enzyme Activity Assay

The Fut8 enzyme activity was measured by using the previous
method (20). Five micrograms cell lysates as the enzyme source
were added to the assay buffer (200 mM MES, 1% Triton X-100)
supplemented with donor (500 µM GDP-L-fucose) and substrate[50 µM GnGn-Asn-4-(2-pyridylamine) butylamine (PABA)].
The mixture was incubated at 37°C for 8 h, and the reaction was
stopped by heating at 100°C for 5 min. The reactive solution
was then centrifuged at 12,000 × g for 10 min, and 10 µL of thereaction products were subjected to high-performance liquid
chromatography (HPLC) with a Fluorescent detector (Waters
Corporation, USA). The excitation and emission wavelengths are
320 and 400 nm, respectively.

Immunoprecipitation

Cell extracts (500 µg) were mixed with 20 µL of Protein
G-Sepharose (50%) and corresponding Abs, and then incubatedat 4°C overnight with continuous rotation. After washing threetimes in lysis buffer, the pull downed samples were boiled for 5 min
in Laemmli sample buffer with or without 2-mercaptoethanol.

PCR Array

Total RNAs were extracted from Fut8+/+ SPLs and Fut8−/− SPLs
with TRIzol reagent (Takara Bio). Mouse T Cell and B Cell
Activation PCR Array (SA Biosciences) was carried out according to the protocol of the manufacturer. The difference of gene
expression between Fut8+/+ SPLs and Fut8−/− SPLs was calculated.

T–B Cell Conjugate Formation

Mouse was immunized by subcutaneous injection with 200 µg
OVA mixed with an equal volume of complete Freund’s adjuvant(CFA) (Sigma). Two weeks later, mice were immunized with
200 µg OVA by subcutaneous injection. Mice sera were collected
at 0, 7, and 14 days post-immunization.

Conjugate formation between T cells and B cells were carried out
as described previously with slight modification (21). T cells and1 µg/mL OVA323–339 (NH2-ISQAVHAAHAEINEAGR-COOH)pulsed B cells were mixed at a 1:1 ratio and a quick centrifugation
to initiate cell–cell contact. To observe conjugate formation,
B cells and T cells were labeled with MHC-II-FITC and TCRβPE-Cy5 before mixing. T–B cell conjugates were then analyzed
by flow cytometry to determine the percentage of T–B cells that
had both TCRβ-PE-Cy5 and MHC-II-FITC positive staining.

Enzyme-Linked Immunosorbent
Assay (ELISA)

Remove of Core Fucose on Surface
of T Cells

Animal Immunization

The concentrations of IL-2 analyzed using mouse IL-2 ELISA
kits (Boster Biological Engineering, Wuhan, China), according
to the manufacturer’s instructions. The concentrations of IL-2
were calculated according to a standard curve prepared using
samples of known concentration. The absorbance was measured
at a test wavelength of 450 nm with a microplate reader.
The immunoglobulin isotypes were measured by mouse mAb
isotyping reagents (Sigma).

Purified CD4+ T cells (4 × 106) were treated with 100 mU Glyko®
α(1-2,3,4,6) Bovine Kidney Fucosidase (GKX-5006, Prozyme),
incubate 3 h at 37°C in the reaction buffer. The enzyme reaction
was terminated by centrifuging at 2,500 × g for 5 min and thecells were collected.

Confocal Microscopy

Conjugate formation between T cells and B cells were carriedout as described previously with slight modification (21). T cells
and 1 µg/mL OVA323–339 pulsed B cells were mixed at a 1:1 ratio
and a quick centrifugation to initiate cell–cell contact. Cell–cellconjugates were subsequently transferred to poly-d-lysine coatedcoverslips and incubated at 37°C for 30 min. Cells were fixedwith 4% PFA for 20 min, and then blocked with 5% BSA andanti-CD16/CD32 (2.4G2) mAb for 30 min. After washing, cells
were stained with anti-MHC-II Ab for 1 h. All images were takenusing a spinning disk confocal microscope (Leica).

Cell Proliferation Assay

The growth rate of cells was measured using MTT assay. CD4+
T cells (1 × 106) were cultured in 96-well culture plate with antiCD3ε (2 µg/mL) and anti-CD28 mAbs (1 µg/mL). After 48 h ofincubation, each well was added 10 µL of MTT solution, and thenthe absorbance was analyzed by a microplate reader (Thermo
Multiskan Ascent, Finland) at 570 nm.
In addition, T cell proliferation with the OVA323–339-loaded
B cells was analyzed by carboxyfluorescein diacetatesuccinimidyl
ester (CFSE, Sigma) dilution methods. CD4+ T cells (1 × 106)
were purified, and then labeled with 5 µM CFSE in PBS for 8 minat room temperature and coincubate with OVA323–339 loaded
Fut8+/+ OT-II B cells (1 × 106) for 48 h, and then analyzed by flow
cytometric analysis.

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MACS Magnetic Cell Sorting

Single splenic cell suspensions were prepared by first grinding
the tissues and then by passage through 30-µm nylon mesh.
Red blood cells were lysed by incubation with 0.14 M NH4Cl
and 20 mM Tris (pH7.4) for 3 min at room temperature.

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Lack of Core Fucosylation Ameliorated
EAE Symptoms with Reduced
CD4+ T Cell Activation

After the lysis of red blood cells, CD4+ T cells and B cells
were positively isolated with anti-CD4 Ab and anti-CD45R
Ab-conjugated magnetic beads (Miltenyi Biotec). The purified
cell populations were detected by fluorescence activated cellsorting analysis.

Wild-type (Fut8+/+) and Fut8−/− mice were generated previously(19). Histological analyses of the splenic architecture of Fut8+/+
and Fut8−/− mice were unremarkable in H&E staining. The Fut8
products, core-fucosylated N-glycans, are ubiquitously expressedin the Fut8+/+ spleen, as confirmed by LCA (Figure 2A), while
those were abolished in Fut8−/− spleens. LCA blot analysis also
confirmed the knockout of Fut8 expression in the whole celllysates of Fut8−/− SPLs (Figure 2B). The FUT8 enzymatic activity was not detected in the Fut8−/− SPLs using HPLC analysis(Figure 2C).
Experimental autoimmune encephalomyelitis is an activatedCD4+ T cell-mediated autoimmune disease model. Peptides(MOG35–55) and PTX could induce the migration of activatedT cells through the blood–brain barrier and caused several
neurologic symptoms. To determine the association between
core fucosylation and the activation of CD4+ T cells, EAE models
were established using Fut8+/+ and Fut8−/− mice. EAE is actively
induced but appear more quickly upon adoptive transfer ofactivated MOG35–55-specific T cells in Fut8+/+ mice (Figure 2D;
Video S1 in Supplementary Material), while Fut8−/− mice showedslight EAE symptoms (Figure 2D; Video S2 in Supplementary
Material). The body weights of Fut8+/+ mice were significantly
reduced, but no change was found in Fut8−/− mice during EAE
induction (Figure 2D). In addition, the proliferation of CD4+
T cells was significantly decreased in Fut8−/− EAE mice. Moreover,the proliferation of CD4+ T cells with MOG35–55-loaded B cells
was remarkably reduced by de-core fucosylation (Figure 2E).

Flow Cytometric Analysis (FACS)

Cells were isolated from tissue and incubated with an antiCD16/CD32 (2.4G2) mAb to block Fcγ receptors. The cells were
stained on ice for 15 min with several combinations of mAbs, as
indicated in the figure legends. Flow cytometry was performed
on a FACS-Calibur (Becton Dickinson, Mountain View, CA,
USA) and analyzed using FlowJo software (Tree Star).

Induction of Experimental Autoimmune
Encephalomyelitis (EAE)

For EAE induction, Fut8+/+ and Fut8−/− mice were subcutaneously injected with 100 µg of myelin oligodendrocyte glycoprotein peptides (MOG35–55) emulsified in CFA. Then, mice were
injected intraperitoneally with 200 µg pertussis toxin (PTX) (List
Biological Laboratories) on 0, 1, and 2 days. Clinical assessment
of EAE was performed according to the following scale: 0, nodisease; 1, limp tail; 2, hind-limb weakness; 3, partial hind-limb
paralysis; 4, complete paralysis of hind-limbs; and 5, moribund
state.

Statistical Analysis

Student’s t-test was used for statistical analysis. Data are presented
as mean values ± SEM, or as mean values ± SD. A probability
value of p < 0.05 was considered significant. *p < 0.05, **p < 0.01,***p < 0.001.

Lack of Core Fucosylation Suppressed
the IgG Class-Switching by Impaired
CD4+ T Cell Activation

RESULTS
Core Fucosylation Is Significantly
Upregulated in the Sera andCD4+ T Cells of SLE Patients

Flow cytometry analysis revealed that, although Fut8−/− micecontained normal proportions of CD4+ and CD8+ T cell
populations in the spleen (Figures 3A,B), they were markedly
reduced after OVA immunization contrast with the Fut8+/+ mice
(Figures 3A,B). Immunoglobulin class-switching is a biologicalmechanism that changes a mature B cell’s production of antibody
via its B cell receptor from one class to another. For example,
from an isotype called IgM to an isotype called IgGs. To illustrate
the effects of Fut8 in the class-switching of immunoglobulin, we
measured the class-switched (IgGs of different subclasses) andnon-switched (IgM) in the sera of Fut8+/+ and Fut8−/− mice using
mouse mAb isotyping reagents. In 4-week-old Fut8−/− mice, theamounts of IgG1, IgG2a, IgG2b, and IgG3 were significantly lower
than those in Fut8+/+ mice after OVA immunization, while thoseof IgM were relatively normal (Figure 3C). The cytokines, suchas IL-4, IL-5, IL-6, and IFNγ secreted by CD4+ T cells, contributeto the different IgG class-switching in the mice and human. It
is reasonable to consider that the reduced IgG class-switching
attributed to the low levels of cytokines secreted by CD4+ T cells
in Fut8−/− mice (Table 1). In addition, since the TGF receptor
signaling was attenuated in the Fut8−/− mice (19), the IgG3

Higher circulating levels of ANA were detected in sera from
SLE patients. In this study, we found that core fucosylation
was dramatically increased in the sera from patients with
SLE, as evidenced by LCA, which preferentially recognizes
the core fucose structure (22) (p < 0.001) (Figures 1A,B).
The expression of IgGs was also upregulated in sera of the SLE
patients (p < 0.05) (Figure 1B). These observations pinpoint
the contribution of hyper core fucosylation to SLE severity andpathogenesis.
The hyperactivity of B cells in SLE is T cell dependent, andCD4+ T cell activation plays a crucial role in SLE pathogenesis(23). We found that the percentage of CD4+ T cells in the peripheral blood of SLE patients is similar to those of healthy control(Figure 1C). However, the percentage of CD4+CD69+ T cells was
significantly increased in the SLE (n = 17) (Figure 1D). Moreover,the enzyme activity of Fut8 was dramatically increased in the
CD4+ T cells isolated from the SLE patients (Figures 1E,F), indicated that increased core fucosylation in SLE patients correlateswith CD4+ T cell activation.

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FIGURE 1 | Core fucosylation was significantly increased in the systemic lupus erythematosus (SLE) patients. (A) The sera of SLE patients were analyzed by lensculinaris agglutinin (LCA) blot and Western blot. Plates were incubated with biotin-conjugated LCA (1:20,000). Comparable results were obtained in three
independent experiments. (B) Densitometric analysis of the bands of IgG and LCA in sera in SLE patients. Data are shown as mean values ± SEM (*p < 0.05;***p < 0.001). (C) The percentage of CD4+ T cells in the peripheral blood of SLE patients. (D) The percentage of CD4+CD69+ T cells in the peripheral blood of SLE
patients (n = 17). (E,F) FUT8 activities in the CD4+ T cells of SLE patients by high-performance liquid chromatography (HPLC). CD4+ T cells were isolated with
anti-CD4 Ab-conjugated magnetic beads. The sorted cell populations were routinely more than 96% pure. Five micrograms cell lysates as the enzyme source weremixed with the assay buffer. After incubation at 37°C for 8 h, 10 µL of the supernatant was subjected to HPLC. Activity was expressed as pmol of GDP-fucose
transferred to the acceptor per hour per milligram of protein. Data are shown as mean values ± SD (n = 17; ns, not significant; *p < 0.05; **p < 0.01). S is the
peptide substrate and P is the product of fucosylation.

class-switching regulated by TGF signaling was also suppressedin the Fut8−/− mice.

FACS analysis showed that core fucosylation on the cellularsurfaces was abolished in Fut8−/− CD4+ T cells (Figure 4C). We
compared the phosphorylation levels of Fut8+/+CD4+ T cellswith Fut8−/−CD4+ T cells in response to costimulations with
anti-CD3/CD28 Abs. In those comparisons, the levels of pZAP70 in Fut8−/−CD4+ T cells were significantly lower than those in
Fut8+/+CD4+ T cells (Figure 4D). Moreover, the populations of
CD69+ cells (activated T cells) in Fut8−/−CD4+ T cells were lower
than those in Fut8+/+CD4+ T cells following OVA immunization,
while these were similar before immunization (Figure 4E).
Furthermore, cell proliferation of Fut8−/−CD4+ T cells was significantly reduced in response to the stimulation of anti-CD3/
CD28 Abs (Figure 4F).

Loss of Core Fucosylation Impaired the
Signal Transduction via TCR

T cell receptor signaling is very important for T cell activation.
As illustrated in Figure 4A, no different expression of TCR was
found in the Fut8+/+CD4+ T and Fut8−/−CD4+ T cells. Nonetheless,the core fucose of the N-glycans in the molecules was eliminatedby a disruption of the Fut8 (Figure 4B). Moreover, the MS spectra of N-glycans released from TCRs were analyzed. It is notablethat the high levels of signals were corresponded to the corefucosylated glycans bearing non-, mono-, or di-galactose in the
Fut8+/+CD4+ T cells, while those were completely disappearedin Fut8−/−CD4+ T cells (Figure S2 in Supplementary Material).
These results further confirmed in mice that TCRs are highly
core-fucosylated proteins and contributes to its activities.
In order to examine the role of core fucosylation in the activation of CD4+ T cells, we isolated CD4+ T cells from Fut8+/+
and Fut8−/− mice, and checked the level of core fucosylation.

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Core Fucosylation Is Essential for
TCR–pMHC Conjugates in CD4+ T Cell
Activation

B cells present antigenic peptide with MHC-II molecule as
APCs, and CD4+ T cells physiologically recognize a complex
of a peptide-loaded MHC-II in T–B cell interaction (18).

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FIGURE 2 | Continued

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FIGURE 2 | The experimental autoimmune encephalomyelitis (EAE) model was slightly induced in Fut8−/− mice. (A) Immunohistochemical analysis of Fut8+/+ spleen
and Fut8−/− spleen. The paraffin sections of spleens were deparaffinized and hydrated through a graded series of ethanol to phosphate-buffered saline. One section
was assessed by hematoxylin–eosin staining (magnification ×200). RP, red pulp; WP, white pulp. Another section was incubated with biotin-conjugated lens culinaris
agglutinin (LCA) (1:200) for 1 h. Finally, the slides were visualized with 3,3′-diaminobenzidine. The staining with LCA was showed by integrated optical density
analysis. Data are shown as mean values ± SD (***p < 0.001). (B) Lectin bolt of Fut8+/+ SPL and Fut8−/− SPL. The SPL lysates were run on 10% SDS-PAGE gel andstained with Coomassie blue staining and LCA (1:5,000). (C) High-performance liquid chromatography (HPLC) analysis of Fut8 activity. Fut8 activities were
examined using fluorescence-labeled sugar chain, GnGn-Asn-PABA, as an acceptor substrate, as described in Section “Materials and Methods.” The substrate (S)and Fut8 product (P) were eluted at 8 and 15 min, respectively. (D) Disease score of mice in Fut8+/+ and Fut8−/− mice EAE model. EAE induction of Fut8+/+ andFut8−/− mice (n = 7). Mice were immunized with 100 µg MOG35–55 peptide in complete Freund’s adjuvant and injected with 200 µg pertussis toxin, and detected thesigns of EAE daily for 30 days. Comparable results were obtained in four independent experiments. Body weights of mice were measured every 2 days after EAE
induction of Fut8+/+ and Fut8−/− mice (n = 7). (E) Cell proliferation from EAE mice. Purified splenic CD4+ T cells and/or MOG35–55 peptide-loaded B cells from EAE
models were incubated for 48 h at 37°C, and then the cell proliferation was detected by MTT assay. Comparable results were obtained in four independent
experiments. Data are shown as mean values ± SD (*p < 0.05; **p < 0.01).

FIGURE 3 | Lack of core fucosylation suppressed the IgG class-switching by impaired CD4+ T cell activation. (A,B) Flow cytometry analysis of the proportion of
CD4+ and CD8+ cells. SPLs were obtained from Fut8+/+ and Fut8−/− mice with or without OVA immunization. SPLs were filtered through nylon mesh, andresuspended. Numbers indicate the percentage of the total spleen cells within this quadrant, and 10,000 events were acquired for each analysis. Data arerepresentative of three independent experiments. Data are shown as mean values ± SD (ns, not significant; *p < 0.05; **p < 0.01). The number of Fut8+/+CD4+ T
and Fut8−/−CD4+ T cells were (1.29 ± 0.32) × 107 and (6.38 ± 0.89) × 106 of the SPLs, respectively (n = 5). (C) Comparison of the levels of serum immunoglobulin
isotypes in Fut8+/+ and Fut8−/− mice after OVA immunization. The immunoglobulin isotypes are compared by mouse mAb isotyping reagents (Sigma). Comparable
results were obtained in three independent experiments. Data are shown as mean values ± SD (*p < 0.05; **p < 0.01).

Therefore, we were interested in determining whether corefucosylation is involved in the TCR sensitivity to pMHC-II. To
explore the role of core fucosylation on the TCR interaction

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with pMHC-II ligands, we crossed Fut8+/− mice with OT-II TCR
transgenic mice (expressing CD4+ TCR specific for OVA323–339),
and obtained Fut8+/+OT-II and Fut8−/−OT-II mice. LCA blots

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markedly increased at the site of T–B cell contact in Fut8+/+OT-II
T cells for 30 min and was ubiquitous on the Fut8−/−OT-II T cellsurface (Figures 5D,E). Next, T–B cell conjugates were quantitatively analyzed via flow cytometry analysis. Few conjugatesof T–B cells were observed in the absence of OVA323–339 peptide,whereas peptide-pulsed B cells effectively interacted with CD4+
T cells. The percentages of T–B cell conjugates in Fut8+/+OT-IIand Fut8−/−OT-II MHC-II+ TCRβ+ cells were 29.4 and 7.4%,
respectively (Figures 5F,G), indicated that core fucosylation
affected the T–B cell interaction. Moreover, the secretion of IL-2
was reduced in the culture media of Fut8−/−OT-II T–B cells compared with Fut8+/+OT-II T–B cells (Figure 5H). Furthermore, theT cell proliferation with OVA323–339-loaded B cells was analyzed
by CFSE dilution methods. Compared to the proliferation of
Fut8+/+OT-II CD4+ T cells, those of Fut8−/−OT-II CD4+ T cells
was significantly reduced with the cocultivation of OVA323–339loaded Fut8+/+OT-II B cells (Figure 5I). These results indicatedthat Fut8 deficiency contributes to attenuated T–B cell communication, and follows attenuated T cell activation.
To further elucidate the underlying mechanism of the
reduced T–B cell interaction caused by the disruption of Fut8,
T and B cell Activation PCR Array was used to compare mRNA
expression in Fut8+/+ SPLs with that in Fut8−/− SPLs following
OVA immunization. As illustrated in Table 1, the expression
levels of four genes (CD3e, CD4, CD8, and CD40L) associated with TCR complex formation, eight genes (IL-2Rα, IL-4,
IL-6, IL-10, IL-12, IFNγ, CXCL12, and CXCR4) involved in
T cell activation, and two genes (CD79a and CD81) associatedwith B cell activation, were downregulated in Fut8−/− SPLs.
Moreover, the gene expressions of signal molecules such as
MAPKKK, Vav1, PIK3, PKC, and Cyclin D3 were downregulated in Fut8−/− SPLs. Since core fucosylation of proteins is an
important post-translational process, it is not surprising thatmany molecules involved in T and B cell activation were downregulated in Fut8−/− SPLs.

TABLE 1 | Gene expression of Fut8−/− SPLs after OVA immunization.
Gene name

Gene access number

Fold change (Fut8+/+/Fut8−/−)T-cell activation
CD3e
CD4
CD8
Cd40L
IL-2Rα
IL-4
IL-6
IL-10
IL-12
IFNγ
CXCR4
CXCL12

NM-007648
NM-013488
NM-009858
NM-011616
NM-008367
NM-021283
NM-031168
NM-010548
NM-008352
NM-008337
NM-009911
NM-001012477

2.15
3.16
2.51
2.17
3.04
2.18
2.14
2.25
3.89
3.08
2.04
2.07

B-cell activation
CD79a
CD81

NM-007655
NM-133655

3.35
2.82

Cell signaling
MAPKKK
Vav1
PIK3
PKC
Cyclin D3

NM_009316
NM-011691
NM-001077495
NM-008859
NM-001081636

2.41
3.28
2.36
2.00
3.84

analysis showed that the core fucosylation level of T and B cells
in Fut8−/−OT-II mice (Figure S3 in Supplementary Material).
Since MHC-II on the B cell surface can present peptides forrecognition and activation of T cells, in this study, B cells wereisolated from Fut8+/+OT-II and Fut8−/−OT-II spleen and incubated with monobiotin-labeled OVA323–339. Although electron
density was seen for the structures of I-Ad covalently linked toan OVA323–339, with a single N-glycan (24), the core fucosylation
did not affect the peptide presentation abilities of the MHCs
between Fut8+/+OT-II and Fut8−/−OT-II B cells (Figure S4 in
Supplementary Material). The CD4+ T cells were then stimulated with OVA323–339 loaded B cells and subsequent T cell priming was investigated. The freshly purified ex vivo CD4+ T cells
were stimulated with the OVA323–339-loaded B cells. As shownin Figure 5A, compared with Fut8+/+OT-II CD4+ T cells, thephosphorylation level of ZAP-70 was dramatically decreased in
Fut8−/−OT-II CD4+ T cells with stimulation of OVA323–339-loaded
B cells. Moreover, to remove the cell-surface fucosylation, theFut8+/+OT-II CD4+ T cells were treated with 100 mU Glyko®
α(1-2,3,4,6) Fucosidase, which can cleaves α1,6-linked fucose
more efficiently than other α-fucose linkages (25). The signaling via TCR was significantly suppressed in OT-II CD4+ T cells
treated with this fucosidase, when the T cells were stimulatedwith OVA323–339-loaded B cells (Figure 5B), indicated that the
Fut8 inactivation results in the less responsive for TCR stimulation with pMHC-II, despite similar TCR expression levels(Figure 4A). Moreover, the population of TCR+CD69+ cells was
significantly reduced in Fut8−/−OT-II CD4+ T/OVA323–339-loaded
B cells compared with Fut8+/+OT-II CD4+ T/OVA323–339-loaded
B cells (Figure 5C).
To further determine how Fut8 deficiency affects the T–B cellinteractions, the communication of CD4+ T cells and B cells wasobserved by confocal microscopy. OVA323–339-loaded MHC-II was

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DISCUSSION
The glycosylation and Golgi processing pathways have coevolved
with the larger regulatory network that controls T cell activation.
It is not surprising that changes of glycosylation are linked to
AD pathogenesis, such as galactosylation (26, 27) and sialylation (28, 29). The lower levels of sialylated IgG were foundin rheumatoid arthritis (RA) and Wegener’s granulomatosis
patients, and the sialylated IgG was increased in the sera ofpatients during remission (28, 29). Moreover, loss of galactose
residues on IgG1 is showed in the sera of RA patients (26, 27).
The significant differences of O-glycan on T helper cells weredetected in active SLE patients (30). Core-fucosylated glycans
that contain bisecting GlcNAc was increased on the IgG of SLE(31). In this study, core fucosylations were associated with SLEseverities, and significantly increased in the CD4+ T cells of
SLE patients. Coincidently, previous study by Fujii et al. foundthat the core fucosylation on T cells, required for activation of
TCR signaling with anti-CD3/CD28 Abs and induction in colitis,
is significantly increased in patients with inflammatory bowel
disease (32). Hence, one possible consequence of hyper core

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Core Fucosylation in T-Cell Activation

FIGURE 4 | Core fucosylation is required for the activation of CD4+ T cells. (A) Western blots analysis of T cell receptor (TCR). Whole cell lysates were resolved by
SDS-PAGE on a 8% gel, transferred to a PVDF membrane, and probed with anti-TCRβ Ab. Densitometric analysis of the bands of TCRβ normalized against
GAPDH. (B) Core fucose of N-glycan on TCRβ in Fut8−/−CD4+ T cells was detected by LCA blot. Whole cell lysates were immunoprecipitated with an anti-TCRαβantibody. The immunoprecipitates were resolved by SDS-PAGE on a 8% gel, and probed with the LCA and anti-TCRβ Ab. (C) Histograms of binding capacity with
LCA. Core fucosylation level on the surface proteins of Fut8+/+CD4+ T and Fut8−/−CD4+ T cells investigated by FACS analysis. (D) Downregulation of phosphorylated
Zap70 in Fut8−/−CD4+ T cells. Purified CD4+ T cells were serum-starved and were stimulated with anti-CD3/CD28 Abs for 5 min at 37°C. Cells were lysated in lysis
buffer for 15 min on ice. Whole cell lysates were subjected to 10% SDS-PAGE. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Densitometricanalysis of the bands of pZAP70 normalized against ZAP70. Data are reported as the mean ± SD from three independent experiments (**p < 0.01; ***p < 0.001).
(E) Loss of Fut8 reduced the CD4+CD69+ cells populations in the SPL after OVA immunization. SPLs were isolated from OVA-immunized and unimmunized mice(n = 5). Cells were stained with anti-CD69 and anti-CD4 Abs, and then detected by FACS. Data are reported as the mean ± SD from three independent experiments(*p < 0.05; ns, not significant). (F) Loss of Fut8 decreased the proliferation of CD4+ T cells. Purified CD4+ T cells were stimulated with anti-CD3ε Ab-coatedmicrobeads and anti-CD28 Ab for 0, 6, 24, and 48 h at 37°C. The growth rates of CD4+ T cell were detected by MTT assay. Data are reported as the mean ± SD
from three replicate cultures (*p < 0.05; **p < 0.01). The absorbance related to the formazan dye level was measured with a microplate reader at 570 nm.

fucosylation-induced T cell activation could be the developmentof SLE. However, the underlying mechanisms of how core fucosylation regulate T cell activation with TCR–pMHC interaction
remain unclear.
T cell recognition of pMHC-II ligands on B cells is thought
to be carefully coordinated in CD4+ T cells. Conformational
flexibility is likely responsible for the high degree of promiscuity or cross-reactivity that is evident in the TCR recognitionof pMHCs (33–35). There are two models to explain how
TCR–pMHC interactions result in T cell activation (36, 37). One

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model involves an activation threshold based on the occupancy
time of the TCR clustering with pMHCs. The alternative modelis that pMHC could induce a specific conformational change
of TCR complex and influence the quality of signal transductions via TCRs. Since the core fucose of N-glycan is located on
the cellular surface of T and B cells, and the core fucosylation
could affect the flexibility of N-glycan antenna (38) as well as
the conformational stability of proteins (39), it is reasonable toassume that core fucosylation of TCR would affect the geometry
and conformation of any TCR–pMHC clusters in the T–B cell

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Core Fucosylation in T-Cell Activationinteractions. Based on a previous study in D. melanogaster cells,
the closeness of the interaction of Cα with Cβ is shown by thehydrogen bonds of the core fucose residue (6, 17), suggested

core fucose possibly strengthens association of Cα with Cβ.
A water molecule also bridges the fucose exocyclic oxygen andthe side chain of Arg150β (17). In this scenario, core fucosylation

FIGURE 5 | Continued

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FIGURE 5 | Lack of core fucosulation reduced interaction ability between T cell receptor (TCR) and pMHC-II and impaired the signaling via TCR. (A) ZAP70
phosphorylation of Fut8+/+OT-II T–B cells. (T) B cell conjugate formation was initiated by centrifuging together CD4+ T cells with or without 1 µg/mL OVA323–339-loaded
B cells. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Data are representative of three independent experiments. Densitometric analysis of the
bands of pZAP70 normalized against ZAP70. Data are shown as the mean ± SD (**p < 0.01). (B) ZAP70 phosphorylation of Fut8+/+OT-II CD4+ T cells with orwithout fucosidase treatment. Purified CD4+ T cells were treated with or without 100 mU Bovine Kidney Fucosidase, and then coincubated with 1 µg/mL OVA323–339
loaded Fut8+/+OT-II B cells for 30 min. The blots were probed by anti-pZAP70 Ab and anti-ZAP70 Ab. Data are representative of three independent experiments.
Densitometric analysis of the bands of pZAP70 normalized against ZAP70. Data are shown as the mean ± SD (**p < 0.01; ***p < 0.001). (C) Compare of activationof Fut8+/+OT-II T + B cells and Fut8−/−OT-II T + B cells. CD4+ T cells were conjugated with OVA323–339 loaded B cell. After 30 min, the T–B cells were fixed and stainedwith anti-TCRβ and anti-CD69 Abs. Data are shown as mean values ± SD (**p < 0.01). (D,E) Distribution of MHC on the T and B cells. The T–B cells were fixed,
permeabilized, and stained with Abs to MHC-II (green). Data are from three separate experiments. The ratio of MHC II intensity at the T–B cell conjugate site relative
to non-conjugate areas. Data are shown as the mean values ± SD (**p < 0.01). (F) FACS analysis of the conjugates between B cells and CD4+ T cells. B cells(OVA323–339-loaded or not loaded) were labeled with anti-MHC-II (FITC) and CD4+ T cells were labeled with anti-TCRβ (PE-Cy5). T and B cells were quickly mixed andconjugated by a brief centrifugation step. They were then incubated at 37°C for 30 min. A representative staining profile with anti-MHC-II and anti-TCRβ mAb isshown. The conjugate cells are double positive (MHC-II+TCRβ+) cells. (G) Percentage of T–B cell conjugates (both MHC-II and TCRβ positive cells) was calculated.
Data are reported as the mean ± SD (**p < 0.01) in four independent experiments. (H) The secrition of IL-2 was downregulated in the culture media of Fut8−/−OT-II
T–B cells. OD values were measured at 492 nm using a microplate reader. Data are reported as the mean ± SD (**p < 0.01) in three independent experiments.
(I) Proliferation of Fut8+/+OT-II CD4+ T cells with OVA323–339 loaded Fut8+/+OT-II B cells. Fut8+/+OT-II CD4+ T cells were purified and labeled with carboxyfluoresceindiacetatesuccinimidyl ester (CFSE). Fut8+/+OT-II CD4+ T cells were cocultivated with 1 µg/mL OVA323–339 loaded Fut8+/+OT-II B cells for 48 h, the divided cells wereanalyzed by FACS. One representative experiment is shown. M1–M4 indicates daughter cell populations which have subsequently lost half of their CFSE signal.

is expected to provide exciting opportunities to control the
TCR function. In the present study, the percentage of T–B cell
conjugates (TCR+MHC-II+) was reduced by a factor of 3.97
(29.4/7.4%) in Fut8−/−OT-II cells, indicated that the core fucosylation has significant functional implications in TCR–pMHC
interaction.
Given the intimate relationship between pMHC recognition
and TCR signaling, the signaling via TCR is also be regulated byglycosylation. It has long been appreciated that Mgat5 deficiency
could enhance TCR recruitment to the synapse and results ingreater TCR internalization/endocytosis (11). Physiologically,

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TCR recognition of pMHC-II ligands on APCs such as dendritic
cells and B cells are the most important checkpoint for CD4+ T cell
activation (40). Engagement of the TCR complexes leads to a signaling cascade of protein tyrosine kinases, such as ZAP70. To explore
the role of core fucosylation on the T cell activation, we generated
Fut8+/+OT-II and Fut8−/−OT-II mice. Fut8 deficiency results inthe attenuated phosphorylation of ZAP70 in Fut8−/−OT-II CD4+
T cells with OVA323–339-loaded B cells. Also, the phosphorylationof ZAP-70 was significantly reduced in Fut8+/+OT-II CD4+ T cellsby the treatment of fucosidase. Moreover, in Fut8−/−OT-II cells,
the number of CD4+ T cells activation (CD69+) was decreased

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Core Fucosylation in T-Cell Activation

by a factor of 3.5. The proliferation of the Fut8−/−OT-II CD4+
T cells cocultivated with OVA323–339-loaded B cells was decreased
compared with the Fut8+/+OT-II CD4+ T cells. It is conceivablethat low T–B cell conjugates was proportional to the decreased
T cell activation and proliferation in Fut8−/− CD4+ T cells. Core
fucosylation is likely to be important in all three proposed stages
involved in the T cell activation. First, core fucosylation is essential for the TCR structural formation. Second, core fucosylationof TCR could regulate the recognition of pMHC and affect T cell
activation threshold. Third, fucose-specific lectins (41) might
participate the events in the T–B cell interaction.
Systemic lupus erythematosus is characterized by the
overproduction of auto antibodies, mainly IgG. However,B lymphocyte hyperactivity in SLE is T cell dependent. T cells
from SLE patients are activated with a decreased activationthreshold and regulated abnormally (32). Indeed, overactive CD4+ T cells had been implicated in the pathogenesis of
SLE (42). Although we know that CD4+ T cell deregulation
contributes to SLE pathogenesis, but the mechanism is stilllargely unknown. Studies in SLE patients and murine models
of lupus have shown enhanced level of IL-4 (43), IFNγ (44),and IL-6 (45, 46). Compared to Fut8+/+ mice, the IgG classswitching was significantly reduced in the sera of Fut8−/− mice
after OVA immunization due to low levels of IL-4, IL-5, IL-6,
IFNγ, and TGF secreted by Fut8−/−CD4+ T cells. Developmentof SLE is associated with genetic, hormonal, environmental,
and immunological factors, mainly those related to the helper
T cell activation. To investigate the mechanisms of the CD4+
T cell activation that is important for SLE pathogenesis, we
induced the EAE model using Fut8+/+ and Fut8−/− mice. Loss of
Fut8 reduced CD4+ T cell activation and ameliorated the EAE
in Fut8−/− mice. Fut8−/− mice are resistant to the induction of
EAE, whereas Mgat5−/− mice are hypersensitive to it (47). In
this regard, Fut8 and Mgat5 function as opposing regulators ofT cell activation thresholds and susceptibility to AD. The hyper
core fucosylations are associated with SLE severities, however,whether altered core fucosylation is a consequence or an underlying cause of the SLE cascade remains unclear.
FUT8 is able to modify multiple proteins, followed by thechange of their functions. Because the most of immune response
molecules are glycoproteins, FUT8 knockdown/knock out
affected their function associated with immune response. Okada
et al. (10) showed that loss of core fucosylation caused an inhibitory receptor PD-1 deprivation on the cellular surface and augmented T cell activation, when Fut8−/−CD4+ T cells transferred

into Rag2−/− mice. Although we did not measure the cell-surface
PD-1 expression in the T cell activation and T–B cell interaction,
the core fucosylation could regulate the PD-1 expression bymodulating TCR signaling strength. Physiologically, there may
be some equilibria of the core fucosylation on the TCR and PD-1
to regulate T cell activation. The complexity of in vivo condition
made us cannot conclude the inability for TCR signaling pathway
is the sole reason for the T cell activation, but our study firstrevealed that Fut8 is essential for TCR–pMHC contact and thefollowing CD4+ T cell activation. The balance between specificand degenerate T cell recognition of pMHC-II holds important
implications for protective immunity versus autoimmunity. Witha better understanding of how core fucosylation regulates of theadaptive immune system, its use in therapy for SLE may prove to
be a useful intervention.

ETHICS STATEMENT
All animal work was approved by the Ethics Committee at the
Dalian Medical University. The Ethics Committee at the hospital
approved the study protocol.

AUTHOR CONTRIBUTIONSWL, SM, and SS designed research and performed experiment;
ML, ZL, and RY analyzed the experimental data; TM, JG, JZ, andNT corrected paper; WL designed research and wrote paper. All
authors reviewed the results and approved the final version of themanuscript.

FUNDING
This work was supported by the National Nature Science
Foundation of China (31570797, 31270864, 30972675), andNatural Science Foundation of Liaoning Province (2015020253),
China.

SUPPLEMENTARY MATERIALThe Supplementary Material for this article can be found online at
http://www.frontiersin.org/articles/10.3389/fimmu.2018.00078/
full#supplementary-material.
VIDEO S1 | Experimental autoimmune encephalomyelitis symptoms in Fut8+/+
mice.
VIDEO S2 | Experimental autoimmune encephalomyelitis symptoms in
Fut8−/− mice.

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Core Fucosylation in T-Cell Activation

Copyright © 2018 Liang, Mao, Sun, Li, Li, Yu, Ma, Gu, Zhang, Taniguchi and Li.
This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does notcomply with these terms.

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Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.

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14

January 2018 | Volume 9 | Article 78

Glycoconj J (2017) 34:127–138
DOI 10.1007/s10719-016-9739-2

ORIGINAL ARTICLE

Catfish rhamnose-binding lectin induces G0/1 cell cycle arrest
in Burkitt’s lymphoma cells via membrane surface Gb3
Shigeki Sugawara 1 & Changhun Im 1 & Tasuku Kawano 1 & Takeo Tatsuta 1 &Yasuhiro Koide 2 & Daiki Yamamoto 2 & Yasuhiro Ozeki 2 & Kazuo Nitta 1 &Masahiro Hosono 1

Received: 15 June 2016 / Revised: 24 August 2016 / Accepted: 3 October 2016 / Published online: 28 October 2016
# Springer Science+Business Media New York 2016

Abstract Silurus asotus egg lectin (SAL), an α-galactosidebinding protein isolated from the eggs of catfish, is a memberof the rhamnose-binding lectin family that binds to Gb3 glycan (Galα1–4Galβ1–4Glc). We have previously demonstrated that SAL reduces the proliferation of Gb3-expressing
Burkitt’s lymphoma Raji cells and confirm here that it does
not reduce their viability, indicating that unlike other lectins, it
is not cytotoxic. The aim of this study was to determine thesignal transduction mechanism(s) underlying this novel SAL/
Gb3 binding-mediated effect profile. SAL/Gb3 interactionarrested the cell cycle through increasing the G0/1 phase population of Raji cells. SAL suppressed the transcription of cell
cycle-related factors such as c-MYC, cyclin D3, and cyclindependent protein kinase (CDK)-4. Conversely, the CDK inhibitors p21 and p27 were elevated by treatment with SAL. In
particular, the production of p27 in response to SAL treatment
increased steadily, whereas p21 production was maximal at
12 h and lower at 24 h. Activation of Ras-MEK-ERK pathway
led to an increase in expression of p21. Notably, treatment ofRaji cells with anti-Gb3 mAb alone did not produce the above
Electronic supplementary material The online version of this article(doi:10.1007/s10719-016-9739-2) contains supplementary material,
which is available to authorized users.
* Masahiro Hosono
mhosono@tohoku-mpu.ac.jp
1

Division of Cell Recognition Study, Institute of Molecular
Biomembrane and Glycobiology, Tohoku Medical andPharmaceutical University, 4-4-1 Komatsushima, Aoba-ku,
Sendai 981-8558, Japan

2

Laboratory of Glycobiology and Marine Biochemistry, Departmentof Life and Environmental System Science, Graduate School of
NanoBio Sciences, Yokohama City University, 22-2 Seto,
Kanazawa-ku, Yokohama 236-0027, Japaneffects. Taken together, our findings suggest that Gb3 on theRaji cell surface interacts with SAL to trigger sequential GDPRas phosphorylation, Ras-MEK-ERK pathway activation,
p21 production, and cell cycle arrest at the G0/1 phase.
Keywords Cell cycle arrest . Extracellular signal-regulated
kinase . Globotriaosylceramide . p21 . SUEL/
rhamnose-binding lectin

Abbreviations
BrdU
Bromodeoxy uridine
CCND3
Cyclin D3
CDK
Cyclin-dependent protein kinase
ERK
Extracellular signal-regulated kinase
FBS
Fetal bovine serum
FITC
Fluorescein isothiocyanate
Gb3
Galα1–4Galβ1–4Glc-Cer
GDP
Guanosine diphosphate
GEM
Glycosphingolipid-enriched microdomains
GTP
Guanosine triphosphate
GSL
Glycosphingolipid
mAb
Monoclonal antibody
JNK
c-Jun N-terminal kinase
MAPK
Mitogen-activated protein kinase
MEK
MAPK/ERK kinase
PVDF
Polyvinylidene difluoride
qRT-PCR
Quantitative reverse transcriptionpolymerase chain reaction
SAL
Silurus asotus egg lectin
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis
SUEL
Sea urchin egg lectin

128

Introduction
Rhamnose-binding lectins (RBLs) have been found in theeggs (oocytes) of various fish species [1–3] and are capableof binding to L-rhamnose and α-D-galactosides. The primarystructure of rhamnose-binding lectins is similar to that of a Dgalactose-binding lectin termed SUEL that was purified fromsea urchin (Anthocidaris crassispina) eggs and is composed of
105 amino acid residues [4]. The symbolic term BRBLs^ isnamed after their carbohydrate-binding specificity against Lrhamnose. Indeed, it shows the highest affinity among common
saccharides, however, actual ligands of RBL are thought to be αgalactosyl sugar chains such as human type B oligosaccharide
and globotriaosylceramide (Gb3) being on the cell surface.
Silurus asotus egg lectin (SAL) is a 32-kDa protein comprisedof three tandem repeats of 95 amino acid residues [5]. Previously,
we demonstrated that SAL agglutinated Raji Burkitt’s lymphoma
cells (which express glycosphingolipids [GSLs] containing Gb3(or CD77; Galα1–4Galβ1–4Glc) in GSL-enriched microdomains on the cell surface) but does not agglutinate
erythroleukemia K562 cells (which do not express Gb3 on thecell surface) [6]. Additionally, SAL caused a reduction in cell size
and increased annexin-V binding to and propidium iodide incorporation into Raji cells. Although this effect on Raji cells might
represent damage at the late apoptosis or necrosis stage, the SALtreated Raji cells remained viable [7]. In contrast, the lectin CSL3
purified from the eggs of the chum salmon (Oncorhynchus keta)
was found to induce cell death of Gb3-displaying Caco-2 cells
via an apoptotic pathway [8]. Watanabe et al. reported that Gb3expressing HeLa cells were killed by pierisin, a toxic lectin isolated from cabbage butterfly (Pieris rapae) wings [9]. Pierisin
contains a triple tandem repeat of ricin B-like domains and an
ADP-ribosyltransferase domain in the polypeptide [10].
Treatment of HeLa cells with pierisin caused a delay in the cell
cycle as well as apoptosis [11]. Therefore, SAL appears to have a
different biological function as compared to CSL3 and pierisin.
Much research has focused on Gb3 because it comprises oneof the components of glycosphingolipid-enriched microdomains(GEMs) on the cell surface, which has been shown to function as
trigger of signal transduction. The glycosphingolipids including
Gb3 in GEM were reported to associate with signal transducers
such as Src family kinases and small G-proteins localized in thecytosol through the lipid bilayer [12]. Therefore, in this study wefocused on identifying the signal transduction pathways stimulated by the SAL-Gb3 interaction. We found that the interaction
between SAL and Gb3 enhanced the expression level of cyclindependent protein kinase inhibitors. This enhancement promoted
Raji cell cycle arrest at the G0/1 phase through phosphorylation of
GDP-Ras, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), and ERK.
Taken together, these findings suggest that Gb3 plays an important role in cell signal regulation through its ability to bind tolectins. Elucidation of the mechanism of cell cycle arrest by SAL

Glycoconj J (2017) 34:127–138

and Gb3 might lead to the discovery of novel lectin-based cell
regulatory systems.
Materials and methods
Materials SAL was purified by sequential chromatography
on DE23 (Whatman, Little Chalfont, UK) anion exchange
and D-galactose-Sepharose 6B columns as described previously [13].
Cell lines Burkitt’s lymphoma Raji cells from the Cell
Resource Center of Biomedical Research, Institute ofDevelopment, Aging and Cancer, Tohoku University(Sendai, Japan) were cultured in RPMI-1640 medium(Nissui Pharmaceutical Co., Tokyo, Japan) supplemented
with 10 % v/v fetal bovine serum (FBS) and antibioticantimycotic solution (penicillin [100 IU/ml], streptomycin[100 μg/ml], and amphotericin B [0.25 μg/ml], Life
Technologies, Carlsbad, CA, USA) and maintained at 37 °C
in a 95 % air/5 % CO2 atmosphere.
Cytotoxicity and cell viability assays Raji cells (1 × 106 /ml)
were seeded in 6-well plates and incubated with various concentrations of SAL (0–100 μg/ml) for 24, 36, 48, 72, 96, or
120 h. Cytotoxic activity and cell growth were determined bytrypan blue (0.5 % w/v) exclusion assay [14]. Cell proliferation was determined by WST-8 assay using a Cell Counting
Kit-8 (Dojindo Laboratories; Kumamoto, Japan). After plating the cells into a 96-well flat-bottom plate at 5 × 104 cells/
well (90 μl), the cells were treated with 100 μg/ml SAL (final
concentration) for 24 or 48 h. Then, 10 μl WST-8 solution wasadded into each well and the cells were incubated for 4 h at
37 °C. The absorbance was measured at a wavelength of
450 nm using the GloMax Multi Detection System(Promega, Madison, WI, USA). Cells were treated with rat
anti-Gb3 monoclonal antibody (mAb) (clone 38.13;
Beckman Coulter, Miami, FL, USA) in flat-bottom plastic
plates for 24, 48, 72, 96, or 120 h at 37 °C. Cytotoxic activity
and cell growth were determined as described above. Brightfield images were acquired using an inverted microscope(model IX71; Olympus Co., Tokyo, Japan) with a 10× objective lens.
Detection of cell cycle arrest by SAL Cell cycle arrest was
assayed using a fluorescein isothiocyanate (FITC)
bromodeoxy uridine (BrdU) flow kit (BD Biosciences, San
Jose, CA, USA) according to the manufacturer’s protocol. In
brief, cells (5 × 105) were treated with SAL (100 μg/l) for
24 h, fixed with 4 % paraformaldehyde, and permeabilizedby saponin for 1 h at 37 °C in the presence of 1 U DNase.
Cell division was estimated as the ratio of BrdU incorporation
into cells (detected using a FITC-conjugated anti-BrdU antibody after incubation for 20 min at room temperature) to totalGlycoconj J (2017) 34:127–138

DNA amount in the cells (detected with 7-aminoactinomycinD) using a FACSCalibur (BD Biosciences) with single laser
emitting excitation at 488 nm and analyzed with Cell Quest
pro software (BD Biosciences).
Expression levels of mRNA encoding cell cycle-related
molecules Raji cells (5 × 105) were cultured for 12 or 24 h
in RPMI-1640 medium containing SAL (100 μg/ml), SAL(100 μg/ml)/saccharide (20 mM), or without SAL at 37 °C
in 95 % air/5 % CO2. Total RNA was extracted from cells
using an AllPrep RNA/Protein Kit (Qiagen, Valencia, CA,
USA). cDNA was synthesized from the total RNA (1 μg)
using a SuperScript VILO cDNA Synthesis Kit (Invitrogen,
San Diego, CA, USA). Quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) assays were performed
using a LightCycler 480 system with the LightCycler 480
Probes Master Kit (Roche Diagnostics, Indianapolis, IN,
USA). PCR primers for amplification of CDK4, c-MYC, cyclin D3 (CCND3), p21, and p27 using a TaqMan/probe library
assay were designed by the Universal Probe Library Assay
Design Center (https://www.roche-applied-science.
com/sis/rtpcr/upl/acenter.jsp). The expression levels of thesegenes were standardized relative to the mRNA expression
level of GAPDH (as a housekeeping gene) based on theiraverage crossing point values.
Protein expression of signal transduction molecules, theirphosphorylated forms, and cell cycle-related molecules
Raji cells (5 × 105) were cultured for 12 or 24 h in RPMI1640 with or without SAL (100 μg/ml) at 37 °C in 95 % air/
5 % CO2. Cell lysate was prepared using an AllPrep RNA/
Protein Kit (Qiagen), subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (10 % separation gel), and electrotransferred onto a polyvinylidene
difluoride (PVDF) membrane (pore size 0.45 μm) (HybondP; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) according to the manufacturer’s protocol [15–17]. The membrane was treated with blocking buffer (Blocking One;
Nacalai Tesque Inc., Kyoto, Japan) for 1 h at room temperature and washed with Tris-buffered saline (TBS) containing
0.05 % Tween-20. The primary antibodies used were directed
to p21Waf1/Cip1 (1:1000, rabbit mAb, clone 12D1; Cell
Signaling Technology Inc., Danvers, MA, USA [CST]),
p27Kip1 (1:1000, rabbit mAb; clone D69C12; CST), c-MYC(1:1000, rabbit polyclonal antibody; CST), CDK4 (1:2000,
mouse mAb; clone DCS156; CST), cyclin D3 (1:2000, mouse
mAb; clone DCS22; CST), phospho-MEK1/2 (1:1000, rabbitmAb; CST), MEK1 (1:1000, rabbit mAb; CST), phosphoERK1/2 (1:1000, mouse mAb; BD Biosciences), ERK1
(1:5000, mouse mAb; BD Biosciences), phospho-p38
MAPK (1:2500, mouse mAb; BD Biosciences), p38 MAPK
(1:5000, mouse mAb; BD Biosciences), phospho-JNK(1:250, mouse mAb; BD Biosciences), JNK (1:250, mouse

129

mAb; BD Biosciences), and GAPDH (1:20,000, mouse
mAb; clone 6C5; Ambion/Invitrogen, Carlsbad, CA, USA).
These antibodies were applied in immunoreaction enhancer
solution (Can Get Signal Solution 1; Toyobo Co., Osaka,
Japan). The membrane was incubated for 16 h at 4 °C. The
secondary antibody, horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG (Chemicon International Inc.,
Temecula, CA, USA), was diluted 1:20,000 in immunoreaction enhancer solution. The membrane was incubated for 1 h
at room temperature and then exposed to X-ray film (Fuji FilmCo., Tokyo, Japan) using enhanced chemical luminescenceand western blotting with ECL Prime detection reagent (GEHealthcare Bio-Sciences AB). A synthetic inhibitor of
MEK1/2, 1,4-diamino-2,3-dicyano-1,4-bis (2aminophenylthio) butadiene (U0126) (1 μg) (Calbiochem;
S a n D i e g o , C A , U S A ) w a s d i s s o l ve d i n 2 4 7 μ ldimethylsulfoxide to create a 10 mM stock solution. Cells
were incubated with U0126 (10 μM) in RPMI-1640 with
FBS for 2 h, supplemented with SAL (100 μg/ml), and incubated for another 12 h. Whole cell extracts were separated by
SDS-PAGE on a 10 % gel and blotted onto PVDF membranes. Phosphorylated kinases were identified by western
blotting with anti-phospho-MEK 1/2, anti-MEK 1/2, antiphospho-ERK1/2, anti-ERK1, and HRP-conjugated antimouse IgG antibodies. Signals were detected by X-ray
film exposure and western blotting as described above.
Proteins were quantified by densitometry using NIH
ImageJ software (version 1.440, Bethesda, MD, USA).
The optical densities of phosphorylated MEK 1/2 (PMEK1/2) and phosphorylated ERK1/2 (P-ERK1/2) were
measured and normalized to the optical densities of
MEK1/2 and ERK1, respectively.

Detection of GTP-Ras The active form of Ras exchanged
through GDP to GTP as a result of SAL stimulation was
assayed by affinity precipitation using a Ras activation assay
kit (Upstate Biotechnology; Lake Placid, NY, USA). Raji cells(1 × 106) were cultured for 0.5, 3, 6, 12, or 24 h in RPMI-1640
containing SAL (100 μg/ml) at 37 °C in 95 % air/5 % CO2,
lysed with Mg2+ lysis buffer (125 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid [HEPES], pH 7.5, 750 mM
NaCl, 5 % Igepal® CA-630, 50 mM MgCl2, 5 mM ethylenediaminetetraacetic acid, and 10 % glycerol), and incubatedwith the agarose-conjugated Ras-binding domain of Raf-1(5 μl) for 30 min at 4 °C. The agarose-coated beads wereboiled in sample buffer and the cell lysate was then subjected
to SDS-PAGE on a 10 % separation gel and transferred onto a
PVDF membrane. The GTP-Ras formed following SAL stimulation was detected by anti-Ras mAb (0.05 μg/ml) andHRP-conjugated polyclonal antibodies (1:1000) [18, 19].
Activated Ras was detected using ECL Prime (GEHealthcare Bio-Sciences AB).

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Glycoconj J (2017) 34:127–138

RNA interference-mediated knockdown of alpha-1,4galactosyltransferase (A4GALT) Raji cells (1.2 × 106) cells
were centrifuged and resuspended in 100 μl nucleofection V
solution (Lonza, Basel, Switzerland) containing 20 μM
A4GALT siRNA (Dharmacon, Lafayette, CO, USA) andelectroporated with a Nucleofector (Lonza) using the M-13
program. After nucleofection, the cells were transferred to a
12-well plate containing 1 ml fresh complete medium (RPMI
1640 containing 10 % FBS and antibiotic-antimycotic solution) and incubated at 37 °C for 120 h. After incubation, theexpression of Gb3 was detected by flow cytometry.
Flow cytometric analysis of Gb3 expression Cells (2 × 105)
were treated with or without anti-Gb3 mAb (BGR23, mouse
IgG2b; Tokyo Kasei Co. Ltd., Tokyo, Japan) at a dilution of
1:1000 in PBS (100 μl) at 4 °C for 30 min and washed threetimes with PBS. The cells were then treated with Alexa Fluor(AF) 488-conjugated goat anti-mouse IgG (H + L) (Molecular
Probes, Invitrogen AG, Basel, Switzerland) at a dilution ofaStatistical analysis Experimental results are presented as themeans ± standard error (SE). Differences in means were evaluated using the two-tailed Student’s t-test with P values <0.05
considered to be statistically significant.

Results
Effects of SAL on cell proliferation and viability In thecurrent study, we further expand upon our previous findings
following exposure of Raji cells to SAL (100 μg/ml) for 24 hby examining the concentration- and time-dependent effect of
SAL. According to the trypan blue exclusion assay, SAL did
not reduce the viability of Raji cells even at the concentrationof 100 μg/ml for 120 h (Fig. 1a and S1). Proliferation was

(%)
100

Cell viability

75

50

25

0

0

1

5

b

100 (µg/ml)

50

c
7

1.2

Absorbance (450 nm)

Number of cells (× 105 cells)

Fig. 1 SAL reduces theproliferation but has no effect on
the viability of Raji cells. a. Timeand dose-dependent effects of
SAL treatment on cell viability.
Cells were treated with SAL (0, 1,
5, 50, or 100 μg/ml) for 24 h(white columns), 36 h (black columns), or 48 h (gray columns) at
37 °C. Cell viability was evaluated by trypan blue exclusion assay.
The values shown are the meansof triplicate experiments. Error
bars: standard error (SE). b. Cells
were treated with SAL (0, 1, 5,
50, or 100 μg/ml) for 24, 36, or
48 h at 37 °C. The number ofliving cells was assessed bytrypan blue exclusion assay. ○:0 μg/ml (control). Δ: 1 μg/ml. □:5 μg/ml. ■: 50 μg/ml. ●: 100 μg/
ml. c. Cells were treated with (+)
or without (−) SAL (100 μg/ml)
for 24 or 48 h at 37 °C.
Proliferation was evaluated by the
WST-8 assay. The values shown
are the means of triplicate experiments. Error bars: SE

1:2500 in PBS (100 μl) at 4 °C for 30 min. The degree of
Gb3 expression on the cell surface was analyzed using the
FACSCalibur as described above.

6
5
4
3
2

1
0.8
0.6
0.4
0.2
0

1

0

24 48

SAL (-)

002436Time (h)48

24

48 (h)SAL (+)Glycoconj J (2017) 34:127–138

131
Number of cells (fold against control)

reduced further at higher concentrations and was abolishedcompletely at 50 μg/ml (Fig. 1b). The WST-8 assay showed thatSAL reduced cell proliferation in a time-dependent manner(Fig. 1c). On the other hand, Gb3-binding monoclonal antibody,anti-Gb3, had no such effects to Raji cells including cellagglutination (Fig. 2a~c, S2 and S3). Stimulation with SAL(100 μg/ml) for 72 h caused a 6-fold decrease in the number ofcells compared with control cells (Fig. 3). When the medium was
replaced by SAL-free medium, the cells completely recovered
their proliferation activity after 48 h (Fig. 3, column BSAL free^).
These results suggest that SAL reduced the proliferation of Rajicells but was not cytotoxic.

5
4
3
2

1
0

24 48 72
Raji

SAL
free

phase was not shifted by SAL treatment (Table 1). These findings
suggest that SAL arrests the G0/1 phase of the cell cycle bybinding to Gb3, followed by the loss of cell proliferation.

a

b
1.2
Number of cells (x 105)

100
Cell viability (%)

Raji
+SAL

Fig. 3 Removal of SAL restores cell proliferation. Cells were cultured
without SAL at 37 °C for 0, 24, 48, or 72 h (Raji). Other cells were treatedwith SAL (100 μg/ml) for 72 h (Raji + SAL) or with SAL followed by
SAL-free medium for 48 h (SAL free). The number of living cells wasassessed by trypan blue exclusion assay. The values shown are the meansof triplicate experiments. Error bars: SE

G1 cell cycle arrest by SAL To gain insight into the mechanism of SAL-induced growth inhibition in Raji cells, we analyzed the SAL-dependent changes in cell cycle distribution. Flowcytometric analysis showed that stimulation with SAL (100 μg/ml) for 24 h caused a substantial increase of the ratio of the G0/1
phase to the whole Raji cell population of 20 % with a consequent decrease of the proportion of cells in S phase by 20 %(Fig. 4 and Table 1). The G0/1 cell cycle arrest no longer occurred
when SAL was removed from the medium. In contrast, the G2/M
Fig. 2 Anti-Gb3 mAb does not
induce apoptosis or inhibit cellproliferation. Cells were treatedwith anti-Gb3 mAb (100 or
200 μg/ml) or SAL (100 μg/ml)
for 24 h at 37 °C. Cell viability (a)and proliferation (b) were evaluated by trypan blue exclusion and
WST-8 assays, respectively. The
values shown are the means of
triplicate experiments. Error bars:
SE. c. Cell agglutination inducedby treatment with anti-Gb3 mAb(100 or 200 μg/ml) or SAL(20 μg/ml). Negative control(Cont): addition of PBS instead of
lectin or antibody. The images arefrom bright-field microscopy at
100× magnification

6

80
60
40

20
0

100

200

anti-CD77 Ab(µg/ml)

100

PBS

1
0.8
0.6
0.4
0.2

0

SAL
(µg/ml)

c

100

200

anti-CD77 Ab(µg/ml)anti-Gb3 Ab
100 µg/ml

200 µg/ml

Cont

SAL (20 µg/ml)

100
SAL(µg/ml)PBS

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Glycoconj J (2017) 34:127–138

a

b

104

104

103

103

S

BrdU

BrdU

Fig. 4 Alteration of cell cycle
distribution in SAL-treated Rajicells. Cells were cultured without
SAL at 37 °C for 72 h (SALuntreated) (a). Other cells weretreated with SAL (100 μg/ml) for
72 h (SAL-treated) (b) or subsequently with SAL-free medium
for 48 h (SAL-removed) (c). The
distribution of cells in each cell
cycle phase (G0/1, S, and G2/M)
was determined by FACSCalibur
analysis

102

101

100

G0/1

200

102

101

G2/M

0

S

400
600
7AAD

800

1000

100

G2/M
G0/1

0

200

400
600
7AAD

800

1000

c 104

BrdU

103

S

102

101

G2/M
G0/1

100
0

Down-regulation of c-MYC, CDK4, and CCND3 gene expression levels and the induction of mRNA and protein
expression levels of the CDK inhibitors p21 and p27 by
SAL Numerous studies have shown that CDK acts as an accelerating factor of the cell cycle [20, 21]. In Raji cells treatedwith SAL for 24 h, CDK4 mRNA levels were down-regulated
in comparison with those of untreated cells (Fig. 5a).
However, SAL had no effect on the mRNA expression levelsof CDK2 (data not shown). The expression of CDK4 is regulated by c-Myc. In addition, mammalian cyclin D3 is a member of the G1/S cyclin family and functions as a key protein totransition the cell cycle to the next phase [22–24]. Therefore,
Table. 1 Effect of SAL on cell cycle distribution. The percentages ofcells in each cell cycle phase (G0/1, S, or G2/M) were determined by
FACSCalibur analysis. The values shown are the means ± SE from two
or more independent experiments
G0/1

S

G2/M (%)

SAL-untreated

34.3 ± 2.5

62.0 ± 2.9

2.7 ± 0.4

SAL-treated
SAL-removed

53.5 ± 3.2
27.0 ± 0.7

35.1 ± 2.5
67.5 ± 1.1

7.5 ± 1.7
3.7 ± 0.5

200

400

600
7AAD

800

1000

the alteration of protein expression levels of c-MYC and cyclin D3 in Raji cells were examined following SAL treatment.
As shown Fig. 5a, SAL treatment for 24 h reduced the expression level of c-MYC by 40 % and that of CCND3 by 30 %.
Western blotting confirmed the coordinate decreased expression levels of the c-Myc, CDK4, and cyclin D3 proteins(Fig. 5b). In addition, the inhibitory effects of SAL on gene
expression were partially eliminated in the presence of 10 mM
melibiose or rhamnose (Fig. 5c). These findings indicate thatthe interaction between SAL and Gb3 reduced the expressionof CDK4, c-MYC, and CCND3 mRNAs.
p21 and p27 have been shown to inhibit CDK activity andcell cycle progression [25–27]. We therefore examined whether the expression of p21 and p27 was changed by treatment
with SAL. qRT-PCR analysis showed increases of 130 % and
70 % in the mRNA expression levels of p21 and p27, respectively, in SAL-treated Raji cells (Fig. 5a). In addition, western
blotting analysis showed corresponding increases of p21 andp27 protein expression levels (Fig. 5b). Furthermore,
up-regulation of p21 was specifically inhibited in thepresence of rhamnose or melibiose (10 mM) but notby glucose and lactose. Notably, however, elevation of
p27 was not blocked by these saccharides (Fig. 5c).

Glycoconj J (2017) 34:127–138

133

1.2

1

1

0.8
0.6
0.4

0.8
0.6
0.4

0.2

0.2

0

0

0

12
Time (h)

24

1
0.8
0.6
0.4
0.2

0

12
24
Time (h)012Time (h)0012Time (h)242p27 / GAPDH

p21 / GAPDH

3
2.5
2
1.5
1

1.5
1
0.5

0.5

0

1.2
CCND3 / GAPDH

1.2
c-MYC / GAPDHCDK4 / GAPDH

a

0

0

12
24
Time (h)b0Time (h)
12

24

24

CDK4
c-MYC
cyclin D3
p21
p27
GAPDH
Fig. 5 SAL treatment alters the gene and protein expression of CDK4, cMYC, cyclin D3, p21, and p27. a. Cells (1 × 106) were treated with SAL(100 μg/ml) at 37 °C for 12 or 24 h. Total RNAs extracted from SALuntreated and -treated cells were analyzed by qRT-PCR using specificprimers for CDK4, c-MYC, CCND3, p21, p27, and GAPDH, respectively.
The control value was defined as one. Fold increases in the target genes
relative to the control were normalized to GAPDH. Results are expressed
as n-fold increase over the control; means ± SE of three independent
triplicate experiments. b. Cells (1 × 106) were treated with SAL(100 μg/ml) at 37 °C for 12 or 24 h. Whole cell extracts obtained fromSAL-untreated and -treated cells were subjected to western blotting using

anti-CDK4, anti-c-MYC, anti-cyclinD3, anti-p21, anti-p27, and antiGAPDH antibodies. c. Cells (1 × 106) were exposed to SAL (100 μg/ml) and SAL/saccharide (20 mM) co-treatment for 24 h. Total RNAs
extracted from SAL-treated, SAL/saccharide co-treated, and untreatedRaji cells were analyzed by qRT-PCR using the specific primers for
CDK4, c-MYC, CCND3, p21, p27, and GAPDH, respectively. The control value was defined as one. Fold increases in the target genes relative tothe control were normalized to GAPDH. Results are expressed as n-foldincrease over the control; means ± SE of three independent triplicate
experiments

These findings suggest that the up-regulation of the CDK
inhibitors p21 and p27 by SAL treatment induced G0/1 phasecell cycle arrest.

dependent manner as indicated by enhanced phosphorylationof MEK1/2 and ERK1/2 (Fig. 6). Conversely, SAL treatment
had no enhancing effect on the phosphorylation of p38 MAPKor c-Jun N-terminal kinase, which are MAPK family membersin the broad sense (Fig. S4). A4GALT siRNA-treatment ofRaji cells suppressed Gb3 cell surface expression (Fig. 7aand b). Consequently, A4GALT-knockdown abolished SALinduced cell agglutination and also inhibited the phosphorylation of MEK1/2 and ERK1/2 following SAL treatment (Fig. 7c

Activation of the MEK-ERK signaling pathway by Gb3/
lectin interaction at the cell surface The MEK-ERK signaling pathway has been reported to participate in the regulationof cell growth [28]. Treatment with SAL resulted in the formation and accumulation of GTP-Ras in Raji cells in a time-

134

Glycoconj J (2017) 34:127–138

2

2
c-MYC / GAPDHCDK4 / GAPDH

c

1.5
1
0.5
0

1
0.5
0

SAL
Saccharide

-

+
-

+
Mel

+
Lac

+
Rha

+
Glc

SAL
Saccharide

-

+
-

+
Mel

+
Lac

+
Rha

+
Glc

-

+
-

+
Mel

+
Lac

+
Rha

+
Glc

3
p21 / GAPDH

1.5

CCND3 / GAPDH

1.5

1

0.5

0

SAL Saccharide -

+
-

+
Mel

+
Lac

+
Rha

+
Glc

+
-

+
Mel

+
Lac

+
Rha

+
Glc

2.5
2
1.5
1
0.5

0
SAL
Saccharide

p27 / GAPDH

4
3
2
1

0
SAL
Saccharide-

Fig. 5 (continued)

and d). Specifically, the levels of MEK1/2 and ERK1/2 phosphorylation were significantly increased by about 2 and 2.5fold following SAL treatment in A4GALT siRNA-untreatedRaji cells, whereas only 1.1 and 1.2-fold increases were
SAL (100 µg/ml)
0.5

3

6

12

24

0

(h)

12

24

(h)

GTP-RasSAL (100 µg/ml)
0

0.5

3

6

P-MEK1/2
MEK1/2P-ERK1/2

observed upon SAL treatment in A4GALT siRNA-treatedRaji cells (data not shown). These results suggest that thedirect interaction between Gb3 and SAL induced the MEKERK signaling pathway.
Up-regulation of p21 by SAL via phosphorylation of the
Ras-MEK-ERK signaling pathway We tested whether
ERK1/2 phosphorylation was associated with the increase of
p21 expression. Treatment with SAL in combination with the
MEK inhibitor U0126 down-regulated p21 mRNA expression(Fig. 8a) and restored cell growth (Fig. 8b). U0126 by itself
had no significant effect on cell growth (Fig. 8b). Preincubation with U0126 significantly decreased SAL-induced
phosphorylation of ERK1/2 (Fig. 8c). SAL-induced enhancement of p21 protein expression was also inhibited in the presence of U0126 (Fig. 8c).

ERK1

Fig. 6 SAL treatment causes activation of Ras and phosphorylation of
MEK, and ERK. Cells (1 × 106) were treated with SAL (100 μg/ml) at
37 °C for various durations from 0 to 24 h. Whole cell extracts were
subjected to western blotting using antibodies directed to GTP-Ras, PMEK1/2, MEK1/2, P-ERK1/2, and ERK1. Representative results from
triplicate experiments are shown

Discussion
Taken together, the results from this study indicate that the
SAL/Gb3 interaction on the surface of Raji cells promotes
activation of Ras (to form GTP-Ras) and phosphorylation ofGlycoconj J (2017) 34:127–138

a

b

100

Raji

80

100

A4GALT siRNA Raji

80

60

CountsCountsFig. 7 Gb3 knockdown inhibits
the phosphorylation of MEK and
ERK. For the detection of Gb3,
untreated Raji cells (a) and
A4GALT siRNA-treated Rajicells (b) were stained with antiGb3 mAb and AF488-conjugatedgoat anti-mouse Ab (black line).
The degree of Gb3 expression on
Raji cell membranes was determined by FACSCalibur.
Fluorescence intensity of control
cells: gray line. c. Cell agglutination induced by treatment withSAL (100 μg/ml). The images arefrom bright-field microscopy at
100× magnification. d. Degrees
of expression and phosphorylation of MEK and ERK proteins
were detected by western blotting
as described in Materials andMethods

135

40
20

60
40
20

0

0
100

101

102

103

100

104

102

103

104

Fluorescence intensity

Fluorescence intensityc101

SAL (-)SAL (+)Raji

A4GALT
siRNA Rajid

SAL

A4GALT siRNA

-

+
-

+

+
+

P-MEK1/2

MEK1/2P-ERK1/2

ERK1
GAPDH

MEK1/2 and ERK1/2. The suppression of cell growth by SAL
was likely due to the enhancement of p21 expression causedby the phosphorylation of ERK1/2, which derived from theRas-MEK-ERK1/2 pathway. In addition, SAL reduced theprotein expression levels of CDK4, c-myc, and cyclin D3.
These results therefore indicate that SAL treatment induced
cell cycle arrest at the G0/1 phase.

Gb3 is the key structure in globo-series GSLs, which contain a galactosyl α1–4 linkage to lactosylceramide. Cell surface Gb3 is involved in the transmission of various signals
into the cytoplasm via α-galactoside-binding lectins such as
SAL, pierisin, and the mussel Mytilus galloprovincialis lectin(MytiLec) [9–11, 29]. Park et al. elucidated that the otherglobo-series GSL, globotetoraosylceramide (Gb4), interacts

136

b
1.6

Cell growth rate of Rajiap21 / GAPDH

Fig. 8 SAL treatment enhances
p21 protein expression levels
through a Ras-MEK-ERK pathway. a. Quantitative RT-PCR
showing p21 expression, normalized relative to GAPDH, following treatment with SAL (100 μg/ml) and/or the MEK inhibitor
U0126 (10 μM). The values
shown are the means of triplicate
experiments. Asterisk denotes
P < 0.05 versus untreated control
cells. NS denotes not significant
versus untreated control cells. b.
Growth of cells treated or untreated with SAL and/or U0126,
measured by the WST-8 assay.
The values shown are the meansof triplicate experiments. Asterisk
denotes P < 0.05 versus untreatedcontrol cells. NS denotes not significant versus untreated control
cells. c. Degrees of expression
and phosphorylation of RasMEK-ERK pathway-related proteins as detected by western blotting as described in Materials andMethods

Glycoconj J (2017) 34:127–138

*

1.2

NS

NS

0.8

0.4
0
SAL

U0126

-

+
-

+
+

1
NS
0.8
0.6

0.2
0

SAL

+

*

0.4

U0126

-

+
-

+
+

+

c
SAL
U0126

-

+
-

+
+

+

P-MEK1/2
MEK1/2P-ERK1/2
ERK1

p21
GAPDH

with the epidermal growth factor receptor to promote activation of the MAPK pathway [30]. However, it has not yet beenclarified whether Gb3 is involved in the activation of such apathway. In the present study, direct interaction between Gb3
and SAL was found to transmit a regulatory signal in Raji cells
through the MEK-ERK signaling pathway (Figs. 6 and 7).
Phosphorylation of ERK initiates synthesis of the CDK inhibitor p21, which induces cell cycle arrest at the G0/1 phase.
External stimulation through p21 prevents damage to cellular
DNA by irradiation and harmful toxins by arresting the cell
cycle at the G1/S phase transition. In comparison, p27 is involved in cell differentiation and proliferation in addition tocell cycle arrest [31]. p21 binds to the cyclin D3-CDK4 complex and suppresses cell growth. Conversely, recent reports
have shown that the induction of p21 phosphorylation by
ERK2 promotes cell proliferation [32]. Consistent with theseproperties, it has been reported that kinase-deficient forms of
ERK1/2 were able to up-regulate the expression of p21 andsubsequently induce G0/1 phase cell cycle arrest [33]. Previous
studies have suggested that Ras undergoes post-translational
processing to be able to associate with cell membranes [34].
Thus, GDP-Ras might be located in the vicinity of GEMs onwhich Gb3 molecules are present. It is therefore important toelucidate the mechanism of SAL/Gb3 interaction in the initiation of Ras activation to understand the cellular functions oflectin-induced, glycan-mediated signal transduction.
Previous reports have indicated that Ras is activated by theexchange of GTP for GDP, leading to the GTP-dependent
binding of target proteins to Ras [35]. In analogy to the present
findings, the activation of Ras is caused by binding between
galectin-1 and the N-acetyllactosamine of α5β1 integrin,
which is followed by the induction of cell cycle arrest at theG0/1 phase [36]. Thus, Ras activation might involve multiple
pathways associated with various glycans and lectins. Several
types of rhamnose-binding lectins having distinctive domainarchitectures are known [37, 38]. SAL has a unique domain
construction with 9 binding sites based on a trimer of polypeptides, each consisting of triple tandem repeat domains [39].
For example, mAb raised against Gb3, which has divalenthigh-affinity binding sites, did not cause phosphorylation of
GDP-Ras-MEK-ERK1/2 or induce Raji cell cycle arrest(Fig. 2). Binding of inadequate ligands to Gb3 therefore evidently do not cause the transmission of a phosphorylation
signal to GDP-Ras. Studies of the cell regulatory effects ofvarious Gb3-binding lectins will likely help to elucidate thenovel mechanisms whereby multiple lectins are able toGlycoconj J (2017) 34:127–138

transmit different growth-regulatory signals through the same
glycan. This phenomenon is highlighted by our recent discoveries regarding MytiLec, which has a completely different
primary sequence than SAL and causes direct apoptosis bybinding to Gb3 [29]. This report raises the possibility thatthese lectins lead to different cell-regulatory signals [29].
In the current study, phosphorylated ERK1/2 increased theexpression of the CDK inhibitor p21. This protein contains
CDK-binding domains at its N-terminus and interacts with
CDK to inhibit its activity, resulting in cell cycle arrest at theG0/1 phase [40]. In general, the expression of CDK inhibitors isregulated by the tumor suppressor p53, a transcription factor thatis expressed consequent to DNA damage [41]. The p21 and p27
peptides have differing molecular masses of 21 and 27 kDa,
respectively, and their C-terminal domains also play different
roles. We observed that the time-dependent up-regulation of
p27 occurred simultaneously with the down-regulation of cyclin
D3 during a 24-h period to promote cell cycle arrest at the G1
phase. In contrast, p21 was strongly up-regulated during a 12-h
period following treatment with SAL and then decreased slightly
during the subsequent 24-h period (Fig. 5).
Consistently with the phenomena described above, we
found that SAL treatment is affected by the up-regulation of
p21 (Fig. 5) and that production of p21 interfered with cyclin
D3/CDK4 complex formation. Oligosaccharides containing
α-galactoside are not a common type of glycan in humancells. Gb3 has been identified as the target of hemorrhagic
toxins (e.g., verotoxin secreted by pathogenic bacteria, O157
strain) and has been proposed as a rejection antigen of pig
organs that carry an α-galactose residue 1–3 linked tolactosylceramide following xenotransplantation into humans.
The present results, using a lectin isolated from a fish, indicate
a novel role of Gb3 as the trigger for activation of signaltransduction molecules to induce cell cycle arrest of human
lymphoma cells. Notably, α-galactoside-binding lectins (withdiverse primary structures) are isolated more frequently fromlower vertebrates and invertebrates than from higher vertebrates (mammals and birds) [42–47]. The physiological functions of these Bprimitive^ lectins remain to be clarified inalmost all cases. However, it is becoming increasingly clearthat such knowledge will greatly promote biomedical studies
focused on novel regulatory systems in human cells.
Acknowledgments This study was supported by the BAcademic
Frontier^ Project for Private Universities and the BStrategic Project toSupport the Formation of Research Bases at Private Universities(SENRYAKU)^ from the Ministry of Education, Culture, Sports,
Science, and Technology (MEXT), Japan. We would like to thank
Editage (www.editage.jp) for English language editing.
Compliance with ethical standards
Conflict of interest The authors declare that there are no conflicts ofinterest regarding the publication of this paper.

137
Ethical approval This article does not contain any studies with humanparticipants or animals performed by any of the authors.

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Articlepubs.acs.org/jnp

Lissoclibadin 1, a Polysulfur Aromatic Alkaloid from the Indonesian
Ascidian Lissoclinum cf. badium, Induces Caspase-Dependent
Apoptosis in Human Colon Cancer Cells and Suppresses Tumor
Growth in Nude Mice
Takeo Tatsuta,‡ Masahiro Hosono,*,‡ Henki Rotinsulu,†,§ Defny S. Wewengkang,†,§ Deiske A. Sumilat,†,⊥Michio Namikoshi,† and Hiroyuki Yamazaki*,††Department of Natural Product Chemistry, Faculty of Pharmaceutical Sciences and ‡Division of Cell Recognition Study, Institute of
Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Aoba-ku, Sendai 981-8558, Japan§
Faculty of Mathematic and Natural Sciences and ⊥Faculty of Fisheries and Marine Science, Sam Ratulangi University, Kampus Bahu,
Manado 95115, Indonesia
S Supporting Information*

ABSTRACT: Lissoclibadins, polysulfur aromatic alkaloids, were isolated
from the Indonesian ascidian Lissoclinum cf. badium. Lissoclibadins 1 (1),
3 (2), 4 (3), 7 (4), 8 (5), and 14 (6) inhibited the growth of four humansolid cancer cell lines: HCT-15 (colon adenocarcinoma), HeLa-S3 (cervix
adenocarcinoma), MCF-7 (breast adenocarcinoma), and NCI-H28(mesothelioma). Lissoclibadin 1 (1) exerted the most potent cytotoxic
eﬀects in vitro and mainly promoted apoptosis through an intrinsic
pathway with the activation of a caspase-dependent pathway in HCT-15
cells. In vivo studies demonstrated that 1 suppressed tumor growth innude mice carrying HCT-15 cells without signiﬁcant secondary adverse
eﬀects. In conclusion, the results obtained in the present study
demonstrate that 1 has potential as a chemotherapeutic candidate forpreclinical investigations.

M

exerted suppressive eﬀects on HCT-15 cells in antitumor
experiments using xenograft model nude mice.

arine natural products possess unique structural featuresand various biological activities.1−3 A number of marinederived compounds have exhibited potent inhibitory activities
against human tumor cells in in vitro and in vivo experiments.
Some marine substances have entered clinical trials for thetreatment of cancers, and cytarabine, trabectedin (ET743),
eribulin (a synthetic derivative of halichondrin B), andmonomethylauristatin E (MMAE) as the warhead of a
monoclonal antibody (Adcetris) have been approved as
anticancer agents.4−7
In the course of our studies on bioactive metabolites frommarine organisms, we have reported the isolation and structuresof three novel sulfur-containing alkaloids, lissoclibadins 1−3,
together with four known congeners from the Indonesian
ascidian Lissoclinum cf. badium, which have rare polysulfur
aromatic amine structures.8 Further studies on this ascidian led
to the discovery of 11 related compounds, lissoclibadins 4−14.9
We previously reported their antimicrobial activities, inhibitionof cell proliferation in murine and human cancer cell lines, andeﬀects on IL-8 production in PMA-stimulated humanpromyelocytic leukemia (HL-60) cells.10
We have now further evaluated the antitumor properties oflissoclibadins in vitro and in vivo, and the ﬁndings obtained
indicated that lissoclibadin 1 induced caspase-dependent
apoptosis in the human colon cancer cell line HCT-15 and© 2017 American Chemical Society andAmerican Society of Pharmacognosy■

RESULTS AND DISCUSSION
Lissoclibadins 1−3 were obtained as cytotoxic components
against HL-60 cells8 and exhibited inhibitory activities againstcolony formation by Chinese hamster V79 cells and cellproliferation in nine human solid-tumor cell lines.10a The
eﬀects of lissoclibadins 4−14 have also been investigatedagainst V79 and murine leukemia L1210 cells.9
Further cytotoxicity studies were performed using lissoclibadins 1 (1), 3 (2), 4 (3), 7 (4), 8 (5), and 14 (6). The
cytotoxicities of compounds 1−6 were initially investigated infour human cancer cell lines: HCT-15 (colon adenocarcinoma),
HeLa-S3 (cervix adenocarcinoma), MCF-7 (breast adenocarcinoma), and NCI-H28 (mesothelioma). These cancer cells weretreated with each test compound at various concentrations for
24 h, and cell viabilities were measured using the WST-1
assay.11 The eﬀects of compounds 1−6 on the viabilities of fourcancer cell lines are summarized in Table 1. Among theselissoclibadins, compounds 1, 5, and 6 exhibited stronger
Received: November 14, 2016
Published: February 9, 2017499

DOI: 10.1021/acs.jnatprod.6b01051
J. Nat. Prod. 2017, 80, 499−502

Journal of Natural Products

Article(Figure S1). These results suggest that compound 1 possesses
potent cytotoxic eﬀects.
Some anticancer agents are known to eliminate cancer cells
through apoptotic processes.12 Apoptosis is essential for tissue
development and homeostasis and also functions as a defense
system to avoid the eﬀects of many toxic substances.12
Therefore, nuclear morphological observations by ﬂuorescent
staining and a ﬂow cytometric analysis by double staining were
performed in order to investigate whether the cytotoxicity oflissoclibadin 1 (1) is triggered by an apoptotic pathway. HCT15 cells treated with 5 μM 1 for 24 h were stained with
Hoechst-33258, and nuclear morphological eﬀects wereobserved using a ﬂuorescence microscope. Nuclear morphological observations of 5 μM 1 revealed the condensation ofchromatin and fragmentation of nuclei in a few cells, as shownin Figure 1. Moreover, similar nuclear morphological changes
were detected in HeLa-S3, MCF-7, and NCI-H28 cells treatedwith 10 μM 1 for 24 h (Figure 1).

Table 1. Cytotoxicities of Compounds 1−6 against Four
Human Cancer Cell Lines: HCT-15, HeLa-S3, MCF-7, and
NCI-H28a

Figure 1. Nuclear morphological changes induced by lissoclibadin 1(1) in HCT-15, HeLa-S3, MCF-7, and NCI-H28 cells. Each cell line
(5 × 104 cells/mL) was precultured for 24 h, and compound 1 wasthen added at the indicated concentrations and cultured for 24 h. Cells
were stained with Hoechst-33258 and observed using a ﬂuorescent
microscope.

cytotoxicity (IC50, μM)
compound

HCT-15

HeLa-S3

MCF-7

NCI-H28

lissoclibadin 1(1)lissoclibadin 3(2)lissoclibadin 4(3)lissoclibadin 7(4)lissoclibadin 8(5)lissoclibadin 14(6)etoposide

4.0 ± 1.8

6.3 ± 1.4

7.6 ± 4.0

7.1 ± 2.3

13.2 ± 1.4

16.0 ± 1.8

n.d.b

n.d.

17.2 ± 5.2

17.8 ± 5.3

n.d.

n.d.

15.7 ± 1.2

14.2 ± 1.3

n.d.

n.d.

4.9 ± 1.9

6.3 ± 0.5

11.8 ± 3.1

7.1 ± 1.0

4.2 ± 2.4

5.9 ± 1.6

6.4 ± 2.7

6.4 ± 1.6

19.7 ± 5.7

43.1 ± 6.5

n.d.

n.d.

The occurrence of apoptosis was analyzed using ﬂow
cytometry. HCT-15 cells were treated with 5 μM 1 for 3, 6,
12, and 24 h and then stained with ﬂuorescein isothiocyanate(FITC)-conjugated annexin V (annexin V) and propidium
iodide (PI) in order to conﬁrm the apoptotic actions inducedby compound 1. Annexin V staining (lower right) signiﬁed
early apoptotic events, while dual annexin V and PI staining(upper right) indicated the late phase of apoptosis (Figure 2).
On the other hand, PI staining (upper left) showed necrosis,
and nonstained cells (lower left) were viable cells (Figure 2). As
shown in Figure 2, the proportion of early apoptotic cells(lower right) signiﬁcantly increased to 11%, 37%, 43%, and
40% after the incubation with 1 at 5 μM for 3, 6, 12, and 24 h,
respectively. The late apoptotic cell proportion (upper right)
was also increased after the 24 h incubation with 5 μM 1 in atime-dependent manner (Figure 2). These results indicate thatthe cytotoxic eﬀects of lissoclibadin 1 are attributable to the
induction of apoptosis.
The apoptotic response in HCT-15 cells to 1 wassigniﬁcantly decreased by the addition of the pan-caspase
inhibitor z-VAD-fmk (Figure 3). The percentage of apoptotic
cells (upper right and lower right) decreased from 64% to 11%.
Therefore, compound 1 induced apoptotic death in HCT-15
cells through a caspase-dependent pathway.

aEach value indicates the mean ± SD of three diﬀerent experiments
performed in triplicate. bNot determined.

cytotoxicity against all cancer cell lines and were more potent
than etoposide, an anticancer drug. Moreover, compounds 1, 5,
and 6 showed the lowest IC50 values of 4.0, 4.9, and 4.2 μM,
respectively, against HCT-15 cells (Table 1). On the other
hand, compounds 2−4 exhibited weaker activities than 1, 5,
and 6 (Table 1).
Among the six lissoclibadins tested, lissoclibadins 1 (1) and14 (6) were roughly equipotent, but compound 1 was selected
for further studies. Thus, morphological changes in cancer cells
treated with 1 (0−10 μM) were examined using lightmicroscopy. Lissoclibadin 1 (1) inﬂuenced cell adhesion andmorphology in a dose-dependent manner in the four cell lines
500

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J. Nat. Prod. 2017, 80, 499−502

Journal of Natural Products

Articleeﬀects of 1 on cell adhesion and morphology (Figure S2). It
may be demonstrated that the treatment with 1 causes anirreversible lethal stress in cancer cells upstream of caspasecascade activation or prevention of 1-induced caspase activation
triggers induction of other cell death signaling. Taken together,
we have concluded that compound 1 induces potent
cytotoxicity attributed to strong activation of caspase cascade
originating in caspase-9 activation.
BALB/c nu/nu nude mice were used as a xenograft model toevaluate the in vivo antitumor eﬀects of lissoclibadin 1 (1) on
the HCT-15 cell line. Tumor growth on day 28 in the grouptreated with 1 at 25 mg/kg per day (n = 6) was approximately
60% less than that in the control group, whereas theadministration group at 5 mg/kg per day (n = 6) showed noeﬀect (Figure 4A). Neither obvious toxicities nor body weightchanges were observed during the experimental period (Figure
4B). These results show that compound 1 suppressed tumor
growth in nude mice.
The present study revealed that lissoclibadin 1 (1) induced
cell death via apoptosis due to the mitochondrial cytochrome cdependent activation (intrinsic pathway) of the caspase-9 andcaspase-3 cascade pathway. Moreover, compound 1 exhibited
antineoplastic activity in HCT-15 cells in vivo at 25 mg/kg per
day with relative safety. This is the ﬁrst study on the in vivo
antitumor eﬃcacy of 1, and, accordingly, lissoclibadin 1 has
potential as a lead compound for further development as acancer chemotherapy agent.

Figure 2. Analysis of apoptosis stages in HCT-15 cells assessed by ﬂow
cytometry with double staining (annexin V and PI). HCT-15 cells (5× 104 cells/mL) were precultured for 24 h in 1 mL, lissoclibadin 1 in
DMSO (5.0 μM) or DMSO alone (ﬁnal concentration 0.1%) was thenadded, and the cells were incubated for the indicated time. Analyses ofannexin V binding and PI incorporation were performed by
FACSCalibur. The percentages of annexin V-positive cells arepresented in the right panel. Each value is the mean ± SD of three
independent experiments.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Lissoclibadins 1 (1), 3 (2), 4(3), 7 (4), 8 (5), and 14 (6) were isolated from the ascidian L. cf.
badium collected in Indonesia as described previously.8,9 A cellcounting kit (WST-assay) was purchased from Dojindo MolecularTechnologies, Inc. Caspase inhibitors (z-VAD-fmk, z-IETD-fmk, andz-LEHD-fmk) were purchased from Medical & Biological Laboratories
Co., Ltd. (MBL).
Cell Cultures. HCT-15 (colon adenocarcinoma), HeLa-S3 (cervix
adenocarcinoma), and MCF-7 (breast adenocarcinoma) cell lines wereobtained from the Cell Resource Center for Biomedical Research,Institute of Development, Aging and Cancer, Tohoku University. The
NCI-H28 cell line (malignant mesothelioma) was purchased from theAmerican Type Culture Collection. Cells were routinely kept in RPMI
1640 medium (Nissui Pharmaceutical Co. Ltd.) supplemented with
10% fetal calf serum, penicillin (100 U/mL), and streptomycin (100μg/mL) at 37 °C in a 95% air and 5% CO2 atmosphere.
WST Assay. WST-1 assays were performed in accordance with the
manufacturer’s instructions. Brieﬂy, cells were plated at 5 × 103 cells/
well on 96-well plates and grown for 24 h. Various concentrations oflissoclibadins (0.1−40 μM) and etoposide (1.0−100 μM) were addedin triplicate to the cultures and incubated for 24 h before adding the
WST-1 solution. The absorbance of the resulting product wasmeasured 4 h later at a wavelength of 450 nm with backgroundsubtraction at 650 nm. The cytostatic/cytotoxic eﬀects of compounds
were expressed as relative cell viability (% of control). The
experiments were conducted three times, and the IC50 value showing
the compound concentration required for the 50% inhibition of cell
viability was calculated employing GraphPad Prism 3.0 software.
Observation of Nuclear Morphology. Cells (5 × 104 cells/mL)
were cultured in 5 mL on six-well plates and grown for 24 h. After thetreatment, cells were trypsinized, collected by centrifugation, andwashed with PBS. Cells were then ﬁxed with 100 μL of 1.0%
paraformaldehyde at 4 °C for 15 min and stained with 50 μL of 1 mg/
mL Hoechst-33258 at 4 °C for 15 min. After three washes with PBS,
cells were mounted on glass slides using Prolong gold antifade
reagents (Molecular Probes). Fluorescence was visualized with a Zeiss
Axioscope 2 ﬂuorescence microscope (Carl Zeiss).

Figure 3. Eﬀects of pan-caspase, caspase-8, and caspase-9 inhibitors on
apoptosis induced by lissoclibadin 1 (1). HCT-15 cells (5 × 104 cells/mL) were precultured for 24 h in 1 mL. Each caspase inhibitor wasadded at 50 μM, and the cells were incubated for 30 min. Lissoclibadin
1 in DMSO (5.0 μM) or DMSO alone (ﬁnal concentration 0.1%) was
then added, and the cells were incubated for the indicated time.
Analyses of annexin V binding and PI incorporation were performedby FACSCalibur. The percentages of annexin V-positive cells arepresented in the right panel. Each value is the mean ± SD of three
independent experiments.

The caspase cascade is transmitted through two kinds ofpathways: intrinsic and extrinsic pathways.13 Therefore, thepathway activated by the treatment with 1 was investigated in
the annexin V−PI dual-staining analysis using speciﬁc caspase-8(z-IETD-fmk) and caspase-9 (z-LEHD-fmk) inhibitors. As
shown in Figure 3, the prevention of annexin V-positive
staining by z-LEHD-fmk was more potent than that by z-IETDfmk, which indicated that apoptosis induced by 1 was caused by
the intrinsic pathway rather than the extrinsic pathway. zLEHD-fmk could not completely prevent the apoptosis incontrast to z-VAD-fmk, indicating that 1 triggers the apoptotic
signal cascade so strongly that inhibition of caspase-9 alone is
not suﬃcient for complete prevention of 1-induced apoptosis.
The treatment with caspase inhibitors did not suppress the501

DOI: 10.1021/acs.jnatprod.6b01051
J. Nat. Prod. 2017, 80, 499−502

Journal of Natural Products

ArticleFigure 4. Antitumor eﬀects of lissoclibadin 1 (1) in a tumor xenograft model bearing HCT-15 cells. HCT-15 cells (5 × 106 cells) were implanted
into each nude mouse. When the tumor size reached 50−80 mm3, mice were injected intratumorally with 5 mg/kg of 1, 25 mg/kg of 1, and 25 mg/
kg of irinotecan (as a positive control) six times every second day from day 0 (indicated by arrowhead). Tumor sizes for calculating the volume (A)and body weight (B) were measured every second day. The body weight was monitored for toxicity.

*Tel/fax: +81 22 727 0218. E-mail: yamazaki@tohoku-mpu.ac.
jp (H. Yamazaki).

Annexin V/PI Staining. Annexin V binding and PI incorporation
were detected with a MEBCYTO apoptosis kit (MBL) according to
the manufacturer’s directions. Brieﬂy, cells (5 × 104 cells/mL) were
cultured in 1 mL on 24-well plates. After the treatment with 5 μM
lissoclibadin 1 (1), adherent cells were trypsinized, pooled, and stainedwith annexin V-FITC and PI. The ﬂuorescence intensities of FITCannexin V and PI were measured using a FACSCalibur ﬂow cytometer(Becton Dickinson).
The role of caspase activation in this process was examined by theaddition of the pan-caspase inhibitor z-VAD-fmk, caspase-8-speciﬁc
inhibitor z-IETD-fmk, and caspase-9-speciﬁc inhibitor z-LEHD-fmk.
Each caspase inhibitor (50 μM) was added to cultured cells 30 min
before the addition of 1 (5 μM). The analysis of annexin V binding
and PI incorporation was then completed, as described above. The
results from three independent experiments were expressed as themean ± SD. Statistical analyses were conducted using GraphPad Prism
3.0, and comparisons were made using one-way analysis of variance(ANOVA) followed by Bonferroni’s post hoc tests.
In Vivo Antitumor Experiment. All animal experiments were
performed in accordance with the Guidelines for Animal Experimentsof the Tohoku Medical and Pharmaceutical University (permission
number 08029). HCT-15 cells (1 × 108/mL) were mixed with anequal volume of ice-cooled Matrigel (Becton Dickinson Labware), andan aliquot of the cell suspension (100 μL) was injected subcutaneously
into the lower backs of 6-week-old female athymic nude mice (Balb/c
nu/nu, Charles River Lab. Inc.). One week after the inoculation (day0), mice bearing tumor volumes from 50 to 100 mm3 were randomly
divided into 4 groups with 6 mice in each group. Lissoclibadin 1 (1)and irinotecan, used as a reference antitumor drug, were dissolved insterile PBS containing 5% DMSO and 5% glucose (solvent). Groups 1,
2, and 3 were injected intratumorally with 5 and 25 mg/kg of 1 and 25
mg/kg of irinotecan, respectively, on days 0, 2, 4, 6, 8, and 10. Group 4
was administered vehicle (solvent only). Body weights and tumor sizes
were measured every other day from day 2 until 28. Solid tumor
volumes were calculated as follows: length (mm) × width (mm) ×
depth (mm) × π/6. Tumor growth and body weight changes were
evaluated by the ratio of each value against the initial values on day 0.

■

ORCID

Hiroyuki Yamazaki: 0000-0002-1540-7255
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
This work was supported in part by the Japan Science andTechnology Agency (JST) (2007 Research for Promoting
Technological Seeds) to M.N., Kanae Foundation for the
Promotion of Medical Science to H.Y., and a Grant for Basic
Science Research Projects from The Sumitomo Foundation to
H.Y.

■

ASSOCIATED CONTENT* Supporting InformationS

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01051.
Experimental data for antitumor eﬀects of 1 and 1H
NMR spectra for 1−6 (PDF)■

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AUTHOR INFORMATION

Corresponding Authors*Tel/fax: +81 22 727 0114. E-mail: mhosono@tohoku-mpu.ac.
jp (M. Hosono).
502

DOI: 10.1021/acs.jnatprod.6b01051
J. Nat. Prod. 2017, 80, 499−502

www.impactjournals.com/oncotarget/

Oncotarget, 2017, Vol. 8, (No. 26), pp: 42466-42477
Research Paper

Synergistic anti-tumor effect of bullfrog sialic acid-binding lectin
and pemetrexed in malignant mesothelioma
Toshiyuki Satoh1,2, Takeo Tatsuta2, Shigeki Sugawara2, Akiyoshi Hara1 andMasahiro Hosono2
1

Department of Clinical Pharmacotherapeutics, Tohoku Medical and Pharmaceutical University, Aobaku, Sendai, Miyagi
981-8558, Japan

2

Division of Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical
University, Aobaku, Sendai, Miyagi 981-8558, Japan

Correspondence to: Masahiro Hosono, email: mhosono@tohoku-mpu.ac.jp
Keywords: malignant mesothelioma, apoptosis, cell cycle, sialic acid-binding lectin, combination treatment
Received: December 28, 2016

Accepted: April 06, 2017

Published: April 18, 2017

Copyright: Satoh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT
Malignant mesothelioma is an aggressive cancer with limited therapeutic
options. Sialic acid-binding lectin isolated from Rana catesbeiana oocytes (cSBL)
is a multifunctional protein with anti-cancer activity. The effects of pemetrexed,
cisplatin, and cSBL were evaluated in mesothelioma and normal mesothelial celllines. We evaluated cytotoxicity, apoptosis, caspase-3 cleavage and activation, cellproliferation, cell cycle arrest, and levels of cell cycle proteins in H28 cells treated withpemetrexed, cisplatin, and cSBL alone or in combination. Treatment with cSBL alone
was cytotoxic to mesothelioma cells. The anti-cancer effect of cSBL was observedin a broader range of cell lines and exhibited greater cancer cell selectivity thanpemetrexed or cisplatin. Combination treatment with pemetrexed + cSBL resulted
in greater dose-dependent cytotoxicity than pemetrexed + cisplatin, the standard ofcare in mesothelioma. The synergistic effect of pemetrexed + cSBL was mediated by
the cytostatic effect of pemetrexed and the cytotoxic effect of cSBL. It thus appears
that cSBL has therapeutic potential for the treatment of mesothelioma.

INTRODUCTION

also commonly observed [14]. Therefore, alternative
therapeutic agents for mesothelioma are needed.
Sialic acid-binding lectin isolated from Ranacatesbeiana oocytes (cSBL) is a multifunctional proteinwith lectin-binding [15, 16], ribonuclease (RNase) [17], andanti-tumor activity [16]. cSBL is cytotoxic to cancer cells
including leukemia [18–21], breast carcinoma [21–24],
mesothelioma [25], and hepatoma cells [21, 26, 27].
,W KDV OLWWOH HIIHFW RQ QRUPDO FHOOV VXFK DV ¿EUREODVWV
melanocytes, keratinocytes, and mesothelial cells[20, 21, 25, 26, 28]. cSBL-induced cell death involves
at least three steps: (1) binding to the cell surface via
carbohydrate chain containing sialic acid, (2) cellinternalization, and (3) RNA cleavage and activation of
apoptosis. The cytotoxic effects of cSBL are mediated by
the induction of apoptosis in response to mitochondrial
perturbation. RNase activity is essential for cSBL-induced
cytotoxicity [24]. Treatment of tumor-bearing mice(transplanted with sarcoma 180 cells, Ehrlich, or Mep 2
ascites cells) with cSBL at a non-toxic dose prolonged

Malignant mesothelioma is an aggressive cancerof mesothelial cell origin that results from exposure
to asbestos [1, 2]. Asbestos was extensively used inindustry and construction during the 20th century. It
ZDV¿UVWDVVRFLDWHGZLWKWKHLQFLGHQFHRIPHVRWKHOLRPD
in the 1960s [3–6]. Because mesothelioma develops
20–30 years after asbestos exposure, the number ofmesothelioma patients is expected to increase [7–9]. There
are few therapeutic options for mesothelioma. The folate
antimetabolite pemetrexed is a chemotherapeutic that is
typically used in combination with platinum-containing
drugs such as cisplatin [10, 11]. Compared to cisplatinmonotherapy, combination treatment with pemetrexed +cisplatin improves response rate, progression-free survival,
overall survival, and quality of life in mesothelioma
patients [10]. However, most patients treated withpemetrexed and cisplatin experience tumor progression
or relapse within a year [12, 13]. Drug resistance iswww.impactjournals.com/oncotarget

42466

Oncotargetsurvival [16]. In contrast to commonly used DNAtargeting agents, the cytotoxic effects of RNases are nongenotoxic [29]. Thus, cSBL has therapeutic potential as anovel RNA-targeting anti-cancer agent.
Combination chemotherapy is the standard of care formany cancers. It allows for the use of doses that maximize
the therapeutic effects while preventing chemoresistance.
cSBL has an anti-cancer effect in mesothelioma cell lines(e.g. NCI-H28 [H28], ACC-MESO-1 [MESO-1], and ACCMESO-4 [MESO-4]), and exhibited synergistic effects withtumor necrosis factor-related apoptosis-inducing ligand
75$,/ LQ+FHOOV>@DQGLQWHUIHURQȖLQKHSDWRPD
cell lines [27]. We investigated whether cSBL exhibited
greater tumor selectivity than pemetrexed and cisplatin, andwhether combination treatment with cSBL + pemetrexed
was comparable or superior to combination treatment withpemetrexed + cisplatin.

sensitive to pemetrexed (Figure 1 and Table 1). We
previously demonstrated that combination treatment withcSBL + TRAIL has a synergistic effect H28 cells [25].
The concentration of each drug in the combination regimen
was based on the IC50 value of each agent as a single
treatment. Pemetrexed + cisplatin and pemetrexed + cSBL
reduced cell viability to a similar extent (Figure 2A). To
evaluate the synergistic effect of each drug combination,
we calculated combination index (CI) values. The
CI curves shown in Figure 2B indicated that pemetrexed +cSBL had a stronger synergistic effect and broader fraction
affected (Fa) range than the other combinations. Cisplatin+ cSBL exhibited the weakest cytotoxic and synergistic
effects. We also calculated the concentration of each agent
at Fa = 0.5 (i.e., the concentration predicted to reduce cell
viability by 50%) (Table 2). Lower concentrations ofeach agent were required to inhibit cell viability by 50%
when they were combined rather than administered asingle agents. The concentration of pemetrexed decreasedby nearly 50% when used in combination with cSBL
(0.38 µM) compared to cisplatin (0.65 µM).

RESULTScSBL exhibits greater cancer cell selectivity thanpemetrexed and cisplatin

Pemetrexed and cSBL induce apoptosis inmesothelioma cells

We evaluated the effects of cSBL, pemetrexed, andcisplatin on the viability of epithelioid mesothelioma cells(NCI-H2452 [H2452], MESO-1, and MESO-4), biphasic
mesothelioma cells MSTO-211H (MSTO) and sarcomatoid
mesothelioma cells (H28), and non-malignant mesothelial
cells (MeT5A) using WST-8 assays. All three agents
reduced mesothelioma cell viability. However, cSBL had
the least effect on MeT5A cells (Figure 1). Even at thehighest concentration (20 µM), cSBL only inhibited MeT5A
cell viability by 40% (Figure 1C). In contrast, pemetrexed
decreased Met5A cell viability by 50% at 0.01 µM andcisplatin decreased viability by 70% at 10 µM. We calculated
the half maximal inhibitory concentration (IC50 GH¿QHGDV
the concentration required to inhibit cell growth by 50%,
from dose-response curves. The relative sensitivity (RS) of
each agent represents the ratio of the IC50 value in a cancer
cell line to the IC50 value in MeT5A cells (Table 1). H2452,
MESO-1, and MESO-4 cells were resistant to pemetrexed(RS: 0.37, 0.06, and 0.06, respectively), and H28, H2452,
and MESO-1 cells were resistant to cisplatin (RS: 0.66, 0.24,
and 0.26, respectively). In contrast, cSBL was cytotoxic inthese drug-resistant cell lines. The RS of cSBL was higher(9.48–247.02) than the RS values of pemetrexed andcisplatin in mesothelioma cells, indicating that the cytotoxic
effect of cSBL was more selective to cancer cells.

We previously demonstrated that cSBL induces
apoptosis in H28 (sarcomatoid histological type) as well
as MESO-1 and MESO-4 (epithelioid type) cells, and thatthe synergistic anti-tumor effect of cSBL + TRAIL in H28
cells was mediated by an increase in apoptosis [25]. To
elucidate the mechanism underlying the synergistic effect ofpemetrexed + cSBL in H28 cells, we evaluated markers of
apoptosis. In the initial combination treatment experiments(Figure 2A), pemetrexed (20 µM), cisplatin (40 µM), andcSBL (1 µM) reduced the viability of H28 cell lines tosimilar levels (approximately 30%). Therefore, we used theseconcentrations in all subsequent experiments. After 72 h oftreatment, the percentage of annexin V-positive cells was
27.3%, 38.7%, and 44.3% in cells treated with pemetrexed,
cisplatin, and cSBL, respectively, and 44.8%, 47.3%,
and 46.0% in cells treated with pemetrexed + cisplatin,
pemetrexed + cSBL, and cisplatin + cSBL, respectively)LJXUH 7KHUHZHUHQRVWDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHV
between the individual and combination treatments.

The synergistic effect of pemetrexed + cSBL is
not mediated by changes in caspase-3 activity
To investigate whether the synergistic anti-tumor
effect of pemetrexed + cSBL was mediated by apoptosis,
we analyzed activated caspase-3 levels. Western blot
analysis demonstrated that all of the treatments increased
activated caspase-3 levels (Figure 4A). Caspase-Glo™ 3/7
assays indicated pemetrexed and cisplatin did not induce
FDVSDVH DFWLYDWLRQ ,Q FRQWUDVW D VLJQL¿FDQW LQFUHDVH
in activated caspase-3 was observed in cells treatedcSBL and pemetrexed exert a strong synergistic
effectWe investigated the pharmacological interaction
between the three agents by evaluating the viability of
H28 cells treated with pemetrexed + cisplatin, pemetrexed+ cSBL, or cisplatin + cSBL. H28 cells are moderately
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with cSBL alone or with any of the three combination
WUHDWPHQWV )LJXUH %  7KHUH ZHUH QR VLJQL¿FDQW
differences in caspase-3 activity between cells treated withcSBL alone or the combination treatments.

or cisplatin treatment, whereas the number of dead cells
VLJQL¿FDQWO\LQFUHDVHGZLWKF6%/WUHDWPHQW )LJXUH% 

The anti-tumor activity of pemetrexed + cSBL ismediated by cytostatic and cytotoxic effects

Pemetrexed and cisplatin inhibit cellproliferation, but cSBL has a cytotoxic effect in
H28 cells

Cell viability assays suggested that pemetrexed
and cisplatin inhibited proliferation. Therefore, we
analyzed cell cycle progression in H28 cells treatedwith these agents. Flow cytometry analysis revealed that
pemetrexed and cisplatin induced cell cycle arrest in S
phase and the S or G2 phase, respectively. In contrast,
cSBL had a minimal effect on cell cycle progression.
+RZHYHULWSURPRWHGDVLJQL¿FDQWLQFUHDVHLQWKHQXPEHU
of cells in the sub-G1 phase, indicative of apoptosis.
DNA histograms of cells treated with the combination
treatments resembled the histograms of cells treatedwith the individual agents (pemetrexed + cisplatin: Sphase arrest, pemetrexed + cSBL: S phase arrest and
sub-G1 increment, and cisplatin + cSBL: S or G2 phasearrest and sub-G1 increment) (Figure 6A and 6B). To
investigate the molecular mechanisms underlying theseeffects, we assessed the levels of proteins that regulate

Because apoptosis was not upregulated with theaddition of pemetrexed to cSBL, we investigated themechanism by which pemetrexed and cSBL inhibited
mesothelioma cell viability. Previous reports have indicatedthat the anti-tumor effect of both pemetrexed and cisplatinis mediated by the induction of apoptosis in response tocell cycle arrest [30–35]. Therefore, we analyzed the effectof pemetrexed and cisplatin on cell proliferation. The total
number of cells decreased by 10.4%, 14.2%, and 32.7%
in cells treated with pemetrexed, cisplatin, and cSBL,
respectively, for 72 h compared to control (PBS-treated)
cells (Figure 5A). The ratio of annexin V- and propidium
iodide (PI)-positive cells indicated that the number ofdead cells barely increased in response to pemetrexed-

Figure 1: Dose-response curves in the mesothelioma cell lines (H28, H2452, MESO-1, MESO-4, and MSTO), and MeT5A mesothelial
cells treated with pemetrexed (A), cisplatin (B), or cSBL (C). Cells were treated with pemetrexed (0.1 nM–20 mM), cisplatin (1 nM–1 mM),
or cSBL (1 nM–30 µM) for 72 h. The dots and bars represent the mean and SD, respectively. Dose-response curves are depicted as lines ordotted lines. Each data point represents the mean ± SD of at least three independent WST-8 assays. Each sample was plated in triplicate.
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Table 1: IC50 values (µM) and RS of pemetrexed, cisplatin, and cSBL in mesothelioma cells
Drugs
Pemetrexed

Drug targets

MeT5A

H28

H2452

MESO-1

MESO-4TS

129.5011.27

353.002267.00

2077.00

0.28(24.20–693.20)(5.67–22.39)(208.6- 597.3)(1393–3691)(1634–2639)(0.23–0.34)
465.99DHFR
GRAFT

Cisplatin

DNA

IC50
RS
IC50
RS

cSBL

RNA

IC50
RS

MSTO

1.00

11.49

0.37

0.06

0.06

11.27

17.18

47.62

44.14

4.54

2.23

(8.07–15.73)(15.14–19.50)(41.23–55.00)(38.62–50.46)(3.87–5.33)(1.62–3.06)
5.06

1.00

0.66

0.24

0.26

2.48

52.22

0.46

0.52

5.51

1.54

0.21

(33.94–80.36)(0.35–0.68)(0.41–0.66)(4.67–6.50.)(1.10–2.17)(0.15–0.29)

1.00

113.89100.00

9.48

33.91

247.02

7KHFRQ¿GHQFHLQWHUYDOVIRUHDFK,&50 value are shown in parentheses. The RS value was calculated as the IC50 value of each agent in MeT5A cells divided by the IC50 value
in each cancer cell line.

DISCUSSION

cell cycle progression (cyclin, p21Waf1/Cip1, and Akt) bywestern blotting. The levels of cyclin A and p21Waf1/Cip1
were unchanged in cells treated with pemetrexed alone,ZKLOH WKH OHYHOV RI SKRVSKRU\ODWHG $NW VLJQL¿FDQWO\LQFUHDVHG&\FOLQ$DQG%OHYHOVVLJQL¿FDQWO\GHFUHDVHG
in cisplatin-treated cells, while p21Waf1/Cip1 levels increased.
In contrast, cyclin A, B1, D1, and E levels, and as wellas p21Waf1/Cip1DQGSKRVSKRU\ODWHG$NWOHYHOVVLJQL¿FDQWO\decreased in cSBL-treated cells. The levels of cyclin A
and B1 decreased, whereas p21Waf1/Cip1 levels increased incells treated with pemetrexed + cisplatin. Pemetrexed +F6%/DQGFLVSODWLQF6%/VLJQL¿FDQWO\GHFUHDVHGF\FOLQ
B1, p21Waf1/Cip1, and phosphorylated Akt levels, similar to
those observed in cells treated with cSBL alone, whereas
pemetrexed + cSBL had the same effect on cyclin A levels
as treatment with pemetrexed alone (Figure 6C).

Mesothelioma is categorized as one of threehistological subtypes: epithelioid, biphasic, or sarcomatoid[1, 36]. Previous studies have demonstrated that theepithelioid subtype is associated with more favorable
survival outcomes compared to non-epithelioid subtypes[1, 37, 38]. We found that cSBL had strong cytotoxic
effects in a broader range of mesothelioma cell types
including pemetrexed- or cisplatin-resistant cells. We
previously demonstrated that cSBL selectively boundto 20 human and animal cancer cell lines but not to 10
normal cell lines [15]. A comprehensive analysis ofcSBL cytotoxicity and cancer selectivity was performed
previously [39, 40]. We found that cSBL preferentiallybinds and internalizes into cancer cells compared to

Figure 2: Pharmacological interactions between pemetrexed, cisplatin, and cSBL in H28 cells. (A) The drug concentration
ratios were as follows: pemetrexed + cisplatin (1:2), pemetrexed + cSBL (20:1), cisplatin + cSBL (40:1). Cells were treated with pemetrexed(2 nM–400 µM), cisplatin (4 nM–800 µM), or cSBL (0.1 nM–20 µM) for 72 h. The horizontal axis indicates the concentration of pemetrexed
in the pemetrexed + cisplatin or pemetrexed + cSBL combination or the concentration of cisplatin in the cisplatin + cSBL combination.
(B) CI-Fa curves for H28 cells treated with pemetrexed + cisplatin, pemetrexed + cSBL, or cisplatin + cSBL. CI values < 1 indicate asynergistic effect, and CI values > 1 indicate an antagonistic effect. Each data point represents the mean ± SD of three independent WST-8
assays. Each sample was plated in triplicate.
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Table 2: CI values and drug concentrations at Fa = 0.5 in H28 cells

SingleCombination

Drug/Combo

CI value

Pemetrexed
CisplatincSBL
Pemetrexed + Cisplatin
Pemetrexed + cSBL
Cisplatin + cSBL

í
í
í
0.12
0.05
0.47

Concentration at Fa = 0.5
Pemetrexed (µM) Cisplatin (µM)
20.44
í
í
0.65
0.38
í

í
15.2
í
1.31
í
4.6

cSBL (µM)
í
í
0.69
í
0.02
0.12

Figure 3: Pemetrexed, cisplatin, and cSBL, either alone or in combination, induced apoptosis in H28 cells. Cells weretreated with pemetrexed (20 µM), cisplatin (40 µM), or cSBL (1 µM) for 72 h. The y-axis indicates the percentage of annexin V-positive
FHOOV7KH SHUFHQWDJH RI 3,SRVLWLYH DQG QHJDWLYH FHOOV LV LQGLFDWHG E\ WKH GLIIHUHQW FROXPQ SDWWHUQV7KH VWDWLVWLFDO VLJQL¿FDQFH RI WKHpercentage of annexin V-positive cells compared to the control is shown. **p < 0.01, ***pQVQRWVLJQL¿FDQW

Figure 4: Caspase-3 activation is not enhanced by combination treatment. Cells were treated with pemetrexed (20 µM),
cisplatin (40 µM), or cSBL (1 µM) for 72 h. (A) Cleaved (activated) caspase-3 was detected using western blot analysis. (B) Caspase-3
activity was analyzed using a Caspase-Glo™ 3/7 assay. *pQVQRWVLJQL¿FDQW
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RNase activity [16]. Internalization-defective P-388
mutant (RC-150) cells, which are cSBL-resistant, canbind cSBL but show no cytotoxicity [41]. Therefore, we
K\SRWKHVL]H WKDW WKH ODFN RI VSHFL¿F LQWHUQDOL]DWLRQ RI
cSBL could make normal cells non-sensitive.
Although the effects of cSBL in combination withother agents have been investigated in various cancer celllines [21, 25, 27], there have been no reports describing
the effects of cSBL in combination with pemetrexed or
cisplatin. We have demonstrated that the synergistic effectof pemetrexed + cSBL is comparable to that of pemetrexed+ cisplatin (Figure 2A and 2B). Treatment with pemetrexed+ cSBL could decrease the risk of dose-dependent adverse
effects associated with pemetrexed and/or the developmentof pemetrexed resistance. The synergistic effect ofpemetrexed + cSBL is not mediated by increased apoptosis(Figures 3, 4). Apoptosis is commonly observed in cancer

cells treated with relatively high concentrations of anticancer agents, whereas a cytostatic effect (i.e. transient
growth arrest) is typically observed with relatively low
concentrations [42]. We found that the percentages ofannexin V/PI double-positive cells (Figure 3) and deadcells in the Muse™ analysis (Figure 5) of pemetrexedor cisplatin-treated cells were comparable and relatively
low (13–17%). Cell cycle analysis (Figure 6) indicatedthat pemetrexed and cisplatin exert cytostatic effects,
whereas cSBL exerts cytotoxic effects. These data suggestthat pemetrexed- and cisplatin-treated cells proceed toan early apoptotic stage, but that cSBL is required forcompletion of apoptosis. The differential effects of eachagent on cell cycle proteins suggest that the molecularmechanisms underlying cell cycle arrest are dependent
RQWKHFHOOW\SHDQGWKHVSHFL¿FWUHDWPHQW3HPHWUH[HGreportedly induces S-phase arrest in A549 lung cancer
cells by prolonging Akt activation, thereby sustaining

Figure 5: Pemetrexed and cisplatin inhibit proliferation, whereas cSBL has a cytotoxic effect in H28 cells. Cells weretreated with pemetrexed (20 µM), cisplatin (40 µM), or cSBL (1 µM) for 0–72 h. The number of cells was estimated using a Muse™ Count &Viability Kit. (A) Cell growth rates were calculated as the ratio of the cell number at 72 h to the cell number at 0 h and presented as a fractionof the controls. (B 7KHQXPEHURIOLYHDQGGHDGFHOOVHYHU\KLVVKRZQ6WDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHVLQWKHOLYHFHOOQXPEHUDWK
ZHUHREVHUYHGLQWKHWUHDWPHQWJURXSVFRPSDUHGWRWKHFRQWURO6WDWLVWLFDOO\VLJQL¿FDQWGLIIHUHQFHVLQWKHGHDGFHOOQXPEHUDWK
were observed in cSBL-treated cells compared to cisplatin- or pemetrexed-treated cells (††). *p < 0.05, **p < 0.01, ***p < 0.001, ††p < 0.01.
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SHPHWUH[HGWUHDWPHQWGLGQRWVLJQL¿FDQWO\DOWHUWKHOHYHOVof cyclin A. However, pemetrexed induced an increasein phosphorylated Akt levels and arrested cells cycle in
S-phase (Figure 6). The levels of the cyclins evaluated aswell as p21Waf1/Cip1OHYHOVVLJQL¿FDQWO\GHFUHDVHGLQF6%/

treated cells. Since cSBL inhibits RNA translation throughdegradation, short-lived proteins such as cyclins are likely
to be sensitive to cSBL treatment. Interestingly, in cells
treated with pemetrexed + cSBL, although the levels of
cyclin B1, D1, and E, p21Waf1/Cip1, and phosphorylated Akt
VLJQL¿FDQWO\GHFUHDVHGWROHYHOVVLPLODUWRWKRVHREVHUYHG

Figure 6: Pemetrexed, cisplatin, and cSBL, either alone or in combination, alter cell cycle dynamics in H28 cells.
Cells were treated with pemetrexed (20 µM), cisplatin (40 µM), or cSBL (1 µM) for 72 h. (A, B) Flow cytometry analysis of cell cycle
progression in H28 cell lines after 72 h of treatment. (C) Western blot analysis of cyclin (A, B1, D1, and E), p21Waf1/Cip1, pan-Akt, andphospho-Akt levels.
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in cells treated with cSBL alone, cyclin A levels did not
VLJQL¿FDQWO\FKDQJH$OWKRXJKF6%/PD\LQKLELWF\FOLQ$
translation, pemetrexed might stabilize cyclin A levels andenhance CDK2/cyclin A kinase activity, thereby inducing
S-phase arrest and/or apoptosis when used in combination
with cSBL [43–45]. The strong synergism of pemetrexed+ cSBL is mediated by the cytostatic action of pemetrexed
triggered by sustained CDK2/cyclin A activation, and theproapoptotic effect of cSBL.
The levels of p21Waf1/Cip1 were differentially affectedby each treatment. The tumor suppressor p21Waf1/Cip1
arrests cell cycle progression by inhibiting the functionof cyclin-CDK complexes or DNA polymerase [46–48].
Overexpression of p21Waf1/Cip1 induces cell cycle arrestin G1-, G2- [49], or S-phase [50, 51]. Pemetrexed andcisplatin induce cell cycle arrest by increasing p21Waf1/Cip1
levels [30, 52]. As a single treatment, pemetrexed did notchange p21Waf1/Cip1 levels. However, cisplatin and cSBL
VLJQL¿FDQWO\ LQFUHDVHG DQG GHFUHDVHG SWaf1/Cip1 levels,
respectively. Of the combination treatments evaluated, only
pemetrexed + cisplatin substantially increased p21Waf1/Cip1
levels. Therefore, p21Waf1/Cip1 may be important for cisplatininduced cell cycle arrest in H28 cells. Indeed, p21Waf1/Cip1
induced apoptosis in MSTO cells treated with a combinationof cisplatin and piroxicam. Silencing p21Waf1/Cip1 inhibited
this effect [53]. Because cisplatin strongly increased p21Waf1/
Cip1
in H28 cells (Figure 6), we propose that the decreaseof p21Waf1/Cip1 levels in cSBL-treated cells might explain
the lack of synergy between cisplatin and cSBL. Lazzarini
et al. reported that shRNA-mediated inhibition of p21Waf1/
Cip1
enhanced the anti-tumor effects of DNA-damaging
agents such as doxorubicin, etoposide, and CPT11 in H28

and H2052 cells [54]. Inoue et al. reported that sorafenib
downregulated p21Waf1/Cip1 levels and promoted cell deathin renal cell carcinoma and hepatocellular carcinoma whenused in combination with DNA-damaging agents such as
paclitaxel or doxorubicin [55]. Therefore, cSBL + DNAdamaging agents (with the exception of cisplatin) might
be an effective therapeutic strategy for mesothelioma.
The proposed mechanisms of action of the combination
treatments are shown in Figure 7.
In conclusion, cSBL exhibits a potent anti-tumor
effect in multiple mesothelioma cell lines due to its
cytotoxic activity and high selectivity for cancer cells
compared to either pemetrexed or cisplatin. Pemetrexed+ cSBL exhibited a strong synergistic effect that wascomparable or even superior to the standard regimen ofpemetrexed + cisplatin. We propose that the synergistic
effect results from the combination of the cytostatic effectof pemetrexed and the cytotoxic effect of cSBL. Therefore,cSBL has therapeutic potential for mesothelioma.

MATERIALS AND METHODS
Cell culture
The H28, H2452, and MSTO mesothelioma
cell lines and immortalized, non-malignant MeT-5A
mesothelial cell line were purchased from American
Type Cell Culture Collection (Manassas, VA, USA).
The MESO-1 and MESO-4 mesothelioma cell lines
were obtained from Riken Cell Bank (Tsukuba, Japan).
H28, H2452, MSTO, MESO-1, and MESO-4 cells were
cultured in RPMI-1640 medium supplemented with 10%

Figure 7: Schematic representation of the molecular mechanisms underlying the synergistic effects of each combination
treatment in H28 cells.
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Annexin V staining and PI incorporation assays

fetal bovine serum (FBS). MeT-5A cells were cultured in
Medium 199 with Earle’s balanced salt solution (75 mM
L-Gln and 1.25 g/L sodium bicarbonate) supplemented
with 3.3 nM epidermal growth factor (EGF), 400 nMhydrocortisone, 870 nM insulin, 20 mM HEPES, and 10%
FBS. All cells were cultured with 100 U/mL penicillin and100 µg/mL streptomycin at 37°C in a 95% air and 5% CO2
atmosphere.

To evaluate apoptosis, we evaluated annexin V
binding and PI incorporation using a MEBCYTO apoptosiskit (MBL, Nagoya, Japan) according to the manufacturer’s
instructions. Cells (5 × 104 cells/mL) cultured in 12-wellplates (1 mL/well) were treated with pemetrexed (20 µM),
cisplatin (40 µM), or cSBL (1 µM). Fluorescence intensity
ZDV GHWHFWHG XVLQJ D )$&6FDOLEXU ÀRZ F\WRPHWHU DQGthe data analyzed using the CELLQuest software (BD
Biosciences, Franklin Lakes, NJ, USA).

Reagents
cSBL was isolated using sequential chromatography
with Sephadex G75, DEAE-cellulose, hydroxyapatite, and
SP-Sepharose as previously described [15]. Pemetrexed
disodium heptahydrate was purchased from LC
Laboratories (Woburn, MA, USA). Cisplatin was purchasedfrom WAKO Pure Chemical Industries, Ltd. (Osaka, Japan).
The caspase-3 polyclonal antibody and cyclin B1 (D5C10),
cyclin D1 (92G2), p21Waf1/Cip1 (12D1), Akt (pan) (11E7), andphospho-Akt (Ser473) rabbit monoclonal antibodies werepurchased from Cell Signaling Technology (Beverly, MA,
USA). The cyclin A rabbit polyclonal antibody (H-432)
and cyclin E mouse monoclonal antibody (HE12) werepurchased from Santa Cruz Biotechnology Inc. (CA,86$ 7KHȕDFWLQDQWLERG\IURP6LJPD$OGULFK 7RN\R
Japan) and horseradish peroxidase (HRP)-conjugatedanti-mouse IgG were purchased from Zymed (South San
Francisco, CA, USA). HRP-conjugated anti-rabbit IgG waspurchased from Cedarlane (Hornby, Ontario, Canada), andthe Caspase-Glo™ 3/7 Assay was purchased from Promega(Madison, WI, USA).

Detection of caspase-3 activity
The level of activated caspase-3 was analyzed using
western blot assays with an antibody against cleaved(activated) caspase-3. Cells (5 × 104 cells/mL) cultured in
6-well plates (2 mL/well) were treated with pemetrexed(20 µM), cisplatin (40 µM), or cSBL (1 µM) for 72 h.
Whole cell lysates were prepared using extraction buffer(150 mM NaCl, 10 mM Tris-HCl [pH 7.4], 5 mM EDTA,
1% Nonidet P-40, 0.1% sodium deoxycholate, and 0.1%
sodium dodecyl sulfate) supplemented with 1 tablet/10 mL
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail(Roche Applied Science, Indianapolis, IN, USA) and 1
tablet/10 mL PhosSTOP phosphatase inhibitor (Roche
Applied Science). Soluble proteins were collected andprotein concentration measured using a BCA Protein$VVD\.LW 7KHUPR)LVKHU6FLHQWL¿F:DOWKDP0$86$ 
according to the manufacturer’s instructions. The proteins
were separated using SDS-PAGE and transferred to
Immobilon-P Transfer Membranes (Merck Millipore,
Billerica, MA, USA). Membranes were sequentially
incubated with primary and secondary antibodies dilutedin Can Get Signal (Toyobo CO., LTD., Osaka, Japan). The
protein bands were detected using ECL Prime Western
Blotting Detection Reagent (GE Healthcare, Little
Chalfont, UK).
Caspase-3 enzymatic activity was measured usinga Caspase-Glo™ Assay. Cells (5 × 104 cells/mL) cultured
in white 96-well plates (25 µL/well) were treated withpemetrexed (20 µM), cisplatin (40 µM), or cSBL (1 µM)
for 72 h in triplicate. Caspase-Glo™ Reagent (25 µL) was
added to each well and the contents of the wells mixedusing a plate shaker for 30 seconds. The cells wereincubated at 37°C in a 5% CO2 atmosphere for 1 h. The
luminescence in each well was measured using GloMax™
Multi Detection System (Promega, Madison, WI, USA).

Cell viability assays
Cell viability was determined using the WST-8
assay. Cells (5 × 104 cells/mL) cultured in 96-well plates(100 µL/well) were treated with various concentration ofpemetrexed, cisplatin, or cSBL for 72 h. The cells wereincubated with Cell Count Reagent SF (Nacalai TesqueInc., Kyoto, Japan) at 37°C in a 5% CO2 atmosphere for
1–4 h. The absorbance of the resulting product at 450 nm
was measured and the background absorbance at 650 nm
subtracted. The IC50 was calculated using GraphPad Prism
5.0 (San Diego, CA, USA). Experiments were conductedin triplicate.

Drug combination studies
The effect of combination treatment on cell viability
was assessed using WST-8 assays. The concentrationof the individual components was based on IC50 values
obtained in the single treatment experiments. CI values
were calculated using the CompuSyn software (ComboSyn
Inc., Paramus, NJ, USA) as previously described [56]. CI
values < 1 indicated a synergistic effect and CI values > 1
indicated an antagonistic effect.
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Cell proliferation assays&HOO SUROLIHUDWLRQ ZDV TXDQWL¿HG XVLQJ D 0XVH™
Count & Viability Kit (Merck Millipore, Billerica, MA,
USA). Cells (5 × 104 cells/mL) cultured in 24-well plates(500 µL/well) were treated with pemetrexed (20 µM),
cisplatin (40 µM), or cSBL (1 µM) for 0–72 h. The cells
42474

Oncotargetwere collected at 24 h intervals and combined with Muse™
Count & Viability Reagent in which both viable andnon-viable cells are differentially stained based on theirpermeability to the DNA-binding dyes (cells:reagent = 1:9).
The total number of viable or dead cells was counted usinga Muse™ Cell Analyzer (Merck Millipore).

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LQWHQVLW\ZDVGHWHFWHGXVLQJD)$&6FDOLEXUÀRZF\WRPHWHU
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CONFLICTS OF INTEREST

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FUNDING
This study was supported by the ‘Strategic
Research’ Project (2012–2017) for Private Universities
from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan and a Grant-in-Aid for Young
Scientists (B) (No. 26870527 to Takeo Tatsuta).

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Contents lists available at ScienceDirect

BBA - General Subjects
journal homepage: www.elsevier.com/locate/bbagen

Sialidase NEU3 deﬁnes invasive potential of human glioblastoma cells by
regulating calpain-mediated proteolysis of focal adhesion proteins

0$5.

Kohta Takahashia,b, Sergei Proshinc, Kazunori Yamaguchid, Yoji Yamashitae, Ryuichi Katakurae,
Koji Yamamotoa,f, Hiroshi Shimaf, Masahiro Hosonob,⁎, Taeko Miyagia,f,⁎⁎a

Division of Cancer Glycosylation Research, Tohoku Medical and Pharmaceutical University, Sendai, Japan
Division of Cell Recognition Study, Tohoku Medical and Pharmaceutical University, Sendai, Japanc
St Petersburg State Pediatric Medical University, Litovskaya ul., 2, St Petersburg, 194100, RussiadDivision of Molecular and Cellular Oncology, Miyagi Cancer Center Research Institute, Natori, Japane
Department of Neurosurgery, Miyagi Cancer Center, Natori, Japanf
Division of Cancer Chemotherapy, Miyagi Cancer Center Research Institute, Natori, Japanb

A R T I C L E I N F O

A B S T R A C T

Keywords:
Sialidase
Glioblastoma
Invasion
Calpain
Focal adhesion
Gangliosides

Background: Glioblastoma multiforme is one of the most malignant tumors of the human central nervous systemcharacterized by high degree of invasiveness. Focusing on this invasive nature, we investigated whether ganglioside-speciﬁc sialidase NEU3 might be involved, because gangliosides are major components of brain tissues,and cell surface sialic acids, as target residues of sialidase catalysis, are thought to be closely related to cell
invasion.
Methods: NEU3 mRNA levels of human glioblastoma specimens were evaluated by quantitative RT-PCR. Humanglioblastoma cell lines, U251, A172, and T98G were used for cell invasion and migration by transwell and cell
scratching assay. The molecules involved in the signaling cascade were investigated by western blot and immunoﬂuorescent microscopy.
Results: NEU3 expression was down-regulated in human glioblastoma specimens. In the human glioblastoma celllines, NEU3 overexpression reduced invasion and migration by promoting the assembly of focal adhesions
through reduced calpain-dependent proteolysis, but NEU3 silencing resulted in accelerating cell invasion via
disassembly of focal adhesions. In NEU3-silenced cells, elevation of calpain activity and GM3 accumulation wereobserved, as results of reduced sialidase hydrolysis, localization of calpain and GM3 at the cell lamellipodium
being demonstrated by immunoﬂuorescence microscopy.
Conclusion: Sialidase NEU3 was found to exert a great inﬂuence on cell invasion in regulation of calpain activity
and focal adhesion disassembly and consequent invasive potential of glioblastoma cells.
General signiﬁcance: This ﬁrst demonstration of sialidase involvement in invasive potential of gliolastoma cells
may point to NEU3 as an attractive treatment target of human gliomas.

1. Introduction
Glioblastoma multiforme is the commonest and most malignant
tumor of the human central nervous system, and characterized bymassive and diﬀuse inﬁltration into surrounding normal brain tissues.
Even with intensive treatment, median survival of aﬀected patients isonly in the order of 14 months after diagnosis [1,2]. Local invasion anddistant migration are considered leading causes of tumor recurrence
and treatment failure, but the underlying mechanisms in glioblastomas
are not fully understood, despite reports of potential targets [3]. With
the aim of providing pointers to novel eﬀective treatments, we havefocused on the highly invasive nature of this tumor by investigating
sialidase (EC 3.2.1.18), a key enzyme for the control of cellular sialic
acid contents through the removal of sialic acid residues from

Abbreviations: FAK, Focal adhesion kinase; FA, Focal adhesion; ECM, Extracellular matrix; Suc-LLVY-AMC, Succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine 4-methylcoumaryl-7-amide;
PIP2, Phosphatidylinositol 4, 5-bisphosphate; TLC, Thin-layer chromatography; Sulfo-NHS-biotin, Sulfosuccinimidyl-6-[biotin-amido]hexanoate; PBS, Phosphate buﬀered saline; RIPA,
Radio-immunoprecipitation assay; Phalloidin-TRITC, Phalloidin-tetramethylrhodamine B isothiocyanate; Lac-Cer, Lactsylceramide; Gb3, Globotriaosylceramide; PLCdelta, Phospholipase
C delta; PH, Pleckstrin homology⁎
Correspondence to: M. Hosono, Divisions of Cell Recognition Study, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai 981-8558, Japan.
⁎⁎
Correspondence to: T. Miyagi, Division of Cancer Chemotherapy, Miyagi Cancer Center Research Institute, 47-1 Nodayama, Medeshima-Shiode, Natori 981-1293, Japan.
E-mail addresses: mhosono@tohoku-mpu.ac.jp (M. Hosono), miyagi-ta@nifty.com (T. Miyagi).
http://dx.doi.org/10.1016/j.bbagen.2017.07.023
Received 3 February 2017; Received in revised form 26 July 2017; Accepted 27 July 2017
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K. Takahashi et al.

glycoproteins and glycolipids [4,5], aberrant sialylation being implicated in invasion and metastasis of cancer cells. Sialidases regulate
cell surface sialylation in collaboration with sialyltransferases thattransfer sialic acid residues to glycoconjugates. With the context of
sialylation of glycoproteins, there is evidence that sialyltransferases
may thereby participate in glioma malignancy [6,7]. Understanding of
the roles of sialic acid-containing glycosphingolipids is clearly important given their involvement in neuronal function.
Gangliosides are ubiquitously expressed in mammalian cells and areparticularly abundant in the nervous system. Accumulating evidence
indicates that they play speciﬁc functional roles essential for cell survival, proliferation, and diﬀerentiation during brain development under
desialylation control by sialidase catalytic reactions. In very earlystages of brain development, the pattern of ganglioside expression islimited to simple gangliosides, predominantly GM3 and GD3, but theseare soon lost after birth, becoming replaced by more complex forms,
particularly GM1, GD1a, GD1b, and GT1b, as major components [8,9].
In human gliomas, however, re-expression of simple gangliosides has
been reported [10,11]. While functional roles have yet to be fully
clariﬁed, it was recently reported that GD3 enhances invasiveness bycomplex formation with PDGF receptor and Yes kinase [12], andplaying an important role in tumorigenicity [13].
Among the four types of human sialidases so far identiﬁed (NEU1NEU4), diﬀering in major subcellular localization and enzymatic
properties, as well as position of the encoding genes [4], NEU3 andNEU4 appear to be involved in neuronal diﬀerentiation, neuritogenesis,
and axonal growth in opposing ways. The level of NEU3 expression
increases during the development of the mouse brain and during retinoic acid-induced neuronal diﬀerentiation whereas NEU4 decreases
and is known to suppress diﬀerentiation [14,15]. Additionally, in a
recent study, involvement of NEU4 in stem cell survival of some glioblastoma cells was indicated [16]. NEU3 would be mostly expected toparticipate in cell invasion and motility, because it hydrolyzes almost
speciﬁcally gangliosides and is up-regulated in various carcinomas [5].
In fact, it has been observed to participate in promotion of cell invasion
and motility in renal cell carcinoma [17] and head and neck squamouscell carcinoma [18]. In the present study, we focused on NEU3 by observing whether and how it is involved in invasiveness and motility of
glioblastoma cells, and obtained evidence that it modulates invasion
and migration through regulation of calpain-dependent disassembly of
focal adhesion proteins.

Collection Research Bioresources) were maintained in Dulbecco's
modiﬁed Eagle's medium with 10% heat-inactivated fetal bovine serumat 37 °C in a 5% CO2 atmosphere. For astrocyte culture, Gibco Human
Astrocytes (Life Technologies, N7805-100) were cultured in Gibco
Astrocyte Medium (A1261301), and Geltrex matrix-coated plates(A1413202) with 25,000 cells per well in 6-well plates were used according to the manufacturer's instructions. For NEU3 gene transfection,
the entire open reading frame (1.2 kb) of the human NEU3 gene wasinserted into a retrovirus vector pMXs-neo and the plasmid was introduced into PlatA, and targeted cells were then incubated with culture media containing infectious viruses for two days and selected bycultivation in the presence of 300 μg/ml G418 for 10–14 days, as described previously [19]. Several experiments were performed withtransient transfection of the pCAGGS-NEU3 expression plasmid [20] by
Fugene HD (Promega). GFP-PLCdelta-PH was obtained from Addgene(Plasmid 21,179). For silencing, siRNAs targeting NEU3 was synthesized by Dharmacon Inc. (Thermo Fisher Scientiﬁc) as described [20],and transfected using Lipofectamine RNAiMAX reagent (Invitrogen).
One sample was 5′-AAGGGAGTGTGGTAAGTTT-3′ beginning at nucleotide 839 of the NEU3 open reading frame sequence, and thescrambled control was 5′-GCGATTAATGTAGGTT CGA-3′. AnothersiRNA targeting NEU3 synthesized by iGENE Therapeutics (Tsukuba,
Japan) featured 5′-GGTTACAGTAGAATGTGAAGTGGCA-3′. These two
siRNAs targeting NEU3 caused 75–95% NEU3 reduction and almost thesame results in various experiments, and therefore, the former was
routinely utilized for the present study. The eﬃciency of NEU3 silencing was evaluated by real time PCR as described [21]. Transiently
transfected cells were used for experimentation at 48–72 h.
2.4. Sialidase activity assays
Crude extracts were used for sialidase assays with bovine brain GM3(Alexis Biochemicals, Lansen, Switzerland) as the substrate in the presence of Triton X-100. After incubation at 37 °C for 10–30 min, theamount of sialic acid released was determined by a modiﬁed thiobarbituric acid method or by ﬂuorometric high-performance liquid
chromatography with malononitrile as previously described [20]. One
unit of activity was deﬁned as the amount of enzyme that cleaved
1 nmol sialic acid from the substrates. Protein concentrations were
determined by dye-binding assay (Bio-Rad Laboratories, Hercules, CA,
USA).

2. Material and methods
2.5. Cell adhesion, migration and invasion assays
2.1. Patient samples
For cell adhesion assays, extracellular matrix-coated 96-well plates(BD Biosciences) were blocked with 1% bovine serum albumin in PBS.
Cell suspensions in serum-free medium were then applied to thesecoated 96-well plates. After incubation for 30 min and ﬁxation, attached cells were stained with 0.5% crystal violet, and incorporated dye
was eluted with 2% deoxycholate, and measured spectrophotometrically at A550. In cell-adhesion-inhibition assays, suspended
cells were incubated with 1 μg/ml neutralizing anti-β1 integrin antibody at 37 °C for 30 min before plating. Migration and invasion assays
were carried out using ﬁbronectin-coated cell culture inserts (Falcon)
and BD Biocoat Matrigel invasion chambers (BD Bioscience), respectively. Cells were seeded at 2 × 104 cells/well with serum-free medium
onto the upper chamber, and lower chamber was ﬁlled with medium
containing 10% fetal bovine serum. After 24 h, cells were ﬁxed andstained with 0.1% crystal violet and the invading or migrating cells on
the lower membrane surface were counted under a microscope. For
wound healing assay, cells were grown on 24-well plate. Once conﬂuence was reached, wound was made by using a pipette tip. Woundscratches were imaged immediately after scratching and overnight
migration. The area covered by the cell sheets during migration wasmeasured. At least three experiments per conditions were performed.

Surgical specimens of human glioblastoma, classiﬁed as grade IV
using the criteria of the World Health Organization, were obtained at
the time of surgical removal at the Division of Neurosurgery, Miyagi
Cancer Center. Informed consent was obtained from each patient toallow the use of portions of tissue for research purposes, and the study
was approved by the Committee on Human Rights in Research at
Miyagi Cancer Center. Histological diagnoses were based on WHO(World Health Organization) criteria by a neuropathologist.
2.2. Antibodies and reagents
Antibodies for integrin α3 (ASC-1), α5 (P1D6), β1 (6S6) and β4(ASC-8) were obtained from Millipore, for transferrin receptor (H-300),
paxillin (H-114), and FAK (A17) from Santa Cruz Biotechnology, andfor Talin (8d4) from Sigma. Fibronectin was from BD Bioscienses, and
Suc-LLVY-AMC and calpeptin were from Sigma.
2.3. Cell culture and transfection
Human glioblastoma cell lines, U251, A172 and T98G (Japan

%%$*HQHUDO6XEMHFWV  ²

K. Takahashi et al.

Fig. 1. Down-regulation of sialidase NEU3 expression in humanglioblastoma specimens. Sialidase NEU3 mRNA levels were
evaluated in human glioma tissues and non-neoplastic brain
tissues (A) by quantitative real-time PCR according to themethods described previously [21], and statistical signiﬁcanceof the diﬀerences in levels was determined (B).

were deﬁned as having an arc shape and size of the F-actin structure,and cells bearing lamellipodia with a greater than 20 μm width fromone end of a lamellipodia to another were counted as lamellipodiabearing cells. At least 200 cells per coverslip were analyzed and values
are means ± SD from three independent experiments.

2.6. Cell surface biotinylation assay
Cells were washed three times with chilled PBS containing 1 mM
MgCl2 and 0.1 mM CaCl2, and then incubated with sulfo-NHS-biotin(Piece) (0.5 mg/ml) in the same buﬀer at 4 °C for 30 min. Reactions
were terminated by incubation of the cells with 100 mM glycine in PBS.
Cells were lysed in modiﬁed RIPA buﬀer (50 mM Hepes, pH 7.5,
150 mM NaCl. 1% Nonidet P-40. 2 mM EDTA, 7.5 μg/ml of aprotinin,
10 μg/ml of leupeptin, 10 mM NaF, 2 mM PMSF), and the biotinylated
proteins were precipitated with streptavidin-conjugated agarose(Sigma) for analysis by immunoblotting.

2.9. Intracellular calcium release
U251 cells were seeded in 96-well-plate at a density of 20,000 per
well and cultured for 24 h. The cells were rinsed three times with assay
buﬀer (130 mM NaCl, 5 mM KCl, 10 mM HEPES, 8 mM D-glucose,
1.2 mM MgCl2 and 1.5 mM CaCl2, pH 7.4), and then incubated with thisbuﬀer supplemented with the organic anion transport inhibitor probenecid (2.5 mM), 1 μM Fluo 4-AM and 0.1% Pluronic F-127 for 60 min at
37 °C. Before measurement ﬂuorescence emission at 525 nm following
excitation at 480 nm, cells were rinsed a further three times with assay
buﬀer.

2.7. Immunoblotting
Cells were lysed with modiﬁed RIPA buﬀer. Lysates were clariﬁed
by centrifugation at 12000 × g for 10 min and then resolved on SDSPAGE. After transfer to PVDF membranes, speciﬁc proteins were detected using appropriate primary antibodies, and peroxidase-coupledsecondary antibodies with ECL Plus Western blotting reagent (GEHealthcare). For the immunoprecipitation assay with anti-FAK antibodies, cells were allowed to adhere for 1 h to cell-culture dishes coatedwith ﬁbronectin, and then lysed with modiﬁed RIPA buﬀer. The lysates
were incubated with anti-FAK antibody at 4 °C for overnight, and thenincubated with protein G-Sepharose at 4 °C for 2 h. The immunoprecipitates were analyzed by immunoblotting as describedabove.

2.10. Calpain activity
Cells from conﬂuent dishes were harvested with 0.1% trypsin-EDTA
solution, neutralized with 10% fetal bovine serum containing media,
and washed twice with HEPES-buﬀered Hank's balanced salt solution,
pH 7.4, without phenol red. Equal numbers of cells were resuspended in
100 μl of HEPES-buﬀered Hank's balanced salt solution at 5 × 104 cells
in 96-well plate at 37 °C in a 5% CO2 incubator for 10 min. To assay
calpain activity, calpain speciﬁc hydrolysis of the peptidyl 7-aminobond of the cell-permeable ﬂuorogenic calpain substrate, Suc-LLVYAMC ﬂuorescence emission at 525 nm following excitation at 480 nm
was added at 20 μM to cell suspensions, followed by incubation at 37 °C
for 15–30 min. AMC ﬂuorescence was measured by ﬂuorometer(GloMax-Multi detection system; Promega) with excitation/emission
wave lengths of 365 nm/410 to 460 nm.

2.8. Immunoﬂuorescence microscopy
Cells grown on ﬁbronectin-coated coverslip were ﬁxed with 4%paraformaldehyde for 15 min and then incubated with 0.1% Triton X100. After blocking with 1% BSA, cells were incubated with anti-paxillin antibody for 1 h, followed by incubation with Alexa 488 anti-rabbit
IgG. The cells were then washed and mounted under Fluoromount/Plus(Diagnostic BioSystems) as an anti-fading reagent. Fluorescence images
were obtained using a confocal laser-scanning microscope (FluoView
FV1000, Olympus). To visualize F-actin and nucleus, phalloidin-tetramethylrhodamine B isothiocyanate (phalloidin-TRITC, Sigma) andDRAQ5 were used. To assess localization of calpain and GM3, cells wereseeded at 0.5 × 105 cells/well onto ﬁbronectin-coated cover slips, after
24 h transfected with siRNA targeting NEU3 by RNAiMAX, and on thefollowing day treated with EGCase (EGCase II ACT RE, TAKARA 4463)
at the concentration of 20 min/ml in 5% FBS containing DMEM at 37 °C
overnight. Cells were ﬁxed with 4% PFA for 20 min, permeabilized andblocked by 0.2% saponin and 0.2% gelatin in PBS, each for 30 min,respectively, at room temperature, and then stained using anti-GM3(M2590) 1/100 or anti-Calpain (CST) 1/25 antibodies. Lamellipodia

2.11. Calpain translocation
GM3 dissolved in sterile water was sonicated in serum-free medium,
kept on ice for 10 min and added to the cells in serum-free medium atconcentration of 50 μM. After incubation at 37 °C for 24 h, cells werelysed in the buﬀer (20 mM HEPES, pH 7.4, 20 mM NaCl, 1.5 mM MgCl2,
250 mM sucrose, 1 mM EDTA, 2 mM PMSF, 7.5 μg/ml aprotinin and10 μg/ml leupeptin) by passing through 27G needle for 10 times andleft on ice for 20 min. After homogenization, the lysates were centrifuged at 4 °C 1000 × g for 5 min and supernatants were collected as
total protein fractions. The supernatants were then centrifuged at
150,000 × g for 30 min at 4 °C in an Optima MAX-XP ultracentrifuge(Beckman coulter). The resulting supernatants were collected as

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K. Takahashi et al.

A

NEU1

6

NEU3

0.8

0.5

p < 0.001

0.6

p < 0.001
p < 0.001

4

0.4

0.5

p < 0.002

0.4

3

0.3

0.3

0.2

2
0.2

0.1

1

0.1

2
A

T9

17

1

te
as

U

t ro

25

cy

8G

2

T9

A

17

1

te
as

t ro

25

cy

8G

2

T9

17

1

A

U

25

cy
te
as
t ro

8G

0.0

0.0

0

U

Relative mRNA level

0.7
5

Fig. 2. Sialidase expression of human glioblastoma cell lines andthe inﬂuence of alteration of sialidase NEU3 expression on cell
invasiveness. The mRNA levels of the four human sialidases
were evaluated in the three glioblastoma cell lines as comparedto those of astrocytes (A) by real-time PCR, as described earlier[21]. NEU2 expression was undetectable in these human cells.
Decreased invasiveness was shown in NEU3 overexpressing cells(B), whereas enhanced invasiveness was noted in the siRNAmediated silenced cells (C). Sialidase activity was determinedwith GM3 as substrate in NEU3-transfected cells compared with
control cells, and the eﬃciency of siRNA-mediated NEU3 silencing was evaluated by real time PCR in U251 cells.

NEU4

0.6

B
p < 0.05

p < 0.05

150
100
50

50
40
30
20
10

vec

NEU3

40

20

0

siNEU3

U251

40
30
20
10
vec

40
30
20

10

NEU3

T98G

p < 0.01

0

siCtrl50

NEU3

p < 0.01

100

invaded cells / fields

50

invaded cells / fields

invaded cells / fields

p < 0.01

60

60

A172

U251

80

80
70

0

0
vec

100

invaded cells / fields

invaded cells / fields

invaded cells / fields

200

0

C

p < 0.05

60

250

80

60
40

20

0

siCtrlsiNEU3siCtrlA172

siNEU3

T98G

cytosolic fractions, and the pellets containing the membrane fractions
were resuspended in the lysis buﬀer with 1% Nondet P-40 and homogenized for 10 min. The amount of calpain was determined by immunotting as described above using anti-calpain antibody.

2.13. Statistical analysis

2.12. Thin-layer chromatography

3. Results

Glycolipids were extracted from cells (1 × 107) in sequence with
2 ml of isopropanol/hexane/water (55:25:20, v/v/v) and hydrolyzed
with 0.1 M NaOH/methanol. After desalting with a Sep-Pak C18 cartridge, total lipid extracts were applied to DEAE-Sephadex A-25 minicolumns, fractionated into neutral and acidic glycolipids, and analyzed
by thin-layer chromatography on high-performance thin-layer chromatography plates (Merck) with orcinol-H2SO4 reagent for detection, as
described elsewhere [17]. Densitometric analyses were performed with
VarsaDoc and Quantity One software (Bio-Rad).

3.1. Sialidase expression in human glioma tissues and glioblastoma cell lines

Results are expressed as mean ± SD. All values were comparedusing the Student's t-test or the Welch's t-test.

Fig. 1 shows the results of NEU3 expression in human glioblastoma
and non-neoplastic brain tissues as assessed by quantitative RT-PCR.
Signiﬁcant decrease in NEU3 mRNA levels in tumor compared with
non-tumor tissues was detected (relative levels were 0.55 ± 0.27 and1.04 ± 0.48, respectively). To determine whether decrease of NEU3
expression might be involved in cell invasion, we employed threeglioma cell lines, U251, A172 and T98G. The expression levels of four

%%$*HQHUDO6XEMHFWV  ²

K. Takahashi et al.

Fig. 3. Sialidase NEU3 stabilizes cell adhesion and suppresses cell invasion in glioblastoma cells. Enhanced adhesion to collagen IV and ﬁbronectin (A), and suppression of cell migration
on ﬁbronectin (B). Changes in cell surface expression of integrin β1 and β4 by cell surface biotinylation assay followed by immunoblotting (C) and its quantiﬁcation (n = 3) (D).
Attenuation of the NEU3-mediated enhanced adhesion to ﬁbronectin by treatment with a blocking antibody for integrin β1 binding (E).

human sialidases were ﬁrstly evaluated in these cell lines (Fig. 2A).
When compared to those of astrocytes derived from normal humanbrain, U251 and A172 cells clearly showed reduced levels of NEU3 andNEU4, but no signiﬁcant change in the NEU1 level, whereas T98G cells
possessed the relatively similar levels of the three forms as to astrocytes.
In line with our previous report that NEU2 expression is hardly detectable in human cells [22], these cells also exhibited undetectable
NEU2. To clarify the inﬂuence of NEU3 on glioma cell invasiveness, we
examined invasion by three glioma cell lines, U251, A172 and T98G,
using Transwell Matrigel invasion assays. Transfection of NEU3 resulted
in marked decrease in the number of invaded cells (Fig. 2B), and on theother hand, NEU3 siRNA-mediated silencing increased invasiveness(Fig. 2C). NEU3 transient expression in these cells yielded approximately 25–60-fold increase in the sialidase activity with gangliosideGM3 as substrate as compared with control cells, and silencing yielded
35–15% mRNA level of the controls. Stable U251 cell NEU3 transfectants showed 31-fold increase in the activity, and the gene-silencing
eﬃciency was about 80% relative to control cells.

signiﬁcantly suppressed (Fig. 3B). As integrins have been implicated inglioma invasion [23], cell surface integrin expression was then examined by cell surface biotinylation analysis. In agreement with increased cell adhesion, NEU3 transfection yielded a slight but signiﬁcant
increase of integrin β1, a receptor for collagen IV and ﬁbronectin,
whereas integrin β4 showed reduction, although the levels in whole celllysates were unaﬀected (Fig. 3C, D). NEU3 did not give an inﬂuence on
the cell surface expression of other integrins, and there was no change
in the level of biotin-labeled transferrin receptors. Since several previous observations [24,25] suggested integrin β4 to not actually participate in invasion of glioma cells, we next focused on integrin β1 inglioma U251 cells. On treatment with an antibody blocking binding ofintegrin β1 to the ECM, NEU3-mediated enhanced adhesion to ﬁbronectin was almost fully abrogated (Fig. 3E), indicating stabilizationof cell surface integrin β1. It should be noted here that similar results
were also obtained with NEU3-transiently expressed U251 cells.

3.2. NEU3 stabilizes cell adhesion to collagen IV and ﬁbronectin andsuppresses invasion of glioma cells

Attachment of cells to ECM components induces clustering of integrins on cell surface, triggers formation of focal adhesion (FA) complexes, and then is followed by rapid turnover of the FA assembly anddisassembly, essential for cell spreading and migration. The change inthe level of integrin β1 localized at surface membranes, therefore,
might inﬂuence on the FA assembly and disassembly. To verify thepossibility, we analyzed FAs of the cells by immunocytochemistry using
antibodies for FA-resident protein, paxillin. NEU3 overexpressing cells
showed prominent increase in the number of FAs and in formation ofactin stress ﬁbers on a ﬁbronectin matrix (Fig. 4A, B). Formation of celllamellipodia was also signiﬁcantly suppressed compared with control

3.3. NEU3 stabilizes focal adhesion and suppresses cell spreading

Cellular invasiveness is generally dependent on multi-processes including adhesion, movement, and penetration involving the extracellular matrix (ECM). To understand the molecular basis of NEU3mediated suppression of invasion, we then examined the eﬀect of alteration of NEU3 expression using stably transfected U251 cells. Whencell adhesion to ECM, as an initial step of invasion, was analyzed, NEU3
overexpression was found to enhance the adhesion to collagen IV andﬁbronectin (Fig. 3A), while cell migration on ﬁbronectin was

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K. Takahashi et al.

A

B

F-actinpaxillin

paxillin

F-actinpaxillin

nuclearnuclearnuclearnuclearsiNEU3

paxillin

NEU3

vec

p < 0.001
90

paxillin

F-actinpaxillin

nuclearnuclear

20µM

paxillin

F-actinpaxillin

nuclearnuclear80
70
60

p < 0.01

50
40
30
20
10
0

vec

NEU3

siCtrlsiNEU3siCtrlD

spreading cells (%)

p < 0.001

siNEU3

vec

C

NEU3

focal adhesion / cell

vec

siCtrl

A

70
60
50
40
30

p < 0.05

20
10
0

vec

NEU3

siCtrlsiNEU3Fig. 4. Sialidase NEU3 enhances focal adhesion formation on ﬁbronectin and reduces glioblastoma cell spreading. The number of focal adhesions on ﬁbronectin matrix in NEU3
overexpressing cells and in its silenced cells (A), and the representative images are shown. Means ± SD values from three independent experiments are graphically illustrated in (B). Cellspreading and lamellipodium formation in the NEU3 overexpressing and its silenced cells, and the representative images are shown in (C). The number of spreading cells was normalized
to the total number of cells (D). Values are means ± SD from three independent experiments.

cells (Fig. 4C, D). Furthermore, NEU3 silencing markedly enhanced thelamellipodia formation on leading edges (Fig. 4C, D). NEU3 thus appeared to down-regulate glioma cell migration and invasion by FA
stabilization and suppression of turnover, whereas NEU3 silencing
causes the opposite.

3.5. Regulation of calpain activity by NEU3-mediated glycolipid alteration
To elucidate the mechanism of calpain regulation by NEU3, weanalyzed intracellular calcium ion, an activator of calpain, but no inﬂuence was evident (data not shown). Since calpain might be activatedby interaction with phosphatidylinositides such as phosphatidylinositol
4, 5-bisphosphate (PIP2) at inner leaﬂets of plasma membrane [26,27],
we then examined if NEU3-mediated glycolipid alteration might aﬀect
PIP2 localization and subsequent calpain activation at plasma membranes. To observe what glycolipids are expressed in glioblastoma cells
and how they are altered by NEU3 expression, the glycolipid pattern of
U251 cells was analyzed by thin-layer chromatography (TLC). The
proﬁle of neutral glycolipids revealed clearly detectable glycolipids,with TLC bands having the similar mobility to lactosyl-ceramide (LacCer), globotriaosylceramide (Gb3), and globoside (Gb4) (Fig. 6A, left).
The acidic glycolipid proﬁle exhibited a very pronounced band showing
similar TLC mobility to GM2, others distinctly recognized with similar
mobility to GM3 and GM1, and more hydrophilic gangliosides modestly
detectable (Fig. 6A, middle). Consistent with the recent reports by
Fabris et al. [28] describing glioblastoma gangliosides compared tothose of healthy brain tissues to be characterized by diverse ceramide
composition with fatty acyl chains, the doublets of glycolipids observed

3.4. NEU3 suppresses calpain activity and FA protein degradation
To elucidate how NEU3 regulates FAs turnover, we focused on
calpain, catalyzing degradation of FAs proteins. The amount of cleaved
form of Talin and FAK, major substrates of calpain, was ﬁrstly observed.
When U251 cells were attached on ﬁbronectin-coated tissue culture for
1 h, and then the level of Talin and FAK were quantiﬁed by western
blotting with anti-Talin and anti-FAK antibodies. Cleaved forms observed at 190 and 80 kDa respectively, were reduced by NEU3 overexpression (Fig. 5A). Diminution of the cleaved products in the presence of the calpain inhibitor, calpeptin, conﬁrmed their origin throughproteolytic cleavage by calpain (Fig. 5A). In contrast, NEU3 silencing
promoted degradation of FAK and Talin, which was inhibited by calpeptin treatment (Fig. 5B). With a cell permeable ﬂuorogenic calpain
substrate, Suc-LLVY-AMC, NEU3 was found to suppress calpain activity(Fig. 5C) whereas NEU3 silencing exerted the opposite eﬀects (Fig. 5D).


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K. Takahashi et al.

Fig. 5. Sialidase NEU3 suppresses calpain activity and degradation of FA proteins. Note reduced amount of cleaved formsof Talin and FAK in ﬁbronectin-coated tissue culture with NEU3,
and attenuation of the NEU3-mediated eﬀects in the presence of
calpeptin (A). Enhanced degradation of FAK and Talin by NEU3
silencing, and its attenuation by calpeptin (B). Calpain activity
in intact cells was determined with the cell permeable ﬂuorogenic substrate, Suc-LLVY-AMC. Suppression of the calpain activity by NEU3 (C) and its promotion by NEU3 silencing that
was attenuated by calpeptin (D).

in this glioblastoma cells are likely due to the heterogeneity on theceramide moiety. In comparison of the glycolipids in NEU3-overexpressing and -silenced cells with those of respective control cells,changes in Lac-cer and Gb4 in neutral glycolipids and in GM3 and GM1
in gangliosides were recognizable. Interestingly, modiﬁcations inducedby NEU3 silencing were a decrease in both Lac-cer and Gb4, and on theother hand, an increase in GM3 and GM1, whereas NEU3 overexpression mostly caused the opposite changes. Decreased Gb4 has
been suggested to reduce cell proliferation via suppressing EGFR -induced ERK pathway but less eﬀects on cell motility of cancer cells aspresented by Park et al. [29], and in neuroblastoma cells, switchingfrom cell proliferation to migratory signaling has been proposed
through phosphorylation of PDGF receptors mainly in GM1-rich lipid
microdomains [30]. Although the mechanism of the metabolic changes
in Gb4 and GM1 in U251 cells remains to be elucidated, they may beclosely related to exert eﬀect on a cell motility and invasiveness rather
than on cell proliferation, which is not contradictory to the phenomena
induced concomitantly by GM3 resulting in promotion of cell motility
and invasion described below.
As shown in our previous studies with various cells [4], the changes
in Lac-Cer and GM3, a product and a substrate of NEU3, respectively,
were conﬁrmed here in the neuroblastoma cells (Fig. 6A, left andmiddle), as quantiﬁed in the graph (right). Since Morford et al. andNuzzi et al. have suggested in Jurkat T-cells and in neutrophils, respectively [31,32], that calpain might exist in the raft fraction together
with GM3 and get activated, we examined whether the amount of
calpain in the lipid raft fraction is aﬀected by NEU3 silencing, andfound that any signiﬁcant change in calpain distribution was not detectable in U251 cells as assessed by raft fractionation followed byimmunoblotting (data not shown). Then we performed immunostaining
analysis by immunoﬂuorescence microscopy. Interestingly, in NEU3

silenced cells, strongly polarized GM3 and calpain on the lamellipodium were observed, and the lamellipodia and polarization were abrogated after treatment of EGCase, which hydrolyses the linkage between the oligosaccharide and ceramide (Fig. 6B, C). Furthermore, we
detected PIP2 production in the cells using the PH-green ﬂuorescentprotein (GFP) probe, which contains the PH domain from PLCdelta[33]. By NEU3 silencing, GFP-PLCdelta-PH and calpain also strongly colocalized at lammelipodia and it diminished by EGCase treatment forovernight, (Fig. 6D). The treatment for 6–7 h hardly showed any eﬀect,
as GM3 observed to be almost hydrolyzed for 12–16 h. Because PIP2 is
one of the activator for calpain, polarized GM3 and lamellipodia formation may support PIP2 production at and/or localization to plasma
membrane and subsequent activation of calpain at the membranes.
Cellular calpain activity was, in fact, increased by exogenous additionof GM3 (Fig. 6E). To verify that calpain translocates to plasma membrane and is activated by GM3, the eﬀect of GM3 exogenously added tothe cell culture was observed. As shown in Fig. 6F, a larger amount of
calpain was indeed detected in the membrane fractions by NEU3 silencing or GM3 addition. These results suggest that NEU3 down-regulation leads to accumulation of GM3, which may be important for
NEU3-mediated lamellipodia formation and calpain regulation resulting promotion of cell motility and invasion.

3.6. Calpain inhibitor abrogates invasion by NEU3 silencing
As shown in Fig. 2, in contrast to reduction of cell invasion in NEU3
overexpressing glioblastoma cells, silencing exerted an enhancing effect. To verify the eﬀect of calpain activity on the cell invasion, woundhealing assay and invasion assay were carried out in NEU3-silenced
U251 cells. As shown in Fig. 7A and B, wound healing assays revealed
that NEU3 down-regulation resulted in promotion of cell migration as

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K. Takahashi et al.

A

Acidic Glycolipids

Neutral Glycolipids
Std

vec

Std

NEU3

siCtrl

siN3

Std

vec

NEU3 siCtrl

siN3
Lac-Cer

Lac-Cer

Glc-Cer

GM3

NEU3 siN3 NEU3 siN3

GM3

Lac-Cer and GM3 level(relative to vec or siCtrl)

Lac-Cer

1.8

GM2
GM1

Gb3

GD3
Gb4
GD1a

p< 0.01

1.6
1.4

p< 0.05

1.2
1.0
-1.2
-1.4

p< 0.05-1.6-1.8

EGCaseB

siCtrlsiNEU3GM3

GM3

siCtrlsiNEU3GM3

D
p< 0.0001

GM3

calpaincalpaincalpain

Cell with lamellipodia (%)

70

calpain

vec

60
50
40
30
n.s.
20
10
0
siCtrl

Merge

Merge

Merge

siNEU3 siCtrl

siNEU3

EGCaseMerge

E
p< 0.002
Calpain activity (fluorescence)

5000

EGCaseC

siCtrlsiNEU3siCtrlsiNEU3

GFP-PLCdelta PH

GFP-PLCdelta PH

GFP-PLCdelta PH

GFP-PLCdelta PH

calpaincalpaincalpaincalpain

4000
3000
2000
1000
0
-

exogenous
GM3

F
Merge

Merge

Merge

Merge

-

GM3
Membrane calpain-+

Total calpain

FBS
GM3

-

+

-

-

+

Membrane calpain

Total calpain(caption on next page)

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K. Takahashi et al.

Fig. 6. Regulation of calpain activity by NEU3-mediated glycolipid modulation(A) Alteration of glycolipid pattern in NEU3 overexpressing or NEU3-silenced U251 cells (1 × 107) analyzed by thin-layer chromatography (TLC). Neutral glycolipids were separated in asolvent system of chloroform/methanol/0.02% aqueous CaCl2 (60:40:9, v/v/v), and acidic glycolipids in chloroform/methanol/0.02% aqueous CaCl2 (60:35:8, v/v/v). The doublets are
due to the heterogeneity of the ceramide moiety. The values in the graph (right panel) indicate changes in the amounts of Lac-cer and GM3 in NEU3 overexpressing and silenced cells,
relative to vector and siRNA controls, respectively, of four independent experiments, as analyzed with Quantity One software (Bio-Rad).
(B) Polarized GM3 and calpain at lamellipodia in NEU3-silenced cells as assessed by immunoﬂuorescent microscopy. Note abrogation of the polarization of GM3 and calpain (right in B)
and lamellipodia formation (C) after EGCase treatment for overnight. (D) GFP-PLCdelta-PH, PIP2 probe, was transiently expressed in U251 cells, and its co-localization with calpain was
analyzed. Scale bar, 20 μm. (E) Stimulation of cellular calpain activity by GM3 (50 μM) exogenously added and incubated for 16 h. (F) Translocation of calpain to membranes by NEU3
silencing or addition of GM3, as assessed by immunoblotting.

Fig. 7. Calpain inhibitor abrogation of enhanced cell invasion by NEU3 down-regulation.
(A) Promotion of cell migration in NEU3-silenced cells as assessed by wound healing assays and abrogation of the eﬀects by treatment with the calpain inhibitor, calpeptin. (B) The values
in the graph indicate changes in the wounded area in NEU3-silenced cells, obtained from three independent experiments. (C) Promotion of cell invasion in NEU3-silenced cells assessedusing Biocoat Matrigel invasion chambers, and inhibition of the eﬀects by calpeptin.

compared to control cells, but the augmentation was abrogated by thecalpain inhibitor, calpeptin. Similarly, invasion assays using Biocoat
Matrigel invasion chambers demonstrated promotion of cell invasion,
which was also inhibited by calpeptin (Fig. 7C). These results suggestthat NEU3 down-regulation observed in human glioblastoma tissues(Fig. 1) would contribute to the invasiveness, a characteristic feature ofthis malignancy, probably by causing calpain activation and acceleration of focal adhesion turnover through ganglioside modulation including GM3 accumulation as outlined in Fig. 8.

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K. Takahashi et al.

Fig. 8. A possible pathway of sialidase NEU3 contributions toinvasiveness of human glioblastoma. NEU3 down-regulation inhuman glioblastoma tissues and glioblastoma cells could contribute to the increased invasiveness, by causing calpain activation and acceleration of FAs turnover through gangliosidemodulation, including GM3 accumulation.

4. Discussion

pathological phenomena. Calpain activity is altered during carcinogenesis and may be involved in processes contributing to cancer development and progression [36,37] by proteolysis of various substrates
including focal adhesion proteins and proto-oncogene products. Interestingly, in our U251 cells, NEU3 down-regulation was found to activate calpain activity and cause accelerated degradation of focal adhesion proteins, such as Talin and FAK, leading to enhanced cell migrationand invasion. This calpain activation was likely induced by increased
GM3, as a result of decreased sialidase reactions. Previous work by Jan
et al. also pointed to a requirement of calpain for glioblastoma cell
invasion in calpain 2-silenced cells, calpain maintaining the level ofextracellular metalloprotease 2 [38]. Our present study indicated that
NEU3 gives rise to glioblastoma invasion by regulating the activity andcellular distribution of calpain through ganglioside modulation. Although the detailed mechanisms remains unclear at present, ganglioside changes including GM3 accumulation as the result of altered NEU3
expression might cause translocation of calpain to the cell surface andbring about potentiation of its activity. Consistent with our results,
Chakrabarti et al. have described a ganglioside mixture to stimulate
calpain activity [39], and Nuzzi et al. also have suggested in neutrophilsthat GM3-containing lipid rafts at the leading edge recruit phosphoinositides, such as PIP3, and translocate calpain to membrane and leadto its activation [32]. It has also been reported that formation of
sphingolipid-rich domain in the outer leaﬂet is required for accumulation of PIP2 in the inner leaﬂet of the plasma membrane [40,41]. Ourpresent ﬁndings that GM3 accumulation on NEU3 down-regulation cancontribute to enhanced glioblastoma cell invasion by calpain activation
point to a new mechanism of NEU3 for cell invasion and may provide abasis for the development of novel therapeutics targeting NEU3 inglioma patients.

In the present study, we examined whether sialidase is involved in
the invasiveness of human glioblastoma cells, because a gangliosidehydrolyzing sialidase, NEU3, is known to be associated with invasion
and migration of carcinoma cells. As expected, but opposite to various
carcinomas, human glioblastoma tissues showed down-regulation of
NEU3, and siRNA-mediated NEU3 silencing of glioblastoma cells actually caused enhancement of invasiveness as compared with control
cells. In this context, it is especially interest that NEU3 silencing resulted in decreased cell motility of renal cell carcinoma through inhibition of IL6-mediated Akt /Rho signaling pathway [17], and also
through blocking of β1 integrin recycling and FAK/AKT pathway [34].
The mechanism here for the increased cell invasion on down-regulationof the sialidase was found to involve promotion of degradation of focal
adhesion proteins subsequent to calpain activation, with GM3 accumulation. It is uncertain at present what factors are responsible for the
NEU3 eﬀects diﬀerent from those in other epithelial cancers, but may
be attributed to the origin of most gliomas. According to current consensus [35], neural stem cells located in the subventricular zone of thebrain are thought to be the source of glioma. They accumulate enoughgenomic mutations leading to diverse forms of glioma and undergo
epithelial to mesenchymal transition, which is required for migratory
phenotype and characterized by loss of epithelial morphology. Thisfeature is likely to be the one of the factors for the diﬀerential eﬀects of
NEU3.
Previous studies have generally reported [10,11] that increase of
GD3 and GM3 and decrease of gangliosides normally present in adultbrain (GM1, GD1a, GD1b and GT1b) are characteristic features ofhuman glioblastomas, correlating with a higher grade of malignancy. It
is feasible that proportions of simple gangliosides such as GD3 and GM3
in glioma tissues would increase, because these are good substrates for
NEU3 and would be expected to accumulate on its down-regulation.
GD3 has recently been proposed as a key molecule contributing toglioblastoma invasiveness [12] and tumorigenicity [13]. In addition tocausing calpain activation through GM3 accumulation, the NEU3
down-regulation observed in human gliomas might inﬂuence malignant
properties through GD3 accumulation with GD3 synthase up-regulation.
Calpains are known to play roles in numerous physiological andTransparency document
The http://dx.doi.org/10.1016/j.bbagen.2017.07.023 associatedwith this article can be found, in the online version.
Acknowledgments
We appreciate Dr. Akemi Suzuki (Tohoku Medical andPharmaceutical University) for his helpful discussion, and also Mrs.
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K. Takahashi et al.

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Setsuko Moriya (Tohoku Medical and Pharmaceutical University) and
Mr. Tadashi Wada (Miyagi Cancer Center Research Institute) for theirvaluable technical assistance. This study was supported in part by
Grants-in-Aid for Scientiﬁc Research on Innovative Areas (No. 3301)
from the Ministry of Education, Culture, Sports, Science andTechnology of Japan (24110514).
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2018年3月1日発行
発行者・発行所 東北医科薬科大学分子生体膜研究所
宮城県仙台市青葉区小松島4­4­1