東北医科薬科大学 分子生体膜研究所年報

東北医科薬科大学
分子生体膜研究所年報
2021年度 研究成果報告書

2021 Annual Report ofInstitute of Molecular Biomembrane and Glycobiology

二○二一年度【令和３年度】

目次■ 所長挨拶 …………………………………………………………………………………2■ 箱守仙一郎賞……………………………………………………………………..……4「箱守仙一郎賞」規約
第５回箱守仙一郎賞（2021 年度）
奨励賞：ナマズ卵レクチンはヒト子宮頸がん由来 HeLa におけるスニチニブの取り込みおよび排出に影響をおよぼす菅原栄紀（東北医科薬科大学 分子生体膜研究所 分子認識学・講師）

優秀論文賞：α1,6-Fucosyltransferase contributes to cell migration and proliferation as well as
to cancer stemness features in pancreatic carcinoma.
梁 彩霞 （Liang Caixia, 東北医科薬科大学・分子生体膜研究所・細胞制御学）■ 研究報告…………………………………………………………………………………10＜機能病態分子部門＞「炎症・炎症性細胞死におけるスフィンゴ糖脂質 Gal2Cer の役割」
井ノ口仁一、稲森啓一郎、永福正和、狩野裕考、新田昂大＜分子認識部門＞「レクチンの抗腫瘍メカニズムの解明と糖脂質のかかわり 2021」
細野雅祐、菅原栄紀、立田岳生＜細胞制御学部門＞「糖鎖間の相互作用と糖鎖生合成の制御機構の解析」
顧 建国、福田 友彦、伊左治 知弥、黒田 喜幸＜糖鎖構造生物学部門＞「糖鎖構造解析のための技術基盤の構築」
山口 芳樹、真鍋 法義、大野 詩歩

1

ご挨拶
2021 年度は、新型コロナウイルス感染拡大の影響の２年目となり、１年目の完全遠隔授業（オンライン）から５割対面と５割遠隔に切り替わりました。感染状況により緊急事態宣言が発出され、種々の行動制限が再び始まりました。賛否両論のあった、一年遅れの東京オリンピックが前代未聞の無観客下での開催となってことは、まだ記憶に新しい所であります。
一方で、驚異的な速さで開発された mRNA ワクチンの接種が日本でも本格的に進み始めていました。効果が 90%以上と言われたワクチンが広まれば、COVID-19 は１、２年の間に収束して行くのではないかと個人的には大きな期待を抱いていた時期がありました。ところが、非常に感染力の強いオミクロン株が主流となって更に大きな感染の波になってしまいました。幸いなことに、重症化したり死亡したりするリスクは、昨年猛威を振るったデルタ株よりは世界的に見ても低下しているようです。そのため世界の多くの国ではコロナ前の日常を取り戻すべく、経済活動とコロナを両立させるウイズコロ
ナに舵を切った国が多く、日本でも従来のような厳しい行動制限はかなり緩和されました。これまでに水際対策として新規留学生の入国が難しくしていた措置も 2021 年 1２月から大きく緩和され、本来 2021 年 4 月から入学する留学生達が１年遅れて 2022 年４月に入学できるようになりました。
この１年間、多くの学会は Web 開催かハイブリッド形式での開催でした。皆さんも対面で人と接する大切さを再確認されたのではないでしょうか。対面がかなわない情況で、オンライン講義・会議、テレワークなどの活用は、その不便さをある程度補ってくれました。しかしながら、研究活動や教育あるいは仕事を進める上で不可欠な人との信頼関係や共感を、リモートの環境だけで醸成するのは難しいことも実感しました。来年度からは全面的に対面で活動できるようになることを切に願っています。
研究面については、長年の蓄積と実績のある糖鎖構造と機能解析に関して、糖鎖科学を牽引する研究拠点を目指します。上述のような厳しい状況下において、コロナ感染対策を取り対面講義をこなしながら学問への情熱を持ち続け教員と、研究を続けた大学院生のみなさんの熱意と努力により、分子生体膜研究所（分生研）は研究教育活動を活発に行うことができました。その結果、第 40 回日本糖質学会でポスター賞（宋 万里, D3）、第 15 回東北糖鎖研究会でみちのく糖鎖
ポスター賞（宋 万里, D3）、第５回箱守奨励賞（菅原 栄紀,
講師）と優秀論文賞（梁 彩霞, D3）(上図, 左から梁さん、顧と菅原先生)などが受賞さ2

れました。また、細胞制御学に所属する黒田助教は、来年度から古巣の愛知県立大学の講師として栄転することになっています。おめでとうございます！
今年度から分生研 journal club & progress report を４教室の持ち回りの隔週開催でスタートさせました。教室間の交流のみならず、英語で発表することで若手教員や大学院生が国際学会で発表する機会にも繋がると信じています。また、8 月には、研究所全体で科研費申請内容に対する意見交換を行い、採択率の向上を目指しています。来年度はこれまで国内で例がない大型国家プロジェクトである「ヒューマングライコームプロジェ
クト」が始まります。今年度は準備組織であるネットワーク拠点から、日本における独創的専門技術を有する糖鎖研究者として、当研究所の 4 名の教授が Collaborative Fellow(CF 相談役)に任命されました。今後、本プロジェクトに積極的に参画していきたいと考えています。
今回の年報では、各研究部門の研究活動報告および第５回箱守仙一郎賞の奨励賞および優秀論文賞をご紹介致します。
皆様からのご指導ご鞭撻のほど心よりお願い申しあげます。
令和４年３月東北医科薬科大学分子生体膜研究所・所長
顧

3

建国

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

令和 2 年 4 月
東北医科薬科大学分子生体膜研究所
顧 建国
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

対象

1~２名 顕彰楯および副賞
1~２名 顕彰楯および副賞（１）奨励賞：東北エリア（新潟県および群馬県を含む）で研究に従事する応募時 45 歳までの研究者（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(各年度内)に発表（accepted でも可）された学術原著論文に対して審査・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。

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応募と審査方法（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 歳以
下の糖鎖科学研究者（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 所長 顧 建国
メールアドレス：jgu@tohoku-mpu.ac.jp； 応募締め切り：5 月 31 日
（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（下記基礎点と推薦者の評価点の合計による）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＋推薦者の評価点から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 (IF):IF× 5 点

推薦者（自薦または他薦）の評価点（推薦書に記入、最高点 10 点）
共同研究者の貢献を考慮し、以下の項目を参考して該当研究に対する応募者の貢献度を客観的にみて公正に評価する。
１） 研究の考案（最高点 2 点）
２） 実験の実行（最高点 5 点）
３） 論文作成（最高点 3 点）
評価最高点
独創性
インパクト（重要性）
論文構成
将来性

10 点
10 点
10 点
10 点

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第５回箱守仙一郎賞（2021 年度）

奨励賞
ナマズ卵レクチンはヒト子宮頸がん由来 HeLa におけるスニチニブの取り込みおよび排出に影響をおよぼす
Catfish egg lectin affects influx and efflux rates of sunitinib in humancervical carcinoma HeLa cells
菅原

栄紀（東北医科薬科大学 分子生体膜研究所 分子認識学・講師）

ナマズ卵由来レクチン（SAL）は，グロボトリアオシルセラミド（Gb3）の糖鎖を介してバーキットリンパ腫細胞株 Raji に結合し，ヨウ化プロピジウム（PI）およびドキソルビシンの細胞内取り込み量を増加させて細胞生存率を著しく減少させることを報告している。本研究では，SAL が Gb3 を発現している HeLa において分子標的薬であるスニチニブ（スーテント，SU）の細胞毒性作用を増強するか否かを検討した．SAL
を前処理することにより SU による HeLa の生存率が有意に減少し，この時の SU の取り込みは，30 分後で有意に増加していた．次に，培地から薬剤を除去し細胞内の SUの残存量を観察したところ，SAL 未処理細胞では 3 時間後に大部分の SU が排出されているが，処理細胞では 24 時間経過しても多くの SU が細胞内に残存していた．また，Gb3 欠損 HeLa では，SAL 処理による SU の取り込み促進および排出抑制のどちらも認められなかった．次に，SU の集積する部位を，共焦点レーザー顕微鏡を用いて調べた結果，SU は SAL 処理細胞内に形成される油滴状の膜構造物の周囲に集まる像が確認された．さらに，この構造物の帰属を明らかにするために，リソソームのマーカーである LAMP1 と RFP の融合タンパク質を HeLa に強制発現させた後，SAL 処理した細胞に SU を取り込ませたところ，SU と LAMP1 は共局在していることが明らかとなった．以上の結果から，SAL は HeLa 細胞内にリソソーム様の構造物形成を促進
し，細胞内に取り込まれた SU はこの構造物にトラップされ細胞内に蓄積し，HeLa に対する細胞毒性を増加させている可能性が示唆された（１）．
(1) Sugawara S, Takayanagi M, Honda S, Tatsuta T, Fujii Y, Ozeki Y, Ito J, Sato M, HosonoM.
Catfish egg lectin affects influx and efflux rates of sunitinib in human cervical carcinoma
HeLa cells. Glycobiology, 30, 802-816 (2020).

6

【経歴】
2000 年：東北薬科大学薬学部卒業
2002 年：東北大学大学院薬学研究科 博士課程前期課程修了（自然免疫におけるペプチドグリカン認識タンパク質の機能解析に従事）2005 年：東北薬科大学大学院薬学研究科 博士課程後期課程修了（糖鎖あるいは DNA を認識する魚卵由来タンパク質の生化学的解析に従事）2005 年：東北薬科大学附属癌研究所第一部

助手

2007 年：東北薬科大学 分子生体膜研究所 分子認識学教室

助教

2017 年〜現在：東北医科薬科大学 分子生体膜研究所 分子認識学教室

講師（ナマズ卵レクチンを利用した新規がん治療法の開発に従事）【受賞の御礼ならびに抱負】
この度は、第５回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とともに箱守仙一郎賞審査員長の顧 建国先生ならびに審査員の先生方に厚く御礼申し上げます．仙台の地で糖鎖研究を行う一研究者として箱守仙一郎先生のお名前が冠された賞を受賞することは私の目標でもありました．これからは頂いた賞に恥じぬように，
これまで以上に糖鎖科学の発展に努めていきたいと思っております．
私の研究テーマは，大学院博士後期課程入学時から一貫して，ナマズ卵レクチン（SAL）のがん治療への応用を目指した基礎研究であります．中でも，糖脂質の一種であるグロボトリアオシルセラミド（Gb3）を高発現しているがん細胞に対する効果的な治療法の開発を思考して研究に取り組み，これまでに１）Gb3 を発現しているバーキットリンパ腫細胞はナマズ卵レクチン処理により初期アポトーシス初期状態に変化する，２）ナマズ卵レクチンはバーキットリンパ腫細胞 Raji の増殖を抑制するな
どの成果を報告してきました．研究生活の中で様々な難題にあたり困難を極めたこともありましたが，箱守先生が来学された際に行われる研究報告会では，いいデータは褒めていただき，研究に足りないところは⽂献や研究⽅法などを含めて詳細にお話しいただき，これからの研究に光がさしたような感じがしてとても有り難かったです．
受賞論文では，Gb3 を発現している子宮頸がん由来細胞株 HeLa おいて抗がん剤とSAL を併用することにより薬剤の濃度を下げても高濃度と同じ効果が得られ，この時，細胞内への薬剤の取り込み量が増加していることを明らかにしました．抗がん剤治療で最も問題となるのは副作用の発現でありますが，SAL は，がん細胞の薬剤感受性を高める効果を発揮することでこの問題を解決でき，また Gb3 を発現しているがん細胞であれば，SAL は併用する抗がん剤の用量低減に貢献できる可能性も有すると考えております．しかしながら，SAL 処理による薬剤の取り込み促進の分子メカニズム

7など不明な点は残されているので，これらのことを少しずつ明らかにしてレクチンによるがん治療実現に向けて研究を進めていきたいと考えております．
最後に，研究のご指導を頂きました細野雅祐教授をはじめ本研究にご協力いただきました先生方，研究室員の皆様に深く感謝申し上げます．これからも日々の努力を怠らず仙台の地から糖鎖科学に関する新しい知見を多く発信できるように精進していきたいと存じます．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．

優秀論文賞
α1,6-Fucosyltransferase contributes to cell migration and proliferation as well as to cancer
stemness features in pancreatic carcinoma.

Liang C., Fukuda, T., Isaji, T., Duan, C., Song, W., Wang, Y. and Gu, J.
Biochim Biophys Acta Gen Subj., 1865, 3239-3252, 2021

梁 彩霞 （Liang Caixia, 東北医科薬科大学・分子生体膜研究所・細胞制御学）

論文要旨
膵がんでは FUT8 の発現が上昇するが、膵がんの特徴である浸潤・転移しやすさとコ
アフコシル化の関係は未解明である。膵がんの予後不良に対する FUT8 の役割を明らかにするため、膵がんの研究に広く用いられている MIA PaCa-2 や PANC-1 細胞株に
CRISPR/Cas9 を用いて FUT8 を欠損させた細胞を作成し、膵がんにおける FUT8 の機能を明らかにした。細胞遊走能を検討したところ、FUT8 欠損 MIA PaCa-2 細胞は野生型に比べて、約 80%と顕著な遊走能の阻害が認められた。また、MTT およびコロニー形成による細胞増能を検討したところ、いずれの方法でも FUT8 欠損細胞は 50%以下に減少し、細胞表面における増殖因子受容体数も減少していた。さらに、フコース誘導体(2FF)の添加によりコアフコシル化を阻害しても、FUT8 欠損細胞と同様に、増殖・遊走に対する抑制が確認された。FUT8 欠損により、腫瘍増殖抑制作用が期待されたため、免疫不全マウス皮下に作成した細胞を移植したところ、生着・増殖は認められものの、有意な腫瘍増殖抑制作用が認められた。そこで、再発・転移に重要な役割を果たすと考えられているがん幹細胞性に与える影響を検討した。まず、がん幹細8

胞マーカーの発現を検討したところ、FUT8 欠損細胞では、CD133, CXCR4, C-Met,
EpCAM などの発現が減少していた。次に、がん細胞同士の相互作用を検討するため、スフェロイドを形成させたところ、FUT8 欠損細胞では細胞間の凝集が弱く不安定であることが分かった。さらに、がん幹細胞の特徴の一つである薬物耐性に対しても検討した。その結果、FUT8 の欠失は、膵がんの治療薬である Gemcitabine に対する応答性を高めた。これらの結果は、FUT8 の発現が無くなると、幹細胞性が失われることを示唆している。実験的ではあるものの、2FF の投与でも FUT8 欠損細胞と同様の効果が得られたことは、フコース誘導体をもちいて FUT8 の機能制御を介して、膵
がん幹細胞を標的とした治療などの可能性が示された。

受賞の御礼ならびに抱負
I am both excited and deep humbled when I was informed by Prof. Gu that I have received
the Sen-itiroh Hakomori glycoscience award. I couldn’t believe my ears, I was not so sure if I
really deserved this honor, because I knew many of researchers in the field even worked moreharder than I did. It is a great honor for me to receive the award, which is not only a recognitionof my existing work, but also an encouragement for the following research work. I am sure that
this achievement couldn’t be received without a wonderful and strong productive research team.
I’d like to express my sincere appreciation to all members of Institute of Molecular
Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University.
I’d like also to express my sincere appreciation to the prize committee members. Prof.
Hakomori was an outstanding scientist in the field of glycobiology. His academic reports andpassion for scientific research are a deep inspiration to our younger generation. The Fall of theStar brings regret and deep pain to us, but it is more about nostalgia and admiration. His hard
work, boundless love and dedication to science will always inspire us to move forward.
The three years of doctoral life were very important and memorable experiences for me. I
owe my sincerely gratitude to President Motoaki Takayanagi for providing the opportunity to
study at Tohoku Medical and Pharmaceutical University and provide financial assistance.
Thanks to my mentor, Prof. Gu, who gave me great guidance and help in my studies and life.
What is more, I would like to thank Dr. Tomohiko Fukuda and Dr. Isaji Tomoya for theirconsultation and technical support, and Ms. Yan Hao as well as other members of the laboratory.
Thank you for taking me on the most enjoyable journey of my life.

9During the three years of studying in Japan, many kind people gave me great support,
encouragement and help. I want to take this opportunity to thank them all. Thank you very
much! Now, I was already graduated from Tohoku medical and pharmaceutical university andworked in the clinical medical research center of The Second Affiliated Hospital of Nantong
University. I am still engaged in basic research on glycobiology. I will continue to do my best tocontribute to the glycobiology society.
Thank you very much!
梁彩霞 (Liang Caixia)

10

研究報告

＜機能病態分子部門＞
特任教授

井ノ口仁一

准教授

稲森啓一郎

講師

永福正和

助教

狩野裕考

博士研究員

新田昂大
炎症・炎症性細胞死におけるスフィンゴ糖脂質 Gal2Cer の役割

糖尿病性腎症は、いまだ根治を可能とする治療法・治療薬が存在しない疾患であり、詳細な発症・増悪メカニズムの解明ならびに治療ターゲットとなる分子の同定が求められている。
細胞膜構成成分のひとつであるスフィンゴ糖脂質は、糖尿病性腎症において病態に関連した腎臓での発現変化が観察されることは、すでに知られていたが、その病態生理学的な役割については、ほとんど分かっていなかった。我々は、2 型糖尿病性腎症
病態で、グロボ系スフィンゴ糖脂質の腎発現が変化することに加え、炎症促進に関与していることを明らかにし、2019 年に報告した(Glycobiology, 29: 260-268, 2019)。最近、1 型糖尿病性腎症においても、特徴的なスフィンゴ糖脂質の腎発現が変化していること、そして、その糖脂質がマクロファージにおける炎症・炎症性細胞死(パイロトーシス)に関与することを新たに見出した。
【1 型糖尿病性腎症病態における腎スフィンゴ糖脂質の発現変化】
薬剤(Streptozotocin)誘導性マウスモデルならびに、自然発症マウスモデル(Akita)を用いて、腎臓のスフィンゴ糖脂質発現を薄層クロマトグラフィー(TLC)にて解析を行った。これまで行ってきた 2 型糖尿病性腎症モデルマウスの解析では、グロボ系スフィ
ンゴ糖脂質の発現が顕著に増加していたが、上記の 1 型のモデルマウスにおいては、
グロボ系の発現は減少しており、それとは異なる糖脂質の発現増加が認められた。レクチン(PA-1L)を用いたさらなる解析によって、この増加していた糖脂質はガラビオシ
ルセラミド(Gal2Cer)であることが明らかとなった(図 1)。

11

図1

1 型糖尿病性腎症モデル。マウスの腎糖脂質発現変化【GalCer の持つ炎症ならびに炎症性細胞死の促進活性】
当研究室では、これまでにスフィンゴ糖脂質 Gb3Cer、Gb4Cer ならびにガングリオ
シド GM3 が、Toll-like receptor 4(TLR4)を介したマクロファージの活性化促進能を有することを明らかにしてきた。(EMBO J, 39: e101732, 2020)。Gal2Cer についても、上記と同様にマクロファージ(BMDMs)の活性化を促進するかどうかを今回検討した。TLR4リガンドであるリポ多糖(LPS)と Gal2Cer でマクロファージを共刺激し、培養上清中の炎症性サイトカイン(TNFα)を ELISA にて定量した。その結果、LPS 単独よりもGal2Cer との共刺激の方が、より TNFα の検出量が高値であった(図 2A)。これは、Gal2Cer においても炎症を促進する活性があることを示唆しており、マクロファージ
活性化に寄与していることが明らかとなった。
また、近年注目集めている炎症性細胞死(パイロトーシス)についても Gal2Cer の関与を検討した。パイロトーシスの誘導経路には、TLR4 を介したメカニズムが報告されており、TLR4 を介した炎症促進に関与していた Gal2Cer が、パイロトーシスの誘導促進に寄与する可能性は高いと考えられた。評価方法については、上記と同様にマクロファージを共刺激し、培養上清中に分泌されるパイロトーシスの指標である IL-1(α, β)の量を ELSIA にて定量した。その結果、IL-1 の検出量は LPS 単独と比較して共刺激の方が高かった(図 2B)ことから、Gal2Cer にはパイロトーシス誘導促進活性が存在する
ことが示唆された。興味深いことに、Gal2Cer 単独でのパイロトーシス惹起は誘導されないことから（図 2B）、これまで知られている誘導メカニズムとは異なる可能性が考えられる。したがって、今後はその詳細な機序について明らかにし、パイロトーシ
スの関与する疾患における治療法・予防法の開発につなげたい。

12

図 2 A: マウス骨髄由来マクロファージ(BMDMs)を LPS/Gal2Cer で刺激。48 時間
後、培養上清中の TNFα 量を ELSIA にて定量。 B: マウス骨髄由来マクロファー
ジ(BMDMs)を LPS/Gal2Cer で刺激。48 時間後、培養上清中の IL(α, β)量を ELSIA にて定量。

＜発表論文＞
1. Homeostatic and pathogenic roles of the GM3 ganglioside
Jin-ichi Inokuchi, Hirotaka Kanoh, Kei-ichiro Inamori, Masakazu Nagafuku, Takahiro Nitta,
and Koichi Fukase a(Department of Chemistry, Graduate School of Science, Osaka University a )The FEBS Journal (2021) PMID: 34125497 DOI: 10.1111/febs.16076

2. GRASP55 regulates intra-Golgi localization of glycosylation enzymes to control
glycosphingolipid biosynthesis
Prathyush Pothukuchi a, Ilenia Agliarulo a, Marinella Pirozzi a, Riccardo Rizzo a, Domenico
Russo a, Gabriele Turacchio a, Julian Nüchel b, Jia-Shu Yang c, Charlotte Gehin d, Laura
Capolupo d, Maria Jose Hernandez-Corbacho e, Ansuman Biswas f, Giovanna Vanacore a, Nina
Dathan a, Takahiro Nitta, Petra Henklein g, Mukund Thattai f, Jin-Ichi Inokuchi, Victor W Hsu c,
Markus Plomann b, Lina M Obeid e, Yusuf A Hannun e, Alberto Luini a, Giovanni D'Angelo a d,
Seetharaman Parashuraman a(Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome Italy a,
Medical Faculty, Center for Biochemistry, University of Cologne, Cologne Germany b, Divisionof Rheumatology, Inflammation and Immunity, Department of Medicine, Brigham and Women's
Hospital Harvard Medical School, Boston MA, USA c, École Polytechnique Fédérale de13

Lausanne (EPFL), Lausanne Switzerland d, Stony Brook University Medical Center, Stony
Brook NY, USA e, National Center of Biological Sciences, Bengaluru India f,
Universitätsmedizin Berlin Institut für Biochemie Charité CrossOver Charitéplatz 1 / Sitz,
Berlin Germany g)
EMBO J (2021) PMID: 34516001 DOI: 10.15252/embj.2021107766＜学会発表＞
・ Glycosphingolipids in central and peripheral leptin signaling
Kei-ichiro Inamori
日本薬学会第 141 年会，Web 開催，2021 年 3 月，S44-3
・ Homeostatic and Pathogenic Balance Regulated by Ganglioside GM3
Jin-ichi Inokuchi
3rd Australasian Glycoscience Symposium
June 3-4, 2021, Web 開催
・ 1 型糖尿病性腎症における糖脂質発現変化
新田昂大、狩野裕考、稲森啓一郎、鈴木明身、井ノ口仁一
第 63 回日本脂質生化学会 Web 開催 2021 年 6 月 要旨集 P100-101
・ 潰瘍性大腸炎におけるスフィンゴ糖脂質の発現変化
稲森啓一郎，小川仁 a，新田昴大，大川光，狩野裕考，遠藤克哉 b，佐藤賢一 b，中村保宏 c，柴田近 a，鈴木明身，井ノ口仁一（東北医薬大・医・外科学第一（消化器外科）a、東北医薬大・医・内科学第二（消化器内科）b、東北医薬大・医・病理学 c）
第 15 回東北糖鎖研究会，Web 開催，2021 年 9 月，要旨集 14-15
・ 潰瘍性大腸炎におけるスフィンゴ糖脂質の発現変化
稲森啓一郎，小川仁 a，新田昴大，大川光，狩野裕考，遠藤克哉 b，佐藤賢一 b，中村保宏 c，柴田近 a，鈴木明身，井ノ口仁一（東北医薬大・医・外科学第一（消化器外科）a、東北医薬大・医・内科学第二（消化器内科）b、東北医薬大・医・病理学 c）
第 14 回セラミド研究会学術集会，ハイブリッド開催，2021 年 10 月，
要旨集 2314

・ 糖尿病性腎症におけるスフィンゴ糖脂質の発現変化解析
新田昂大, 狩野裕考, 稲森啓一郎, 鈴木明身, 井ノ口仁一
第 14 回セラミド研究会学術集会 Web 開催 2021 年 10 月 要旨集 P17
・ 新規ミスセンス変異を認めた本邦初の B4GALNT1 関連神経変性症の 1 例
中村勝哉 a,b, 土田奈緒美 c, 井ノ口仁一, 小島朋美 a), 池田淳司 b,
小平農 b, 稲森啓一郎, 永福正和,新田昂大, 真鍋法義, 大野詩歩 d,
山口芳樹 d, 下畑享良 e, 松本直通 c, 古庄知己 a,f, 関島良樹 b（信州大学医学部附属病院遺伝子医療研究センター a, 同 脳神経内科 b リウマ
チ・膠原病内科、横浜市立大学大学院医学研究科遺伝学 c,東北医科薬科大学分子生体膜研究所糖鎖構造生物学 d,岐阜大学大学院医学系研究科脳神経内科学分野 e,
信州大学医学部遺伝医学教室 f）
日本人類遺伝学会第 66 回大会 2021 年 10 月 WEB 開催

・ 全ヒト糖鎖関連遺伝子 Variant の網羅的抽出と解析
西原祥子 a,木下聖子 a,栂谷内晶 a,細田正恵 a,伊藤和義 a,山口芳樹 b,真鍋法義 b,大野詩歩 b,稲森啓一郎,井ノ口仁一,灘中里美 c,北川裕之 c,城田松之 d,木下賢吾 d（創価大学 糖鎖生命システム融合研究所 a, 東北医科薬科大学 分子生体膜研究所b, 神戸薬科大学 c, 東北大学 東北メディカル・メガバンク機構 d）

第 40 回日本糖質学会年会（2021 年 10 月 27～29 日、鹿児島）口頭発表
要旨集 120
・ 潰瘍性大腸炎患者におけるスフィンゴ糖脂質の発現変化
稲森啓一郎，小川仁 a，新田昴大，大川光，狩野裕考，遠藤克哉 b，佐藤賢一 b，中村保宏 c，柴田近 a，髙橋伸一郎 d，鈴木明身，井ノ口仁一（東北医薬大・医・外科
学第一（消化器外科）a、東北医薬大・医・内科学第二（消化器内科）b、東北医薬大・医・病理学 c、東北医薬大・医・臨床検査医学 d）
第 15 回スフィンゴテラピィ研究会，Web 開催，2021 年 11 月，要旨集 15
・ GM3 Ganglioside in Insulin Resistance and Inflammation
Jin-ichi Inokuchi
16th Meeting of the International Endotoxin and Innate Immunity Society (IEIIS) Oct 1215, 2021 Kobe Japan Invited Oral presentation15

・ 1 型と 2 型糖尿病性腎症における腎スフィンゴ糖脂質発現変化の比較
新田昂大、狩野裕考、稲森啓一郎、鈴木明身、井ノ口仁一
第 94 回 日本生化学会大会 Web 開催 2021 年 11 月 プログラム集 P86
・ 細胞膜スフィンゴミエリンによる CD8T 細胞の分化・機能制御機構の解明
豊島 かおる a, 井ノ口 仁一, 大野 勲 a（東北医薬大・医・医学教育推進センター a)
第 44 回 日本分子生物学会年会 Web 開催，2021 年 12 月
・ TLR リガンドの機能評価～レポーターアッセイとイメージングによる解析～
大垣 仁之 a、 樺山 一哉 a,b、 内藤 ひかり a、 下山 敦史 a,b、 狩野 裕考、井ノ口 仁一、 深瀬 浩一 a,b(阪大・院理・化学 a, 阪大・院理・PRC b)

第 44 回 日本分子生物学会年会 Web 開催，2021 年 12 月

16Article

GRASP55 regulates intra-Golgi localizationof glycosylation enzymes to control
glycosphingolipid biosynthesis
Prathyush Pothukuchi1 , Ilenia Agliarulo1,‡, Marinella Pirozzi1,‡, Riccardo Rizzo1,§, Domenico Russo1,€chel2 , Jia-Shu Yang3, Charlotte Gehin4 , Laura Capolupo4,
Gabriele Turacchio1, Julian Nu
Maria Jose Hernandez-Corbacho5, Ansuman Biswas6, Giovanna Vanacore1, Nina Dathan1,
Takahiro Nitta7, Petra Henklein8, Mukund Thattai6, Jin-Ichi Inokuchi7 , Victor W Hsu3 ,Markus Plomann2 , Lina M Obeid5,† , Yusuf A Hannun5, Alberto Luini1, Giovanni D’Angelo1,4 &Seetharaman Parashuraman1,*

Abstract
The Golgi apparatus, the main glycosylation station of the cell,consists of a stack of discontinuous cisternae. Glycosylation
enzymes are usually concentrated in one or two specific cisternaealong the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes toglycosylation are not clear. Here, we show that the Golgi matrix
protein GRASP55 directs the compartmentalized localization of key
enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55
binds to these enzymes and prevents their entry into COPI-based
retrograde transport vesicles, thus concentrating them in thetrans-Golgi. In genome-edited cells lacking GRASP55, or in cells
expressing mutant enzymes without GRASP55 binding sites, these
enzymes relocate to the cis-Golgi, which affects glycosphingolipid
biosynthesis by changing flux across metabolic branch points.
These findings reveal a mechanism by which a matrix protein
regulates polarized localization of glycosylation enzymes in the
Golgi and controls competition in glycan biosynthesis.
Keywords glucosylceramide synthase; glycosphingolipids; glycosylation; Golgi
apparatus; GRASP55
Subject Categories Membranes & Trafficking; Metabolism
DOI 10.15252/embj.2021107766 | Received 18 January 2021 | Revised 26 July
2021 | Accepted 6 August 2021 | Published online 13 September 2021

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5
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The EMBO Journal (2021) 40: e107766

Introduction
Glycans are one of the fundamental building blocks of the cell andplay key roles in development and physiology (Bishop et al, 2007;
Kohyama-Koganeya et al, 2011; Ryczko et al, 2016; Varki, 2017;
Akintayo & Stanley, 2019). Cellular glycan profiles are sensitive tochanges in cell state and/or differentiation and are also important
contributors to the process (Russo et al, 2018b). Indeed, several
developmental disorders are associated with impaired production ofglycans (Chang et al, 2018). Thus, how the glycan biosynthesis isregulated to achieve specific cellular glycan profiles is an importantbiological problem. In eukaryotes, glycans are assembled mainly by
the Golgi apparatus on cargo proteins and lipids that traverse theorganelle (Stanley, 2011). Glycan biosynthesis happens in atemplate-independent fashion (Varki & Kornfeld, 2015), yet the
products are not random polymers of sugars but a defined distribution of glycans that is cell-type and cargo-specific (Rudd et al, 2015;
Varki & Kornfeld, 2015). This suggests that their biosynthesis isguided by regulated program(s). Transcriptional programs havebeen identified that contribute to defining the glycome of a cell, butthey only partially account for it (Nairn et al, 2008, 2012; Varki &Kornfeld, 2015). An obviously important but unexplored factor that

Institute of Biochemistry and Cell Biology, National Research Council of Italy, Rome, Italy
Medical Faculty, Center for Biochemistry, University of Cologne, Cologne, Germany
Division of Rheumatology, Inflammation and Immunity, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
Ecole
Polytechnique Federale de Lausanne (EPFL), Lausanne, Switzerland
Stony Brook University Medical Center, Stony Brook, NY, USA
National Center of Biological Sciences, Bengaluru, India
Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan€r Biochemie Charite CrossOver Chariteplatz 1 / Sitz, Berlin, Germany
Universit€
atsmedizin Berlin Institut fu*Corresponding author. Tel: +39-081-6132283; E-mail: raman@ibbc.cnr.it†
Deceased.
‡
These authors contributed equally to this work§
Present address: Institute of Nanotechnology, National Research Council (CNR-NANOTEC), Lecce, Italyª 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license

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influences glycosylation is the Golgi apparatus itself (Varki, 1998;
Maccioni et al, 2002).
The Golgi apparatus is a central organelle of the secretory pathway that processes newly synthesized cargoes coming from theendoplasmic reticulum (ER), primarily by glycosylation, before sorting them toward their correct destination in the cell. It consists of a
stack of 4–11 cisternae (Klumperman, 2011), populated by enzymes
and accessory proteins that maintain a suitable milieu for the
enzymes to act on biosynthetic cargoes. The stack is polarized witha cis-side where cargoes arrive and a trans-side from where theyleave. The enzymes are not homogeneously distributed across theGolgi stack but are restricted or compartmentalized to 1–3 specificcisternae. The cisternal maturation model provides a conceptual
framework for understanding Golgi enzyme compartmentalization(Nakano & Luini, 2010; Glick & Luini, 2011). According to themodel, secretory cargoes are transported forward by the anterograde
flux mediated by cisternal progression, which consists of constantformation and consumption of cis and trans cisternae, respectively.
The retention of Golgi glycosylation enzymes in the face of thiscontinuous forward flux is mediated by their retrograde transportthat acts as counterbalance for the forward transport. The retrograde
transport is promoted by coat protein complex I (COPI) machinery(Rabouille & Klumperman, 2005; Popoff et al, 2011; Papanikou
et al, 2015; Ishii et al, 2016; Liu et al, 2018) and is assisted in thisprocess by adaptor molecules like GOLPH3 (Tu et al, 2008, 2012;preprint: Rizzo et al, 2019), conserved oligomeric complex (COG)
proteins, and Golgi matrix proteins especially Golgins (Eckert et al,2014; Wong & Munro, 2014; Blackburn et al, 2019). However, thespecific molecular mechanisms and processes by which the same
retrograde transport pathway promotes localization of enzymes todistinct cisternae remain unknown.
The compartmentalized localization of enzymes has beensuggested to influence both sequential as well as competing glycosylation reactions. The localization of enzymes along the cis-trans axis
reflecting their order of action (Dunphy & Rothman, 1985) has beensuggested to influence the efficiency of sequential processing reactions (Fisher et al, 2019). On the other hand, the promiscuity ofglycosylation enzymes (Biswas & Thattai, 2020) makes compartmentalized localization of competing enzymes a critical factor in
determining the specificity in glycan output (i.e., the type and quantity of glycans produced) (Dunphy & Rothman, 1985; Pothukuchi
et al, 2019; Jaiman & Thattai, 2020). When two or more enzymes
compete for a substrate, the order in which they get access to it can
substantially influence the glycans produced and subsequently thephysiological outcomes. Competing reactions are frequent in glycosylation pathways, and all known glycosylation pathways have one
or more competing glycosylation steps. Nevertheless, how thecompartmentalized localization of competing enzymes is achieved,
how it is regulated to influence glycosylation reactions, and what
the physiological relevance of this regulation is remain unexplored.
To evaluate and understand the contribution of Golgi compartmentalization in regulating glycosylation, we have focused ourstudy on sphingolipid (SL) glycosylation. We chose this model
system for several reasons: a. It is well characterized from bothbiochemical and transcriptional perspectives (Halter et al, 2007;
D’Angelo et al, 2013; Russo et al, 2018b); b. the glycosylation reaction is less influenced by the cargo structure in contrast to protein
glycosylation and thus is a cleaner system to study effects of Golgi

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Prathyush Pothukuchi et alprocesses on glycosylation; c. there are simple biochemical methods
available to analyze SL glycosylation (D’Angelo et al, 2013); and d.
finally, SLs have important roles in physiology and development(Hannun & Obeid, 2018; Russo et al, 2018a). The SL glycosylation
pathway exhibits the essential features of glycosylation pathways
like localization of enzymes reflecting their order of action and also
at least two competing reaction steps that are important in determining the metabolic outcome of the pathway (see below). Further,while enzymes of the pathway are well characterized, molecular
players regulating their sub-Golgi compartmentalization are
unknown. By studying SL glycosylation, we identify GRASP55 as animportant factor that compartmentalizes two enzymes catalyzing
critical branch points of the SL glycosylation pathway. GRASP55
binds to and prevents the entry of these enzymes into retrograde
transport carriers. This retaining action of GRASP55 is essential for
dynamic compartmentalization of these enzymes in the Golgi stack.
The competing enzymes thus positioned at appropriate levels in the
Golgi stack regulate cargo flux across competing reactions of thepathway and determine the metabolic outcome viz. sphingolipid
produced by the cell. These results delineate a molecular mechanism of enzyme compartmentalization and how it controls cellsurface glycan profile.

Results
Disruption of Golgi organization alters SL biosynthesis
SL biosynthesis starts with the production of ceramide (Cer) in theER, which is then processed in the Golgi to sphingomyelin (SM) or
glycosphingolipids (GSLs). The model cell system we use, HeLa
cells, produces two species of GSLs—globosides (Gb3) and gangliosides (GM1 and GM3) (Halter et al, 2007; D’Angelo et al, 2013;
Russo et al, 2018b) (See Fig 1A for schematic of the SL system in
HeLa cells). This SL pathway includes sequential processing of Cer
to complex GSLs as well as two bifurcating steps where thesubstrates get differentially channeled. The first is the bifurcation
between SM and glucosylceramide (GlcCer) biosynthesis, where thesubstrate Cer is channeled into either of the pathways. The second
is the biosynthesis of Gb3 or GM3 from lactosylceramide (LacCer).
These two critical steps determine the amount and type of SLs
produced by the cell. We first examined the localization of SL
biosynthetic enzymes and found that they localize to three distinct
zones in the secretory pathway (Fig 1B, Appendix Fig S1): (i) the
early secretory pathway including the ER and the cis/medial-Golgi(C1, C2 cisternae), where Cer biosynthetic enzymes are localized(33), have little if any SL biosynthetic enzymes except for a slightly
elevated amount of GM3S and GlcCer synthase (GCS) in thecis/medial-Golgi compared with other GSL biosynthetic enzymes;(ii) medial/trans-Golgi (C3, C4 cisternae) where most of the GSL
biosynthetic enzymes are present alongside substantial amounts of
Sphingomyelin synthase 1 (SMS1) and (iii) trans-Golgi network(TGN), where SMS1 predominates. While all the GSL biosynthetic
enzymes show a gradient of increasing concentration from cis- totrans-Golgi, the gradient is much sharper in the case of GB3S and
LacCer synthase (LCS) compared with GCS and GM3S
(Appendix Fig S1). Thus, the SL biosynthetic enzymes are distributed reflecting their order of action with precursor (Cer) producingª 2021 The Authors

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A

B

D

C

E

F

Figure 1. Disruption of SL biosynthetic machinery organization alters SL output.
A

Schematic representation of GSL biosynthetic pathway in HeLa cells (Glu, glucose; Gal, galactose; Sia, N-acetylneuraminic acid; Cer, ceramide). Products ofbiosynthesis are represented in bold and enzymes that catalyze the reactions in gray. The arrows represent the SL metabolic flux from ceramide.
B
Schematic representation of GSL biosynthetic zones in HeLa, SM biosynthesis predominates in TGN, whereas GSL and SM productions happen in medial/trans-Golgi(C3 and C4 cisternae). Cis-Golgi/ER is where Ceramide biosynthesis happens with little, if any, SL production. CerS* refers to the group of Ceramide synthases
localized to the ER. The size of the lipid label arbitrarily represents the proportion of the lipid expected to be synthesized in the compartment based on thelocalization of corresponding enzymes.
C
High-performance thin-layer chromatography (HPTLC) profile of HeLa cells pulsed for 2 h with [3H]-sphingosine and chased for 24 h. The peaks corresponding toeach SL species are indicated, and numbers represent each SL species as percentage of total SL.
D
The total radioactivity associated with Cer, SM, and GSLs (GluCer, LacCer, Gb, and GM), or GM and Gb were quantified and presented as percentages relative tototal. Data represented as means  SD of three independent experiments.
E, F Biosynthesis of SL in HeLa cells expressing GTP-locked mutants of Sar1 or ARF1 or treated with Brefeldin A (BFA; 5 lg/ml) was measured by [3H]-sphingosine pulsechase assay. Radioactivity associated with GSLs was quantified and represented as fold change with respect to control. (E) For BFA-treated cells, the SL output wasmeasured 8 h after pulse. Data represented as means  SD of two independent experiments. *P < 0.05, **P < 0.01 (Student’s t-test). (F) The ratio of GM/Gb isrepresented. Data represented as means  SD of two independent experiments. *P < 0.05, **P < 0.01 (Student’s t-test).

enzymes in the early secretory pathway and the Cer processing
enzymes in late secretory pathway, which is in turn divided into
two distinct zones where GSL and SM biosynthesis predominate. Ofª 2021 The Authors

note, we expressed HA-tagged enzymes (see Materials and Methods) for our studies since the endogenous enzymes were barely
detectable and efficient antibodies for EM studies of endogenous

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enzymes were not available. Nevertheless, the localization mostly
reflects expected localization based on enzyme activity and previously published evidence (Parashuraman & D’Angelo, 2019). A
notable exception is the localization of GCS that was shown to beon the cis-side of the Golgi (Halter et al, 2007) contrary to what wereport here. This is because the earlier studies had used a constructwith a tag that blocks the signal for intra-Golgi localization that we
identify and describe here. When this signal is blocked, localizationof GCS is altered resulting in localization to cis-Golgi (see below).
Next, SL output of this system was measured by metabolic labeling with 3H-sphingosine, a precursor of ceramide. This revealed thefollowing distribution of products at quasi steady state i.e., 24 hafter labeling: SM (70%), globosides (10%), and gangliosides (5%)
and rest remaining as precursors (Cer, GlcCer or LacCer; 15%)
(Fig 1C and D). The GSLs (globosides, gangliosides, and GSL
precursors GlcCer and LacCer) together constituted 25% of total SLs
produced. We will refer to the ratio of GSL:SM::25:70 as SL output
and the ratio of gangliosides (GM) to Globosides (Gb), GM:Gb::5:10
as GSL output (Fig 1D). For simplicity, the SL output will be represented as GSL fraction since a change in GSLs is always associatedwith a proportional change in SM in the opposite direction. For GSL
output, the situation is complex since a substantial portion of signal
remains as precursors (GlcCer and LacCer), and so GSL output will
be represented as a GM/Gb ratio which under the control conditions
corresponds to 0.5 (GM:Gb::5:10). To summarize, the SL machinery
has a compartmentalized localization across the Golgi in HeLa cells
and produces a SL output such that 70% of the Cer is directed
toward the production of SM and 25% toward the production of
GSLs. Within this 25, 5% is directed toward the production of
gangliosides and 10% toward the production of globosides.
This distribution of glycoforms produced by the Golgi apparatus
has largely been ascribed to the expression of the corresponding
glycosylation enzymes (Maccioni et al, 2002; Nairn et al, 2008,2012). To assess the contribution of enzyme compartmentalization
to this, we monitored SL output after disrupting the spatial organization of SL biosynthetic enzymes by a) overexpressing GTP-locked
mutants of monomeric GTPases——secretion-associated Ras-related
GTPase (Sar1 H79G) and ADP ribosylation factor 1 (ARF1 Q71L)
that are well known to disorganize the secretory pathway (Zhang
et al, 1994; Aridor et al, 1995) and b) by treating the cells with
Brefeldin A, which causes relocation of Golgi enzymes back to the
ER. Overexpression of Sar1 H79G led to collapse of the Golgi apparatus into the ER with SL biosynthetic enzymes showing a reticularER pattern (Appendix Fig S2A). On the other hand, overexpression

Prathyush Pothukuchi et alof ARF1 Q71L mutant led to disruption of stacked cisternal structureof the Golgi, which was replaced by tubulo-vesicular clusters(Appendix Fig S2B), with no separation between cis- and transGolgi markers (Appendix Fig S2C) (List of recombinant DNA usedin this study are listed in Appendix Table S2). The treatment with
Brefeldin A led to the translocation of the enzymes back into the ER
as expected, apart from SMS1 which while present in the ER also
displayed presence in some punctate structures (Appendix Fig S2A).
The SL output was altered in these cells, and consistently, in all
three conditions there was an increased production of GSLs over SM
and gangliosides over globosides (Appendix Fig S2D and E). The SL
output represented as fold change in GSL fraction showed that GSL
production in these cells increased by 1.5–1.9 fold over control cells(Fig 1E). Similarly, GSL output measured as GM/Gb ratio changed
from 0.5 in control cells to 1.3–1.5 in treated cells (Fig 1F). These
data suggest that impaired spatial organization of enzymes correlates with altered SL output, and especially, the output from steps
involving competing reactions is sensitive to disorganization of theGolgi. The contribution of enzyme expression to determination of
glycosylation is well established (Nairn et al, 2012) but the contribution of the Golgi organization and its importance to this process
was not clear. These results underscore a significant and substantive
role played by the Golgi apparatus in determining the glycan output
of a cell.
GRASP55 regulates SL output by controlling substrate flux
between competing glycosylation pathways
Given the importance of the organization of the Golgi apparatus,and likely of the SL biosynthetic machinery localized to the organelle, to determining SL output, we wanted to identify the molecular
players involved in this process. Retention of enzymes in the Golgi
depends on their COPI-dependent retrograde transport. Golgi matrix
proteins especially Golgins contribute to specificity in this process(Wong & Munro, 2014) and thus to compartmentalization of
enzymes. So, to identify specific regulators of compartmentalizationof SL biosynthetic enzymes, we systematically silenced Golgi matrix
proteins and studied the effect on SL production. Among the 14
matrix proteins tested by depletion, downregulation of GRASP55
significantly increased the production of GSLs (a 40% increase in
GSLs compared with control) while downregulation of GOPC and
GCC2 led to a decrease in GSL levels (Fig 2A) (siRNA sequencesused in this study to downregulate indicated human gene expression
are listed in Appendix Table S3). We followed up on GRASP55 since

Figure 2. GRASP55 regulates SL biosynthesis.
HeLa cells were treated with control or indicated siRNA (pool of 4 or 2 as indicated in methods) for 72 h and SL biosynthesis measured by [3H]-sphingosine pulsechase assay. GSL levels are expressed as fold changes with respect to control. CERT and FAPP2 knockdowns (blue bars) were used as controls. Data represented aremean  SD of three independent experiments **P < 0.01 (Student’s t-test).
B, C Effect of GRASP55 depletion on SL biosynthesis monitored by [3H]-sphingosine pulse-chase assay in GRASP55 KO cells or cells treated with GRASP55 siRNA or
following expression of GRASP55-GFP in GRASP55 depleted cells. GSL levels are expressed as fold changes with respect to control. Data represented are mean  SDof three independent experiments *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test). (C) The levels of GM and Gb were quantified and represented as GM/Gb
ratio. Data represented are mean  SD of three independent experiments *P < 0.05, ***P < 0.001 (Student’s t-test).
D
Control and GRASP55KO cells were processed for Cy3-conjugated Shiga Toxin (ShTxB) and Alexa488-conjugated Cholera Toxin (ChTxB) staining followed by flow
cytometry analysis. Mean fluorescence intensity was measured and represented. Data represented are mean  SD of three independent experiments *P < 0.05,***P < 0.001 (Student’s t-test). Scale bar: 10 lm.
E, F SL levels as assessed by LC/MS or MALDI-MS (Gb3) in control and GRASP55 KO (#2) cells. Data represented are mean  SD of three independent experiments*P < 0.05, ***P < 0.001 (Student’s t-test).

A

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Figure 2.

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its depletion altered SL output similar to that obtained by disorganization of the Golgi apparatus (Fig 1E).
GRASP55 was originally identified as a Golgi stacking proteinusing in vitro studies (Shorter et al, 1999; Xiang & Wang, 2010), but
recent studies with knockout (KO) animals and acute protein degradation have demonstrated that removing Grasp proteins does notaffect stacking of Golgi (Grond et al, 2020; Zhang & Seemann,
2021). Other studies have shown that it plays a role in secretion(both conventional and unconventional) (D’Angelo et al, 2009; Kimet al, 2016; Chiritoiu et al, 2019) and glycosylation (Xiang et al,2013; Jarvela & Linstedt, 2014). So, the function of GRASP55 at theGolgi apparatus and the mechanistic details remains unclear. We
generated GRASP55 KO HeLa cells (3 independent clones) using
clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique (Appendix Fig S3A)
(gRNAs used in this study to generate GRASP55 knockout cell lines
are listed in Appendix Table S1) (see Materials and Methods).
Western blotting and immunofluorescence confirmed the complete
abolishment of GRASP55 expression in these KO clones(Appendix Fig S3B and C) while expression levels of other Golgi
matrix proteins were not altered (Appendix Fig S3D) (antibodies
used in this study are listed in Appendix Table S4). Fragmentationof Golgi ribbon architecture was confirmed using both cis- andtrans-Golgi markers (Appendix Fig S4A–C). The Golgi stack itself
did not reveal any obvious alterations with the length of thecisterna, the number of peri-Golgi vesicles, or the fraction of
membrane in vesicles and cisternae (Appendix Fig S4D–G). Metabolic labeling experiments showed that biosynthesis of GSLs significantly increased in all three clones (by 30–50%) with acorresponding decrease in SM (Fig 2B, Appendix Fig S5A and B)
suggesting a bias toward GSL production in the absence of
GRASP55. The total levels of complex GSLs (GM and Gb) also
increased and GM/Gb ratio was altered favoring ganglioside production in 2 out of 3 clones, with a strong effect observed only in one
clone (G55KO #1) and GRASP55 siRNA-treated cells (Fig 2C). We
also analyzed the kinetics of GSL production in GRASP55KO cells(G55 KO#2). SM was produced at similar but faster rate than GSLs
in both control and GRASP55 KO cells (Appendix Fig S5A and B)
likely because of faster CERT-mediated transport of ceramide for SM
biosynthesis (Hanada et al, 2003). We also observed differences in
the rate of Gb and GM biosynthesis between control and GRASP55
KO cells (Appendix Fig S5A and B). While amounts of Gb wereconsistently more in GRASP55 KO cells across all time points, alarge increase in GM in GRASP55 KO cells compared with control
was observed only at the final time point (Appendix Fig S5A and
B). Given the very low levels of GM in HeLa cells, the difference
between control and GRASP55 KO cells is likely seen only after 24 hof chase. Next, we sought to confirm the increased levels of GSLs in
GRASP55 KO cells by complementary methods. GRASP55 KO cells
showed increased binding to bacterial toxins (Shiga and cholera
toxins that bind Gb3 and GM1 respectively) compared with control
cells (Fig 2D, Appendix Fig S5D) (all the Software used for analysis
and figure preparation are listed in Appendix Table S8). Mass spectrometry analysis showed that there was an increase in the levels of
several species of GlcCer, LacCer, and globosides in GRASP55 KO
cell line (Fig 2E and F). A similar increase in GSLs was also
observed in GRASP55 KO human fibroblast cell line (Wi-26)(Appendix Figs S3E and S5C). Finally, the change in SL output

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Prathyush Pothukuchi et alassociated with GRASP55 abolition was rescued by re-expressing
GRASP55-GFP in these cell lines (Fig 2B, Appendix Fig S3C)
suggesting that the observed changes in GSLs were specific. We
conclude that GRASP55 acts as a regulator of substrate (Cer and
LacCer) flux at the two steps that involve competing reactions—SMversus GSLs and gangliosides versus globosides in the SL biosynthetic pathway.
GRASP55 regulates the intra-Golgi localization of GSL
biosynthetic enzymes functioning at metabolic branch points
GRASP55 has been proposed to regulate glycosylation by regulating
the kinetics of intra-Golgi transport (Xiang et al, 2013) and/or
ribbon formation (Jarvela & Linstedt, 2014). For GRASP55-mediatedregulation of GSL biosynthesis, neither is a likely explanation since
the kinetics of GSL biosynthesis (Appendix Fig S5A) is very different
from that of protein glycosylation (Xiang et al, 2013) and downregulation of several matrix proteins known to fragment Golgi ribbon
does not affect GSL biosynthesis (Fig 2A). So, to understand how
GRASP55 regulates GSL biosynthesis we first studied the consequences of GRASP55 deletion on the SM biosynthetic machinery(ceramide transfer protein—CERT and SMS1). GRASP55 deletion
did not reduce the levels of CERT, and there was surprisingly a
consistent increase in SMS1 transcript levels in all 3 clones(Appendix Fig S6A and B). GRASP55 deletion also did not alter theirlocalization to Golgi (Appendix Fig S6C and D) or change thedynamics of CERT (Appendix Fig S6D and E). The kinetics of ceramide transport to the Golgi also remained unaltered (Appendix Fig
S6F). These data suggest that SM biosynthesis is not directly
affected by GRASP55 deletion.
We next examined the effect of GRASP55 deletion on enzymes of
the GSL biosynthetic branch (primers used in this study to determine mRNA levels of indicated gene are listed inAppendix Table S6). There were no consistent changes in theirlevels (Appendix Fig S7A) or their presence in the Golgi(Appendix Fig S7B) in GRASP55 KO cells (all the commercial kits
used for cDNA extraction and mRNA analysis are listed inAppendix Table S7). Their intra-Golgi localization was then examined in nocodazole-induced ministacks with GM130 as a marker for
cis-Golgi/ cis-Golgi network (CGN) compartment and TGN46 as amarker for TGN. Nocodazole-induced ministacks were used since
they show a clearer separation of cis- and trans-Golgi markers andfacilitate the intra-Golgi localization of proteins (Rizzo et al, 2013;
Beznoussenko et al, 2014). The peak localization of GSL biosynthetic enzymes was found in the medial/trans part of the Golgi incontrol cells (Appendix Fig S8). When GRASP55 was deleted, theintra-Golgi localizations of GlcCer synthase (GCS) and LacCer
synthase (LCS) were shifted in the direction of cis-Golgi (Fig 3A and
B, Appendix Fig S8A and B) while the localizations of SMS1, Gb3
synthase (Gb3S), and GM3 synthase (GM3S) were not altered(Appendix Fig S8C–E).
The shift toward the cis-Golgi was also confirmed by electron
microscopy. GCS which is localized mostly to medial/trans-Golgi(C3, C4 cisterna) with peak localization in C4 cisterna became
evenly distributed across the stack in GRASP55 KO conditions(Fig 3C and D). The cis-most cisterna (C1) which had minimal
amount of GCS in control cells showed increased levels of GCS in
GRASP55 KO cells, where it reached almost the same level as inª 2021 The Authors

Prathyush Pothukuchi et al

other cisternae and in one clone (#2) even resulted in having thepeak amount of enzyme (Fig 3D).
In case of LCS, there was a redistribution of the enzyme fromtrans-Golgi (C4 cisterna) to medial-Golgi (C2-C3 cisternae) in
GRASP55 KO cells (Fig 3C and E). Unlike GCS, LCS levels in the C1
cisterna did not change significantly (Fig 3E), and thus, the shift in
intra-Golgi localization was less pronounced in case of LCS. As acontrol, the localization of SMS1 was not altered under the same
conditions (Appendix Fig S8F). To conclude, GRASP55 deletionchanged the intra-Golgi localization of two core enzymes of the GSL
biosynthetic pathway involved in metabolic branching steps viz.
GCS and LCS, shifting them from their mainly trans-Golgi localization to more cis/medial-Golgi localization. These observations raise
the following questions: a. What is the mechanism by which thedepletion of GRASP55 causes the shift in localization of GCS andLCS? and b. Is the displacement of enzymes responsible for metabolic effects observed after GRASP55 depletion?
GRASP55 interacts directly with GCS to promote its
intra-Golgi localization
To understand how GRASP55 regulates the localization of enzymes,
we first studied whether GRASP55 interacts with enzymes of the
GSL biosynthetic pathway. We expressed GRASP55-GFP and HAtagged versions of GSL biosynthetic enzymes in HeLa cells,
immunoprecipitated GRASP55-GFP and analyzed for coimmunoprecipitation of HA-tagged enzymes by Western blotting.
We found that both GCS and LCS co-immunoprecipitated with
GRASP55-GFP while GM3S and Gb3S do not interact (Fig 4A). Theinteraction of GCS and LCS with endogenous GRASP55 was also
observed in human fibroblast cells (Fig 4B). Surprisingly, in spite ofsignificant homology between GRASP55 and GRASP65 proteins(Shorter et al, 1999), the enzymes do not interact with GRASP65,
underscoring the specificity of the interaction (Fig 4B).
GRASP55 is a peripheral membrane protein that is anchored to
the Golgi membrane through its myristoylated N-terminus. So, interaction with GRASP55 is likely mediated by cytosolically exposed
portions of the enzymes. Many Golgi glycosylation enzymes aretype II membrane proteins with a short N-terminal cytosolic tail, atransmembrane domain, and the luminal enzymatic domain. Thus,
the likely part to interact with GRASP55 is the short N-terminal tail.
GCS is an exception to this rule and is a multi-transmembraneprotein with both N- and C- terminal cytosolic tails and a large catalytic portion facing the cytosol. We noticed that the C-terminal tailof GCS ends with a consensus class II PDZ domain interacting motif(Φ-X-Φ-COOH, where Φ refers to hydrophobic amino acids and X toany amino acid) that can potentially bind to the N-terminal tandem
PDZ domains of GRASP55. We chemically synthesized the Cterminal cytosolic tail of GCS and N-terminal cytosolic tails of other
GSL biosynthetic enzymes and studied their interaction with
GRASP55 present in cell lysates as well as purified His-tagged
protein (list of cytosolic peptides used in this study for immunoprecipitation assays are listed in Appendix Table S5).
We found that GCS tail strongly bound to GRASP55 from bothcell lysates and the purified protein unlike the tails of other GSL
biosynthetic enzymes (Gb3S or GM3S) or the tail of B4GALT1 (seebelow for description of LCS binding) (Fig 4C and D). We used
B4GALT1 a galactosyltransferase involved in protein glycosylationª 2021 The Authors

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since it was unrelated to the GSL biosynthetic pathway. GRASP55
consists of two domains—N-terminal tandem PDZ domains (GRASP
domain) followed by a serine proline-rich (SPR) region (30). The
cytosolic tail of GCS interacted with the GST-tagged GRASP domain
of GRASP55 but not with GST-tagged SPR region (Fig 4E). Deletingthe Φ-X-Φ-COOH motif in GCS cytosolic tail (GCS-D3C tail) impaired
its interaction with GST-tagged GRASP domain of GRASP55(Fig 4E). Further, deleting this motif also impaired the interaction of
full-length GCS (GCS-D3C) with GRASP55 in cells as evidenced byco-immunoprecipitation analysis (Fig 4B). Next to gain further
insights into this interaction, we studied it by using isothermal titration calorimetry (ITC). By ITC, we found that the cytosolic tail of
GCS interacts strongly with the GRASP domain of GRASP55 with a
Kd value of 2 µM (Fig 4F). Of note, the Kd values of interaction
between GRASP55 and the Golgin-45 tail were approx. 0.27 µM(Zhao et al, 2017) and that of the LCS tail and GOLPH3 (another
Golgi matrix protein that interacts with Golgi enzymes) wereapprox. 60 µM (preprint: Rizzo et al, 2019). There was no significant interaction of the GRASP domain of GRASP55 with the GCS taildeleted of Φ-X-Φ-COOH motif or B4GALT1 tail (Fig 4F). These studies in sum suggest that the GCS C-terminal tail directly and specifically interacts with the GRASP domain of GRASP55.
To identify the amino acid residues in GRASP55 that are critical
for this interaction, we resorted to modeling. The crystal structureof the GRASP domain of GRASP55 bound to Golgin45 C-terminal
region (which contains a hydrophobic amino acid similar to GCS)
showed that the peptide binds to a cleft between the PDZ1 and
PDZ2 domains of the proteins (Zhao et al, 2017). A conserved
pocket in the GRASP domain consisting of 95LLGV98 corresponding
to the X-Φ1-G-Φ2 motif (X: any amino acid, Φ: hydrophobic aminoacid, G: glycine) acts as a binding site for the C-terminal end of theinteracting peptide forming hydrogen bonds with the last four residues of Golgin-45 peptide. The X-Φ1-G-Φ2 peptide has a strained
left-handed helix conformation, which is usually populated byglycine residues in the Ramachandran plot (Lee & Zheng, 2010). Bfactor analysis of the crystal structure of the GRASP55:Golgin-45
complex revealed that the X-Φ1-G-Φ2 motif forms a rigid loop
(Appendix Fig S9A) where usually Gly is favored over Asp. Indeed,
substitution of Gly to Asp abolished the GRASP55-Golgin-45 peptide
interaction (Zhao et al, 2017). A model of the GCS peptide was firstbuilt using the backbone conformation of the Golgin-45 peptide as atemplate and docked onto the cleft between PDZ1 and PDZ2
domains. The lowest energy model generated was analyzed to probe
the protein:peptide interaction. The structural analysis also indicated the important contribution of the 95LLGV98 pocket to binding
with the C-terminal amino acids of the GCS peptide (Fig 4G). The
predicted binding affinity (DG) between protein and peptide was –13.1 kcal/mol, indicating a favorable interaction, and when “LDV”
was removed from the peptide, the binding affinity was reduced(DDG = 1.5 kcal/mol) indicating the importance of the backbonemediated conserved hydrogen bonds in peptide binding. Given theoverall similarity between the GRASP55-Golgin-45 peptide and
GRASP55-GCS peptide interactions, we tested whether the interactions show similar sensitivities to mutations. As mentioned before,
the GRASP55-Golgin-45 peptide interaction is sensitive to the substitution of Gly97 to Asp (Zhao et al, 2017) and so we introduced thecorresponding G97D mutation in the GRASP55 PDZ domain to
examine its effect on the interaction with the GCS tail. The

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Figure 3. GRASP55 regulates the intra-Golgi localization of GSL biosynthetic enzymes.
A, B Control and GRASP55 KO clones were transfected with HA-tagged GSL biosynthetic enzymes, treated with nocodazole (33 µM) for 3 h, and processed for
immunofluorescence with anti-HA (green), anti-GM130 (red), and anti-TGN46 (blue) antibodies. The relative position of HA-tagged enzymes with respect to GM130
and TGN46 was measured by line scanning and expressed as normalized positions of the peak intensity with the start of GM130 peak indicated as Cis and the endof TGN46 peak indicated as trans in the graph. The double-headed arrows show the distance between the peak localization of GM130 and the enzymes. Scale bar
1 lm.
CControl and GRASP55 KO cells were transfected for 16 h with GCS-HA or LCS-HA and processed for cryoimmunolabeling with anti-HA antibody (10-nm gold
particles) and anti-GM130 antibody (15-nm gold particles) in case of GCS-HA and anti-HA antibody (15-nm gold particles) and anti-GM130 antibody (10-nm gold
particles) in case of LCS-HA. Representative images of the distribution of GCS-HA and LCS-HA are shown. Scale bar 200 nm. Red asterisk marks C4 cisterna andblue circles indicate GM130 labeling.
D, E Distribution of indicated enzymes across the Golgi stack was quantified and represented as fraction of Gold particles in each cisternae for GCS-HA (D) and LCS- HA(E) (n indicated in the graph; data are Mean  SEM).

interaction of the mutant protein GCS C-terminal tail was greatly
diminished (Fig 4H). Thus, the interaction between GCS and
GRASP55 is likely mediated by the C-terminal LDV motif of GCS
interacting with the 95LLGV98 pocket in the GRASP domain of
GRASP55 with Gly97 playing a critical role in the process (Fig 4H).
Next, we examined whether binding to GRASP55 is essential for
intra-Golgi localization of GCS. The last 3 C-terminal amino acids of
GCS were deleted (GCS-D3C) and the localization of the mutant
protein was studied. The intra-Golgi localization of GCS-D3C wasaltered, with it displaying a more cis-Golgi localization compared with
the WT enzyme (Fig 4I and J). Thus, direct interaction with GRASP55
is essential for the correct sub-compartmentalization of GCS.
Compared with GCS, the LCS tail showed a qualitatively weaker
interaction with GRASP55 both for endogenous and recombinant
protein (Fig 4C). Nevertheless, it was significantly above that ofother GSL biosynthetic enzymes (GM3S or GB3S) and that of
B4GALT1. This weak interaction was mediated by the GRASP
domain of GRASP55 (Appendix Fig S9B). Nevertheless, ITC studies
did not show a significant interaction between purified LCS cytosolic
tail and the GRASP domain of GRASP55 (Appendix Fig S9C). Thus,in the case of LCS, while the full-length enzyme coimmunoprecipitated with GRASP55, the cytosolic tail of the enzyme
itself shows only a weak or no interaction with the protein suggesting that the interaction between LCS and GRASP55 is likely to beindirect. The change in localization of LCS in the absence of
GRASP55 correlates with the observed interaction with GRASP55(Fig 4A and B). In summary, we conclude that GCS and LCS interact
with GRASP55, and in the case of GCS, this interaction is direct andis essential for its intra-Golgi localization.
GRASP55 compartmentalizes the enzymes by preventing theirentry into retrograde carriers
According to the cisternal maturation model, the retention of resident proteins in the Golgi is due to their continuous retrograde
transport mediated by COPI in the face of anterograde flux ofcargoes (Nakano & Luini, 2010; Glick & Luini, 2011). In the framework of this model, compartmentalization of enzymes to specificcisternae of the Golgi is achieved by a balance between the anterograde and retrograde flux of enzymes. Indeed, impairing the retrograde transport of enzymes promotes their forward transportleading to their localization to post-Golgi compartments (Rizzo et al,2013). So, to understand how this balance is affected in GRASP55depleted cells to change the steady-state localization of GCS andª 2021 The Authors

LCS, we examined the distribution of GCS and LCS in peri-Golgi
vesicles/carriers. These vesicles depend on COPI for their formation,and entry into these vesicles is essential for the intra-Golgi retrograde transport of proteins (Rizzo et al, 2013). In control HeLa cells,
the distribution of enzymes in peri-Golgi vesicles varied. For
instance, the density of LCS in peri-Golgi carriers was nearly thesame as that of the cisterna while the density of GCS in vesicles was
1.8-fold more than that of the cisterna. Further SMS1, an enzyme
unaffected by GRASP55 depletion was depleted in peri-Golgi vesicles compared with cisterna (density in vesicles was 0.3-fold that of
the cisterna) (Fig 5A–C). These differences in density of enzymes invesicles correlate well with their observed distribution in the Golgi
at steady state, i.e., an increased presence in peri-Golgi vesicles
correlates with increased cis/medial-Golgi localization. For instance,
GCS with higher relative density in peri-Golgi vesicles also has ahigher cis-Golgi to TGN ratio than SMS1, which has a lower relative
density in peri-Golgi vesicles (Appendix Fig S1). This suggests thatthe presence of proteins in retrograde transport carriers could be areliable indicator of intra-Golgi distribution of proteins. Next, westudied the density of enzymes in peri-Golgi vesicles in the absenceof GRASP55. We found that the density of GCS in vesicles increased
to 2.5-fold compared with that present in the cisterna and that of
LCS increased to twofold over that of the cisterna, while that of
SMS1 was unaltered (Fig 5A–C). Thus, GCS and LCS that show anincreased localization to cis/medial-Golgi in the absence of
GRASP55 also show an increased presence in vesicles in GRASP55
KO cells compared with control, while vesicle distribution of SMS1,
whose intra-Golgi localization is unaltered by the absence of
GRASP55, remains unchanged. To further validate the increased
entry of GCS and LCS into peri-Golgi carriers in the absence of
GRASP55, we resorted to in vitro budding assays to purify COPIcoated vesicles from Golgi membranes using well-established methods (Yang et al, 2002). The Golgi apparatus was purified fromcontrol and GRASP55 KO (#2) cells and was incubated with purified
coatomer, myristoylated ARF1, and BARS (Brefeldin A ADP ribosylation substrate) to promote budding of COPI retrograde carriers
from the Golgi. The budded vesicles were then separated from theGolgi apparatus by centrifugation. The Golgi apparatus was recovered in the pellet fraction while purified COPI vesicles remained in
the supernatant fraction (Appendix Fig S10A). We then analyzed
the presence of LCS and GCS in the supernatant fractions byWestern blotting. We found that there were increased amounts of
LCS and GCS in COPI vesicles that budded from the Golgi apparatus
purified from GRASP55 KO cells as compared to control cells

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Figure 4. GRASP55 interacts with GCS and LCS.
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HeLa cells co-transfected with indicated HA-tagged enzymes (GCS, LCS, GM3S, and Gb3S) and GRASP55-GFP were lysed, immunoprecipitated with anti-GFP antibody
or control IgG, and were analyzed by Western blotting for interaction by immunoblotting (IB) with anti-HA antibody. I represents 5% on the input lysate, U 5% of theunbound fraction, and IP the immunoprecipitate. Red asterisks indicate IgG bands, and arrow heads indicate the expected position of HA-tagged enzymes
WI-26 fibroblasts transfected with the indicated HA-tagged enzymes were lysed, immunoprecipitated with anti-HA antibody or control IgG, and were analyzed byWestern blotting for interaction by immunoblotting with the indicated antibodies.
Chemically synthesized biotinylated peptides corresponding to cytosolic portions of glycosylation enzymes were bound to avidin beads and were used to pull downinteractors from HeLa cell lysates and subjected to immunoblotting with anti-GRASP55 antibody.
The interaction of chemically synthesized biotinylated peptides, corresponding to cytosolic portions of glycosylation enzymes, with purified His-tagged full-length
GRASP55 and their interaction was monitored by pulling down the biotinylated peptides bound to avidin beads followed by Western blotting with anti-His tag
antibody.
Chemically synthesized biotinylated peptides corresponding to cytosolic portions of GCS (WT and D3C) and indicated purified GST-tagged GRASP domain or SPR
region of GRASP55 were incubated together, and their interaction was monitored by pulling down the biotinylated peptides with avidin beads followed by Western
blotting with an anti-GST tag antibody.
ITC profile, representative of at least two independent experiments, for biotinylated GCS and GCS D3C cytosolic tails with recombinant GRASP55.
The molecular basis of interaction between GRASP55 and ceramide glucosyltransferase C-terminal peptide is studied by building a model of GRASP55:GCS peptide
structure in the absence of the complex crystal structure. The carboxylate group of Leu of “LDV” motif retains conserved hydrogen bonds with the backbone of
95LLGV98motif of GRASP55. Gly97 residue which crucial to GRASP:GCS interaction is highlighted (pink).
The interactions of chemically synthesized biotinylated peptides corresponding to cytosolic portions of GCS (WT) with the indicated purified GST-tagged GRASP
domain (WT) or GRASP domain (G97D) were monitored by pulling down the biotinylated peptides with avidin beads followed by Western blotting with an anti-GST
tag antibody.
HeLa cells were transfected with either WT GCS or GCS D3C, treated with nocodazole (33 µM) for 3 h and labeled for enzymes, GM130, and TGN46 (to mark CGN and
TGN respectively). Line scan analysis was performed as in Fig 3A and B, and the relative position of enzymes was quantitated and plotted. The data are mean  SD
(n = 30) representative of two experiments. **P < 0.01, ***P < 0.001(Student’s t-test) and ns signifies not statistically significant.
HeLa cells were transfected with either WT GCS or GCS D3C for 16 h and processed for cryoimmunolabeling. Distribution of indicated enzymes across the Golgi stack
was quantified and represented as fraction of Gold particles in each cisterna (n indicated in the graph; data are Mean  SEM).

Source data are available online for this figure.

(Fig 5D and E), thus confirming our EM observations. The formation of these COPI vesicles depended on the presence of ARF1,
ARFGAP, and BARS and did not contain transferrin receptor (acargo that does not enter COPI vesicles) suggesting that the vesicles
purified were indeed genuine COPI vesicles (Fig 5D, Appendix Fig
S10A). This specific increase in amounts of GCS and LCS in the periGolgi carriers in the absence of GRASP55 is consistent with the
hypothesis that GRASP55 limits the retrograde transport of these
enzymes.
If absence of GRASP55 alters the distribution of the protein, does
an increase in GRASP55 levels also influence their distribution? To
test this, we overexpressed GRASP55-GFP in cells along with GSL
biosynthetic enzymes LCS and GCS. In the case of LCS, when
GRASP55 was overexpressed, there was a change in localization of
the enzyme, which was now present in endosome-like structures
along with the Golgi (Appendix Fig S10C). These structures weresimilar to those to which LCS localizes in the absence of GOLPH3,
an adaptor that links LCS to COPI and thus promoting its retrograde
transport through peri-Golgi vesicles (46). This suggests that overexpression of GRASP55 likely inhibits retrograde transport in a waythat is similar to the absence of GOLPH3. On the other hand, overexpression of GRASP55 did not shift GCS to a post-Golgi localization(Appendix Fig S10B) but GCS was increasingly found in the TGN
under these conditions (Fig 5F). This suggests that GCS behaves
similar to LCS upon GRASP55 overexpression, i.e., its localization
shifts to a forward position along the secretory pathway. This distribution of enzymes to TGN or post-Golgi compartment caused by
GRASP55 overexpression is consistent with an impairment of theirretrograde transport. Thus, GRASP55 likely acts to inhibit retrograde
transport of LCS and GCS, such that in its absence the enzymes shift
to a cis/medial-Golgi localization and when GRASP55 levels are
increased, they shift to a TGN/post-Golgi localization.

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These observations suggest that GRASP55 prevents retrograde
transport of GCS and LCS. In case of LCS, it is known that GOLPH3
promotes its retrograde transport. So we studied what happens to
LCS when both GRASP55 and GOLPH3 are removed. GOLPH3KD
led to the localization of LCS in endosomal structures (Appendix Fig
S10C) as reported earlier (Rizzo et al, 2021). Double knockdown of
GRASP55 and GOLPH3 also led to a similar phenotype(Appendix Fig S10C) suggesting that GOLPH3 likely acts downstream of GRASP55 such that the effect of GOLPH3 KD dominates
over the effect of GRASP55 reduction.
We propose that GRASP55 inhibits retrograde transport of LCS
and GCS by acting as a “retainer” that binds to and prevents theirentry into retrograde transport carriers. While the action of adaptors, exemplified by GOLPH3, that promote entry into retrograde
transport vesicles is well accepted, a retainer action in the Golgi
apparatus has not been hypothesized before. A retainer moleculethat prevents the entry of its interactors into peri-Golgi vesicles canbe expected to be absent from the peri-Golgi vesicles unlike adaptor
molecules (GOLPH3) that promote the sorting of their interactors
into peri-Golgi vesicles (Eckert et al, 2014). So, we analyzed the
distribution of GRASP55 on the cisterna and on peri-Golgi vesicles.
We find that while nearly half of the Golgi-localized GOLPH3 wasfound in peri-Golgi carriers, only about 8% of the Golgi-associatedstaining of GRASP55 is associated with peri-Golgi vesicles, suggesting that GRASP55 is likely excluded from peri-Golgi vesicles(Appendix Fig S10D).
Rationalizing these observations into a model, we propose thatrecycling adaptors and retainers act in an opposing manner topromote compartmentalization of enzymes in the Golgi apparatus.
The binding to COPI either directly (Liu et al, 2018) or through recycling adaptors like GOLPH3 (Tu et al, 2008, 2012) promotes theretrograde transport of enzymes thus preventing their exit from the

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Figure 5. GRASP55 compartmentalizes the enzymes by preventing their entry into retrograde carriers.
A–C Control and GRASP55 KO (#2) cells were transfected for 16 h with the indicated HA-tagged enzymes and processed for cryoimmunolabeling with anti-HA antibody(10-nm gold particles) and anti-GM130 antibody (15-nm gold particles). Representative images of the distribution of HA-tagged enzymes are shown. Red circles
indicate GM130 labeling. Arrow heads represent the peri-Golgi vesicles. Scale bar 150 nm. Quantification of the distribution of enzymes in vesicles represented as
normalized linear density (n indicated in the graph; data are Mean  SEM).
D
COPI vesicles were reconstituted using the two-stage incubation system (detailed in the Methods section). After the second-stage incubation, samples were
centrifuged to obtain the pellet fraction that contains Golgi membranes and the supernatant fraction that contains reconstituted COPI vesicles. Both fractions were
immunoblotted for LCS, GCS, and transferrin receptor (Tfr) to show their relative distributions on Golgi membranes and in COPI vesicles.
E
The COPI vesicle reconstitution system was performed as described above (in D), and then the fraction of LCS and GCS in COPI vesicles versus their total
distribution (on Golgi membranes and in COPI vesicles) was calculated. The mean and standard error from three independent experiments are shown, *P < 0.05,
Student’s t-test.
F
HeLa cells co-transfected with HA-tagged GCS and GRASP55-GFP were incubated for 16 h and were processed for cryoimmunolabeling with anti-HA antibody (10nm gold particles) and anti-GFP antibody (15-nm gold particles). Representative images of the distribution of GCS and GRASP55-GFP are shown. Red arrowhead
indicates the clathrin-coated vesicle that marks the TGN area, and black arrowheads indicate the presence of GCS-HA in TGN. Scale bar 150 nm. Distribution of
GCS across the Golgi stack and TGN was quantified and represented as fraction of gold particles (n indicated in the graph; data are Mean  SEM).
G
Model represents GRASP55-mediated compartmentalization of GCS and LCS. A cyclical and balanced activity of GRASP55 and GOLPH3 compartmentalizes LCS/GCS
to the trans-Golgi. The anterograde transport of enzymes (forward direction arrow; cis to trans direction) counterbalances their retrograde transport (reverse
direction arrow; trans to cis direction) resulting in the compartmentalization of these enzymes.
Source data are available online for this figure.

Golgi. On the other hand, retainers bind to their client molecules toprevent their entry into retrograde transport carriers and thus indirectly promote their anterograde transport by cisternal progression(Fig 5G). Thus, a cyclical and balanced transport of enzymes—in
the retrograde direction by COPI machinery assisted by recycling
adaptors and in the anterograde direction by cisternal flow assisted
by retainers—compartmentalizes them to specific cisterna of theGolgi apparatus. Of note, this model explains how distinct molecularcompositions of cisternae are achieved within the Golgi stack, which
includes cis- to trans-Golgi cisterna where COPI operates (Oprinset al, 1993) and not TGN where COPI coat is absent (Ladinsky et al,1999) (Fig 5G).
Change in intra-Golgi localization of GSL biosynthetic enzymes
changes GSL output
We then examined whether the change in localization of enzymes
following reduction in GRASP55 levels contributes to the associatedchanges in GSL biosynthesis. When GSL biosynthesis was examined
in Brefeldin-A-treated cells, the increased GSL production in
GRASP55 KO cells and the increased GM/Gb ratio were notobserved (Fig 6A and B), suggesting that compartmentalized localization of enzymes is essential to manifest GRASP55-deletioninduced alteration in GSL production. Of note, while data in Fig 2Bsuggested that the Gm/Gb ratio was strongly increased only in one
clone (G55KO#1), here we observed an apparent increase of GM/Gb
ratio in all three clones. This is probably due to the differences intime of chase. While data in Fig 2B were obtained after 24 h ofchase, the data in Fig 6B were obtained after 8 h chase (toxicity of
BFA prevented longer chase periods). Nevertheless, in both cases
the GM/Gb ratio showed similar tendency with G55KO #1 > G55KO#2 > G55KO #3. Next, we examined GSL biosynthesis after expression of GCS and LCS mutants that have altered intra-Golgi localization. We expressed three GCS mutants: a. GCS-D3C that shows
more cis-Golgi presence than the WT enzyme (Fig 4J), b. GCS witha HA-tag at the C-terminus (GCS-HAC) that is expected to block
accessibility to the C-terminal valine. GCS-HAC distributes acrossthe stack with significant presence in cis-Golgi (30), and unlike GCS-

ª 2021 The Authors

D3C, it was also partially localized to the ER (Appendix Fig S11A)
and c. GCS with a HA-tag in the N-terminal tail (GCS-HAN), which
localizes to ER (Appendix Fig S11A). Thus, they have distinct but
overlapping distributions along the secretory pathway. Expressionof the wild-type GCS construct in HeLa cells led to a 10% increasein the total GSLs produced compared with non-transfected cells
suggesting that expression of the construct does not overwhelm thebiosynthetic system. The expression of GCS-D3C, which localizes tothe cis-Golgi unlike wild-type GCS, led to a 30% increase in GSLs
produced, a difference that is quantitatively similar to what isobserved between control and GRASP55 KO cells. This suggests thatthe increased production of GSLs under GRASP55 KO conditions
can be explained by the shift in localization of GCS. Interestingly, an
increasing ER localization of GCS (GCS-HAC and GCS-HAN) led to alarger increase in GSL production (2.3- and 2.6-fold increase, respectively) (Fig 6C). The observed differences in GSL production are notdue to differences in expression since addition of Brefeldin A
neutralized these differences (Fig 6C), implying that changes inlocalization were indeed the cause for the observed increase in GSL
production.
Next, we examined whether the next branch point in the SL
metabolic pathway viz GB3-GM3 branch was also sensitive toenzyme localization. The key enzyme regulating this branch point is
LCS, whose localization is again controlled by GRASP55. While wefind that GRASP55 and LCS interact, there is no convincing evidence
for their direct interaction so mutating key residues to decompartmentalize the protein similar to what was achieved for GCS is notpossible. While studying the molecular basis of LCS localization, it
was found that the 14 amino acid cytosolic tail of LCS contains theinformation needed to localize the protein in the Golgi (preprint:
Rizzo et al, 2019). So, we performed alanine mutagenesis of most of
the cytosolic tail of LCS (LCS9A) except for the membrane proximal
5 amino acid region essential for interaction with GOLPH3 and thesubsequent retention of LCS in Golgi. We found that while LCS9A
was still retained in the Golgi apparatus as expected, it localized ina de-compartmentalized manner with no clear trans-Golgi localization as observed with LCS-WT (Appendix Fig S11B). We do notknow the reason why these mutations lead to a de-compartmentalized

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Figure 6. Change in intra-Golgi localization of GSL biosynthetic enzymes changes GSL output.
A, B Control and GRASP55 KO clones were pre-treated with DMSO or BFA (5 lg/ml) for 30 min and SL output monitored by [3H]-sphingosine pulse-chase assay (8 hchase). Total GSL levels were quantified and expressed as fold changes with respect to control (A). The GM/Gb ratio was calculated and represented (B). Data aremean  SD of two independent experiments.
C
HeLa cells were transfected with indicated GCS constructs for 16 h and SL output monitored by [3H]-sphingosine pulse-chase assay (8 h chase) in the presence orabsence of BFA (5 lg/ml). Total GSL levels were quantified and expressed as fold changes with respect to control. NT refers to non-transfected cells. Data aremean  SD of two independent experiments. *P < 0.05, **P < 0.01, (Student’s t-test).
D
HeLa cells KO for LCS were transfected with indicated LCS RUSH constructs for 16 h, and expressed enzymes were retained in the ER (-biotin) or placed in the Golgi
by the addition of biotin (40 lM) (+biotin) and SL output monitored by [3H]-sphingosine pulse-chase assay. The ratio of GM/Gb is represented. Data aremean  SD of two independent experiments.
E
Schematic representation of how the change in localization of GCS in GRASP55 KO with respect to control (pink dashed box versus blue dashed box) from trans- to
cis-Golgi results in preferential access to ceramide and thus an increased production of GSLs.
F
CERT was silenced using siRNAs for 72 h in control and GRASP55 KO (#2) clone before subjecting to [3H]-sphingosine pulse-chase assay. GSLs were quantified andrepresented as fold change with respect to control. Values are mean  SD (n = 3). *P < 0.05, (Student’s t-test).

localization of LCS, but they provide an opportunity to test whether
compartmentalization of LCS regulates the flux across Gb3-GM3
branches similar to what was observed with GCS. To this end, weanalyzed the GSL output following the expression of LCS-WT and
LCS9A constructs in LCS-KO HeLa cells (Yamaji & Hanada, 2014).
We found that the expression of LCS9A in Golgi favored the production of gangliosides over globosides as compared to LCS-WT. Thus,
the GM/Gb ratio of 0.12 that is observed in case of LCS-WTexpressing cells changed to 0.47 in case of LCS9A-expressing cells(Fig 6D). The differences were nullified when the enzymes wereretained in the ER (Fig 6D) suggesting that the observed differenceswere likely due to an altered intra-Golgi localization of these
enzymes. Thus, a change in LCS localization observed in GRASP55
KO conditions may contribute to the observed increase in GM/Gb
ratio in these cells. From these data, we conclude that altered localization of key enzymes of the GSL pathway can reproduce theeffects on SL biosynthesis following GRASP55 deletion and that
localization of enzymes in the Golgi can control cargo flux acrosscompeting biosynthetic pathways.
To explain the effects of GRASP55 KO on GSL biosynthesis basedon these results, we thus considered the localization of these respective enzymes. As discussed earlier, most of the GSL biosynthesis
likely happens in the medial/trans-Golgi where along with the GSL
enzymes, a substantial portion of SMS1 is also localized (Fig 1B).
This likely leads to a competition between SMS1 and GCS for Cer
that is transported by vesicular transport to the medial/trans-Golgi(Fig 6E). By moving GCS to the cis-Golgi, as happening in GRASP55
KO cells, the enzyme now gets preferential access to Cer and resulting in an increased production of GSLs (Fig 6E). Indeed, in CERT
KD cells where the non-vesicular transport of Cer is blocked and Cer
reaches the Golgi mostly by vesicular transport, the effect on GSL
biosynthesis resulting from the absence of GRASP55 is further
increased (Fig 6F) suggesting that GCS and SMS1 compete for Cer
that is transported by vesicular transport to the Golgi. The effect of
GRASP55 KO on GSL output can also be explained by a similar
logic. While both GM3S and Gb3S are localized mainly to themedial/trans-Golgi, significant amounts of GM3S can also be seenin cis/medial-Golgi unlike Gb3S (Appendix Fig S1). Thus, when LCS
is moved toward the cis/medial-Golgi, ganglioside biosynthesis by
GM3S is favored over globoside biosynthesis likely due to preferential access of GM3S to the substrate LacCer.
Thus, these data provide a model of how compartmentalizationof enzymes in the Golgi contributes to faithful glycan output. Whenª 2021 The Authors

two enzymes compete for a common substrate and are localized in
the same compartment, the product of a glycoenzyme with moreaffinity for substrate and/or increased expression dominates theglycan output. On the other hand, if one of the competing enzymes
is present in an earlier part of the secretory pathway relative to theother, it gets preferential access to the substrate before its competitor and thus promotes flux across the pathway catalyzed by it resulting in a glycan output that has a bias toward the product of thisenzyme. Thus, positioning of enzymes in the Golgi regulates thepattern of glycan distribution in the output.

Discussion
Here, we describe how GRASP55 compartmentalizes two key GSL
biosynthetic enzymes—GCS and LCS by binding to these enzymes
and preventing their entry into retrograde transport carriers. Thisaction of GRASP55 opposes COPI-mediated retrograde transport ofresidents, and a balance between these two actions compartmentalizes the enzymes to trans-Golgi. Translocation of the enzymes to
cis-Golgi in the absence of GRASP55 provides them a privileged
access to cargoes thus favoring the reactions catalyzed by them.
This is the first time a molecule that specifically regulates intraGolgi localization of glycosylation enzymes is described and thatacts by preventing the entry of cargoes into COPI carriers. This
study has important implications for the cell biology of Golgi organization as well as glycobiology which we discuss below.
Mechanisms regulating Golgi compartmentalization
The retention of glycosylation enzymes in the Golgi apparatus isdue to their continuous retrograde transport by the COPI machinery.
How the same retrograde transport machinery achieves thecompartmentalization of enzymes to different cisterna was notclear. The discovery of an enzyme adaptor—GOLPH3 acting at thetrans-Golgi—suggested that trans- and cis-Golgi-localized enzymes
may have differential modes of interacting with the COPI machinery. While the cis-Golgi-localized proteins interacted with COPI
directly (Liu et al, 2018), the trans-Golgi residents may require an
adaptor recruited specifically to the trans-Golgi, thus allowing thesame COPI machinery to compartmentalize enzymes to two different sub-Golgi compartments. Our discovery of GRASP55 actively
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process is more elaborate and with both retaining and recycling
adaptors required for appropriate localization to the Golgi. Indeed,in the absence of GRASP55 and with only GOLPH3 present, thecompartmentalization of LCS is altered. Thus, compartmentalizationof Golgi residents is a dynamic process resulting from a balance
between their anterograde and retrograde flux mediated by retaining
and recycling adaptors, respectively (Fig 5G). Recently, GRASP
proteins have been suggested to regulate COPI vesicle dynamics(Xiang et al, 2013; Grond et al, 2020). This activity of GRASP55
may further contribute to oppose the retrograde transport of residents. Of note, GRASP proteins have been linked to the Golgi localization of p24 (Barr et al, 2001) and the transport of cargoes
through the Golgi (D’Angelo et al, 2009). To prove, whether theseeffects are also linked to the role of GRASP55-mediated sorting into
COPI vesicles needs to be investigated. Given the well-known regulation of GRASP55 and GOLPH3 activities by phosphorylation in
response to various stimuli, we expect that regulation of glycosylation by altering Golgi compartmentalization may turn out to be a
regulated physiological phenomenon.
We also note that there are several other Golgi-resident proteins
with canonical PDZ domain-binding motifs, which includes otherenzymes and channels. Thus, GRASP55 (and possibly GRASP65)
may play a wider role in the localization of several proteins in
the Golgi.
Regulation of glycosylation by GRASP55
GRASP55 was originally discovered as Golgi stacking protein(Shorter et al, 1999; Xiang & Wang, 2010), but recent studies haveunequivocally shown that absence of GRASP proteins does notaffect Golgi stacking (Grond et al, 2020; Zhang & Seemann, 2021)
leaving the role of this protein obscure. In this light, the contributionof GRASP55 to conventional as well as unconventional secretion(D’Angelo et al, 2009; Kim et al, 2016; Chiritoiu et al, 2019) andglycosylation (Xiang et al, 2013; Jarvela & Linstedt, 2014) become
prominent areas to investigate. Its effect on glycosylation was
proposed to be indirect through regulation of the kinetics of intraGolgi transport of cargoes (Xiang et al, 2013) and/or ribbon formation (Jarvela & Linstedt, 2014). For GRASP55-mediated regulation of
GSL biosynthesis, neither is a likely explanation since kinetics of
GSL biosynthesis (Appendix Fig S5A) is very different from that ofprotein glycosylation and downregulation of several matrix proteins
known to fragment Golgi ribbon does not affect GSL biosynthesis(Fig 2A). Instead, we show that absence of GRASP55 leads to achange in the localization of LCS and GCS (Fig 3). This alters SL
production by providing a privileged early access of substrates tothese enzymes and/or providing them a more permissive environment (Hayashi et al, 2018; Ishibashi et al, 2018) and thus increases
GSL production (Fig 6E). Thus, our study here provides a mechanistic view of how GRASP55 regulates glycosylation and identifies animportant role for this protein in the Golgi function.
It is well known that presence of two Grasp proteins is a feature ofvertebrates while other organisms have only one Grasp protein andplants have none at all (Vinke et al 2011). Since this single Grasp
protein is mostly localized in the cis-Golgi similar to GRASP65 (Vinke
et al 2011), the medial-Golgi-localized GRASP55 is probably an
evolutionary innovation of vertebrates. Analysis of GCS sequences
showed that the C-terminal 3 amino acid PDZ domain-binding motif

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is conserved only in vertebrates (Appendix Fig S9D) thus parallelingthe evolution of GRASP55. This co-evolution of a protein and theinteraction motif on its client suggests the evolution of a regulatory
mechanism for GSL biosynthesis. The absence of classical PDZ binding motifs in other organisms may likely reflect the absence of such aregulation in these organisms.
Given the close link between GSL metabolism and cancers, weanalyzed whether GRASP55 expression is altered in cancers. There
were no significant alterations at the genomic level (mutations or
copy number alterations) at the GRASP55 loci, but expression levelsof GRASP55 were moderately low in acute myeloid leukemia,
kidney cancers, and thymoma compared with other cancer types(www.cbioportal.org). Acute myeloid leukemic cells are known tohave increased levels of GSLs compared with non-transformed
lymphocytes (Wang et al, 2012). Whether GRASP55 contributes tothis phenomenon is worthy of exploration. Further, mouse insertional mutagenesis studies have found that inactivation of GRASP55
promotes tumorigenesis in liver and colorectal cancer models (Ref:
Candidate Cancer Gene Database). Thus, studying the contributionof GRASP55 to cancer through its role in the regulation of GSL
biosynthesis could be an interesting area to explore.
SL biosynthesis
SL biosynthesis depends on CERT-mediated transfer of ceramide
from ER to TGN for SM biosynthesis (Hanada et al, 2003) and
FAPP2-mediated transfer of GlcCer from cis-Golgi to TGN for Gb3
biosynthesis (D’Angelo et al, 2013). Earlier reports had shown that
GCS and SMS1 are present in cis- and trans-Golgi respectively (Halter et al, 2007). Surprisingly, we observe that GCS is present in the
medial/trans-Golgi. We ascribe the observed difference in localization of GCS to the use of constructs with a tag that blocks the Cterminus (important for interaction with PDZ domain of GRASP55)
in the previous studies (Halter et al, 2007). This revised localizationof GCS has implications for the model of SL biosynthesis: If GCS islocalized to trans-side why is the activity of GCS not sensitive to
CERT KD? While GCS is indeed present in the trans-Golgi, a large
fraction of SMS1 is present in a distinct compartment, the TGN.
CERT-mediated transfer of ceramide likely happens at the TGN, andthus, SMS1 depends on ceramide delivered by CERT unlike GCS.
To sum up, we identify a novel molecular pathway regulated by
GRASP55 that compartmentalizes specific glycosylation enzymes
and by this action modulates the competition between reactions toachieve a specific cellular glycan profile.

Materials and Methods
Reagents
All reagents and chemicals were molecular biology grade. Methanol(Cat # 9093) and chloroform (Cat # 9180) were purchased from JT
Baker, USA. Silica-gel high-performance TLC (HPTLC) plates (Cat#1055830001) were purchased from Merck, Germany. Fatty acidfree Bovine Serum Albumin (Cat #A8806), HA Peptide (Cat #I2149),
and Brefeldin A (Cat #B7651) were purchased from Sigma-Aldrich,Germany. Protein A Sepharose CL-4B (Cat #17-0780-01) was
purchased from GE Healthcare Life Sciences, USA. Anti-HAª 2021 The Authors

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Prathyush Pothukuchi et al

magnetic beads (Cat #88836) were purchased from ThermoFisher
Scientific, USA. Sphingosine, [3-3H]-, D-erythro > 97% (Cat#NET1072050UC) was purchased from PerkinElmer, USA. Lipofectamine 2000 (Cat #11668027) and Lipofectamine LTX with PLUS
(Cat #15338100) were purchased from ThermoFisher Scientific,
USA. TransIT-LT1 (Cat #MIR 2305) was purchased from Mirus,
USA. RPMI 1640 (Cat #21875), DMEM (Cat #41965), DMEM/F-12(Cat #11320033), and FBS (Cat #10437036) were purchased from
Gibco/ThermoFisher Scientific, USA. BODIPYTM FL C5-Ceramide(Cat #D3521) was purchased from ThermoFisher Scientific, USA.
Protein A gold 15 nm and Protein A gold 10 nm were acquired from
Cell Microscopy Core, UMC Utrecht. Biotin (Cat #29129) was
purchased from Pierce, USA. Bacterial strains E. coli (DH5a) (Cat#18265017) and E. coli (BL-21-DE3) (Cat #C600003) were purchasedfrom Thermo Fisher Scientific, USA. All the siRNAs and qPCR
primers indicated in Appendix Tables S3 and S6 respectively werepurchased from Sigma-Aldrich, Germany.
Cell lines
HeLa-M (human cervical cancer cells, female origin) was a kind giftfrom Prof. Paul Lehner, University of Cambridge. Wild-type and
GRASP55 knockout Human Fibroblasts (WI-26) were a kind gift from
Markus Plomann, Institute for Biochemistry, University of Cologne.
TALEN LCS-KO HeLa cell line (human cervical cancer cells, female
origin) was kind gift from Kentaro Hanada, National Institute of Infectious Diseases, Japan. HeLa-M and GRASP55 KO HeLa cell lines (seebelow) were cultured in RPMI-1640 supplemented with 10% FCS.
TALEN LCS-KO cell lines were cultured in DMEM supplemented with
10% FCS. Wild-type and GRASP55 knockout Human Fibroblasts (WI26) were cultured in DMEM: Nutrient Mixture F-12 (DMEM/F-12)
supplemented with 10% FCS. All media were supplemented with
100 U/ml penicillin/streptomycin and 2 mM L-glutamine. All cells
were grown in controlled atmosphere (5% CO2 and 95% air) at 37°C.
Mycoplasma contamination was not observed in cell cultures as
observed by DAPI staining. Cell cultures between 3 and 15 passages
were used for the experiments, and the cells were cultured to 80%
confluence for the experiments unless indicated otherwise.
Generation of GRASP55 Knockout cell lines by CRISPR-CAS9
To generate HeLa-M cell lines in which GRASP55 expression was
abolished, we performed genome editing using CRISPR/Cas9
system. We obtained a pool of three plasmids each encoding guide
RNA (gRNA) sequence designed to target GRASP55 coding sequenceand pSpCas9 ribonuclease (Cat # SC-401106; Santa Cruz Biotechnology, Inc.). The list of gRNA sequences are reported in Appendix
Table S1. These plasmids also encoded EGFP allowing positive
selection of transfected cells. Hela-M cells were transfected withpooled plasmid for 48 h, EGFP-positive cells were isolated by FACS,
and single cells were sorted into each well of 96 well plates. The
single cells were maintained in optimal culture conditions for
10 days, by replenishing fresh media every 48 h. After 10 days,
colonies formed from single cells were trypsinized and moved to 48
well plates and expanded to 6 well plates. Clones were collected,
and protein lysates were subjected to SDS–PAGE analysis andWestern blotting analysis using GRASP55-specific antibody to assess
the presence of GRASP55 protein.

ª 2021 The Authors

Generation of expression constructs
GCS-HA was generated by inserting 9aa HA- tag into the C-terminal
cytoplasmic tail of GCS between the two indicated Gly residues
DPTISWRTGRYRLRCGGTAEEILDV. The following oligonucleotide
primers used:
F:50 GCTGGAGAACTGGTCGCTACAGATTACGCTGTGGGTACCCATACGATGTTCCAGATTACGCTGGTACAGCAGAGGAAATCCTAGATGTATGATAACTCG-30 R:50 CGAGTTATCATACATCTAGGATTTCCTCTGCTGTACCAGCGTAATCTGGAACATCGTATGGGTACCCACAGCGTAATCTGTAGCGACCAGTTCTCCAGC-30
GCS D3C (del of three C-terminal amino acids) was obtained by
PCR-amplifying GCS with the following primers to introduce astop codon after the isoleucine residue at position 4 (DPTISWRTGRYRLRCGGTAEEILDV):
F: 50 -CATCGCGGATCCATGGCGCTGCTGGACCTG-30 and
R: 50 -GATCCGCTCGAGTTATCAGATTTCCTCTGCTGTACCAGCGTAATC-30
The PCR-generated fragment for both constructs was digested
with EcoRI and XhoI (New England Biolabs, USA) and cloned into
pcDNA4b-3xHA expression vector. LCS9A Mutant (mutation of 9amino acids to alanine in cytosolic tail of LCS) was generated by
PCR amplification of LCS-WT-RUSH with following primers to introduce alanine into 9 amino acids (MAAAAAAAAAPRRSLLA):
F: 50 -CACAACCCGGGAGGCGCGCCATGGCAGCAGCAGCAGCAGCAGCAGCAGCA-30
R: 50 - GCGAGCAGCGAGCGGCGCGGTGCTGCTGCTGCTGCTGCT
GCTGCTGC 30 . The PCR-generated megaprimer was then annealed
into LCS-WT-RUSH at 66°C and extension at 72°C for 25 cycles.
Plasmids and siRNA transfection
HeLa-M cells, Wi-26 cells, LCS-KO cells, and GRASP55 KO cells
were transfected with plasmid vectors using TransIT-LT1 or Lipofectamine LTX reagents. A list of plasmids used in this study can befound in Appendix Table S2. Knockdown experiments for Hela-M
cells were carried out using a pool of 4 siRNAs or 2 siRNAs using
Lipofectamine 2000 according to manufacturer’s instructions (usedat concentration of 100nM for the target gene). Expression or knockdown efficiencies (> 85%) were checked after every experiment
either by indirect immunofluorescence or immunoblotting or by
qPCR analysis. A list of siRNA sequences used in this study can befound in Appendix Table S3.
Cell lysis, Western blotting, and analysis
Cells were washed three times with ice-cold PBS and lysed immediately at 4°C in RIPA lysis buffer (0.1% Triton X-100, 20 mM TrisHCl, pH 8.0, 0.1% SDS, 0.05% sodium deoxycholate, 150 mM NaCl,
10 mM Na3VO4, 40 mM b-glycerophosphate, and 10 mM NaF) andcomplete protease/phosphates inhibitors (Roche). Cell lysates wereclarified at 20,000 g for 10 min at 4°C to eliminate detergentinsoluble pellet. To visualize GCS on SDS–PAGE, the protein lysates
prepared as described above were treated with 250 mM of DTT insample buffer (62.5 mM Tris-HCL, pH 6.8, 2% SDS, 10% glycerol,0.001% bromophenol, and 125 mM dithiothreitol) at 37°C for
30 min. The lysate was immediately processed for SDS–PAGE andimmunoblotting with antibodies. A complete list of primary and

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secondary antibodies are given in Appendix Table S4. The Western
blots were then exposed to X-ray films, and exposure time wasvaried to obtain optimal signal.
Immunoprecipitation and peptide pull down assay
Total lysates were prepared using IP lysis buffer (150 mM NaCl,
25 mM Tris-HCl pH 7.5, 1% Triton X, 10 mM Na3VO4, 40 mM bglycerophosphate, 10 mM NaF, and protease cocktail inhibitor from
Roche). The protein concentrations were quantified using BCA
Protein Assay kit (Pierce). 1mg of protein was used for precipitation
with antibodies conjugated to either Protein A sepharose ormagnetic dynabeads or to monomeric avidin beads (Pierce) in caseof cytosolic peptides, and incubated at 4°C overnight. The beads
were then washed 5 times in IP lysis buffer, and bound proteins
were subjected to SDS–PAGE and immunoblotted. The list of cytosolic peptides used in this study are reported in Appendix Table S5.
Reconstitution of COPI vesicle formation
A two-stage incubation system was performed essentially as previously described (Yang et al, 2005). In brief, the first stage involved
incubating prewashed (3 M KCl) Golgi membrane (0.4 mg/ml) withcoatomer (6 lg/ml), ARF1 (6 lg/ml), and 2 mM GTP in 500 ll of
assay buffer (25 mM Hepes-KOH, pH 7.2, 50 mM KCl, 2.5 mM Mg(OAc)2, 1 mg/ml soybean trypsin inhibitor, and 200 mM sucrose)for 15 min at 37°C. Afterward, the Golgi membrane was pelleted(12,000× g at 4°C for 10 min) and then resuspended in 100 ll of
assay buffer for the second-stage incubation, which had ARFGAP1(6 lg/ml) and BARS (6 lg/ml) added for 10 min at 37°C. Samples
were then centrifuged for 10 min at 12,000 g and 4°C, with thepellet fraction containing the Golgi membrane and the supernatantfraction containing COPI vesicles.
Protein purification
Recombinant proteins were induced to express with 0.3 mM
isopropyl b-D-thiogalactoside in BL21 (DE3) competent bacterial cells
for 16 h at 22°C. Cells were harvested at 7,519 g for 30 min at 4°C,
lysed in lysis buffer (cold PBS with 1 mM DTT, 1% triton X-100,
phosphatase, and protease inhibitors), and purified with Glutathione
Sepharose 4B beads (GE Healthcare, USA). Recombinant GST-tagged
proteins were eluted in elution buffer (50 mM Tris, 100 mM NaCl,
50 mM reduced glutathione, and pH 8.0); purified recombinant
proteins have been concentrated and the buffer has been exchanged
by using Vivaspin TURBO 4 filters (Sartorius, UK) at 4°C and
4,000× g. The recombinant purified proteins were quantified by
Bradford assay and were assessed for contaminants by SDS–PAGE.
Isothermal titration calorimetry (ITC)
ITC experiments were performed in a buffer containing 300 mM
NaCl, 10 mM Bicine pH 8.5, and 1 mM DTT. Biotinylated peptides
were synthesized and delivered as lyophilized powder with a biotin
moiety located at the N or C-terminus (Charite Universitaetsmedizin
Berlin, Germany). The peptides were dissolved in buffer, centrifuged
at 14,000× g for 10 min, and only the supernatant was used. The
dissolved peptide concentrations were calculated based upon their

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absorbance at 280 nm and their corresponding molar extinction
coefficient. Experiments consisted of titrations of 20 injections of
2 ll of titrant (peptides) into the cell containing GRASP domain
protein at a 25-fold lower concentration. Typical concentrations forthe titrant were around 2.5 mM for experiments depending on theaffinity. Experiments were performed at 25°C and a stirring speed of
1,000 rpm on a MicroCal PEAQ-ITC (Malvern Panalytic). All data
were processed using MicroCal PEAQ-ITC Analysis Software and fit
to a one-site binding model after background buffer subtraction.
GRASP55-GCS peptide modeling
Model of GCS peptide was first built using backbone conformationof Golgin45 peptide as a template and fit into the same cleft of
GRASP55, where Golgin45 peptide is located using COOT (Emsley &Cowtan, 2004). This initial complex structure was further refined by
the Docking2 module of Rosetta protocols through local docking
search (Lyskov & Gray, 2008; Chaudhury et al, 2011; Lyskov et al,2013). The lowest energy model generated by this protocol was
analyzed to probe the protein:peptide interaction. PRODIGY software was used to calculate the protein–protein binding energy (Vangone & Bonvin, 2015; Xue et al, 2016).
Immunofluorescence and confocal microscopy
Indirect Immunofluorescence was performed as follows: cells weregrown on 24 mm coverslips, washed with PBS, and fixed in 4%
paraformaldehyde (Electron Microscopy Sciences, Hatfield, USA) for
30 min at room temperature (RT). The cells were then permeabilized and blocked in blocking buffer (0.05% saponin and 0.5% BSA
in PBS) for 30 min at RT followed by incubation with specificprimary antibodies (see Appendix Table S4) for 1 h at RT andwashed with PBS. Cells were subsequently labeled with appropriate
Alexa Fluor-conjugated secondary antibodies (Appendix Table S4).
The coverslips were mounted using mowiol, and images are
acquired using confocal microscope Zeiss LSM700.
Line scan analysis
The images were acquired with a pinhole set to 1 airy unit andunder non-saturation conditions using a 63× objective (1.4 NA).
Images were 8 bit with dimensions of 512 × 512 pixels, and eachpixel corresponded to an area of 132 × 132 nm2. The line scan analysis was performed as described previously (Rizzo et al, 2013) using
the Zen software system (Carl Zeiss). In brief, images of stacks
stained for GM130, TGN46, and enzyme of interest tagged with HA
were acquired as described earlier. Only cells with a moderate levelof expression were considered for the analysis. Golgi stacks withclearly separated GM130-stained and TGN46-stained zones wereidentified and used for the analysis. A line was drawn in the middleof the stacks along the cis-trans direction, and the fluorescenceintensity of each stained marker along this line was plotted. At least
30 stacks were examined per treatment, and a representative data
are shown for analysis. The normalization of the distances was
carried out by considering the start of the GM130 peak as 0, and theend of the TGN peak as 1. The images of Golgi stacks were
processed using the “Image with Zoom” function of Metamorph
7.7.3.0 (Universal Imaging), for presentation.

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Cell profiler analysis
Control and GRASP55 KO cells were stained ShTxB-Cy3 and ChTxBAlexaFluor 488 and images were acquired as described above using
a 20× objective (NA 0.5). Images were 8 bit with dimensions of
512 × 512 pixels, and each pixel corresponded to an area of
623 × 623 nm2. For quantification experiments, 10–15 random
fields were imaged with the same microscope settings (i.e., laser
power and detector gain). The integrated intensity of fluorescence ofeach cell was calculated for each channel after the cells weresegmented by Cell Profiler (Carpenter et al, 2006). Since only a fraction of the cells were positive for staining, the normalized intensity
analysis was performed by taking top 10% of total cells in all theconditions and normalized on the background intensity.
Flow cytometry analysis
Control and GRASP55 KO cells were subjected to trypsin digestion,
fixed with 4% paraformaldehyde, washed, and resuspended with
PBS. Cells were incubated with bacterial toxins for 1 h at 4°C. Then,cells were extensively washed with PBS and incubated withfluorescence-labeled secondary antibodies when required or directly
analyzed by BD FACS ARIAIII cell sorter (BD Biosciences). Cells
incubated with secondary antibody alone, or unlabeled cells, wereused as a negative control. The cell surface expression of GSLs ofselected cells was further analyzed in the gated region by BD FACS
ARIAIII cell sorter (BD Biosciences). Bacterial toxins used aredescribed in (Table S4).
Electron microscopy
Ultrastructural analysis of Golgi morphology
Briefly, control and GRASP55 knockout cells were grown in 35 mm
plastic dishes to 80% confluence. Cells were then fixed with 1%
Glutaraldehyde (Electron Microscopy Sciences) in 0.2 M HEPES pH
7.3 at RT for 1 h. The fixative was replaced with 1% BSA in PBS,
and cells were carefully detached using a plastic cell scraper,collected into microfuge tubes, and centrifuged to obtain the pellet.
All samples were then washed three times in 0.2 M HEPES pH 7.3
and post-fixed 30 min in 1% Osmium Tetroxide in the dark at 4°C
in the same buffer. They were then washed three times in distilledwater and post-fixed 25 min in 1% Osmium Tetroxide and 1.5%
Potassium Ferrocyanide in the dark at room temperature in HEPES
0.2 M pH 7.3. After washing three times in distilled water, they
were stained with 0.5% uranyl acetate over night at 4°C. The
pellets were dehydrated in graded steps of ethanol (50, 70, 90, and100%), 2 times with 100% of acetone, and embedded into Epon.
Thin sections (60 nm) were cut on a Leica UC7 ultramicrotome
and examined with 120 kV Philips Tecnai 12 Biotwin electron
microscope (FEI, Eindhoven, The Netherlands) using a VELETA
digital camera.
Cryo-immuno EM
Control or GRASP55 Knockout cells were transfected using indicated HA-tagged construct for 16 h. The cells were then fixed with
2% formaldehyde and 0.2% glutaraldehyde in PHEM buffer(0.1 M) pelleted by centrifugation, embedded in 12% gelatin,
cooled on ice, and cut into 1-mm3 cubes at 4°C. The cubes wereª 2021 The Authors

immersed in 2.3 M sucrose at 4°C overnight and then frozen inliquid nitrogen. Fifty-nanometer sections were cut with a diamondknife on a UC7 Leica cryo-ultramicrotome. The sections were
picked up in a mix of 2% methyl cellulose and 2.3 M sucrose(1:1), as previously described (Pulvirenti et al, 2008), andcollected on grids covered with Formvar carbon supporting film
(Electron Microscopy Sciences, PA, USA). The grids were first
incubated with the rabbit anti-GM130 and/or mouse anti-HA polyclonal antibodies and then incubated with different sizes of Protein
A gold (10 and 15 nm) to reveal antigen staining. After labeling,
the sections were treated with 1% glutaraldehyde and embedded
in methyl cellulose uranyl acetate for 10 min on ice. The excess of
methyl cellulose uranyl acetate was removed, and the sections
were dried at room temperature before their analysis at 120 kV ina Philips Tecnai 12 Biotwin electron microscope (FEI, Eindhoven,
The Netherlands) using a VELETA digital camera. The polarity of
the Golgi stacks was defined by compositional (cis-Golgi marker
GM130) parameter. For quantitation (performed with iTEM image
acquisition software), cis, medial, or trans-Golgi was defined aspreviously described (Rizzo et al, 2013). TGN was defined as thearea in front of the trans cisterna up to a distance that equals the
thickness of the Golgi stack. Vesicles were round profiles of 50–
80 nm in diameter, present within 200 nm from the rims of thestack. The distribution of enzyme in the Golgi stack was expressedas the fraction of gold particles in each cisterna of the Golgi stackand TGN.
Lipid analysis
HPLC-Mass spectrometry
Sphingolipids of control and GRASP55 KO cells were analyzed byliquid chromatography–tandem mass spectrometry (LC-MS/MS) as
described earlier (Bielawski et al, 2006). Briefly, cells were washedtwice with ice-cold PBS, and lipids were extracted with 2 ml ethyl
acetate/isopropyl alcohol/water (60:30:10%, v/v/v) solvent
mixture (Bielawski et al, 2006). The lipid extracts were analyzed
with a Quantum Ultra triple quadrupole mass spectrometer
connected to an Accela HPLC and Accela autosampler using asolvent gradient at the Lipidomics Core at Stony Brook University.
Ceramides were identified through MRM analysis with soft fragmentation. Calibration curves were generated for each lipid andused for quantitative analysis of lipids in the samples. Inorganic
phosphate (Pi) released from total phospholipids was measured byusing a colorimetric method. Briefly, following a lipid extraction byusing the Bligh and Dyer method, samples were dried under astream of nitrogen gas. Then, a mixture of sulfuric and hydrochloric acid was added to the sample to ash the organic content.
Samples were heated overnight at 160°C. Then, water, ammonium
molybdate, and ascorbic acid were subsequently added to samples
and incubated for 30 min at 45°C. The absorbance was measured
at 600 nm by using a Spectramax M5 plate reader. A calibration
curve was created and utilized to quantify the inorganic lipid
content for each sample and used as normalization for relative
quantification of sphingolipids.
Radioactive pulse-chase assay to monitor SL biosynthesis
Radioactive pulse-chase assay was performed as described earlier(D’Angelo et al, 2013). Briefly, HeLa-M cells or human fibroblasts

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wi-26 or GRASP55 KO cells were pulse labeled for 2 h with [3H]sphingosine (final concentration of 0.1 lCi/ml; Perkin Elmer) inserum-free DMEM containing 1% fatty acid-free BSA followed by achase for indicated times in complete media. Lipids were thenextracted from the cells, resuspended in chloroform, spotted onto
silica-gel high-performance TLC (HPTLC) plates (Merck, Germany),
resolved using a mixture of chloroform, methanol, and water(65:25:4 v/v/v) and quantified using GINA (Raytest, Germany)
software analysis.
MALDI-MSTotal lipid extracts were prepared using a standard MTBE protocol
followed by a methylamine treatment for sphingo- and glycosphingolipids analysis by mass spectrometry. Briefly, cell pellet was
resuspended in 100 ll H2O. 360 ll methanol and 1.2 ml of MTBE
were added and samples were placed for 10 min on a vortex at 4°C
followed by incubation for 1 h at room temperature on a shaker.
Phase separation was induced by the addition of 200 ll of H2O.
After 10 min at room temperature, samples were centrifuged at
1,000 g for 10 min. The upper (organic) phase was transferred intoa glass tube, and the lower phase was re-extracted with 400 ll artificial upper phase [MTBE/methanol/H2O (10:3:1.5, v/v/v)]. The
combined organic phases were dried in a vacuum concentrator.
Lipids were then resuspended in 500 ll of CHCl3 and divided into
two aliquots for a further methylamine treatment. 500 µl of freshly
prepared monomethylamine reagent [methylamine/H2O/n-butanol/
methanol (5:3:1:4, (v/v/v/v)] was added to the dried lipid extract
and then incubated at 53°C for 1 h in a water bath. Lipids werecooled to room temperature and then dried. The dried lipid extract
was then extracted by n-butanol extraction using 300 ll watersaturated n-butanol and 150 ll H2O. The organic phase wascollected, and the aqueous phase was re-extracted twice with 300 ll
water-saturated n-butanol. The organic phases were pooled anddried in a vacuum concentrator. Lipids were then resuspended in
500 µl of CHCl3 and analyzed by MALDI-MS. 30 mg/ml 2,5-DHB
was freshly prepared in acetonitrile/water solution (50:50 v/v) with
0.1% TFA. An equivalent volume of sample solution (50 µl) was
then mixed with matrix before deposition on the MALDI target. All
mass spectrometry analysis for the identification of lipids (m/z 600–
1,800) was obtained using an Applied Biosystems 4800 MALDITOF/TOF mass spectrometer equipped with a 200 Hz tripledfrequency Nd:YAG pulsed laser with 355 nm wavelength. Measurements were performed in positive ion reflection mode at an accelerating potential of 20 kV. Each mass spectrum was obtained byapplying a laser energy of 4,600 watts/cm2, averaging 4,000 single
laser shots/spectrum.
FRAP assay
Control and GRASP55 (#2) knockout cells were plated on 35-mm
glass bottom microwell dishes (MatTech, USA) and transfected with
CERT-YFP (1 lg) for 16 h. FRAP was performed using Zeiss LSM
710 confocal microscope equipped with an environmental control
system set to 37°C and 5% CO2. FRAP experiments were performedby bleaching CERT-YFP in the Golgi area (50 bleaching iterations)followed by imaging every 7.5 s for 4 min (2% laser power). Recovery of fluorescence was calculated as ratio of Golgi intensity andthat of total cell after background correction.

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Ceramide transport assay
Control and GRASP55 knockout cells were grown on coverslips to80% confluence. Cells were washed with DMEM-HEPES containing
10% FCS for three times and incubated with BODIPY ceramide(5 µM) in fatty acid-free BSA for 30 min on ice. After incubation,
cells were washed with DMEM-HEPES for three times and followed
by imaging. The laser conditions used were 488 nm (excitation) and
620 nm (emission), and fluorescence was recorded every 2 min fora period of 20 min. The fluorescence of bodipy Ceramide in the
Golgi was determined followed by normalization to the maximum.
RNA extraction and Real-Time PCR
Total RNA was extracted from HeLa-M cells (Control and GRASP55
Knockout) using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions. The yield and the integrity of RNA were determined by spectrophotometer NanoDrop 2000c (Thermo Scientific)
and by TAE agarose gel electrophoresis, respectively. RNA (1 lg)
was reverse transcribed using QuantiTect Reverse Transcription Kit(Qiagen) according to the manufacturer’s instructions and subjected
to qPCR with gene-specific primers (Appendix Table S6) in the presence of LightCycler480 SYBR Green I Master Mix (Roche, Switzerland) on a LightCycler480 II detection system (Roche, Switzerland).
Analyses were carried out on biological triplicate samples for eachexperiment, and they were processed separately. The thermal profile
consisted of 10 min at 95°C pre-incubation, and 40 cycles at 10 s at
95°C, 10 s at 60°C, and 10 s at 72°C. The qPCR data were normalized
to the average of the reference gene human hypoxanthine–guanine
phosphoribosyltransferase 1 (HPRT1). The fold changes in the relative quantifications were calculated according to the DDCt method.
All the commercial kits used are reported in (Appendix Table S7).
Experimental conditions for RUSH experiments
LCS-KO cells were transiently transfected with LCS or LCS9A
constructs at 37°C in absence of biotin for 16 h, to localize theproteins to endoplasmic reticulum (ER). Their ER exit was promotedby the addition of biotin (40 µM) for 6 h. For pulse-chase assay
experiments (Fig 4C), biotin was replaced every 3 h and maintained
throughout chase period for a total of 24 h.
Statistics
Error bars correspond to either standard deviation (SD) or standard
error of mean (SEM) as indicated in figure legends. All the statisticalevaluations were done using GraphPad Prism built-in tests (unpaired two-tailed Student’s t-tests), and as indicated, significancevalues are all marked as follows *P < 0.05, **P < 0.01, and***P < 0.001 (ns, not significant). All the measurements reported
are from distinct samples.

Data availability
Source data are provided with the manuscript, and if not, they areavailable from the corresponding author upon reasonable request.
Targeted lipidomics data from this study are available atª 2021 The Authors

The EMBO Journal

Prathyush Pothukuchi et al

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Acknowledgements

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We thank Antonella De Matteis for valuable discussions, Nina Dathan for help

Beznoussenko GV, Parashuraman S, Rizzo R, Polishchuk R, Martella O, Di

with cloning and construct preparation, and Francesco Russo for assistance

Giandomenico D, Fusella A, Spaar A, Sallese M, Capestrano MG et alwith statistical analysis. We thank the Bioimaging Facility of the Institute of(2014) Transport of soluble proteins through the Golgi occurs by diffusion

Biochemistry and Cell Biology and the joint FACS Facility of the Institute of
Genetics and Biophysics (IGB-CNR, Naples) and IBBC-CNR for access to technologies and Pasquale Barba for assistance with FACS. PP thanks Samba Siva
Rao. We thank Dr. Bruno Correia, EPFL, Switzerland, for help with the analysis ofprotein–protein interaction studies. We thank Drs. Ahamarshan, Corda, Colanzi,
Grimaldi, Jung, Krishnamoorthy, Polishchuk, Qadri, and Roy for criticalcomments on the manuscript. We thank MEDINTECH, the Italian Node of EuroBioimaging (Preparatory Phase II –INFRADEV), Italian Cystic Fibrosis Research
Foundation (FFC #6-2019), POR Campania project 2014-2020 (C.I.R.O.), and CTC
project for financial support. R.R. acknowledges financial support from Fondazione Italiana per la Ricerca sul Cancro (FIRC Fellowship 15111). A.L. acknowledges financial support from the, AIRC (Projects IG 15767 and IG 20786),

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Author contributions

Transl Med 6: 477

PP contributed to development of the idea, conducted most of the experi-

Chaudhury S, Berrondo M, Weitzner BD, Muthu P, Bergman H, Gray JJ (2011)

ments, analysis and interpretation of data, and contributed to writing theBenchmarking and analysis of protein docking performance in Rosetta

manuscript; IA conducted the experiments on peptide binding using purified
proteins; MP was involved in labeling and acquisition of EM and confocal

v3.2. PLoS One 6: e22477
Chiritoiu M, Brouwers N, Turacchio G, Pirozzi M, Malhotra V (2019) GRASP55

microscopy images; RR assisted in acquisition and analyses of electron micro-

and UPR control interleukin-1beta aggregation and secretion. Dev Cell 49:

graphs; DR conducted mRNA analysis; GT was involved in EM sample prepara-

145 – 155 e4tion and acquisition of images; LC conducted MALDI-MS and lipidomics

D’Angelo G, Uemura T, Chuang C-C, Polishchuk E, Santoro M, Ohvo-Rekil€a H,measurements; CG performed the ITC experiments; GV assisted PP in morpho-

Sato T, Di Tullio G, Varriale A, D’Auria S et al (2013) Vesicular and non-logical analysis of Golgi; ND was involved in cloning and construct prepara-vesicular transport feed distinct glycosylation pathways in the Golgi.

tion; J-SY performed vesicle budding assay and was guided in this by VWH; PH

Nature 501: 116 – 120

was involved in synthesis of biotinylated peptides; JN provided the WT and

D’Angelo G, Prencipe L, Iodice L, Beznoussenko G, Savarese M, Marra P, Di

GRASP55 KO fibroblasts, performed the protein–protein interaction studies,

Tullio G, Martire G, De Matteis MA, Bonatti S (2009) GRASP65 andand was guided by MP; TN, J-II, MJH-C, LMO, and YAH contributed to MS anal-

GRASP55 sequentially promote the transport of C-terminal valine-bearing

ysis of lipids; AB performed in silico modeling of GRASP55-GCS peptide interaction and was guided by MT; AL contributed to development of the idea of how
Golgi organization may play role in regulating glycosylation and the mode ofaction of GRASP55 in regulating enzyme compartmentalization, critical analy-

cargos to and through the Golgi complex. J Biol Chem 284: 34849 – 34860
Dunphy WG, Rothman JE (1985) Compartmental organization of the Golgi
stack. Cell 42: 13 – 21
Eckert ES, Reckmann I, Hellwig A, Rohling S, El-Battari A, Wieland FT, Popoff

sis of the data throughout the entire project and also provided inputs inV (2014) Golgi phosphoprotein 3 triggers signal-mediated incorporation ofmanuscript preparation. GDA contributed to setting up the system to monitor

glycosyltransferases into coatomer-coated (COPI) vesicles. J Biol Chem 289:

SL biosynthesis, provided important inputs on the data to understand andinterpret changes in lipid biosynthesis, and also provided critical inputs inmanuscript preparation. SP developed the idea, designed and supervised theentire project, and wrote the manuscript.

31319 – 31329
Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics.
Acta Crystallogr D Biol Crystallogr 60: 2126 – 2132
Fisher P, Spencer H, Thomas-Oates J, Wood AJ, Ungar D (2019) Modeling
Glycan processing reveals golgi-enzyme homeostasis upon trafficking

Conflict of interest
The authors declare that they have no conflict of interest.

defects and cellular differentiation. Cell Rep 27: 1231 – 1243.e6
Glick BS, Luini A (2011) Models for Golgi traffic: a critical assessment. Cold
Spring Harb Perspect Biol 3: a005215
Grond R, Veenendaal T, Duran JM, Raote I, van Es JH, Corstjens S, Delfgou L,

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VIEWPOINT

Homeostatic and pathogenic roles of the GM3 ganglioside
Jin-ichi Inokuchi1,2 , Hirotaka Kanoh1, Kei-ichiro Inamori1, Masakazu Nagafuku1, Takahiro Nitta1and Koichi Fukase2,3
1 Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University,
Sendai, Japan
2 Core for Medicine and Science Collaborative Research and Education (MS-CORE), Project Research Center for Fundamental Sciences,
Osaka University, Japan
3 Department of Chemistry, Graduate School of Science, Osaka University, Japan

Keywords
adipocytes; cholesterol transport; chronic
inflammation; GM3 ganglioside; innate
immunity; insulin resistance; leptin
resistance; metabolic syndrome; NPC1L1;
obesity; TLR4
Correspondence
J. Inokuchi, Division of Glycopathology,
Institute of Molecular Biomembrane and
Glycobiology, Tohoku Medical andPharmaceutical University, 4-4-1
Komatsushima, Aoba-ku, Sendai 981-8558,
Japan
Tel: +81 22 727 0117
E-mail: jin@tohoku-mpu.ac.jp(Received 12 March 2021, revised 10 May
2021, accepted 14 June 2021)
doi:10.1111/febs.16076

Two decades ago, we achieved molecular cloning of ganglioside GM3 synthase (GM3S; ST3GAL5), the enzyme responsible for initiating biosynthesis of complex gangliosides. The efforts of our research group since thenhave been focused on clarifying the physiological and pathological roles ofgangliosides, particularly GM3. This review summarizes our long-term
studies on the roles of GM3 in insulin resistance and adipogenesis in adipose tissues, cholesterol uptake in intestine, and leptin resistance inhypothalamus. We hypothesized that GM3 plays a role in innate immune
function of macrophages and demonstrated that molecular species of GM3
with differing acyl-chain structures and modifications functioned as proand anti-inflammatory endogenous Toll-like receptor 4 (TLR4) modulators
in macrophages. Very-long-chain and a-hydroxy GM3 species enhanced
TLR4 activation, whereas long-chain and unsaturated GM3 species counteracted this effect. Lipidomic analyses of serum and adipose tissues
revealed that imbalances between such pro- and anti-inflammatory GM3
species promoted progression of metabolic disorders. GM3 thus functions
as a physiological regulatory factor controlling the balance between homeostatic and pathological states. Ongoing studies based on these findings will
clarify the mechanisms underlying ganglioside-dependent control of energy
homeostasis and innate immune responses.

Introduction
Glycosphingolipids (GSLs), and their sialic acidcontaining derivatives, gangliosides (Fig. 1A), aremembrane lipid components having a lipid portion
embedded in the plasma membrane outer leaflet and asugar chain extending into the extracellular space.
Structural features of GSLs affect membrane fluidityand facilitate microdomain formation, thus contributing to cell–cell interaction and receptor-mediated signal transduction. In cultured adipocytes with TNFa-

induced insulin resistance, elimination of GSLs by
inhibition of glucosylceramide (GlcCer) synthase (DPDMP) [1], the initial step in biosynthesis of all
GlcCer-derived GSLs, results in nearly complete recovery of insulin receptor (IR) signaling [2]. Several metabolic diseases are characterized by elevated expressionof GM3, the simplest ganglioside species synthesizedby GM3 synthase (GM3S) (Fig. 1A), [2-5]. SAT-I,
ST3GALlV, and ST3GAL5 are frequently used

Abbreviations
DAMPs, damage-associated molecular patterns; GSLs, glycosphingolipids; PAMPs, pathogen-associated molecular patterns; ST3GAL5, GM3
synthase; TLR4, Toll-like receptor 4.

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1J. Inokuchi et al.

Pathophysiological roles of GM3

Gal
OH

-Cer

GM3

Sialic acid
(NeuAc)-Cer
ST8SIA1-Cer

GM2
B4GALNT1O

GM1
B3GALT4

16:0

Acyl chain (fatty acid)

HN
O

O
O

O

-Cer

O OH

GD1a
ST8SIA5

ST3GAL2/3

-Cer

-Cer

-Cer

-Cer

-Cer

GD3

GD2

GD1b

GT1b

GQ1b

Modifications

ST3GAL5

NeuAc

GalNAcSphingoid base

OH

VLCFA

LacCer

O

HO

-Cer

Galactose(α-2,3)H (β-1,4)HO
AcHN

B4GALT5/6

COOH

GlcCer

OH

-Cer

HO
O

O

d18:1
O

O

Glucose

OH

O

UGCG

OH

Glc
OH

LCFA

B

Ceramide

Cer

HO

A

18:0
20:0
22:0
24:0

O

h24:0 (α-hydroxylation)O

24:1 (ω-9 desaturation)

Fig. 1. Biosynthetic pathway of gangliosides and acyl-chain structures of GM3 molecular species. (A) Biosynthetic pathway (schematic) of
GM3, from ceramide, and to complex gangliosides. (B) Detailed structures of GM3 species: sialyllactose head group, sphingoid base(d18:1), typical acyl-chain lengths (long-chain, very-long-chain), and acyl-chain modifications (a-hydroxylation, x-9 desaturation).

abbreviations for this enzyme; ST3GAL5 is recommended for common use [6]. ST3GAL5 gene expression and GM3 content are upregulated in visceraladipose tissue in obese animal models [2,5]. We proposed a working hypothesis ‘Insulin resistance as amembrane microdomain disorder’ [7-9] based on findings that increase of membrane GM3 in adipocytes
induced by inflammatory cytokine TNFa resulted inelimination of IR from caveolae [10,11] (Fig. 2).
Numerous molecular species of GM3 have beenfound that differ in acyl-chain length (16–24) and modifications (a-hydroxylation, x-9 desaturation) (Fig. 1B).
GM3 species are secreted abundantly into human serumand circulate to all parts of the body, including insulinsensitive organs and major tissues (e.g., liver, muscle,
adipose) [12]. Altered expression of various GM3 species has been observed in patients with metabolic disorders [4], but specific biological functions of these species
are unclear. Our 2020 study demonstrated that GM3
species act as pro- and anti-inflammatory endogenous
TLR4/MD-2 modulators, whose balance and acyl-chain
structures affect progression of metabolic disorders
starting at presymptomatic phase, and potentially serve
as diagnostic and therapeutic targets of chronic inflammation in these disorders [12].

Visceral adipose tissue, particularly mesenteric adipose
tissue, is an important factor in the pathogenesis of

metabolic syndrome [13-21]. To elucidate the fundamental characteristics of mesenteric adipocytes, weestablished a physiologically relevant culture systemfor differentiation of rat mesenteric-stromal vascular
cells (mSVC) into mesenteric-visceral adipocytes(mVAC) [22,23]. Insulin concentration was optimized
at levels comparable to in vivo level (0.85 ngmL 1) byusing physiological concentration (200 ngmL 1) ofinsulin-like growth factor (IGF-1). IGF-1 and insulin
were found to act synergistically; IGF-1 alone was able
to enhance CCAAT/enhancer binding protein alpha(C/EBPa) and adipocyte lipid-binding protein (aP2)mRNA expression, but did not induce lipid droplet
accumulation associated with adipocyte maturation in
the absence of insulin at physiological concentration.
We evaluated the contribution of resident macrophages in SVCs to adipogenesis using this culture system. Removal of resident macrophages led toincreased adipogenesis of SVCs in mesenteric adipose
tissues, accompanied by enhanced insulin signaling
and reduced levels of GSLs (GlcCer, lactosylceramide[LacCer], GM3). Removal of macrophages also
resulted in increases of IR and IR substrate 1 (IRS1)
phosphorylation following insulin stimulation and of
IR protein level in mSVCs [5]. Thus, levels of GSLs,
particularly GM3, in adipocytes are precisely controlled by soluble factors secreted from resident
macrophages that induce adipogenesis. Mouse embryonic fibroblasts (MEFs) prepared from ST3Gal5-nullmice showed increased adipogenesis and insulin signaling [5]. These findings indicate that GM3 is directly
involved in insulin signaling in adipose tissues and that

2

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GM3 functions as a physiologicalregulator of insulin signaling andadipogenesis

J. Inokuchi et al.

Pathophysiological roles of GM3

Fig. 2. Proposed mechanism underlying the shift of insulin receptors (IR) from the caveolae to the glycosphingolipid-enriched microdomains(GEM) in adipocytes during a state of insulin resistance. Caveolae and noncaveolar rafts (GEM) are shown in schematically. Caveolae and
GEM reportedly can be separated by an anti-CAV1 antibody. IR may be constitutively resident in caveolae based on binding to CAV1
scaffolding domain through the caveolin-binding domain in its cytoplasmic region. Proper insulin metabolic signaling requires binding of IR
and CAV1. In adipocytes, localization of IR in caveolae is disrupted by elevated levels of the endogenous GM3 during a state of insulin
resistance induced by TNFa [10,11]. Our findings demonstrate that dissociation of the IR/CAV1 complex is results from interaction of alysine residue at IR944, located just above the transmembrane domain, with increased GM3 clustered at the cell surface.

GM3 levels of preadipocytes and mature adipocytes
maintain a degree of insulin signaling necessary for
adipogenesis (Fig. 3).

Hypercholesterolemia is a key risk factor for the development of atherosclerosis and cardiovascular diseases.
Prevention of these potentially fatal diseases depends
on careful management of blood cholesterol levels,
particularly low-density lipoprotein cholesterol (LDLC) levels, to maintain them in a target range. Statinsare commonly used to inhibit cholesterol biosynthesis
through competitive inhibition of 3-hydroxy-3methylglutaryl coenzyme A [24,25]. Another treatment
option for the reduction in blood cholesterol levels is
application of cholesterol absorption inhibitors in theintestine. LDL-C level was significantly reduced byezetimibe, the first of several new drugs that inhibit
cholesterol absorption by enterocytes in the small
intestine [26].
Several studies have addressed the mechanisms
underlying cholesterol uptake by enterocytes anddemonstrated that intestinal cholesterol absorption ismediated by transport proteins in these cells. Niemann–Pick C1-like protein 1 (NPC1L1), a polytopic
transmembrane protein highly expressed on the apicalmembrane of enterocytes in the jejunum, acts as acholesterol transporter [27]. NPC1L1 was identified as
the molecular target of ezetimibe; this drug recognizes
the extracellular loop of NPC1L1 protein and alters its
conformation, thereby inhibiting cholesterol transport
functions. Intestinal cholesterol absorption was ~ 67%
lower in NPC1L1-null mice than in wild-type (WT)
mice, although the null mice appeared healthy [28].
Degree of such reduction in ezetimibe-treated WT mice
was similar to that in null mice. Cholesterol absorption

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GM3 membrane levels regulate
hypercholesterolemia

Pathophysiological roles of GM3

J. Inokuchi et al.

Fig. 3. Control of homeostatic andpathogenic balance in adipose tissue by
GM3 [5]. (A) Enhanced adipogenesis ofmesenteric preadipocytes following
depletion of resident macrophages. Soluble
factors secreted by resident macrophages
maintain expression of GM3 and other
GSLs in mesenteric preadipocytes andadipocytes, leading to normal adipogenesis.
(B) St3ga15 KO enhances IR signaling in
MEFs and differentiated adipocytes.

in the null mice was not further reduced by ezetimibe
treatment. Ezetimibe treatment was found to enhance
apical cholesterol efflux to intestinal lumen in bothhuman and mouse studies [29,30]. These findings indicate that NPC1L1 inhibition is an effective antihypercholesterolemia therapy.
The molecular pathways of cholesterol uptake by
NPC1L1 involve clathrin-mediated endocytosis [31].
NPC1L1 protein contains three large extracellulardomains and 13 transmembrane segments [32], and its
extracellular N terminus binds cholesterol. A portionof the transmembrane segments termed sterol-sensing
domain (SSD) detects cholesterol levels in the plasma
membrane [33]. When the SSD senses increased cholesterol level, NPC1L1 attaches to a mixed micelle containing cholesterol and other hydrophobic lipids andinternalizes it by clathrin-mediated endocytosis [34,35].

Cholesterol binding to NPC1L1 promotes formationin plasma membrane of cholesterol-enriched microdomains, also known as lipid rafts, by interacting withflotillin proteins preferentially localized in the microdomains [36]. Thus, these membrane microdomains function as a platform for cholesterol uptake into
enterocytes.
Membrane microdomains are enriched in sphingomyelin and GSLs as well as cholesterol. Gangliosides (sialic acid-containing GSLs) are involved in cell–
cell recognition, adhesion, and signal transduction
within microdomains [37]. GM3 (NeuAc-a2,3-Galb1,4-Glc-ceramide), the simplest ganglioside, interacts
with transmembrane receptors, such as the epidermal
growth factor (EGF) receptor and IR, and regulates
receptor functions by creating a specialized lipid environment [10,38]. Intestinal GM3S expression level is

4

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J. Inokuchi et al.

Pathophysiological roles of GM3

high in neonatal mice and declines during the courseof development in mice [39]. Our 2018 study demonstrated small, but significant, GM3 expression inyoung adult mice, based on TLC and LC-MS/MS
analyses [40].
Membrane microdomain organization often involves
close functional relationships between gangliosides andflotillins [39,41]. NPC1L1/flotillin interactions are
mediated by gangliosides present on the apical surfaceof enterocytes. We observed reduction of NPC1L1dependent cholesterol uptake in GM3S-deficient cells[40]. Cholesterol uptake was significantly lower in
NPC1L1-expressing GM3S-null cells than in control
cells, and cholesterol-dependent translocation of
NPC1L1 from the plasma membrane to the intracellular region was impaired. GM3S-null mice, consistently
with these findings, showed reduced cholesterol uptake
and cholesterol-dependent translocation of NPC1L1 in
intestinal enterocytes.
GM3 deficiency was associated with reduction ofboth genetic hypercholesterolemia in Apo E mutationand diet-induced hypercholesterolemia. On the other
hand, GM3S-null mice fed a normal diet had plasmacholesterol levels similar to those of WT mice. Thesefindings indicate involvement of GM3 in the exogenous pathways of cholesterol metabolism, including
intestinal NPC1L1 activity.
Developmental changes in intestinal GSL composition are synchronized with expression levels of intestinal nutrient transporters [42]. KO of intestinal GlcCer
synthase in a mouse model resulted in retarded growthand early death because of defects in intestinal intracellular vesicular transport [43]. These studies indicatedthe physiological importance of GSLs in intestinal
nutrient absorption, but did not address role of GSLs
in the NPC1L1 pathway. Function of NPC1L1 as acholesterol transporter requires a cholesterol-enriched
membrane microdomain [35,36,44]; however, thedetailed mechanisms underlying function of GM3 andrelated gangliosides in NPC1L1-mediated cholesterol
absorption remain unclear.
Gangliosides, particularly GM3, and its synthesizing
enzyme, GM3S, are potential targets for hypercholesterolemia therapy. Genetic variation in NPC1L1 is closely associated with interindividual variation in
response to ezetimibe [45], and studies have revealed
loss of ezetimibe-binding mutations in the extracellularloop of NPC1L1 [46]. Thus, mutations in this region
may result in loss of response to ezetimibe treatment.
Membrane lipid modification, that is, through GM3S
inhibition, is applicable regardless of binding affinity ofcompounds to NPC1L1 and may provide an alternative
therapeutic approach for nonresponsive individuals.

Recent studies by our group and others have demonstrated essential roles of neural gangliosides in central
control of energy homeostasis through regulation of
leptin signaling. Leptin, an adipocyte hormone produced in proportion to body fat mass, serves as an
adiposity signal and helps maintain body weight homeostasis and energy balance through binding to its cognate receptor, LepR [47, 48]. Several LepR isoforms(six in mice and five in humans) have been identified
to date, and only the longest isoform, LepRb, has a
crucial cytoplasmic region necessary for efficient leptin
signaling in central neurons that control feeding andenergy expenditure [49].
Leptins carry information on adiposity status to thebrain, specifically to various hypothalamic nuclei:
arcuate nucleus (ARC), paraventricular nucleus(PVN), ventromedial hypothalamus (VMH), and lateral hypothalamic area (LH). ARC, the primary targetof leptin activity, contains two interconnected groupsof LepRb-expressing neurons: anorexigenic POMC
(proopiomelanocortin) neurons and orexigenic AgRP/NPY (agouti-related peptide/neuropeptide Y) neurons[50]. These two types of neurons regulate melanocortin
signaling in a coordinated manner to maintain energybalance.
Mice lacking functional leptin (ob/ob) or LepRb(db/db) are hyperphagic and display severe early-onset
obesity [51]. Leptin binding to LepRb leads to phosphorylation of Janus kinase 2 (JAK2), which subsequently phosphorylates three conserved Tyr residues
in LepRb (Tyr985, Tyr1077, Tyr1138). Each of the resulting phospho-Tyr (p-Tyr) residues recruits specific Src
homology 2 (SH2) domain-containing proteins to the
LepRb-JAK2 complex (Fig. 4) [52]. p-Tyr1138 is thebinding site for the SH2 domain of signal transducer
and activator of transcription 3 (STAT3), the key
mediator of the well-studied JAK-STAT pathway,
which is essential for anti-obesity activity of leptin in
the brain. After binding, STAT3 is phosphorylated by
JAK2 and translocated to the nucleus, where it activates gene expression of targets such as POMC andsuppressor of cytokine signaling 3 (SOCS3). SOCS3
binds to p-Tyr985 of LepRb and inhibits JAK2 activation and thereby mediates negative feedback inhibitionof leptin signaling [53]. p-Tyr985 also functions as abinding site for the protein Tyr phosphatase 2 (SHP2);
recruited SHP2 is subsequently phosphorylated by
JAK2 and then recruits growth factor receptorbound protein 2 (Grb2), an adaptor protein thatmediates activation of extracellular signal-regulated

The FEBS Journal (2021) ª 2021 Federation of European Biochemical Societies

5

GM3-related gangliosides regulate
hypothalamic leptin signalingPathophysiological roles of GM3

J. Inokuchi et al.

kinase (ERK). Thus, SHP2 positively regulates leptinmediated ERK activation, and the SHP2-ERK
pathway plays an important role in leptin-dependent
regulation of energy balance. Phosphorylation of
LepRb Tyr1077 similarly promotes activation of
STAT5 through phosphorylation by JAK2. STAT5 isinvolved in central regulation of energy balance, but
the LepRb-STAT5 pathway does not play an essential
role in body weight regulation [54].
In vertebrates, gangliosides are expressed mostabundantly in nerve tissues. Complex gangliosides
GM1, GD1a, GD1b, and GT1b are the predominant
species in mammalian brains [55]. Functional involvement of gangliosides in central regulation of energy
homeostasis through LepRb signaling was first demonstrated using a mouse model with inducible KO of
GlcCer synthase (GCS: Ugcg). GCS is essential forneuronal development; therefore, systemic KO mice
were embryonic lethal, and the neuron-specific KO
mice displayed severe developmental defects [56-58].
To circumvent these defects, R. Jennemann’s group
generated a tamoxifen-inducible neuron-specific GCS
KO (GCS icKO) mouse model and used it toinvestigate functional roles of neuronal GSLs in energy
homeostasis [59]. icKO mice displayed progressive obesity, hypothermia, and reduced sympathetic activity.
Their hypothalamic LepRb signaling was also reduced,
as evidenced by neuronal activity (STAT3 phosphorylation and c-fos protein expression) following i.p. leptin administration. Interaction between LepRb and
GM1/GD1a in mouse hypothalamic neuronal cell line
N-41 was demonstrated by in situ proximity ligation
assay and co-immunoprecipitation experiments. Treatment of N-41 cells with n-butyldeoxynojirimycin (NBDNJ), a GCS-specific inhibitor that depletes GlcCer
and its derived gangliosides, strongly reduced leptininduced JAK phosphorylation. These findings indicatethat a-series gangliosides positively regulate leptin
metabolic activity by interacting with LepRb in
hypothalamic neurons.
GD3 synthase (GD3S: St8sia1) KO mice, which lackb-series gangliosides (e.g., GD1b, GT1b), had serum
leptin levels much lower than those of WT mice [60].
Leptin was accumulated in adipose tissues of KO mice,indicating deficient leptin secretion. Consistently, primary culture experiments with adipose-derived stromal
vascular fractions (SVF) showed that secretion of leptin, but not of adiponectin (another important
adipocyte-derived hormone), was notably lower in KOmice than in WT. Flow cytometric analyses revealed
that SVF cells of WT expressed both a- and b-series
gangliosides and that deficient leptin secretion in SVF
cells of KO mice was restored by exogenous additionof b-series gangliosides (GD3, GD1b, GT1b) to culture medium. These findings, and results of experiments using ganglioside-depleted adipocytes andimmunolocalization of leptin and caveolin-1, indicatethat b-series gangliosides regulate leptin secretion from
adipocytes in lipid rafts.
GD3S KO mice had greatly reduced serum leptin
levels, but did not develop obesity; that is, their bodyweight was similar to that of WT. Hypothalamic leptin
signaling was enhanced in the KO mice, as evidenced
by STAT3 phosphorylation levels in ARC [61]. Overexpression of GD3S in N-41 cells, which enhanced
expression of b-series gangliosides and suppressed thatof a-series gangliosides, reduced leptin-induced STAT3
phosphorylation. Leptin-mediated interaction of
LepRb with GM1 or GD1a was demonstrated by coimmunoprecipitation experiments. These findings indicate that increased levels of a-series gangliosides in the
hypothalamus of KO mice enhanced central leptin signaling and that such enhancement compensated for
deficient leptin secretion, resulting in seemingly normalenergy expenditure.

6

The FEBS Journal (2021) ª 2021 Federation of European Biochemical Societies

Fig. 4. Leptin receptor signaling pathways and altered signaling
observed in ganglioside KO mice and cells. The increased levels of
a-series gangliosides in the hypothalamus (i.e., GD3S KO)
enhanced STAT3 phosphorylation, whereas lack of a-series
gangliosides (i.e., GCS icKO and GM3S KO) reduced STAT3
phosphorylation, suggesting that a-series gangliosides positively
regulate LepRb-STAT3 signaling. In contrast, the lack of a-series
gangliosides or GM1b expressed in GM3S KO may enhance
LepRb-ERK signaling.

J. Inokuchi et al.

Studies by our and R.L. Proia’s groups demonstrated that GM3S KO mice with C57BL/6 background have seemingly normal obesity phenotype
similar to WT, even with high-fat diet (HFD) feeding[5,62]. In contrast, obesity phenotype of KO mice withobese KK-Ay background was strikingly different
from that of WT control [63]. WT KK-Ay mice were
hyperphagic and developed severe, early-onset obesity,and diabetic pathology; in contrast, GM3S KO micewith KK-Ay background had significantly lower bodyweight and food intake and improved glucose andinsulin tolerance. Hypothalamic sensitivity to leptin in
WT KK-Ay, evaluated on the basis of c-fos
immunoreactivity in the ARC following i.p. leptin
administration, was greatly reduced by age 10 weeks
because of development of ‘leptin resistance’, a state inwhich elevation or exogenous administration of leptin
is not sufficient to reduce feeding or body weight [64].
In contrast, hypothalamic sensitivity to leptin in
GM3S KO KK-Ay was maintained at age 10 weeks.
GM3S KO in N-41 cells (which lack a-series gangliosides and instead express minor o-series ganglioside
GM1b, whose synthesis does not require GM3S)
resulted in suppression of leptin-induced STAT3 phosphorylation. In contrast, leptin-induced ERK phosphorylation was greatly enhanced in these cells. Leptin
activates SHP2-ERK pathway and controls c-fos
expression [65]; therefore, these findings suggest that
LepRb-SHP2-ERK pathway is upregulated in GM3S
KO and thereby maintains responsiveness to leptin in
GM3S KO KK-Ay mice.
As summarized in Fig. 4, increased levels of a-series
gangliosides in the hypothalamus (i.e., GD3S KO)
enhanced STAT3 phosphorylation, whereas lack of aseries gangliosides (i.e., GCS icKO and GM3S KO)
reduced STAT3 phosphorylation, suggesting that aseries gangliosides positively regulate LepRb-STAT3 signaling. In contrast, the lack of a-series gangliosides orGM1b expressed in GM3S KO may enhance LepRbERK signaling. In conclusion, GM3 and related gangliosides function in LepRb signaling, in a gangliosidespecies-specific manner, by modulating downstream signaling pathways essential for energy homeostasis.
Molecular mechanisms underlying these effects remain
to be elucidated, but likely involve direct interactions
between LepRb and specific gangliosides. It is also possible that crosstalk occurs between LepRb and otherreceptors at the cell surface, whereby specific gangliosides affect hypothalamic neurons. Ongoing studies by
our group and others will address these intriguing possibilities and clarify the mechanisms underlying
ganglioside-dependent control of energy homeostasis.

The FEBS Journal (2021) ª 2021 Federation of European Biochemical Societies

Pathophysiological roles of GM3

Role of GM3 molecular species in
TLR4 signaling
GM3 (Fig. 1A,B) is a predominant ganglioside in
human sera, with concentration 10–15 µgmL 1(~ 10 µM) [12,66]. Fatty acids of serum GM3 are comprise long-chain fatty acid (LCFA), (16:0, 18:0, and20:0); very-long-chain fatty acid (VLCFA), (22:0, 23:0,
24:0); unsaturated VLCFA, (22:1 and 24:1); and ahydroxyl VLCFA (h24:0, h24:1) (Fig. 1B). Humansera contain similar levels of LCFA, VLCFA, andunsaturated VLCFA species and a smaller level of ahydroxyl species. Altered expression patterns of various GM3 species have been observed in patients withmetabolic disorders [4]; however, specific biological
functions of these species are unclear.
We investigated effects of various GM3 species on
innate immune responses of human peripheral blood
CD14-positive cells (monocytes) [12]. GM3 species
alone had no notable effects. VLCFA species (22:0,
24:0, h24:0) synergistically enhanced lipopolysaccharide (LPS)-mediated production of proinflammatory
cytokines (IL-6, TNF-a, IL-12/23 p40), whereas LCFA
species (16:0, 18:0) strongly inhibited this process.
Unsaturated VLCFA (24:1) had no stimulatory effect,suggesting that desaturation is a protective modification against proinflammatory properties of VLCFA
species. These enhancing and inhibitory effects weredose-dependent and were observed at physiologicalconcentration (<10 µM).
Among various types of gangliosides, only VLCFAGM3 species displayed dose-dependent synergistic activation; other complex gangliosides had inhibitory
effects [12]. GM3 species displayed acyl-chain
structure-dependent positive and negative regulation,
but Cer, GlcCer, and LacCer had no such effects.
Thus, innate immune responses are positively and negatively regulated by serum GM3 species in an acylchain-dependent manner (Fig. 5A).
Chronic inflammation is an important factor inpathogenesis of metabolic disorders [67,68]. Prolonged
activation of innate immune responses by patternrecognition receptors such as Toll-like receptors (TLR)[69,70] leads to chronic inflammation. In metabolic
disorders, TLR4 is activated by various ligands: (a)exogenous pathogen-associated molecular patterns(PAMPs), for example, elevated serum LPS levels [71],(b) endogenous damage-associated molecular patterns(DAMPs) released from activated macrophages andadipose tissues, for example, high-mobility group box1 protein (HMGB1) [72,73], free fatty acids (FFAs)[74], and fetuin-A protein [39].

7

J. Inokuchi et al.

Pathophysiological roles of GM3

A

B

C
Fig. 5. Pathophysiological significance ofGM3 molecular species. (A) Regulation ofpro- and anti-inflammatory activity of GM3
species, precursor GSLs, and complex
gangliosides, dependent on acyl-chain andsaccharide-chain structure. (B) Regulation ofhTLR4/MD-2 signaling by GM3 speciesbalance between VLCFA species and
LCFA-/unsaturated VLCFA species. (C)Association between serum pro-/antiinflammatory GM3 species and progressionof metabolic disorders.

We investigated mechanisms underlying GM3mediated innate immune responses were investigatedby screening signaling pathways targeted by GM3 species. Monocytes were co-stimulated by exogenous
PAMPs and endogenous DAMPs (including several
TLR ligands) in combination with GM3 species.
VLCFA species showed selective synergy with LPS, a
TLR4 ligand, but not with other TLR ligands [12].
LCFA species strongly inhibited LPS-mediated cytokine production. Thus, TLR4-mediated cytokine production was selectively modulated by GM3 species.
VLCFA species also strongly enhanced cytokine production mediated by HMGB1, an endogenous TLR4
ligand released from dead cells or visceral adipocytes
in obese patients [72,73]. Interestingly, effects of GM3
22:0 and 24:0 were reversed by increased doses ofGM3 16:0, 18:0, and 24:1. These findings indicate that
GM3 species act as selective endogenous modulators
of TLR4 signaling and that TLR4 activation is positively or negatively controlled by the balance of
LCFA-, VLCFA-, and unsaturated VLCFA-GM3 species in serum (Fig. 5B).
We investigated pro-/anti-inflammatory roles ofGM3 species in metabolic disorders by performing
LC-MS/MS analysis of expression patterns of serum
GM3 species in human sera [4,12]. Progressive increase
in VLCFA-GM3 species and decrease in LCFA-GM3
species were correlated with increases of HOMA-IR(homeostatic model assessment for insulin resistance)and serum C-reactive protein (CRP) and with severityof metabolic disorders. In patients with obesity(presymptomatic), hyperlipidemia, or hyperglycemia,
LCFA species (16:0, 18:0, 20:0) were strongly reduced,
whereas VLCFA species (22:0, 23:0, 24:0, h24:0) were
elevated.
Early- and intermediate-phase increases of bodymass index (BMI) and abdominal circumference wereassociated with strong reduction of LCFA species andincrease of VLCFA species. Elevation of VLCFAGM3 species is evidently characteristic of obesity andplays a pathogenic role in early stages of metabolic
disorders. a-Hydroxy VLCFA-GM3 (h24:0) levels
showed strong linear correlations with BMI andabdominal circumference and also with insulin resistance and chronic inflammation indicators such as
ALT, HOMA-IR, and CRP. On the other hand, moderate reduction of VLCFA species and strong increaseof unsaturated VLCFA species were observed in severe
obesity and following onset of metabolic disorders (hyperlipidemia, hyperglycemia). Desaturation of VLCFA
species evidently occurs following onset of metabolic
disorders.
Our above findings, taken together, clearly indicatethat pathogenesis of metabolic disorders via chronic
inflammatory processes involves increases of VLCFAGM3 species and decreases of LCFA-GM3 species

8

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J. Inokuchi et al.

Pathophysiological roles of GM3(Fig. 5C). In steady state, homeostasis is maintainedby proper balance of GM3 species; in presymptomaticand early phases of metabolic disorders, VLCFA species increase and promote chronic inflammation andinsulin resistance; in late phases, desaturation of
VLCFA species occurs, but a-hydroxyl VLCFA species can still potentiate chronic inflammation.
In view of the notable increases of serum VLCFAGM3 levels in metabolic disorders, we performed comparative studies using mouse models of obesity. ob/ob
mice showed early onset of metabolic disorders andhad an increased GM3 abundance in visceral adipose
tissue [2,12]. We measured levels of various GM3 species by LC-MS/MS [12]. Epididymal adipose tissue ofcontrol C57/BL6 mice had 16:0, 18:0, 20:0, 22:0, 23:0,
24:0, and 24:1 as major GM3 species, and a-hydroxyl
species as a minor component. ob/ob mice showednotably higher level of a-hydroxyl species: strong
increase of VLCFA species (h22:0, h23:0, h24:0) andmoderate increase of LCFA and unsaturated VLCFA
species (h16:0, h18:0, h20:0, h24:1). Thus, increased
VLCFA-GM3 level and a-hydroxylation occur in visceral adipose tissue in obesity and metabolic disorders.
Feeding of HFD for 10 weeks resulted in obesity andincreased GM3 levels [5,12]. LC-MS/MS analysis
revealed greater increase of a-hydroxyl GM3 species in
HFD-fed mice [12]. In comparison with ob/ob mice,
predominant GM3 species in HFD-fed mice had
shorter acyl chains (h18:0, h20:0, h22:0).
Crystal structure analysis of TLR4/MD-2 complex[75,76] indicated formation of two ligand-binding sites
for LPS on the dimerization interface between two
TLR4/MD-2 units. MD-2 forms hydrophobic pockets
that bind to the acyl-chain moiety of LPS, while
TLR4 leucine-rich repeats (LRR) form basic clusters
that recognize the hydrophilic head group of LPS. We
found that mutations of basic residues greatly reduced
human TLR4 activation by VLCFA-GM3 [12]. MD-2dependent recognition of GM3 acyl chain was evaluated by comparing inhibitory effects of GM3 16:0 on
mTLR4/mMD-2, hTLR4/hMD-2, and mTLR4/hMD2, a chimeric complex of mouse TLR4 and humanMD-2. hTLR4/hMD-2 and chimeric mTLR4/hMD-2
activation was inhibited by 16:0 GM3 at physiologicalconcentration, whereas mTLR4/mMD-2 activation
was not. Thus, MD-2 is evidently involved in recognition of GM3 acyl-chain structure and provides a basis
for molecular species selectivity [12].
To elucidate the mechanisms whereby GM3 species
enhance or suppress TLR4 activation in an acyl-chain
structure-dependent manner, we examined a ligandmacromolecular docking on hTLR4/hMD-2 complex.
Binding modes of VLCFA-GM3 (24:0) and LCFA-GM3(16:0) on the molecular surfaces around the hydrophobic pocket of hMD-2 and the basic residues of hTLR4
were determined. Docking models of hTLR4/hMD-2/
GM3 (24:0 or 16:0) complex are shown in Fig. 6A,B.
GM3 24:0 and 16:0 bound to hydrophobic pocket of
hMD-2 via the fatty acid and sphingoid base (Fig 6C–E), similar to LPS and lipid IVa, respectively. The GM3
24:0 binding mode overlapped closely to that of Ra-LPS
in the crystal structure of reference, and the GM3 24:0
saccharide chain was surrounded by basic residues of
TLR4 (Fig. 6C). Basic residues of TLR4 (K341, K362,
and R322) that interacted with LPS saccharide chain
and showed conformational changes upon TLR4 activation [76,77] were closely associated with GM3 24:0 saccharide chain. In contrast, R264, a key residue inrecognition of 4’-phosphate of LPS and triggering
TLR4 activation [76], was far from GM3 24:0 saccharide chain. These findings indicate that VLCFA-GM3
is able to enhance TLR4 signaling without triggering
activation.
The GM3 16:0 binding mode was compared to that oflipid IVa complexed with hMD-2 (Fig. 6E). Lipid Iva
binding mode differed from that of LPS, showing
reverse orientation of 4’-phosphate and acyl chains,which may inhibit hTLR4/hMD-2 dimer formation bypresenting hydrophilic groups (phosphate, glucosamine)
to lipophilic dimer interface [75,76] (Fig. 6C,E). Similarly, GM3 16:0 binding mode was opposite that ofGM3 24:0, showing reverse orientation of saccharide
chain and the acyl chain (Fig. 6D), thereby interferingwith and suppressing TLR4 activation through presentation of saccharide chain to dimer interface (Fig. 6E).
Lipid IVa and lipid A (LPS-core structure), in contrast,show the same binding orientations on mTLR4/mMD-2[75]. These comparative analyses help clarify the mechanisms whereby various GM3 species enhance or reduce
TLR4 activation in an acyl-chain structure-dependent
manner [12].
Molecular mechanisms whereby GM3 species are
secreted and targeted to TLR4/MD-2 complex remain
to be elucidated. GM3 is presumably secreted into
serum as part of a lipoprotein complex [4,66]. TLR4 isactivated by LDL cholesterol [78], and Cer 24:0 ispreferentially incorporated into LDL cholesterol [79].
We showed that serum VLCFA-GM3 level is correlated with non-HDL cholesterol, whereas serum
LCFA-GM3 level is correlated with HDL cholesterol,
indicating species-selective incorporation into lipoproteins [4,12]. Soluble-form CD14, a carrier protein
for LPS, also enhances pro- and anti-inflammatory
activity of GM3 species [12]. a-Hydroxylation ofGM3 species, mediated by fatty acid-2 hydroxylase(FA2H), may promote secretion of GM3 species, since

The FEBS Journal (2021) ª 2021 Federation of European Biochemical Societies

9J. Inokuchi et al.

Pathophysiological roles of GM3

A

B

hTLR4

hTLR4

GM3 24:0

GM3 16:0

hMD-2

C

hMD-2

R322

D

K341 K362
Saccharides

E

NeuAc
NeuAc
R264

Gal
NeuAc
VLCFA
(24:0)

VLCFA(24:0)NeuAc

Sph(d18:1)

Dimer interfaceSph(d18:1)GM3 24:0 vs. Ra-LPS

Sph(d18:1)

LCFA(16:0)

GM3 24:0 vs. GM3 16:0

Sph(d18:1)

Dimer interface

LCFA(16:0)

GM3 16:0 vs. Lipid-IVa

Fig. 6. Ligand macromolecular docking analysis reveals species-specific GM3 recognition by TLR4/MD-2. (A, B) Docking models of GM3
24:0 (A) and 16:0 (B) binding to human TLR4/MD-2 complex (3FXI). Blue color: basic residues of TLR4. (C–E) Superposition of GM3 24:0 (indocking model) vs. Ra-LPS (in 3FXI) (C), GM3 24:0 vs. GM3 16:0 (in docking model) (D), and GM3 16:0 vs. lipid IVa (in 2E59) (E). Basic
residues and the dimer interface are indicated schematically.

a-hydroxylation reduces acyl-chain hydrophobicity
and affects lipid diffusion [80].
Metabolic and biosynthetic pathways of the various
GM3 species are comprised of complex mechanismsthat depend on activities of several enzymes: ceramide
synthases (CerSs), fatty acid elongases (Elovls), acylCoA desaturases, and ST3GAL5. Increased LCFACer (16:0) and decreased VLCFA-Cer (22:0, 24:0)
levels in obesity were correlated with progression of
metabolic disorders [81,82]. Such alterations of Cer
species are presumably due to imbalance of CerS2/6
expression and inhibition of b-oxidation. Imbalanced
expression of Cer species in metabolic disorders results
in reduced LCFA-GM3 and increased VLCFA-GM3
production. Elovl6 KO inhibited progression of metabolic disorders by altering fatty acid composition [83].
Blocking of fatty acid elongation (16:0-to-18:0) inthese Elovl6 KO mice resulted in increased 16:0 anddecreased 18:0-to-24:0 fatty acid content. Elovl6 deficiency may reduce expression levels of VLCFA-GM3
species and help maintain homeostatic balance
between pro- and anti-inflammatory GM3 species. In
resolution phase of innate immune responses, desaturases terminate inflammation by inducing fatty aciddesaturation [84]. We found that desaturation of
VLCFA-GM3 species occurs after onset and in severe
metabolic disorders. Expression of both elongase anddesaturase genes is regulated by SREBP-1, a key transcription factor in lipid signaling which is activatedsimultaneously with NF-jB [83,84]. TNF-a and IL-1b
induce St3gal5 expression and GM3 production in adipocytes [2,5], and production of a-hydroxy VLCFAGM3 in obesity involves TLR4 signaling [12]. Thesefindings, taken together, clearly indicate that fatty acid10

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J. Inokuchi et al.

structures and GM3 species expression levels are controlled by complex, precisely coordinated mechanisms.

Pathophysiological roles of GM3

1 Inokuchi J & Radin NS (1987) Preparation of the active
isomer of 1-phenyl-2-decanoylamino-3-morpholino-1propanol, inhibitor of murine glucocerebrosidesynthetase. J Lipid Res 28, 565–571.

2 Tagami S, Inokuchi Ji J, Kabayama K, Yoshimura H,
Kitamura F, Uemura S, Ogawa C, Ishii A, Saito M,
Ohtsuka Y et al. (2002) Ganglioside GM3 participatesin the pathological conditions of insulin resistance.
J Biol Chem 277, 3085–3092.
3 Sato T, Nihei Y, Nagafuku M, Tagami S, Chin R,
Kawamura M, Miyazaki S, Suzuki M, Sugahara S,
Takahashi Y et al. (2008) Circulating levels ofganglioside GM3 in metabolic syndrome: a pilot study.
Obes Res Clin Pract 2, 231–238.
4 Veillon L, Go S, Matsuyama W, Suzuki A, Nagasaki
M, Yatomi Y & Inokuchi J (2015) Identification of
ganglioside GM3 molecular species in human serum
associated with risk factors of metabolic syndrome.
PLoS One 10, e0129645.
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6 Inokuchi J & Uemura S (2014) ST3 beta-galactosidealpha-2,3-sialyltransferase 5 (ST3GAL5). Handbook of
Glycosyltransferaes and Related Genes (Taniguchi N,
Honke K, Fukuda M, Narimatsu H, Yamaguchi Y &Angata T, eds), Vol. 2, pp. 675–684, 2nd edn. Springer,
Tokyo, Heidelberg, New York, Dordrecht, London.
7 Inokuchi J (2010) Membrane microdomains and insulin
resistance. FEBS Lett 584, 1864–1871.
8 Inokuchi J (2014) GM3 and diabetes. Glycoconj J 31,
193–197.
9 Inokuchi J (2011) Physiopathological function ofhematoside (GM3 ganglioside). Proc Jpn Acad Ser B
Phys Biol Sci 87, 179–198.
10 Kabayama K, Sato T, Saito K, Loberto N, Prinetti
A, Sonnino S, Kinjo M, Igarashi Y & Inokuchi J(2007) Dissociation of the insulin receptor andcaveolin-1 complex by ganglioside GM3 in the stateof insulin resistance. Proc Natl Acad Sci USA 104,
13678–13683.
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BW, Igarashi Y & Inokuchi J (2005) TNFalphainduced insulin resistance in adipocytes as a membrane
microdomain disorder: involvement of gangliosideGM3. Glycobiology 15, 21–29.
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W, Fujii M, Kabayama K, Shimoyama A, Fukase K
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Macrophage-mediated inflammation in metabolic
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14 Bays HE, Gonzalez-Campoy JM, Bray GA, Kitabchi
AE, Bergman DA, Schorr AB, Rodbard HW & Henry
RR (2008) Pathogenic potential of adipose tissue andmetabolic consequences of adipocyte hypertrophy and

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11

Concluding remarks
GM3 is localized on plasma membrane as a componentof membrane microdomains, which constitute signaling
platforms for IR and play essential roles in regulationof signal transduction. GM3 species, varying widely in
acyl-chain structure, are secreted abundantly into
human serum and function as endogenous TLR4 modulators in an acyl-chain structure-dependent manner.
TLR4 activation is selectively enhanced by VLCFAGM3 species, but suppressed by LCFA and unsaturated
VLCFA-GM3 species. Metabolic disorders are often
characterized by increased VLCFA-GM3 and decreased
LCFA-GM3 levels in serum, which contribute to disease progression via chronic inflammatory process. LCMS/MS analysis of serum GM3 species will potentiallyfacilitate evaluation of hidden risks of inflammatory
diseases mediated by TLR4 signaling. The studies
summarized here, by clarifying the pathophysiologicalroles of GM3 species, will lead to novel diagnostic andtherapeutic approaches involving the development ofanti-inflammatory drug.

Acknowledgements
The authors’ studies described in this review were supported by a Grant-in-Aid for Scientific Research (B)from MEXT (to JI) (20H03452), the Takeda Foundation (JI), and the MEXT-Supported Program for theStrategic Research Foundation at Private Universities(S1201031). The authors are grateful to Dr S. Anderson for English editing of the manuscript.

Conflicts of interest
The authors declare no conflict of interest.

Author contributions
JI, KI, MN, and HK wrote the review. TN and KF
contributed the significant parts of research involvedin this review, LC-MS/MS analysis, and insightful
structural/functional analysis of TLR4, respectively.

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＜分子認識部門＞
教授 細野雅祐
講師 菅原栄紀
講師 立田岳生
レクチンの抗腫瘍メカニズムの解明と糖脂質のかかわり 2021
１）シスプラチンの感受性に影響を及ぼす Gb3 の局在について
昨年度，Gb3 発現細胞として HeLa および Gb3 非発現細胞として A4GALT 欠損 HeLa(Gb3-HeLa)を用いて，Gb3 の発現がシスプラチンの感受性に対して影響を与えるか否かについて検討し，Gb3-HeLa に対して HeLa の生存率が約 25% 低下することおよびこのメカニズムの一部に内因性アポトーシス制御分子の発現変化が関与していることを報告した．Gb3 は細胞内および細胞膜上に存在すると考えていることから，本年度はそのどちらに局在している Gb3が抗がん剤の効果増強に寄与しているのかを検討した．まず始めにGb3-HeLa に PiggyBac
Transposon Vector System を用いて A4GALT 遺伝子をゲノム上に組み込むことにより再度
Gb3 を発現させたGb3-HeLa/Gb3 を作製した．この細胞に発現している Gb3 は，HeLa と同様に細胞膜上および細胞内に局在することが示された（図 1）．これらの細胞を用いてシスプラチンの感受性を検討した結果，Gb3-HeLa のアポトーシス細胞の割合は HeLa より約17% 低下した．一方，Gb3-HeLa/Gb3 ではGb3-HeLa より約 20% 増加し，Gb3 を再発現させることによりシスプラチンに対する感受性が回復した．次に，細胞膜上に Gb3 を導入したGb3-HeLa+Gb3 を作製した．細胞膜上に導入された Gb3 は，導入直後と比較すると僅
かな減少は認められるが 48 時間後まで維持されることが分かった（図 1）．この細胞を用いてシスプラチンに対する感受性を調べた結果，Gb3-HeLa およびGb3-HeLa+Gb3 におけるアポトーシス細胞の割合に変化は見られなかった．これらの結果から，シスプラチンに対する感受性には，細胞膜上ではなく細胞内膜系に局在する Gb3 が関与している可能性が示唆された. 今後は，オルガネラマーカーと抗 Gb3 抗体を用いた二重染色法による Gb3 の細胞内局在部位を検討すると共に Gb3 がシスプラチン感受性を増強する作用メカニズムを明らかにする．
HeLa

DGb3-HeLa/Gb3

DGb3-HeLa+Gb3

図 1 . 共焦点顕微鏡による Gb3 発現部位の観察

54２）Gb3 近傍に存在する分子の探索
細胞膜上に Gb3 を高発現していることが知られているバーキットリンパ腫細胞株 Raji において，SAL が Gb3 に結合することによりアポトーシス初期の状態を誘導するものの細胞死を誘導することなく，細胞収縮および細胞増殖抑制を引き起こすことを報告している．これらの現象は，Gb3 の近傍に存在している分子あるいは SAL が Gb3 に結合した後に GEM にリクルートされる分子によって引き起こされるのではないかと考えている．Gb3 近傍の分子を探索する手法として本研究室では，
これまでに抗 Gb3 マウスモノクローナル抗体および抗 Gb3
抗 体 に 反 応 す る Horseradish peroxidase (HRP) 標 識 マ ウ ス IgG 抗 体 を 利 用 し て
Enzyme-mediated activation of radical sources (EMARS)法により検討を行ってきたが，目的とするタンパク質の同定には至っていなかった．本年度は，研究対象である SAL を直接 HRPで標識した HRP-SAL を作製し，この HRP-SAL および EMARS 試薬として開発された FITC
標識 Aryl azide（Ar-Flu）用いて EMARS 法を行った．さらにラフトに局在する分子を標的にする目的で EMARS 反応後に UltraRIPA lipid raft kit を利用してラフト画分の抽出を行い
Gb3 近傍の分子の探索を試みた．HRP-SAL 処理および未処理の細胞から得られたラフト画
分を SDS-PAGE で分離後，抗 FITC 抗体を用いたウエスタンブロッティング法を行い比較したところ，35 kDa 付近に認められるバンドは，HRP-SAL 処理した Raji のラフト画分でのみ検出された．さらにこの分子は，抗 FITC 抗体を共有結合させた磁気ビーズを利用した免疫沈降により精製可能であった．今後は，得られた 35 kDa 付近のタンパク質を質量分析により同定し，SAL が Gb3 に結合することにより引き起こされる現象の詳細な分子機構を明らかにする．

３）ウシガエル (Rana catesbeiana) 卵から単離されたシアル酸結合性レクチン (sialic
acids-binding lectin，cSBL) は，レクチンおよびリボヌクレアーゼ活性を併せ持つ多機能性タンパク質である．正常細胞への毒性は低く，多種類のがん細胞に対して高い抗腫瘍活性を発揮する．本研究室では，cSBL の抗腫瘍効果のさらなる解明を目的に，cSBL 高感受性
悪性中皮腫 H28 細胞を cSBL で持続処理することで cSBL 耐性細胞 (cSR) を樹立し，これを用いて cSBL を低濃度で長期間処理した際に発現が変化する遺伝子の特定を可能にした．耐性度の高い cSR-A1 および B1 株をマイクロアレイ解析した結果，これまでに，cSR細胞における発現変動のあった遺伝子（differentialy expressed genes :DEGs）を多数検出しており，cSR における多面的な遺伝子発現変化が明らかになっている．今回 DEGs の詳細を解析したところ，ATP binding cassette (ABC) transporter である ABCC2 (MRP2) および
ABCA1 の発現が低下していることが見出された (Table 1)．ABCC2 はがん細胞に高発現し薬剤耐性に寄与することが知られている ABC transporter ファミリーの一種である．

55

Table 1. ABC transporter genes down-regulated in cSR cells compared with parent H28 cell line.

そこで，ABCC2 に着目しそのタンパク質レベルの発現を確認したところ，cSR-A1 およびB1 細胞共に ABCC2 の著しい発現の低下が観察され，タンパク質レベルでも ABCC2 の発現が低下されることが確認された (Fig. 1)．また，親株である H28 細胞において，cSBL 72
時間処理における ABCC2 の発現変化を観察したところ，濃度依存的にその発現が低下する傾向が観察された(Fig. 2)．

Fig. 1. ABCC2 protein expression in H28 and cSR cells. The expression of ABCC2 in each cell line
was detected by western blotting. Densitometric quantification was performed using the results ofthree independent experiments (mean ± SD). **P < 0.01. ***P < 0.001. cSR: bullfrog sialic
acid-binding lectin-resistant; SD: standard deviation.

56

Fig. 2. ABCC2 protein expression in cSBL-treated H28 cells. The cells were treated with cSBL at
0.2 or 1.0 µM for 72 hours. Then, the expression of ABCC2 was detected by western blotting.
Densitometric quantification was performed using the results of three independent experiments(mean ± SD). cSR: bullfrog sialic acid-binding lectin-resistant; SD: standard deviation
このことから cSBL は，持続的な長期間処理および 72 時間程度の短時間処理両方において ABCC2 の発現低下を誘導することが示唆された．ABCC2 はシスプラチンやドキソル
ビシンなど基質となる抗がん剤を細胞外に排出するポンプであり，その高発現はがん細胞の薬剤抵抗性へと寄与する．cSBL は ABCC2 の発現低下を介し，がん細胞における抗がん剤への感受性を増大させる可能性があり，cSBL をそれら抗がん剤と併用することが有用な治療戦略になる可能性がある．今回の発見が新しい抗がん治療戦略へつながることが期待される．
＜発表論文＞
1. Transcriptomic alterations in malignant pleural mesothelioma cells in response to long‑term
treatment with bullfrog sialic-acid binding lectin.
T. Tatsuta, A. Nakasato, S. Sugawara, M. Hosono, Mol. Med. Rep., 23., doi:
10.3892/mmr.2021.12106.
2. Discovery of antitumor effects of leczymes.
T. Tatsuta, M. Hosono, Glycoconj. J., 39, 157-165 (2021).
＜学会発表＞
・ナマズ卵レクチンはヒト子宮頸がん由来 HeLa におけるスニチニブの取り込みおよび排出に影響をおよぼす菅原 栄紀，高栁 円, 本田 捷太，立田 岳生，藤井 佑樹 a，大関 泰裕 b，
伊藤 淳 c，佐藤 信 c，細野 雅祐（長崎国際大学・薬学部 a，横浜市大院・生命ナノ b, 東北医薬大・医学部・泌尿器科学講座 c）
第 15 回東北糖鎖研究会，岩手，2021 年 10 月，要旨集 p.8
・抗がん剤で誘導されるアポトーシスへの Gb3 の関与
八木橋 佳章, 菅原 栄紀, 菊地 聡大, 立田 岳生, 細野 雅祐
第 15 回東北糖鎖研究会，岩手，2021 年 10 月，要旨集 p.18
・がん細胞に発現する Gb3 の抗がん剤感受性に与える影響について
八木橋 佳章, 菅原 栄紀, 菊地 聡大, 立田 岳生, 細野 雅祐

57

第 94 回日本生化学大会，横浜，2021 年 11 月, P-753
・確立された RNase 耐性がん細胞における AKR1B10 と ABCC2 の発現に対する cSBLの影響
中里 ありす、立田 岳生、菅原 栄紀、細野 雅祐、第 94 回日本生化学会大会、横浜、2021
年 11 月、演題番号 P-047
・膀胱がん細胞に対する cSBL の抗腫瘍効果について
立田 岳生、中里 ありす、菅原 栄紀、伊藤 淳、佐藤 信、細野 雅祐、第 94 回日本生化学会大会、横浜、2021 年 11 月、演題番号 P-505
・細胞内膜系 Gb3 はシスプラチンによるアポトーシスに関与する
菅原 栄紀，八木橋 佳章，立田 岳生，細野 雅祐
第 44 回日本分子生物学会年会，横浜，2021 年 12 月, 2LBA-017
・悪性中皮腫細胞に発現する AKR1B10 および ABCC2 に対する cSBL の効果
立田 岳生、中里 ありす、佐藤 彬翔、菅原 栄紀、細野 雅祐、日本薬学会第 142 年会、名古屋、2022 年 3 月、演題番号 26PO5-am1-04
・がん治療への応用を目指したウシガエル卵由来シアル酸結合性レクチン(cSBL) の基礎研究
立田 岳生
第 43 回東北薬学セミナー，仙台，令和３年度日本薬学会東北支部奨励賞受賞講演，2021
年 12 月・悪性中皮腫細胞に発現する AKR1B10 および ABCC2 に対する cSBL の効果
立田 岳生，中里 ありす，佐藤 彬翔，菅原 栄紀，細野 雅祐
日本薬学会第 142 年会，名古屋，2022 年 3 月，演題番号 26PO5-am1-04

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Glycoconjugate Journal (2022) 39:157–165
https://doi.org/10.1007/s10719-021-10033-9

MINI REVIEW

Discovery of antitumor effects of leczymes
Takeo Tatsuta1 · Masahiro Hosono1
Received: 29 September 2021 / Revised: 18 November 2021 / Accepted: 8 December 2021 / Published online: 23 January 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Sialic-acid binding lectin from bullfrog (Rana catesbeiana) eggs, cSBL, is a cytotoxic ribonuclease (RNase) belonging to
the RNase A superfamily. cSBL is cytotoxic to tumor cells, such as malignant pleural mesothelioma by inducing apoptotic
cell death caused by the degradation of RNA in tumor cells. In addition, we have reported some data that cSBL could be
involved in the endoplasmic reticulum stress pathway, and it was also assumed to cause apoptotic cell death. The most significant property of cSBL is its specificity toward malignant cells. Furthermore, since the antitumor activity of cSBL wasconfirmed by in vivo experiments using mouse xenograft models, it is expected to be a candidate for clinical chemotherapy.
Here, we summarize the history of cSBL, alternatively called “leczyme,” with its present and future.
Keywords Lectin · Sialic acids · Ribonuclease · Antitumor activity · Apoptosis

Lectin meets ribonuclease
During the 1970s, Kawauchi et al. found some lectins fromfrog eggs belonging to the genus Rana with hemagglutinating activities [1, 2]. Among them, they succeeded inisolating a unique lectin that showed agglutinating activity
for some murine ascites tumor cells, such as Ehrlich and
AH109A, but not for human erythrocytes [3]. Since mostof the investigators’ interests concerning tumor cell agglutinin were limited to plant lectins at that time, animal lectins
were rarely studied in membrane glycoconjugate research.
The egg agglutinins were inhibited by ganglioside but notgloboside, and sialidase-treated tumor cells lost their agglutinability. Therefore, these lectins were estimated to haveaffinities for sialo-glycoconjugates, including both glycolipids and glycoproteins, and reactivity specific for tumor cells[4]. Dr. Sen-itiroh Hakomori, who was our great mentor andthe first director of the Cancer Research Institute of Tohoku
College of Pharmaceutical Sciences (Fig. 1), was interested
in these lectins, and consecutively supported us by providing
ganglioside and globoside preparations from erythrocytes,
This article belongs to the Topical Collection: Tribute to Professor
Sen-itiroh Hakomori* Masahiro Hosono
mhosono@tohoku-mpu.ac.jp
1

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

which were not commercially available during that period,
along with a lot of variable advice. Although the primarystructure of sialic-acid binding lectin from bullfrog (Ranacatesbeiana) eggs (cSBL) was established using the peptide sequencing method by Titani et al., we could not findany similarity in the early protein database [5]. Three years
later, the sequence of the homolog, Rana japonica egg lectin (jSBL) was elucidated and the similarities between frog
sialic acid-binding lectins (SBLs) and ribonuclease (RNase)
A were found to be around 30% [6], and SBLs showedpyrimidine-specific RNase activity [7]. An RNase purified
from R. catesbeiana oocytes collected in Taiwan by Liaoet al., and named RC-RNase, was found to be identical tocSBL [8]. Subsequently, SBLs met RNase for the first time.
In this way, Nitta et al. proposed the name catalytic lecitn(Leczyme) as a multifunctional molecule with lectin andenzymatic activity [9].

Leczymes show antitumor effect
The antitumor effects of leczymes, including cSBL andjSBL, on cultured cell lines were first reported by Nitta et
al. in 1994 [10]. They showed that both leczymes significantly inhibited mouse leukemia P388 and L1210 cell proliferation in vitro. Furthermore, in vivo experiments werecarried out and the survival time of mice inoculated with
cSBL-treated Sarcoma 180 cells was prolonged, and the13Vol.:(0123456789)

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Glycoconjugate Journal (2022) 39:157–165

Fig. 1  The nomenclature plateon the former building of Cancer research institute of Tohoku
pharmaceutical university (left).
Snapshots of Prof. Sen-itiroh
Hakomori (center) and Dr.
Hiroaki Kawauchi (right) intheir youth, in 1965

administered of an injection of cSBL increased the survival
rates of Sarcoma 180- or Mep II ascites-bearing mice; for10 days of continuous cSBL injection, Sarcoma 180- or Mep
II-bearing mice receiving an injection of 0.5 mg/kg of cSBL
had 67% and 50% survival, respectively, after 45 days. As
significant side effects were not observed after a 50 mg/kg
injection of cSBL, 1/3 of the lethal dose for normal mice, themaximum concentration used in this experiment (2 mg/kg)
was estimated to be a safe dose in vivo. Nitta et al. consecutively reported another study concerning the establishment
of cSBL-resistant P388 cells (RC-150) and their behavior
on cSBL treatment [11]. cSBL bound to and agglutinatedboth P388 and RC-150 cells; however, the internalizationof cSBL was observed only in parent P388 cells, but not in
resistant RC-150 cells, indicating that the uptake of cSBL isessential for antitumor activity. At this point, cSBL has beenreported to bind to a wide variety of cancer cells, although it
shows little response to normal erythrocytes, leukocytes, andfibroblasts [12]. Combined with these results, it has attracted
attention as a new anticancer substance with few side effects.

Leczyme as antitumor ribonucleases
Since 1990, as antitumor RNases have been successively
reported based on the new trend of the research, the antitumor activity of cSBL has been studied, focusing on its
properties not only as a lectin but also as an RNase, and
it is now known that cSBL indeed exerts RNase activity
accompanying with cytotoxicity in cancer cells (Fig. 2).
To date, many types of antitumor RNases have been
identified. They are derived from a variety of species, including plants, bacteria, fungi, and animals. RNase MC2 from
Momordica charantia [13, 14], TBN1 from Lycopersiconesculentum [15], barnase from Bacillus amyloliquefaciens[16], binase from Bacillus intermedius [17, 18], α-sarcin
from Aspergillus giganteus [19], and mushroom RNases [20,21] are typical antitumor RNases [22]; however, the mostextensively investigated is vertebrate pancreatic-type RNases(RNase A superfamily), such as BS-RNase from Bos taurus[23, 24], and Onconase from Rana pipiens [25, 26]. Bovine

13pancreatic RNase A (EC 3.1.27.5) [27], the best-known
member of this family, was the first RNase reported to haveantitumor activity in vitro and in vivo [28–33]. However, as
extremely high amounts of RNase A were required to show
a positive effect, RNases could not maintain their positionas candidates for new antitumor agents. The reasons for thisare described below.

Tumoricidal mechanisms of RNases
Although their mechanism of action is still not fully understood, there is a commonly accepted consensus regarding theirmechanisms [34, 35]. The proposed mechanisms of RNaseinduced cytotoxicity are summarized as follows. (i) Binding
to the cell surface: Antitumor RNases interact with specificor nonspecific targets on the cell surface. Some membraneproteins, glycosaminoglycans, or lipids could be the target[36–38]. (ii) Internalization into cells: RNases are internalized by endocytosis. The endocytic mechanism is still poorly
understood. Even though there are controversial reports onwhether the route is mediated by clathrin / AP-2-mediated [39]or clathrin- and caveolae-independent pathways [40], depending on the difference in cells, it has been suggested that RNases
go through diverse routes to cross the membrane of different
cell lines [41]. RNases can translocate through distinct organelles, such as endosomes, lysosomes, ER, and the trans-Golgi
network, and reach the cytosol [42]. Some RNases engineered
to have a nuclear localization sequence can be transported into
the nucleus [43]. (iii) Evasion of the cytosolic RNase inhibitor protein (RI): RNases can be captured by RI and lose their
enzymatic activity. Thus, the antitumor RNases need to beinsensitive to the RI or evade the RI (saturating the RI with
RNase or capturing by other molecules that impair the interaction with RI) [34],. (iv) Degradation of cellular RNA: RNases
hydrolyze RNA in the cytosol or the nucleus. Differences inthe efficiency of these four steps can affect cell susceptibility[44]. The ribotoxic effect of RNase can transduce apoptotic
signals [45] and sometimes autophagy [46]. Some studies
have reported other details about the mechanisms by which
antitumor RNases induce cell death, such as the involvement

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159

of miRNA [47, 48], and pleiotropic effects on transcriptionchanges [49–51]. Signal transduction induced by RNA damage caused by antitumor RNases still needs to be elucidated.
cSBL is a vertebrate pancreatic-type RNase with an antitumor activity that induces apoptosis in cancer cells through theabove-mentioned steps. Some experiences of the antitumor
effect of cSBL obtained so far will be described in the following sections.

and this agglutination was inhibited by glycoconjugates
containing sialic acids. Furthermore, they investigatedcSBL binding on several cancer and normal cells andfound that cSBL binds to a majority of cancer cells, while
normal cells exhibit low reactivity [12, 52]. The bindingof cSBL to the cell surface was inhibited by the coexistence of sialoglycoproteins and sialidase-treated cells,
evading the cytotoxicity of cSBL [10]. By adding benzylα-N-acetylgalactosamine to the culture medium, thecells became resistant to cSBL. Thus, the involvement ofO-linked glycoconjugates containing sialic acids has beensuggested as the target of cSBL on cancer cell membranes[53]. However, the details of the carbohydrate-binding
properties of cSBL remain unclear.

Binding of cSBL on cancer cell surface
As described above, cSBL was found to be an agglutinin in tumor cells [3]. Nitta et al. reported that it agglutinates various types of tumor cells but not normal cells,
acSBL 00.2

1.0

28S rRNA

ControlcSBL

cControlcSBLµM

DegradedRNA

18S rRNA

DegradedRNA

Viability (%)

b

d

e

100

100

80

80

60

60

40

40

20

20

0

0.2

5

2

10

(µM)

Fig. 2  Cytotoxic effect of cSBL on mesothelioma cell lines. (a) Cellular RNA-degrading effect of sialic-acid binding lectin from Ranacatesbeiana eggs (cSBL) in malignant mesothelioma H28 cells. The
cells were treated with cSBL at indicated concentration for 24 h andtotal RNA was extracted using a Direct-zol RNA mini prep kit (Zymo
ResearchCo., CA). Then, electrophoresis was performed on 1.5%
agarose gel. (b) Morphological change of cSBL-treated H28 cells.
The cells were treated with or without cSBL (5 µM) for 72 h. Cellmorphology was observed using light microscope. Magnification;
10 × (scale: 100 µm) (c) Effect of cSBL on H28 cell proliferation.

0

24

48

72

(h)

After cSBL (5 µM) treatment for 24 h, H28 cells (1 × ­103/well) wereplated in 6-well dishes. After 12 days, cells were fixed and stainedwith 0.1% crystal violet. (d) Viability of cSBL-treated mesothelioma
cell lines. Three mesothelioma cell lines, MESO-1 (white), MESO-4(black) and H28 (gray) were treated with cSBL at various concentration for 48 h. The viability was determined by WST-1 assay. The ratioof viability against control cells was calculated. Data are expressed
as mean ± S.D. from three independent experiments in triplicate. (e)Cells were treated with cSBL (5 µM) for indicated time.The viability
was determined by the same method as described above

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Internalization of cSBL to cancer cells

cSBL‑induced apoptosis

RNA cleavage in cSBL-treated Jurkat cells was observed
after 3 h; thus, it was apparent that cSBL exerts RNase activity in the cells [54]. To evaluate the effects of cSBL on breast
cancer, Kariya et al. confirmed the localization of cSBL in
MDA-MB-231 cells by a immunofluorescence analysis [55].
cSBL was observed in the perinuclear region after 6 h oftreatment. Moreover, the cSBL mutant lacking RNase activity by replacing His103 (essential site for its RNase activity)with Ala (H103A) was also transported to the perinuclear
region in the same cells, indicating that cSBL internalizes
into cancer cells regardless of its RNase activity, and theperinuclear area could be somehow the cSBL acting site.

The first report concerning cSBL-induced apoptosis waspresented by Nitta et al. in collaboration with Dr. Hakomori
at the 1999 International Meeting on Ribonucleases. cSBL
induced apoptotic morphological changes, such as nuclear
condensation and the disappearance of microvilli in mouse
leukemia P388 cells. Subsequently, they revealed that theactivation of caspase-8 and -3, enhancement of annexin V
binding, induction of DNA fragmentation, and expressionof Fas antigen and tumor necrosis factor (TNF) receptor
occurred in cSBL-treated p388 cells [65]. In 2001, Hu et al.
reported that cSBL causes caspase-7 activation in humanbreast cancer MCF-7 cells that lack caspase-3 expression,and that apoptosis is prevented by the overexpression of BclXL in the cells [66]. Therefore, even though observations in
the early study on cSBL-induced cell death showed that protein synthesis was reduced in cSBL-treated cells [10], it wassuggested that cSBL elicits an inducing signal of apoptosis.
After 2013, our group clarified the details of cSBLinduced apoptosis in collaboration with Dr. Hakomori's
group [54, 67, 68]. We used human leukemia Jurkat cells
and observed typical apoptosis-like changes, such as nuclear
fragmentation/condensation, the translocation of phosphatidylserine to the outer layer of the cell membrane, the activation of the caspase family, and the fragmentation of DNA
in cSBL-treated cells. DNA fragmentation was completely
suppressed by the total caspase inhibitor z-VAD-fmk, revealing that cSBL induces apoptosis in a caspase-dependent
manner. We performed experiments using 5,5′6,6'tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanine
iodide (JC-1), which is a mitochondrial membrane
depolarization-detecting reagent. The decrease in mitochondrial membrane potential was almost completely suppressed by
z-VAD-fmk pretreatment in TNF-related apoptosis-inducing
ligand (TRAIL) -treated cells, which induced apoptosis
via the extrinsic pathway. In contrast, the decrease in mitochondrial membrane potential in cSBL-treated cells was notaffected by z-VAD-fmk; that is, cSBL caused strong mitochondrial damage upstream of caspase activation, indicating that cSBL induced apoptosis via the intrinsic pathway[54]. Furthermore, a signal transduction analysis revealed
that the endoplasmic reticulum stress pathway may also be
involved in cSBL-induced apoptosis. In cSBL-treated Jurkat cells, the markers of endoplasmic reticulum stress, suchas an increased expression of calnexin and immunoglobulin
heavy chain binding protein/glucose-regulated protein 78
and the activation of caspase-4 was observed. In addition,
pretreatment with the caspase-4 specific inhibitor z-LEVDfmk attenuated cSBL-induced DNA fragmentation. A comparative study using thapsigargin, an inducer of endoplasmic
reticulum stress apoptosis revealed that the mitochondrial

RI refractoriness of cSBL
The RI, an acidic protein with a molecular weight of ca.
50 kDa, which is present in almost all mammalian cells,
has a leucine-rich repeat structure and forms a characteristic horseshoe shape throughout the molecule [56–58]. The
RI non-covalently binds to RNase A at a ratio of 1: 1, andthe dissociation constant is of the order of fM, indicating
its strong affinity [59, 60]. Therefore, the presence of thisinhibitor is an obstacle for intracellular RNases to exert
their RNA-degrading activity and cytotoxicity. However,like other frog RNases, cSBL does not interact with the
mammalian RI, and the RNase and tumor cell agglutination
activities of cSBL are not affected by this inhibitor [61].
This RI-insensitive feature of cSBL is one of the major differences from other mammalian RNases and provides a great
advantage for its antitumor activity.

cSBL‑induced degradation of cellular RNA
cSBL has conserved catalytic amino acid residues at thesame position as other RNase A superfamily members. It
also has eight cysteine residues that can form four disulfide
bonds. cSBL is essentially a pyrimidine-base-specific RNase[7]. While bovine (RNase A), turtle, and chicken RNases are
essentially cytosine base-preferential [62, 63], cSBL appears
to be a uracil-preferential RNase because the hydrolysis rateof the dinucleotide phosphate UpX consistently exceeds thatof CpX (X = A, G, U, and C) [61]. This property is similar tothat of ONC and iguana RNase [64]. As mentioned above,once cSBL internalizes into cancer cells, it exerts its RNase
activity and degrades cellular RNA followed by the execution of apoptosis, as discussed in the next section.

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161

pathway contributes significantly to cSBL-induced apoptosis [68]. RNase activity has been shown to be requiredfor cSBL to degrade RNA and cause cell death in cancer
cells; however, it remains unclear how cSBL-induced RNA
degradation leads to apoptosis. In our study conducted onhuman leukemia, malignant mesothelioma, and breast cancer
cell lines, JNK and p38 mitogen-activated protein kinases(MAPK) were strongly activated by cSBL treatment, suggesting that these activations may be involved in cSBL-induced

apoptosis. Since treatment with p38 inhibitor and knockdownof p38 attenuated cSBL-induced cell death [55], we speculatethat RNA degradation by cSBL activates these stress kinases,
which leads to apoptosis. On the contrary, inhibition of caspases [54] and p38 MAPK [55] did not completely recovercSBL-induced cell death, although there were statisticallysignificant differences between pretreatment with inhibitors and the control. cSBL might be involved in a p38- andcaspase-independent cell death-inducing pathway, or when

Fig. 3  Scheme of cancer treatment strategy using cSBL. The antitumor mechanism and the preferable properties of cSBL as a chemotherapeutics are summarized. Some events caused by cSBL in cancer cells were mapped and the related events are connected by lines.

cSBL may be an alternative anticancer agent. New combinations withother drugs are expected leading to new and more efficient cancer
treatment strategies

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162

the action of stress kinases and caspases is suppressed, thedramatic effect of RNA breakdown may lead to cell death via
a different pathway.

Other features of cSBL‑induced antitumor
effect
The antitumor effects of cSBL have been verified in manycell lines [69–73], and various studies have reported its highselectivity for cancer cells. Although most of these reports
show that cSBL induces apoptosis, a study conducted byYing et al. reported that cSBL can trigger not only apoptosis
but also autophagocytosis in MCF-7 cells [74]. It has beenreported that cSBL has a highly synergistic cytotoxic effectwith other reagents. Combined with the TRAIL, which has
been shown to have cytotoxic effects on malignant mesothelioma cell lines, or pemetrexed, a key drug for the treatment of malignant mesothelioma, effectively decreases theviability of mesothelioma cells [67, 71]. Furthermore, thecombination with interferon gamma (IFN-γ) induced synergistic cytotoxicity in some hepatoma cells with different
differentiation stages. It should be noted that the effects
of cSBL or cSBL/IFN-γ on different hepatoma cells werecorrelated with the differentiation extent but not the proliferation rate of the cells [75]. Antitumor effects have beenreported not only in ascites cancer-bearing mice [10] but
also in human glioma and malignant mesothelioma xenograft mouse models [70, 72]. In every in vivo examination,cSBL exerted significant antitumor effects without undesired
adverse effects. Although few studies have compared cSBL
with other RNases, Tang et al. reported that cSBL has ahigher cancer specificity than ONC because it is toxic tonormal human HS-68 foreskin fibroblasts; however, cSBL
is not toxic as per their study [76]. Attenuation of apoptosisinducing effects by cSBL is observed during the differentiation of cancer cells by retinoic acids or dimethyl sulfoxide
in human leukemia HL60 cells, the overexpression of Bcl-2
in human breast cancer MCF-7 cells, the reduced expressionof Hsp70 by treatment with quercetin, and the disruptionof cell membrane cholesterols by methyl β-d-cyclodextrin
in mouse leukemia P388 cells [77–80]. On the contrary,cSBL internalization into the cells is enhanced by Japanese encephalitis virus (JEV) infection of the baby hamster
kidney BHK-21 cells, which causes enhanced apoptosis in
JEV-infected BHK-21 cells, indicating that cSBL also possesses antiviral activity [81]. Our recent studies revealed thatcSBL induces apoptosis by decreasing estrogen receptor,
progesterone receptor, and ErbB family proteins in several
breast cancer cell lines [73]; long-term treatment with cSBL
causes pleiotropic changes, including a decreased expressionof aldo–keto reductase 1B10 and adenosine triphosphatebinding cassette transporter C2 in malignant mesothelioma

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Glycoconjugate Journal (2022) 39:157–165

H28 cells [82]. The information described above is expected
to lead to useful applications, such as the effective combination of cSBL with other drugs. Furthermore, cSBL has
several potential advantages as a cytotoxic RNase. Members of the pancreatic RNase superfamily, including cSBL,
are expected to be minimally immunogenic because of theircompact structure and homology with human pancreatic
RNase [83]. In addition, they exhibit high thermal stability
and strong resistance to protein denaturants and proteases[84, 85]. Taken together, cSBL is expected to be an anticancer drug in a new field because it shows unique and cancerselective antitumor effects by a unique mechanism targeting
RNA. The potential role of cSBL in anticancer therapy weresummarized in Fig. 3.
In conclusion, an understanding of the total ability ofcSBL or leczyme is still underway. However, RNA-targetedchemotherapeutic strategies are attractive for the development of non-toxic anticancer agents. Cancer cell selectivityof cSBL, in particular, could make it a potential candidate
for future cancer medicine.
Acknowledgements It is well known that Dr. Sen-itiroh Hakomori was
the pioneer in the research fields of membrane glycolipids and cancer cell biology and also the front runner of biomembrane researchers
worldwide. We have been able to get this far on our improved leczyme
research because of Dr. Hakomori’s help. It is our duty to develop our
project toward translational research on clinical cancer chemotherapy,
showing our appreciation for him.
Funding This study was supported by a Grant-in-Aid for Scientific
Research(C) [grant no. 21K07198 (to Takeo Tatsuta)].

Compliance with ethical standards
Ethical approval This article does not contain any unpublished studies
with human or animals performed by any of the authors.
Conflicts of interest The authors have no conflicts of interest to declarethat are relevant to the content of this article.

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Publisher's Note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional affiliations.

13

MOLECULAR MEDICINE REPORTS 23: 467, 2021

Transcriptomic alterations in malignant pleural
mesothelioma cells in response to long‑term treatment
with bullfrog sialic acid‑binding lectin
TAKEO TATSUTA, ARISU NAKASATO, SHIGEKI SUGAWARA and MASAHIRO HOSONO
Division of Cell Recognition, Institute of Molecular Biomembrane and Glycobiology,
Tohoku Medical and Pharmaceutical University, Sendai, Miyagi 981‑8558, Japan
Received February 3, 2021; Accepted March 29, 2021
DOI: 10.3892/mmr.2021.12106
Abstract. Malignant pleural mesothelioma (MPM) is auniversally lethal type of cancer that is increasing in incidence
worldwide; therefore, the development of new drugs for MPM
is an urgent task. Bullfrog sialic acid‑binding lectin (cSBL)
is a multifunctional protein that has carbohydrate‑binding
and ribonuclease activities. cSBL exerts marked antitumor
activity against numerous types of cancer cells, with low
toxicity to normal cells. Although in vitro and in vivo studies
revealed that cSBL was effective against MPM, the mecha‑
nism by which cSBL exerts antitumor effects is not fully
understood. To further understand the mechanism of action ofcSBL, the present study aimed to identify the key molecules
whose expression was affected by cSBL. The present study
established cSBL‑resistant MPM cells. Microarray analyses
revealed that there were significant pleiotropic changes inthe expression profiles of several genes, including multiple
genes involved in metabolic pathways in cSBL‑resistant cells.
Furthermore, the expression of some members of the aldo‑keto

Correspondence

to: Dr Masahiro Hosono, Division of
Cell Recognition, Institute of Molecular Biomembrane and
Glycobiology, Tohoku Medical and Pharmaceutical University,
4‑4‑1 Komatsushima, Aobaku, Sendai, Miyagi 981‑8558, JapanE‑mail: mhosono@tohoku‑mpu.ac.jp
Abbreviations:

RNase,
ribonuclease;
ONC,
onconase;cSBL, bullfrog sialic acid‑binding lectin; DEG, differentiallyexpressed gene; ATF3, activating transcription factor 3; cSR,
cSBL‑resistant; DOX, doxorubicin; AKR, aldo‑keto reductase;
KEGG, Kyoto Encyclopedia of Genes and Genomes; RT‑qPCR,
reverse transcription quantitative polymerase chain reaction;
PPI, protein‑protein interaction; SLC47A2, solute carrierfamily 47 member 2; CBR, carbonyl reductase; GAPDH,
glyceraldehyde‑3‑phosphate dehydrogenase; RR, resistance rate;
RINe, RNA integrity number equivalent; GO, Gene Ontology

Key words: antitumor drug, sialic acid‑binding lectin, cytotoxic

ribonucleases, microarray profiling, metabolism of cancer cells,
aldo‑keto reductase

reductase family was revealed to be markedly downregulated
in these cells. Among these, it was particularly interestingthat cSBL action reduced the level of AKR1B10, which has
been reported as a biomarker candidate for MPM prognosis.
These findings revealed novel aspects of the effect of cSBL,
which may contribute to the development of new therapeuticstrategies for MPM.
Introduction
Malignant pleural mesothelioma (MPM) is an aggressive
cancer of the mesothelial cells lining the pleural surface ofthe chest wall and lung (1). The main cause of carcinogenesisof this disease is thought to be exposure to an environmental
carcinogen, asbestos (2); furthermore, the involvement of
SV40 and exposure to radiation have been suggested as
cofactors (3,4). The MPM patient prognosis is very poor
and available therapies have still a limited impact on MPM
progression (5). Until recently, antifolate and platinum combi‑
nation chemotherapy was the only established treatment (6,7).
These days, the development of immune‑checkpoint inhibi‑
tors has contributed to the improvement of MPM treatment.
The data from the phase III trial (NCT02899299) designed toevaluate nivolumab plus ipilimumab compared with conven‑tional chemotherapy (pemetrexed and cisplatin or carboplatin)
showed a statistically significant overall survival (OS) benefit
in patients with previously untreated, unresectable MPM, and,
only recently, the FDA has finally approved nivolumab plus
ipilimumab for previously untreated unresectable MPM (8).
Although the nivolumab and ipilimumab combination has
extended the OS (a median OS of 18.1 months compared to14.1 months for platinum‑based standard of chemotherapy) (8),the prognosis is still poor, and research such as identificationof new biomarkers in invasive mesothelioma (9), and search
for new drugs for MPM (10) have been actively conducted
for further improvement of MPM care. In particular, thedevelopment of drugs through different mechanisms of action
will continue to be required in the future.
A major focus in cancer research is the development of
new therapeutic agents that induce cell death in malignant
neoplasms but do not increase inflammation or have signifi‑
cant side effects in normal tissue. Cytotoxic ribonucleases

2

TATSUTA et al: cSBL CAUSES DOWNREGULATION OF AKR1B10(RNases) are a new field of anticancer drug candidates thattarget RNA and several cytotoxic RNases have been reported
to have antitumor effects (11,12). The vertebrate‑secreted
RNase superfamily, also called the RNase A superfamily,
includes several cytotoxic RNases, such as onconase (ONC)from Rana pipience (13) and a variant of human pancreatic
ribonuclease carrying a nuclear localization signal (PE5) (14).
Unlike clinically used chemotherapeutic drugs that target DNA
synthesis and transcription, cytotoxic RNases are believed to
be non‑mutagenic because they target RNA functions, such as
RNA translation or gene regulation (15).
Sialic acid‑binding lectin from Rana catesbeiana (cSBL),
also known as RC‑RNase, is a multifunctional protein thatbinds carbohydrates and has a ribonuclease activity (16‑18).
It was originally identified as a lectin that recognizes sialic
acid‑containing complexes (17), and protein sequence analysis
revealed that it belonged to the vertebrate‑secreted RNase
superfamily (19). It has previously been identified that cSBL
has remarkable antitumor activity against many types ofcancer cells and low toxicity in normal cells (20‑25). Thiseffect was observed in not only in vitro experiments but also
in vivo studies (18,20,26). Our own recent studies revealed
that cSBL induced apoptosis in cancer cells via the intrinsic
pathway (27,28), and that the RNase activity of cSBL wasessential for its antitumor effect (29). The effectiveness of cSBL
has also been studied for in MPM. We reported that althoughcSBL had very low cytotoxicity in the normal pleural meso‑
thelial cell line Met5A, it efficiently reduced the viability of
MPM cells including H28, Meso‑1, Meso‑4, H2452 and MSTO
cells (30,31). We found that pemetrexed + cSBL exhibited astrong synergistic effect that was even superior to the standard
regimen of pemetrexed + cisplatin (31). Furthermore, in vivo
study revealed that cSBL showed a significant tumor growth
inhibitory effect in multiple MPM xenograft models without
any adverse effects, even under conditions where previously
established pemetrexed administration had little or noeffect (26). However, the antitumor mechanism of cSBL is still
unclear, especially when the response of cancer cells to cSBL
application is concerned.
Despite the potential of RNases in cancer treatment, few
studies have identified genes whose expression was altered bycytotoxic RNases. This may be because the RNA extracted
from cytotoxic RNase‑treated cells is likely to be degradedby the RNA‑catabolizing action of the RNase. Therefore, it
is technically difficult to assess differentially expressed genes(DEGs) in cytotoxic RNase‑treated cells. In recent years, some
remarkable research breakthroughs have been made in studies
using microarray analysis. Previous studies using microarray
technology have been able to determine that ONC caused
upregulation of activating transcription factor 3 (ATF3), which
was important for its antitumor effect of ONC (32,33), andthat PE5 caused pleiotropic effects, including gene expression
changes mainly related to metabolism (34). These studies
pioneered the study of gene expression after treatment withcytotoxic RNases. However, these findings were reported onlyin conditions in which there was little RNA degradation, thatis, there was very little antitumor effect. Moreover, no gene
expression studies have involved cSBL.
To further understand the antitumor effects of cSBL, we
treated cSBL‑sensitive MPM cells with cSBL to establish

cSBL‑resistant (cSR) cells. Then, microarray analysis was
performed to identify significantly altered genes in thecSBL‑sensitive and cSR cell lines.
Materials and methods
Reagents. cSBL was isolated from acetone‑dried powderof unfertilized bullfrog body‑cavity eggs using sequential
chromatography with Sephadex G75, DEAE‑cellulose,hydroxyapatite, and SP‑Sepharose (Cytiva), as previously
described (17). For the preparation of ONC, ONC cDNA wascloned into the pET‑11d plasmid (Merck KGaA) in conjunction
with the pelB sequence. BL21 (DE3) pLysS cells (Promega)
were transformed with the plasmid, and its expression was
induced by adding isopropyl β ‑D‑1‑thiogalactopyranoside(0.2 mM) at 34˚C for 72 h. ONC recombinant protein was
purified from the culture liquid by sequential chromatog‑
raphy with Sephadex G75, DEAE‑cellulose, hydroxyapatite,
and SP‑Sepharose. Doxorubicin (DOX) was purchased from
Sigma‑Aldrich. The anti‑caspase‑3 antibody (cat. no. #9662),
peroxidase‑conjugated anti‑mouse IgG and anti‑rabbit IgG
antibodies (cat. no. #7074 and #7076, respectively) werepurchased from Cell Signaling Technology. The anti‑aldo‑keto
reductase (AKR) 1B10 antibody (cat. no. ab96417) was
purchased from Abcam (Cambridge, UK) The anti‑β ‑actin
antibody (clone AC‑74, cat. no. A2228) was purchased from
Sigma‑Aldrich.
Establishment of cSBL‑resistant cell lines. H28 cells werepurchased from the American Type Culture Collection. For
the establishment of cSR cell lines, cells were cultured withcomplete medium containing stepwise increasing concentra‑
tions of cSBL. The starting concentration was 0.01 µM in which
H28 cells could manage to survive, and the final concentration
was set as 0.5 µM, which is 50 times higher than the initialconcentration. The cells were passaged approximately 50 times
to reach the final concentration. These resistant cells wereproven to be stably resistant even after at least 10 passages indrug‑free complete medium. After that, cells were cloned using
limiting dilution. We confirmed five resistant clones, whichwere designated cSR‑A1, ‑A2, ‑B1, ‑B2 and ‑C1. To ensure thatthese clones were permanent and stable, they were cultured forfive passages in the presence of 0.5 µM cSBL. For the analyses
described here, cultures of less than 20 passages after thecloning were used. All cell lines were cultured in RPMI 1640
medium (Nissui) supplemented with 10% fetal bovine serum,
penicillin‑G (100 U/ml), and streptomycin (100 g/ml) (Thermo
Fisher Scientific, Inc.). Cells were maintained in a 5% CO2 incu‑
bator at 37˚C under humidified conditions. Cell morphology
was observed using an IX71 microscope (Olympus).
Colony formation assay. Cells were seeded in 6‑well plates(Corning, cat. no. 353046, 1x103 cells per well). After 24 h,cells were treated with medium alone or with cSBL (1, 5, 10,25, or 50 nM) for 12 days. Then, cells were fixed with para‑
formaldehyde for 15 min and stained with crystal violet for
10 min. Colonies were photographed by Gel Doc XR system(Bio‑Rad Laboratories, Inc.) and counted using Quantity One
software (Bio‑Rad Laboratories, Inc.). Each experiment was
performed in triplicate.

MOLECULAR MEDICINE REPORTS 23: 467, 2021

WST‑8 assay. The WST‑8 assay was performed to determine
the cell viability. Cells (5x10 4 cells/ml) cultured in 96‑wellplates (Corning, cat. no. 353072, 100 µl/well) were treated with
cSBL, ONC, or DOX at the indicated concentrations for 72 h.
Then, the cells were incubated with Cell Count Reagent SF(Nacalai Tesque Inc.) for 1‑4 h. The absorbance of the resulting
product at 450 nm was measured, and the background absor‑
bance at 650 nm was subtracted. Experiments were conductedin triplicate.
Western blotting. Cells (5x104 cells/ml) were cultured in 6‑wellplates (Corning, cat. no. 353046, 4 ml/well) and treated withcSBL or control. 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 deoxycho‑late, and 0.1% sodium dodecyl sulfate] supplemented withcomplete™ Mini EDTA‑free protease inhibitor cocktail tablets(one tablet/10 ml; Roche Applied Science). Soluble proteins
were collected, and the protein concentration was measured
using a BCA protein assay kit (Thermo Fisher Scientific, Inc.,
cat. no. 23227) according to the manufacturer's instructions.
Proteins were separated using 10 or 14% SDS‑PAGE andtransferred onto Immobilon‑P transfer membranes (Thermo
Fisher Scientific, Inc.). The membranes were sequentially
incubated with primary and secondary antibodies diluted in
Can Get Signal solution (Toyobo Co., Ltd.). Protein bands
were detected using ECL Prime Western Blotting Detection
Reagent (Cytiva). The relative density of the protein bands wasmeasured using ImageJ 1.51s software (National Institutes of
Health). Experiments were repeated in triplicates.
Total RNA isolation. Total RNA was isolated from cells using
TRI Reagent (Molecular Research Center) and purified using
the SV Total RNA Isolation System (Promega) according to
the manufacturer's instructions. RNA samples were quanti‑
fied using an ND‑1000 spectrophotometer (NanoDrop
Technologies), and the quality was confirmed using a 2200
TapeStation (Agilent Technologies). The RNA integrity
number equivalent (RINe), which was an index of RNA degra‑
dation, was calculated from the 28S and 18S ribosomal RNAband peak values and other band peak values in the electro‑
phoretic image. For the subsequent cDNA labeling, the Agilent
Low‑Input QuickAmp Labeling kit (Agilent Technologies,
cat. no. 5190‑2305) was used.
Gene expression microarrays. cDNA was amplified, labeled,and hybridized to a 60K Agilent 60‑mer oligomicroarrayaccording to the manufacturer's instructions. All hybridized
microarray slides were scanned using an Agilent scanner.
Relative hybridization intensities and background hybridiza‑tion values were calculated using Agilent Feature Extraction
Software (9.5.1.1).
Data analysis and filter criteria. Raw signal intensities andflags for each probe were calculated from hybridization
intensities (gProcessedSignal) and spot information (gIsSat‑
urated, etc.), according to the procedures recommended
by Agilent. [Flag criteria on GeneSpring Software was asfollows: Absent (A): ‘Feature is not positive and significant’and ‘Feature is not above background;’ Marginal (M):3

‘Feature is not Uniform,’ ‘Feature is Saturated,’ and ‘Feature
is a population outlier;’ and Present (P): others.]. The raw
signal intensities of two samples were log2‑transformed andnormalized by quantile algorithm with the Bioconductor
preprocessCore library package (35,36). We selected probesthat called the P flag in at least two samples. To identify
up‑ and downregulated genes, we calculated Z‑scores (37)
and ratios (non‑log scaled fold‑change) from the normal‑
ized signal intensities of each probe to compare control andexperimental samples. Then, we established the following
criteria for differentially regulated genes: Upregulated
genes: Z‑score ≥2.0 and ratio ≥1.5‑fold and downregulated
genes: Z‑score ≤‑2.0 and ratio ≤0.66. Data have been depos‑
ited in NCBI's Gene Expression Omnibus repository (38)(http://www.ncbi.nih.gov/geo) under the accession number:
GSE 162286.
Functional annotation of DEGs in cSR cell lines. DEGs in cSRcells were characterized functionally using a hypergeometric
test to find overrepresented gene ontology terms in the threemain broad ontologies (biological process, molecular function,and cellular component) (39,40). DEGs were also mapped to
the Kyoto Encyclopedia of Genes and Genomes (KEGG) (41),which assigns proteins to pathways, to find overrepresented
pathways. The analyses were done using the Database forAnnotation, Visualization, and Integrated Discovery online
tool (42).
Network analysis. GeneMANIA (43), an online database that
identifies other proteins associated with a set of input genes,
was used to generate protein‑protein interaction (PPI) network
images. The associations between co‑expression, colocaliza‑
tion, predicted related genes, shared protein domains, genetic
interactions, and physical interactions were determined using
GeneMANIA.
Reverse transcription quantitative polymerase chain
reaction (RT‑qPCR). mRNA expression of the top five down‑
regulated genes (THY1, AKR1B15, AKR1B10, SLC47A2, and
CBR1) were examined using RT‑qPCR. Cells (2x105) were
cultured for 48 h, and total RNA was extracted using an
AllPrep RNA/Protein kit (Qiagen, cat. no. 80404). cDNA
was synthesized from total RNA (1 µg) using a SuperScript
VILO cDNA Synthesis Kit (Invitrogen, cat. no. 11754050).
RT‑qPCR was performed using a LightCycler 480 system with
the LightCycler 480 Probes Master Kit (Roche Diagnostics,
cat. no. 04707494001). PCR primers using a TaqMan/probelibrary 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 cycle threshold values.
Statistical analysis. The results from three or more independent
experiments were expressed as the mean ± standard deviation.
Statistical analyses were conducted using GraphPad Prism 5.0(GraphPad Software, Inc.), and comparisons were made using
one‑way analysis of variance followed by Bonferroni's post
hoc test. P<0.05 was considered statistically significant.

4

TATSUTA et al: cSBL CAUSES DOWNREGULATION OF AKR1B10

Results
Establishment of cSR cells. cSR cells were established byadding different cSBL concentrations at the low range to theculture medium. Five clones were obtained by limiting‑dilution
cloning. The sensitivity of the clones to cSBL was assessedusing colony formation assay. As shown in Fig. 1A (upper
panel), no colonies were found in parental H28 cells treated withcSBL at concentrations of 25 nM or higher. In contrast, all fiveclones showed resistance to cSBL. We calculated IC50 values
from dose‑response curves (Fig. 1B). The resistant rate (RR)
represented the ratio of the IC50 value in cSR cells to the IC50
value in H28 cells (Fig. 1A, lower panel). cSR‑A1 and cSR‑B1
had the highest RRs (15.3 and 14.5, respectively); therefore,
these two clones were utilized in subsequent experiments.
Analysis of cSR cell lines characteristics. The growth curvesof H28, cSR‑A1, and cSR‑B1 cells were examined (Fig. 2A).
There was no significant difference in the growth rates among
these three cell lines. However, cSR cells tended to show
shrunken morphology in low‑density culture conditions, eventhough no difference was observed in high‑density culture
conditions (Fig. 2B). The cSBL resistance was then evaluated.
After treating cells with cSBL, ONC, or DOX for 72 h, the cell
viability was measured using the WST‑8 assay, dose‑response
curves were prepared, and the IC50 values and RRs were calcu‑
lated (Fig. 2C). The RRs of cSR cells to cSBL in the WST‑8
assay were 4.4 (A1) and 4.6 (B1), which were lower than thoseobtained in the colony formation assay. Further, the RRs to
ONC and DOX were lower than to cSBL; the RRs to ONC
for cSR‑A1 and cSR‑B1 were 1.6 and 2.0, respectively, andthe corresponding RRs to DOX were 0.7 and 1.0, respectively.
Similar to what we observed for cSBL, cSR cells tended to show
some resistance to RNA‑targeted ONC. However, for DOX,a DNA‑damaging anticancer drug, the RRs of cSR cells did
not exceed 1, i.e., the effect of DOX was not different between
parent H28 and cSR cells. Since it has been shown that the
antitumor effect of cSBL was due to the induction of apoptosis,
we examined whether the cSR cells had reduced apoptosis aftercSBL treatment. Cells were treated with cSBL (1 or 5 µM)
for 72 h, and protein was extracted to examine the levels ofcleaved caspase‑3 (Fig. 2D). There was less cleaved caspase‑3
in cSBL‑treated cSR‑B1 cells than in H28 cells, but the differ‑ence was not significant. Taken together, our results indicatethat cSR cells become resistant to cSBL after long‑term treat‑ment with low concentrations, but they have relatively weakresistance to short‑term treatment with high concentrations. In
addition, there was no significant difference between H28 andcSR cells in terms of proliferation, morphology, or apoptosis
after treatment with high cSBL concentrations.
Altered gene expression in cSR cell lines. Total RNA wasextracted from H28, cSR‑A1, and cSR‑B1 cells and RNA
quality was evaluated. All samples had RINe values of 10.0
(Fig. S1), indicating that RNA could be extracted in a condi‑tion when it was hardly degraded. Therefore, these samples
were used for microarray analyses. A comparison of theexpression profiles in the cSR‑A1, cSR‑B1, and parental H28
cell lines revealed that 1254 genes (623 upregulated and631 downregulated) were dysregulated in cSR‑A1 cells andFigure 1. Effect of cSBL on colony formation in H28 and cSR cells.
(A) Colony formation assays are performed in the absence or presence of
cSBL (1 to 50 nM). Representative images from three independent experi‑ments are shown. IC50 values and RRs of each cell line are indicated below
the images. (B) Colony numbers in (A) are counted and dose‑response curves
are depicted. Each data point represents the mean ± SD of three independent
experiment. The statistical significance of the colony formation of the cells
compared to the control H28 cells were shown. *P<0.05, **P<0.01 vs. H28.
cSBL, bullfrog sialic acid‑binding lectin; cSR, cSBL‑resistant; RR, resis‑
tance rate; SD, standard deviation.

1,225 genes (608 upregulated and 617 downregulated) weredysregulated in cSR‑B1 cells compared to H28 cells. Amongthem, 927 genes (440 upregulated and 487 downregulated)were common DEGs out of 37,756 known coding transcripts
on the microarray (2.46%). The fold change ranged from
1.5‑ to 934.8‑fold for upregulated genes and 1.5‑ to 755.7‑foldfor downregulated genes. The top 20 up‑ and downregulated
genes in cSR cell lines are listed in Table SI.
GO enrichment analysis. To further understand the functional
relevance of DEGs in cSR cell lines, we performed gene
ontology analysis. The 927 DEGs were used to extract theassociated ontologies based on three broad ontology catego‑ries: ‘biological process,’ ‘molecular function,’ and ‘cellularcomponent.’ In all cases, a P‑value ≤0.05 was considered
statistically significant. The 20 most enriched GO terms forthe DEGs are listed in Table SII. There were 123 signifi‑
cantly enriched GO terms in the biological process category.

MOLECULAR MEDICINE REPORTS 23: 467, 2021

5

Figure 2. Analysis of cSR cell line characteristics. (A) Growth of H28 and cSR cells. Cells are seeded at 2x105 cells/well and counted at the timepoints(3 to 17 days) as indicated. Each data point represents the mean ± SD of three independent experiment. (B) Morphology of H28 and cSR cells. Cells areseeded at 1x105 cells/well. After 24 h (low density) or 72 h (high density), cell morphology is observed. Scale bar, 100 µm. (C) Effect of cSBL, ONC, and
DOX on H28 and cSR cells. Viability of cells treated with the drugs are measured by the WST‑8 assay. IC50 values and RRs are calculated from the depicted
dose‑response curves. Each data point represents the mean ± SD of three independent experiment performed in triplicates. (D) Apoptosis induction in H28
and cSR‑B1 cells treated with cSBL. Cells are treated with cSBL (1 or 5 µM), and the expression of cleaved caspase‑3 is detected using western blotting.
Densitometric quantification is performed using the results of three independent experiments (mean ± SD). The statistical significance of the bands comparedto the non‑treated control were shown. *P<0.05, **P<0.01 vs. 0 µM cSBL. cSR, cSBL‑resistant; cSBL, bullfrog sialic acid‑binding lectin; ONC, onconase; DOX,
doxorubicin; RR, resistance rate; SD, standard deviation.

The most significant term was oxidation‑reduction process(GO:0055114). Interestingly, the GO terms included not only
terms related to cancer characteristics, such as cell prolif‑
eration (GO:0008284, GO:0008285), adhesion (GO:0007155,
GO:0007162), migration (GO:0016477), and apoptosis(GO:0006915), but also several metabolic processes related
to lipids (GO:0006869), cellular protein (GO:0044267), anddrugs (GO:00171449). In the molecular function category, 36
functions were enriched, and the most significantly enriched

term was integrin binding (6.87E‑07). There were several
other binding functions, including growth factors [IGF
(GO:0005520), FGF (GO:0017134), TGF (GO:0050431), and
EGF (GO:0005154)] and other cell membrane molecules
such as receptors (GO:0005102), heparin (GO:0005102),and syndecan (GO:0045545). Thirty‑two components wereenriched in the cellular component category. The top four
GO terms in cellular component included ‘extracellular’[extracellular space (GO:0005615), extracellular exosome

6

TATSUTA et al: cSBL CAUSES DOWNREGULATION OF AKR1B10

Figure 3. The protein‑protein interaction networks of the top 10 up‑ and downregulated DEGs (upper panel) and the 57 genes involved in metabolic pathways(lower panel). Different colors represent diverse bioinformatics methods. DEGs, differentially expressed genes.

(GO:0070062), extracellular matrix (GO:0031012), andextracellular region (GO:0005576)]. They were followedby terms related to the cell membrane, such as cell surface(GO:0009986), basement membrane (GO:0005604), andplasma membrane (GO:0005886). Altogether, it appears that
there are many differences between parental H28 cells andcSR cells in cellular functions, especially in association withcancer characteristics and metabolic processes. Extracellular‑
and cell membrane‑associated GO terms were highly enrichedin the DEGs of cSR cell lines.

Pathway analysis. To analyze the signaling pathways
affected by DEGs in cSR cell lines, we analyzed DEGs using
the KEGG database. Table SIII shows the 18 significantly
affected pathways in cSR cell lines. Among the significantly
enriched pathways (determined by a hypergeometric test
where P<0.05), ‘metabolism of xenobiotics by cytochrome
P450’ was the most significant. Additionally, there wereseveral pathways involved in various metabolic processes.
Fifty‑seven genes were involved in metabolic pathways, allrelated to lipid and carbohydrate metabolism. These results

MOLECULAR MEDICINE REPORTS 23: 467, 2021

7

Figure 4. Expression of THY1, AKR1B15, AKR1B10, SLC47A2, and CBR1 mRNA in H28 and cSR cells. Quantitative RT‑PCR is performed using specificprimers and GAPDH (control gene). The expression levels of genes are normalized to the level of GAPDH, and the level of the corresponding gene in H28 cells(control cell line) is set at 1. Data are presented as mean ± SD of three independent experiment performed in triplicates, and the mean values are indicatedon the X axis titles. *P<0.05, **P<0.01, ***P<0.001 vs. H28. cSR, bullfrog sialic acid‑binding lectin‑resistant; RT‑PCR, reverse transcription polymerase chain
reaction; SD, standard deviation; THY1, Thy‑1 Cell Surface Antigen (CD90); AKR, aldo‑keto reductase; SLC47A2, solute carrier family 47 member 2; CBR1,
carbonyl reductase; GAPDH, glyceraldehyde‑3‑phosphate dehydrogenase.

prompted our interest in further analysis of the interactions
among these DEGs.
PPI network analysis. The networks of proteins encoded by
the top 10 up‑ and downregulated DEGs and the 57 genes
in the KEGG metabolic pathways were identified using the
GeneMANIA PPI network (Fig. 3). The color of the line
represents the type of interaction, and the size of the node
indicates the degree of interaction in the PPI, where larger
nodes have more interactions. These data demonstrated there
were some protein families whose expression was affected
in cSR cells, such as AKR or UDP‑glucuronosyltransferase.
However, there were no significant hub proteins in either PPI
network, suggesting that long‑term exposure of cSBL affectedgene expression in a pleiotropic fashion.
RT‑qPCR analysis. As indicated above, we found that theexpressions of some AKR family members were affected
in cSR cells (Fig. 3). Two of these genes, AKR1B15 andAKR1B10, were among the top three most downregulated
genes in cSR cells (Table SI). Therefore, we were interested
in the AKR family, which has been reported to be associatedwith cancer, and conducted subsequent studies focusing on
highly downregulated genes in cSR cells. In order to confirm
the reproducibility of the microarray, we compared gene

expression patterns between parental and cSR cells using
RT‑qPCR. As shown in Fig. 4, THY1, AKR1B15, AKR1B10,
SCL47A2, and CBR1 all had reduced expression in cSR‑A1 andcSR‑B1 cells. These genes had 22.8‑ to 483.2‑fold decreased
expression, and this downregulation was similar in bothcSR‑A1 and cSR‑B1 cells. Although these fold changes weredifferent from those observed in the microarray analysis, they
were in the same direction. Therefore, RT‑qPCR confirmedthat the microarray experiments were valid and showed thatthe changes were highly significant.
Confirmation of AKR1B10 downregulation using westernblot. Next, we further examined protein expression of the
AKR family members that were downregulated in cSR cells.
We found that the expression of six AKR family genes weredecreased from 9.1‑ to 562.0‑fold in the microarray analysis(Table I). We focused on AKR1B10, which has been reported
to be associated with cancer (44) and whose antibody wascommercially available. The expression of AKR1B10 atthe protein level was confirmed using western blotting. A
significant decrease in AKR1B10 expression was observed
in cSR‑A1 and cSR‑B1 cells compared to H28 cells (Fig. 5).
Therefore, the decreased expression of AKR1B10 as detectedby the microarray analysis was also observed at the protein
level.

8

TATSUTA et al: cSBL CAUSES DOWNREGULATION OF AKR1B10

Table I. Gene expression changes of AKR family in cSR cells.
Gene symbol

Fold changea

AKR1B15
AKR1B10
AKR1C1
AKR1C4AKR1B1
AKR1C3‑562.0‑548.5
‑54.0
‑24.7
‑11.4
‑9.1

Fold change in microarray analysis.

aFigure 5. AKR1B10 protein expression in H28 and cSR cells. The expres‑sion of AKR1B10 in each cell line is detected using western blotting.
Densitometric quantification is performed using the results of three inde‑
pendent experiments (mean ± SD). ***P<0.001 vs. H28. cSR, bullfrog sialic
acid‑binding lectin‑resistant; SD, standard deviation.

Further investigation of DEGs other than the AKR family incSR cells. Finally, we further investigated genes that showed
expression fluctuations based on the results of microarray
analysis. We found that the expression of some members of
ATP‑binding cassette (ABC) transporter superfamily wasdecreased in cSR cells (Table SIV). The expression of ABCC2
was ‑17.3 times lower and ABCA1 was ‑4.7 times lower in cSRcells.
Discussion
In this study, we produced cSR cells by treating sensitive H28
cells with low doses of cSBL over a long period. We also
succeeded in identifying genes whose expression was alteredin cSR cells (Table SI). The GO analysis and pathway analysis
indicated that these genes were related to cell proliferationand cell membrane, as well as sugar and lipid metabolism(Tables SII and SIII). We also found that AKR1B10 was
significantly reduced in cSR cells at the protein level (Fig. 5).
These findings indicate that long‑term treatment of cancer
cells with cSBL alters many metabolic genes and reduces theexpression of AKR family genes, revealing a new aspect of theantitumor effect of cSBL.
Analysis of cSR cell lines characteristics revealed that
although cSR cells tended to exhibit shrunken morphology inlow‑density culture conditions, we could not detect significant
differences in morphology in high‑density culture conditions,proliferation or apoptosis after treatment with high cSBL
concentrations (Fig. 2). The reason for the morphological change

observed in low‑density culture conditions is unclear, but we
speculate that this change may have been caused by changes
in expression of genes related to the morphology, adhesion
or migration. Indeed, GO enrichment analysis showed there
were some enriched GO terms such as cell surface and plasma
membrane in cellular component category or integrin binding
in molecular function category. The RRs to cSR were high (15.3
and 14.5 for cSR‑A1 and ‑B1, respectively) in the colony assay
but low (4.4 and 4.6 for cSR‑A1 and ‑B1, respectively) in the
WST‑8 assay (Figs. 1 and 2). By the nature of the experiment,
the colony assay observed colony forming ability for a relatively
long time (12 days) in the presence of low concentrations of cSBL
(1‑50 nM) and the WST‑8 assay monitored survival changes in
the presence of high concentrations of cSBL (0.01‑10 µM) fora short period of time (72 h). This indicates that cSR cells areresistant to long‑term treatment with low concentrations ofcSBL, but under short‑term treatment conditions with relatively
high concentrations, the apoptosis‑inducing effect of cSBL is
also observed in cSR cells. In other words, it is suggested thateven in cSR cells that have acquired resistance by some mecha‑
nism in this long‑term treatment, intracellular RNA is cleaved
and apoptosis is induced by cSBL treatment exceeding a certain
concentration. The dramatic mechanism of action that involves
RNA cleavage may interfere with the ability of cancer cells todevelop high resistance to cSBL.
Experiments that require RNA extracted from cytotoxic
RNase‑treated cells, such as RNA expression analysis, aredifficult to perform. Such experiments often have low accu‑racy because the extracted RNA is presumably not intact in
the cells that are treated with cytotoxic doses of RNase, andthe RNase may degrade RNA even during the cell lysing step.
However, several recent reports identified DEGs in cytotoxic
RNase‑treated cells using microarray technology. Theseexperiments were carried out in very strict conditions, suchas including only RNA with acceptable concentrations andA260/280 ratios or high RIN values, as it was done in this experi‑
ment (32‑34). Some of the identified genes whose expression
was affected by cSBL treatment were also identified in a
microarray examining cells treated with PE5. Since we used
resistant cells, the identified DEGs may have include genes
involved in the resistance to cSBL. However, the decreasein the AKR family detected here was also observed in the
short‑term treatment of PE5; PE5 is known to reduce theexpression of AKR1A1, a member of the AKR family (34).
Therefore, the downregulation of AKR family members might
be a universal response of cancer cells to cytotoxic RNase or
involved in the antitumor effects of cytotoxic RNases. The
microarray analyses revealed that there were significant pleio‑
tropic changes including those in the expression of multiple
genes involved in metabolic pathways in cSBL‑resistant cells(Table SIII). Some of these metabolic pathway related genes
are listed among the top 20 list of genes up‑ or down‑regulated
in cSR cells (Table SI). Among the up‑regulated genes, theincrease in expression of LIPC which catalyzes the hydro‑
lysis of triglycerides and phospholipids (45), was the highest(934.8 fold higher in cSR cells, Table SI). ST6GAL2, whichshowed the third largest change (126.7 fold higher in cSR cells,
Table SI), is an enzyme that transfers sialic acid from the donorof substrate CMP‑sialic acid to galactose containing acceptor
substrates (46). It is interesting to note that the expression

MOLECULAR MEDICINE REPORTS 23: 467, 2021

level of this enzyme was elevated, because the presence of
sialic acids at the cell surface is thought to be important for
the effect of cSBL (20). In this study, because we found thatthe expressions of some AKR family members were affected
in cSR cells, further investigations were focused mainly onstrongly downregulated genes in cSR cells.
The AKR superfamily is a family of enzymes that revers‑
ibly reduce carbonyl groups (47). These proteins catalyze avariety of metabolic oxidation‑reduction reactions, including
reduction of glucose, glucocorticoids, small carbonyl metabo‑lites, glutathione conjugates, and phospholipid aldehydes (48).
More than 150 proteins belonging to this superfamily areclassified into 15 families (AKR1 to AKR15) based on thesimilarity of amino acid sequences. Each family is further
subdivided into subfamilies, which have 60% or higher
similarity at the amino acid level (47). The largest family,
AKR1, is subdivided into six subfamilies (AKR1A, AKR1B,
AKR1C, AKR1D, AKR1E, and AKR1G). In humans, there
are 14 AKR superfamily proteins, nine of which belong to
the AKR1 family (49). Our microarray analysis revealed thatmultiple AKR genes were downregulated in cSR cells. In
addition, when we focused on AKR1B10, which is involved
in resistance to anticancer drugs and has attracted attention asa new target in cancer therapy (49), we found that its expres‑sion was significantly reduced at the protein level in cSR cells.
AKR1B10 has been reported to be overexpressed in lungcancer (50), liver cancer (51,52), breast cancer (50), pancreaticcancer (53), and oral squamous cell carcinoma (54). One of theroles of AKR1B10 in cancer cells is to suppress the produc‑tion of retinoic acid, a cell differentiation‑promoting factor.
Retinoic acid is produced from retinol via retinal and binds
to the nuclear receptors, retinoic acid receptor and retinoid
X receptor, to promote cell differentiation (55). AKR1B10
is thought to promote cancer cell proliferation and survival
by reducing retinal to retinol, thereby decreasing intracel‑lular retinoic acid production (56,57). In addition, AKR1B10
promotes cancer cell survival by reducing cytotoxic aldehydes
produced by lipid peroxidation, such as 4‑hydroxynon‑
enal (58,59), and is involved in resistance to anticancer drugs
such as cisplatin, mitomycin C, anthracyclines (doxorubicinand idarubicin), and docetaxel (60‑62). Therefore, AKR1B10
has potential not only as a cancer biomarker but also as anovel therapeutic target for cancer treatment and may promote
chemosensitization. Although reports on AKR1B10 in MPM
are very limited, AKR1B10 may also be associated withmalignancy in MPM, as in the other cancer cases indicatedabove. A study aimed to search for novel biomarkers inmalignant mesothelioma performed by Mundt et al (63), iden‑tified that AKR1B10 was one of the prognostic mesothelioma
biomarker candidates. Patients with high AKR1B10 levels
had a mean survival time that was 5.5 months shorter thanthat in patients with low AKR1B10 expression levels (5.5 vs.
11.0 months, respectively; N=14 for high and 13 for low expres‑sion level). Usami et al (64) established two morphologicallydistinct MPM cell lines, Y‑MESO‑8A (epithelial‑like) andY‑MESO‑8D (spindle‑like) from the same patient. Microarray
analysis to determine differences in gene expression in thesecells showed that the expression of AKR1B10 and AKR1C3
in Y‑MESO‑8D were 17.8 and 6.35 times higher, respectively,
than that of Y‑MESO‑8A (64). Another report showed that

9under serum starvation conditions, AKT was phosphorylatedin Y‑MESO‑8D but not in Y‑MESO‑8A (65). However, there
are no reports related to the function of AKR1B10 in thosecells. Detailed investigations focusing on the function of
AKR1B10 in MPM are needed. It is interesting to note that
decreased AKR1B10 expression was observed at the protein
level in cells established by long‑term exposure to cSBL. It is
possible that cSBL could be used to promote chemosensitivity
to anticancer drugs. Indeed, Toyooka and Hayakawa's grouphas succeeded in developing a novel AKR1B10 inhibitor thatsuppressed cisplatin resistance in non‑small cell lung cancer
cells. It also blocked the proliferative and metastatic potential
in these cells (66). Now we are working on the comprehensive
investigation of the effect of cSBL on AKR family including
impact of several anticancer drugs in cSR cells,
In addition to AKR1B10, the expression levels of AKR1B1,
AKR1C1, AKR1C3 and AKR1C4 were reduced in cSR, and it
has also been reported that they were involved in resistance tocisplatin, daunorubicin or DOX (52,67,68). Furthermore, we
found that the expression of some members of the ATP‑binding
cassette (ABC) transporter superfamily, which contributed
to chemotherapeutic resistance, was reduced in cSR cells(Table SIV). In humans, there are 49 known ABC genes clas‑sified into seven different families (A‑G) depending on theiramino acid sequence (68). They serve a variety of functions
other than drug resistance and can be expressed as channels,
receptors and transporters (69). The members involved in
drug efflux from human cells do not belong to one particularfamily (69). Among the ABC transporters found to be reduced
in cSR cells, ABCC2 has been reported to contribute toresistance against methotrexate, doxorubicin, cisplatin (68),and ABCA1 is responsible for transporting cholesterol andphospholipids (70), but has also been reported to contribute toresistance to Nitidine, a cytotoxic benzophenanthridine alka‑
loid (71). Shukla et al reported that several ABC transporter
genes were endogenously overexpressed in three MPM celllines as compared to untransformed LP9/TERT1 mesothelial
cells (ABCB1 in MO, ABCC3 in ME‑26, and ABCA2, ABCC5and ABCA7 in HMESO cells) (9). Hudson et al (72) compared
expression of genes involved in the response to chemotherapy
between II‑45 rat MPM cells and normal 4/4 RM.4 mesothe‑lial cells, and between established chemo‑resistant cell lines
and parental II‑45 cells, respectively. They found that ABCB1
and ABCG2 were endogenously overexpressed in II‑45 MPM
cells compared to 4/4 RM.4 normal cells; furthermore, levelsof ABCB1 in cisplatin resistant II‑45 cells, and ABCC2
in pemetrexed or combination (cisplatin plus pemetrexed)
resistant II‑45 cells were significantly increased compared toparental II‑45 cells. Those reports indicate that although themolecular species which are involved in the chemoresistancediffer depending on the cell type, ABC transporter superfamily
members are associated with inherent and acquired drug resis‑
tance in MPM. Furthermore, ABCB5 is now considered as oneof a therapeutic target in MPM, because ABCB5 is upregu‑
lated in MPM‑initiating cells generated from primary MPM
samples (73). Our previous studies have shown that cSBL had astronger apoptosis‑inducing effect on multidrug resistant K562
leukemia cells that overexpressed ABCB1 than on their parent
K562 cells (27). Since we found that the expression of ABCC2
was reduced in cSR, it was suggested that cSBL was effective

10

TATSUTA et al: cSBL CAUSES DOWNREGULATION OF AKR1B10

against MPM regardless of intrinsic drug resistance and may
be able to reduce MPM resistance to chemotherapeutic agentsthat are substrates for ABCC2. Indeed, although the difference
was not statistically significant in our experimental conditions,cSR‑A1 tended to be more sensitive to DOX (Fig. 2).
In conclusion, we found that long‑term treatment withcSBL affected malignant mesothelioma cells by dysregulating
multiple genes. The detected DEGs may include genes otherthan those directly affected by the cSBL application. Currently,
examinations of the direct effect of cSBL treatment andcombination research with other drugs are being conducted.
Because cSBL significantly reduced the expression of AKR
family members, especially AKR1B10, it may offer new
possibilities for cancer therapy. We believe that investigationof other genes whose expression was changed in cSR cells willfurther elucidate the antitumor effect of cSBL. Furthermore,by enhancing the effect of cSBL itself and searching foreffective concomitant drugs using information obtained in thisstudy, our results can be expected to lead to the establishmentof novel, more effective cancer treatments.
Acknowledgements
Not applicable.
Funding
This research was funded by a Grant‑in‑Aid for Young
Scientists (B) (grant no. 17K15029) to Takeo Tatsuta.
Availability of data and materials
The datasets used and/or analyzed in the current study
are available from the corresponding author on reasonable
request.
Authors' contributions
TT and MH conceived and designed the study. TT, AN and
SS acquired and analyzed the data. TT and MH confirmed
the authenticity of all the raw data. TT prepared the draft ofthe manuscript, including the figures. All authors read andapproved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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＜細胞制御学＞
教授

顧 建国

准教授

福田 友彦

准教授

伊左治 知弥助教

黒田喜幸
糖鎖間の相互作用と糖鎖生合成の制御機構の解析

糖鎖修飾は、タンパク質の翻訳後修飾の中で最も多く、哺乳動物細胞において普遍的かつ重要なプロセスである。糖鎖の変化は癌や糖尿病を含む多くの疾患の特徴でもある。しかし、糖鎖の生合成に関わる分子機序は必ずしも明らかになっていない。私達は、N 型糖鎖と O-GlcNAc 修飾の生合成において共通の糖鎖のドナー基質である
UDP-GlcNAc に注目し、これら 2 種類の糖鎖修飾の関係を明らかにした。O-GlcNAc
修飾は O-GlcNAc 転移酵素(OGT)によって Ser/Thr 残基に単一の GlcNAc 残基が UDPGlcNAc から転移することで生合成される可逆的な修飾である。一方、N 型糖鎖は小胞体にてマンノースがトリミングされた後、ゴルジ装置において種々の糖転移酵素によって生合成される複雑な修飾である。私達は、OGT のノックダウン細胞（OGTKD）を用いて解析した。O-GlcNAc 修飾の減少は、GlcNAc 分岐型糖鎖を増加させると考えられた。しかし、予想に反して、レクチンブロット、HPLC および質量分析により、分岐型の N-型糖鎖の生成は野生型細胞と比較して OGT-KD で有意に減少していることがわかった。GlcNAc 分岐型の減少がどのような糖鎖構造で生じているのか検討したところ、興味深いことに、この低下したのは GlcNAc 転移酵素 IV（GnTIV）によって触媒される β1,4-GlcNAc 分岐型であった。OGT-KD と GnT-IV に触媒される構造特異性の関係を明らかにするため、哺乳動物細胞の主要な UDP-GlcNAc 輸送体である SLC35A3 に焦点を当てて検討した。驚いたことに、SLC35A3 を欠損させた細胞(SLC35A3-KO)では、OGT-KD と同様に β1,4-GlcNAc 分岐型の N 型糖鎖の生成が有意的に減少した。さらに、共免疫沈降実験により、
SLC35A3 は GnT-IVと相互作用するが、
β1,6-GlcNAc 分岐型を触媒する GnT-V とは相互作用しないこ80

とが示された。また、ウエスタンブロットおよび化学酵素標識により、OGT がSLC35A3 を修飾し、その修飾が SLC35A3 の安定性向上に大きく寄与することが確認された。さらに、OGT-KD 細胞で観察された現象と同様に、SLC35A3-KO は細胞の伸展を促進し、細胞の遊走と細胞増殖を抑制する。以上の結果は、上図に示したように
O-GlcNAc 化を介した OGT-SLC35A3-GnT-IV の相互作用により N-型糖鎖の生合成と機能が特異的に制御されることを示している。本研究は、O-GlcNAc と N 型糖鎖間のcross-talk を初めて明らかにした研究である。現在、シアル酸とフコースの輸送体である SLC35A1 と SLC35C1 に焦点を当て、輸送体の機能と糖鎖修飾との関連にも着目して検討を行っている。
一方、癌化に伴い糖鎖の発現が変化することはよく知られている。 例えば、癌細胞における O-GlcNAc や N 型糖鎖の分岐の増加のみならず、ヒアルロン酸の増加も知られている。下図に示すようにこの 3 種類の糖鎖は、UDP-GlcNAc を共通のドナー基質として、OGT、 ヒア
ルロン酸合成酵素(HAS)および
GlcNAc 転移酵素
(GnT)によってそれぞれ生合成されている。上述のように共通の基質を利用しているため糖鎖の相互関係が示唆されるが、これらの相互作
用はよくわからない。そこで、私達はこれら 3 つの糖鎖の生合成に相互関係が存在すると作業仮説をたて、検証した。膵癌細胞株である PANC-1 の OGT-遺伝子抑制（KD）細胞と HAS2 遺伝子欠損（KO）細胞株を樹立し、O-GlcNAc 修飾とヒアルロン酸の生合成を阻害した
際、他の糖鎖修飾がどうなるかを検討した。その結果、 O-GlcNAc の合成を低下させた OGT-KD 細胞では全体および細胞表面の N 型糖鎖の分岐が著しく減少していたことをレクチンブロットと FACS で明らかにした。さらに、ヒアルロン酸結合タンパクを用いてヒアルロン酸の合成量を検討したところ、有意に増加していた。
一方、ヒアルロン酸の合成を抑制した HAS2KO 細胞において O-GlcNAc 修飾は減少していたが、分岐型 N 型糖鎖の発現に顕著な変化は確認されなかった。これらの結果

81

から、3 つの糖鎖生合成の相互関係はより複雑であることが示唆される。
現在は HPLC を用いた細胞内 UDP-GlcNAc 濃度の変化の解析を行っている。さらに複合型及び混成型 N 型糖鎖の合成の鍵となる反応を触媒する酵素を欠損させた GnT-I
KO 細胞の樹立を行い、分岐型糖鎖の生合成が阻害された際のヒアルロン酸と OGlcNAc 修飾の変化の検討を行うことで 3 種類の糖鎖生合成の相互関係を明らかにしようとしている。
＜論文発表＞
1.

Regulation of hyaluronan production by β2 adrenergic receptor signaling. Kuroda, Y. andHigashi, H. Biochem. Biophys. Res. Commun. 575, 50-55, 2021
2. α1,6-Fucosyltransferase contributes to cell migration and proliferation as well as to cancer
stemness features in pancreatic carcinoma. Liang C., Fukuda, T., Isaji, T., Duan, C., Song,W., Wang, Y. and Gu, J.

Biochim Biophys Acta Gen Subj., 1865, 3239-3252, 2021

3. Loss of core fucosylation suppressed the humoral immune response in Salmonella
typhimurium infected mice. Zahid, D., Zhang, N., Fang, H., Gu, J., Li, M. and Li, W. J.
Microbiol. Immunol. Infect. 54, 606-615, 2021
4. N-linked Glycans in the Epithelial-Mesenchymal Transition: Implications for Cancer
Metastasis. Gu, J. Comprehensive Glycoscience 2nd edition, Elsevier, 440-443, 2021
5. O-GlcNAcylation Regulates β1,4-GlcNAc-branched N-glycan Biosynthesis Via the
OGT/SLC35A3/GnT-IV Axis. Song, W., Isaji, T., Nakano, M., Liang C., Fukuda, T. and Gu, J.
FASEB J., 36: e22149. doi: 10.1096/fj.202101520R. 2022

＜学会発表＞
糖鎖によるインテグリンとその下流分子の機能制御と特異性
顧建国, 第 1 回オルガネラゾーン 研究会, Web 開催, 2021 年 6 月
膵がん細胞のがん幹細胞性の獲得と細胞移動・増殖における FUT8 の役割
梁 彩霞, 福田 友彦, 伊左治 知弥, 黒田 喜幸, 顧 建国
第 15 回東北糖鎖研究会, Web 開催, 2021 年 9 月, P. 10

82

O-GlcNAcylation Regulates β1,4-GlcNAc-branched N-glycan Biosynthesis Via the
OGT/SLC35A3/GnT-IV Axis
宋 万里, 伊左治 知弥, 中の 三弥子, 福田 友彦, 黒田 喜幸, 顧 建国
第 15 回東北糖鎖研究会, Web 開催, 2021 年 9 月, P. 26
ゴルジ体における N-型糖鎖の多様性の制御とその意義
伊左治 知弥
第 19 回生物化学若手研究者セミナー, Web 開催, 2021 年 10 月
Focal adhesion kinase (FAK)の O-GlcNAc 修飾による細胞接着斑の形成と細胞機能の制
御伊左治 知弥, 大山 嘉順, 孫 玉涵, 福田 友彦, 黒田 喜幸, 顧 建国
第 40 回日本糖質学会年会, 鹿児島, 2021 年 10 月,

3A-11

O-GlcNAcylation regulates β1,4-GlcNAc-branched N-glycan biosynthesis via the
OGT/SLC35A3/GnT-IV Axis
宋 万里, 伊左治 知弥, 中の 三弥子, 福田 友彦, 黒田 喜幸, 顧 建国
第 40 回日本糖質学会年会, 鹿児島, 2021 年 10 月,

P-046

Focal adhesion kinase の O-GlcNAc 修飾による細胞機能の制御
伊左治 知弥, 大山 嘉順, 孫 玉涵, 福田 友彦, 黒田 喜幸, 顧 建国
第 94 回日本生化学会, Web 開催, 2021 年 11 月, 1T13m-08 (P-020)
O-GlcNAcylation regulates β1,4-GlcNAc-branched N-glycan biosynthesis via the
OGT/SLC35A3/GnT-IV Axis
宋 万里, 伊左治 知弥, 中の 三弥子, 福田 友彦, 黒田 喜幸, 顧 建国
第 94 回日本生化学会, Web 開催, 2021 年 11 月, 1T13m-08 (P-020)
Regulatory function of glycosylation in cancer and its application
顧建国

中国科学院西北高原生物研究所学術セミナー, 特別講演,
年 12 月83

西寧市（Web 開催） 2021

N-型糖鎖による膜受容体の機能制御とその特異性
顧建国、第 2 回オルガネラゾーン 研究会, 東京, 2022 年 3 月

84

DOI: 10.1096/fj.202101520R  

| Revised: 14 December 2021 | Accepted: 22 December 2021

RESEARCH ARTICLE

O-­GlcNAcylation regulates β1,4-­GlcNAc-­branched N-­glycan
biosynthesis via the OGT/SLC35A3/GnT-­IV axis
Wanli Song1 | Tomoya Isaji1 | Miyako Nakano2
Tomohiko Fukuda1 | Jianguo Gu1
1

Division of Regulatory Glycobiology,
Institute of Molecular Biomembrane
and Glycobiology, Tohoku Medical andPharmaceutical University, Sendai,
Japan

2

Graduate School of Integrated
Sciences for Life, Hiroshima University,
Higashi-­hiroshima, Japan
Correspondence
Jianguo Gu, 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.
Email: jgu@tohoku-mpu.ac.jp
Funding information
Japan Society for the Promotionof Science, Grant/Award Number:
19H03184 and 19K07049; Ministry of
Education, Culture, Sports, Science,
and Technology of Japan, Grant/Award
Number: 20H04909| Caixia Liang1 |AbstractN-­Linked glycosylation and O-­linked N-­acetylglucosamine (O-­GlcNAc) are important protein post-­translational modifications that are orchestrated by a diverse set of gene products. Thus far, the relationship between these two typesof glycosylation has remained elusive, and it is unclear whether one influences
the other via UDP-­GlcNAc, which is a common donor substrate. Theoretically,a decrease in O-­GlcNAcylation may increase the products of GlcNAc-­branchedN-­glycans. In this study, via examination by lectin blotting, HPLC, and mass spectrometry analysis, however, we found that the amounts of GlcNAc-­branched tri-­
antennary N-­glycans catalyzed by N-­acetylglucosaminyltransferase IV (GnT-­IV)
and tetra-­antennary N-­glycans were significantly decreased in O-­GlcNAc transferase knockdown cells (OGT-­KD) compared with those in wild type cells. We
examined this specific alteration by focusing on SLC35A3, which is the main
UDP-­GlcNAc transporter in mammals that is believed to modulate GnT-­IV activation. It is interesting that a deficiency of SLC35A3 specifically leads to a decrease in the amounts of GlcNAc-­branched tri-­  and tetra-­antennary N-­glycans.
Furthermore, co-­immunoprecipitation experiments have shown that SLC35A3
interacts with GnT-­IV, but not with N-­acetylglucosaminyltransferase V. Western
blot and chemoenzymatic labeling assay have confirmed that OGT modifies
SLC35A3 and that O-­GlcNAcylation contributes to its stability. Furthermore, we
found that SLC35A3-­KO enhances cell spreading and suppresses both cell migration and cell proliferation, which is similar to the phenomena observed in the
OGT-­KD cells. Taken together, these data are the first to demonstrate that O-­
GlcNAcylation specifically governs the biosynthesis of tri-­  and tetra-­antennaryN-­glycans via the OGT-­SLC35A3-­GnT-­IV axis.

Abbreviations: 2-­AB, 2-­aminobenzamide; CRISPR/Cas-­9, clustered regularly interspaced short palindromic repeats/caspase-­9; DMEM, Dulbecco's
modified Eagle's medium; DOX, doxycycline; FBS, fetal bovine serum; GAPDH, glyceraldehyde-­3-­phosphate dehydrogenase; GnGnbi-­PA,
pyridylamine-­labeled GlcNAc-­terminated bi-­antennary glycan; GnT-­IV, N-­acetylglucosaminyltransferase IV; GnT-­V, N-­
acetylglucosaminyltransferase V; GU, glucose units; HBP, hexosamine biosynthetic pathway; KD, knockdown; KO, knockout; PBS, phosphate-­
buffered saline; shRNA, short hairpin RNA; UDP-­GlcNAc, diphosphate β-­D-­N-­acetylglucosamine; VSV, vesicular stomatitis virus glycoprotein; WT,
wild type.
This is an open access article under the terms of the Creat​ive Commo​ns Attri​butio​n-­NonCo​mmerc​ial-­NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-­commercial and no modifications or adaptations are made.
© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.
FASEB J. 2022;36:e22149.
https://doi.org/10.1096/fj.202101520R

﻿

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Received: 25 September 2021

|   

SONG et al.

KEYWORDS

cell adhesion, GlcNAc branching, N-­glycan biosynthesis, O-­GlcNAcylation, SLC35A3

1|

I N T RO DU CT ION

Among multiple post-­translational modifications (PTMs),
protein glycosylation is a common and important process
in cells,1–­3 and altered glycosylation is a hallmark of many
diseases including cancers4 and diabetes.5 N-­Glycans are
extremely complex and conserved PTMs are found in thesecretory pathways of proteins. Thousands of proteins are
known to be N-­glycosylated, which plays a considerable
role in processes such as cell-­cell and cell-­extracellularmatrix recognition, the selective localization of proteins,and immune responses.2,6,7 Biosynthesis of N-­glycans in
the endoplasmic reticulum (ER) and the Golgi apparatus
involves complex machinery and several steps that include
enzymatic reactions and transportation. N-­Glycosylation
is often associated with other PTMs such as phosphorylation7 and ubiquitination.8,9 For example, an increase inN-­acetylglucosaminyltransferase III (GnT-­III) produced byE-­cadherin lead to a reduced level of phosphorylation inβ-­catenin through EGF receptor or Src signaling and contribute to the suppression of cancer metastasis.10,11 One of
the unique features of N-­glycans is the GlcNAc-­branchedcomplex structure, that is, sequentially synthesized firstby N-­acetylglucosaminyltransferase I (GnT-­I) and GnT-­II,and then by GnT-­III, GnT-­IV, or GnT-­V to produce functionally diverse N-­glycosylated proteins. Furthermore,
GlcNAc-­branched structures are reported to be involved in
the pathogenesis of type 2 diabetes,5,12,13 and in the growth,invasion, and metastasis of cancers.14,15 The loss of GnT-­IV
provokes endocytosis, which decreases the cell surface residency of Glut2 and contributes to type 2 diabetes.12 Higher
levels of GnT-­IV expression have been observed in choriocarcinoma,16 hepatocarcinoma,17 and pancreatic cancer,18
which suggests that production by GnT-­IV promotes invasion and metastasis. Another important factor that cannot
be ignored in N-­glycan biosynthesis is UDP-­GlcNAc, adonor substrate that is produced by the hexosamine biosynthetic pathway (HBP) as an essential substrate in protein N-­glycosylation pathways. UDP-­GlcNAc is considered
to be a nutritional sensor and an important regulator of cellsignaling that concerns cell function.19,20
Unlike the complex N-­glycans that decorate proteins,
O-­GlcNAcylation is a simple protein PTM, where a single
moiety of GlcNAc acts on the serine or threonine residueof target proteins without further elongation or modification into more complex structures.21–­23 O-­GlcNAcylation isa widespread and dynamic modification that is controlled
by only a single pair of enzymes: O-­GlcNAc transferase(OGT) and O-­GlcNAcase (OGA). UDP-­GlcNAc as a single
substrate is used by OGT for O-­GlcNAcylation. Previous
studies have shown that O-­GlcNAcylation induces aconformational change that initiates protein folding,24
competes with phosphorylation in the same, or proximal, serine or threonine residual,25,26 disrupts protein-­protein interactions,27,28 serves as a protein recruiting
signal,29 and regulates protein stability.30,31 Furthermore,
O-­GlcNAcylation plays a fundamental role in chronic
diseases such as diabetes, Alzheimer's disease, cardiovascular disease, and cancers.25,32–­34 Numerous cancer cells
show higher levels of O-­GlcNAcylation, which makesthese cancers more aggressive, and noteworthy examples
include prostate cancer,35 leukemia,36 breast cancer,37 hepatocellular carcinoma,38 and pancreatic cancer.39 The
suppression of OGT expression in breast or colon cancer
cells decreases cell migration and invasion.40,41
An increase in the UDP-­GlcNAc pool via HBP flux inhyperglycemic cells upregulates O-­GlcNAcylation42 andincreases the production of GlcNAc-­branched N-­glycans
in mammary carcinoma cells.43 Conversely, the loss of
OGT in Caenorhabditis elegans caused a more than twofold
increase in total UDP-­GlcNAc compared with that foundin wild type.44 Although amounts of UDP-­GlcNAc affectN-­glycosylation as well as O-­GlcNAcylation, it is unclear
whether there is an interplay between O-­GlcNAcylation
and N-­glycosylation. It would be reasonable to speculatethat suppression of O-­GlcNAcylation should increaseamounts of UDP-­GlcNAc for N-­glycosylation pathways,
which would increase the production of GlcNAc-­branchedN-­glycans. In the present study, however, we found that
OGT knockdown did decrease—­rather than increase—­theproduction of β1,4-­GlcNAc-­branched tri-­N-­glycans catalyzed by GnT-­IV. The main molecular mechanism could
be explained by the fact that O-­GlcNAcylation regulates
the stability of SLC35A3, a main UDP-­GlcNAc transporter,
which specifically interacts with GnT-­IV. These findings
are the first to describe how O-­GlcNAcylation specifically
governs the biosynthesis of tri-­antennary N-­glycans.

2
2.1

|

MATERIALS AND METHO D S| Antibodies and reagentsThe experiments were performed using the following
antibodies: biotinylated Datura stramonium agglutinin(DSA; B-­1185), Concanavalin A (ConA; B-­1005), and

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2 of 16

Sambucus nigra agglutinin (SNA; B-­1305) ABC kit was acquired from Vector Laboratories; mAb against O-­GlcNAc(CTD110.6, 9875S) and a peroxidase-­conjugated secondary
antibody against rabbit (7074S) were from Cell Signaling
Technology. Peroxidase-­conjugated secondary antibodies
against mouse (AP124P) were from Millipore; mAb against
α-­tubulin (T6199), an antibody against FLAG (F1804), andanti-­FLAG conjugated to agarose (Sigma, A2220), VSV(V5507) and anti-­VSV-­Glycoprotein-­Agarose antibody(A1970) were from Sigma, doxycycline hyclate (D9891) was
acquired from Sigma–­Aldrich, and cycloheximide (CHX,#037-­20991) was purchased from Wako (Tokyo, Japan).

2.2| Cell lines and cell culture

The HeLa and 293T cells were acquired from the RIKEN
Bioresource Research Center (Japan). All cells were cultured in DMEM containing 10% fetal bovine serum (FBS,
Gibco) under the standard protocol at 37°C and 5% CO2.

2.3 | Expression plasmids andtransfection
We used the Gateway™ cloning system from Invitrogenfor all overexpression experiments. The Gateway™ entry
vectors were constructed as follows. The cDNA sequences
for SLC35A3 were cloned from HeLa cells then inserted
into pENTR/D-­TOPO vectors with 2 × VSV or 3 × FLAGat the N terminus using the in-­fusion method (Takara
Bio Inc.). The cDNA sequences of GnT-­IVa and GnT-­IVb
were cloned from HeLa cells and GnT-­V was amplified
from GnT-­V pENTR/D-­TOPO vectors in our lab45 andinserted into pENTR/D-­TOPO vectors with 3 × FLAG at
the C terminus using the in-­fusion method. The resultant
cDNAs in entry vectors were confirmed by DNA sequencing. Using LR clonase (Invitrogen), the subcloned cDNAs
in entry vectors were transferred into pcDNA3.1-­RfA
for transient expression. Plasmid diagrams for different
tagged proteins were shown in Figure S3. Transfection
was performed using PEI MAX (molecular mass, 40 kDa;
Polysciences Inc.) following the dictates of the UnitedStates patent application (number US20110020927A1)
with minor modifications. Briefly, before transfection,
3–­5 × 106 cells were seeded on either 10-­ or 6-­cm dishes
and were then incubated for 24 h. Expression vectors withPEI MAX (1 mg/ml in 0.2 M hydrochloric acid) were pre-­incubated for 20 min at a 1:3 ratio in 1 ml of a solution that
contained 20 mM CH3COONa buffer and 150 mM NaCl at
pH 4.0. After incubation for 6 h, the conditioned medium
was replaced with a normal culture medium for further
incubation for 48 h.

  | 3 of 16

2.4 | Lectin blot, immunoprecipitation,
and western blot
The labeled cells were washed with cold PBS and thenlysed in the cell lysate buffer (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. Protein concentrations were determined using a
BCA protein assay kit (Pierce). An equal amount of protein from each sample was loaded to either 7.5% or 15%
SDS-­PAGE (sodium dodecyl sulfate-­polyacrylamide gelelectrophoresis) and transferred to PVDF membranes(Millipore Sigma). The samples were then incubated either with the indicated primary and secondary antibodies
or with biotinylated lectins as indicated. Immunoreactive
bands were detected using either an immobilon Western
Chemiluminescent HRP Substrate (Millipore) or a
Vectastain ABC kit (Vector Laboratories) according to
the manufacturer's instructions. For immunoprecipitation, cell lysates (500–­1000 µg) were immunoprecipitatedwith anti-­VSV-­Glycoprotein-­Agarose (5 µl) or anti-­FLAG
conjugated agarose (10 µl) at 4°C for 2 h with rotation,and then the immunoprecipitates were washed twice withlysis buffer and subjected either to 7.5% or 15% SDS-­PAGE,
then transferred to PVDF membranes. The membranes
were incubated with primary and secondary antibodies as
indicated.

2.5| PCR for mRNA expression analysis

Total RNA was extracted from the cells using TRIzol
Reagent (Invitrogen) according to the manufacturer's instructions. Total RNA (1 μg) was reverse-­transcribed using
a PrimeScript RT reagent kit (Takara Bio Inc.). The gene
expression levels were detected via RT-­PCR, and GAPDH
was performed as a control. The PCR primer sequences arelisted in Table S1. PCR amplification was electrophoresedon 1.5% agarose gel in TAE buffer containing 40 mM Tris-­
acetate and 1 mM EDTA at pH 8.0. Finally, the DNA ladder was visualized with ethidium bromide staining under
UV light. The relative expression levels were obtained byquantifying the intensities of the bands from three independent experiments and then normalized to GAPDH,
and the value of WT cells at 4 h was set as 1.

2.6 | Preparation of N-­glycans labeledwith 2-­aminobenzamide (2-­AB)
N-­Glycan analysis was performed with minor modification, as reported previously.46 The cells (4–­6 × 107) were
homogenized in 1.5 ml of PBS containing a protease

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SONG et al.

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inhibitor mixture (Nacalai Tesque, Kyoto, Japan) usinga Potter-­Elvehjem homogenizer. The homogenized celllysates were centrifuged at 1000× g for 10 min at 4°C, andthe supernatants were further centrifuged at 100 000× g
for 30 min at 4°C. Then precipitates were lysed in 100 μl
lysis buffer (20 mM Tris-­HCl, pH 7.4, 150 mM NaCl,
50 mM EPPS, 1% Triton X-­100) containing protease andphosphatase inhibitors. Protein concentrations were
determined using a BCA protein assay kit (Pierce). The
same amounts of proteins from each sample (1 mg) wereincubated with N-­Glycosidase F (Takara Bio Inc.) at 37°C
for 72 h according to the manufacturer's instructions andthen precipitated with cold ethanol at −20°C overnight.
After centrifugation at 25 000× g for 20 min, the supernatants containing N-­glycans were dried via a rotary vacuum evaporator. Those released N-­glycans were added toSupelclean ENVI-­Carb SPE tubes (3.0 ml, 0.25 g; Sigma–­
Aldrich) and eluted with a mixed solution [50-­mM ammonium acetate buffer (pH 7.0)/acetonitrile (40/60)].47 Afterdrying, N-­glycans were incubated with 30 μl of 1 M 2-­AB
and 0.24 M 2-­picoline borane in 10% acetic acid in methanol and purified with HyperSep™ Diol Cartridges (1 ml,
50 mg; Thermo Fisher Scientific).

2.7 | Analysis of N-­glycans
by normal and reversed-­phase HPLC
To avoid the complexity of the N-­glycans produced bysialylation and galactosylation, these N-­glycans werelabeled with 2-­AB, treated with α2-­3, -­6, -­8, and -­9 neuraminidase A (NEB #P0722, cloned from Arthrobacterureafaciens) and β1-­4 galactosidase (NEB #P0745S, cloned
from Streptococcus pneumoniae), and then analyzed on anormal-­phase HPLC system (JASCO) using an amide-­80
HILIC column (250 × 4.6 mm; Tosoh). Elution was performed at a flow rate of 0.8 ml/min at 45°C using 0.05%
TFA in acetonitrile as solvent A and 0.05% TFA in MilliQ
water as solvent B. The column was pre-­equilibrated with
30% solvent B, and after injection of a sample for 8 min, the2-­AB-­labeled N-­glycans were separated using a linear gradient of 30%–­60% solvent B for 120 min. The 2-­AB-­labeledN-­glycans were detected using a fluorescence detector(JASCO) at excitation and emission wavelengths of 330
and 420 nm, respectively. Fractions corresponding to fluorescence peaks were collected and then subjected either
to a reversed-­phase HPLC or to MS for further analysis. A
glucose oligomer ladder (GU) labeled with 2-­AB (Agilent)
was used as a standard to separate the peaks in the same
column under the same conditions, and the GU values areshown at the top of the figure.
ODS-­80TM columns (150 × 4.6 mm and 250 × 4.6 mm;
Tosoh) were used for reversed-­phase HPLC, and elution

SONG et al.

was performed at a flow rate of 1.0 ml/min at 55°C using
20-­mM ammonium acetate buffer (pH 4.0) as solvent
A and the same buffer containing 1% 1-­butanol as solvent B. The column was pre-­equilibrated with 4% of solvent B, and after injection of a sample for 10 min, the2-­AB-­labeled N-­glycans were separated employing a linear gradient of 4%–­100% of solvent B for 60 min. Factions
corresponding to fluorescence peaks were collected
and then subjected either to a normal-­phase HPLC or
to MS for further analysis. The elution patterns of two-­
dimensional 2-­pyridylaminated N-­glycans on normal andreversed-­phase HPLC are very useful for identifying specific N-­glycan structures.48,49 For example, when using
normal-­phase HPLC, the elution time of β1,4-­GlcNAc-­
branched (tri-­antennary) N-­glycans approximates thatof β1,6-­GlcNAc-­branched (tri'-­antennary) N-­glycans,while those elution times are quite different in reversed-­phase HPLC. The elution time for bi-­antennary GlcNAc
N-­glycans was between the times for tri'-­antennary andtri-­antennary GlcNAc N-­glycans, as shown in Figure S2B.

2.8 | Analysis of N-­glycan structures bymass spectrometry (MS)When the 2-­AB-­labeled N-­glycans separated using LC(ODS or Amide column)-­FD (fluorescence detection)
were dried, each dried residue was dissolved using 20 μlof a solution of 0.05% trifluoroacetic acid/50% acetonitrile, and a portion of the solution was used for MS and
MS/MS analysis as follows. The 2-­AB-­labeled N-­glycans
were introduced into an ESI source using an Accela
HPLC system (Thermo Fisher Scientific) with an injection volume of 5 μl of a flow solvent solution of 0.05%
trifluoroacetic acid/50% acetonitrile with a flow rate of
200 μl/min without column separation. The N-­glycan
structures were analyzed via LTQ Orbitrap XL (hybrid
linear ion trap-­orbitrap mass spectrometer; Thermo
Fisher Scientific). In the MS setting, the voltage of thecapillary source was set at 5.0 kV, and the temperatureof the transfer capillary was maintained at 330°C. The
capillary and tube lens voltages were set at 35 and 130 V,
respectively. MS data were obtained in a positive ion
mode over a mass that ranged from m/z 350 to m/z 2500,
as measured using orbitrap (resolution, 60 000; mass accuracy, 5 ppm). MS/MS data were obtained via an ion
trap (data-­dependent top 3, CID). Xcalibur softwarever. 2.2. (Thermo Fisher Scientific) was used to analyze
the MS and MS/MS data. Monoisotopic masses wereassigned with possible monosaccharide compositions
using a GlycoMod software tool (the mass tolerance forprecursor ions is ±0.005 Da, https://web.expasy.org/
glyco​mod/), and the proposed glycan structures were15306860, 2022, 2, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202101520R by Tohoku Ika Yakka Daigaku, Wiley Online Library on [14/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

4 of 16further verified via annotation using a fragmentation
mass-­matching approach based on the MS/MS data, although MS/MS data were not sufficiently obtained due
to an insufficient quantity. These glycomic raw data andthe identification results for glycan-­structure analysis
by MS were shown in Tables S2 and S3.

2.9| Chemoenzymatic labeling assay

The chemoenzymatic labeling and biotinylation of proteins in cell lysates were carried out using the Click-­iT O-­
GlcNAc enzymatic labeling system (Invitrogen). Briefly,
the whole cell lysate of 293T cells transfected with an
expression plasmid for VSV-­SLC35A3 was immunoprecipitated and then labeled with labeling enzymes GalT
and UDP-­GalNAz according to the Click-­iT O-­GlcNAc
enzymatic labeling system protocol (Invitrogen).
Labeled proteins were conjugated with an alkyne-­biotin
compound, once again following the Click-­iT protein
analysis detection kit protocol (Invitrogen). Control experiments were performed in the absence of GalT and
UDP-­GalNAz. Biotinylated and control samples werethen subjected to 15% SDS-­PAGE and transferred to a
PVDF membrane for further detection using an ABC kit(Vector Laboratories).

2.10| Assay for protein stability

Analysis of SLC35A3 stability was carried out using a cycloheximide chase assay, as reported previously.50 The
inducible OGT-­KD-­293T cells were transfected with
VSV-­SLC35A3 for 48 h with or without DOX, and thenthese cells were cultured with cycloheximide (CHX,#037-­20991; Wako, Tokyo, Japan) at a final concentrationof 50 µg/ml and harvested at indicated times. The band
densities of VSV-­SLC35A3 and α-­tubulin on western blots
were scanned. Normalization was performed by dividing
the VSV signal by the α-­tubulin signal at each time point
to determine the percentage of the initial SLC35A3 level(0 h of CHX treatment with the density at 0 h set as 1).

2.11 | Establishment of SLC35A3-­KO
HeLa cells
Lenti-­CRISPR v2 was a gift from Feng Zhang (Addgene
plasmid #52961).51 The target site on human SLC35A3
Exon 5 (5′-­TCAGCTGGTTCTCAATTTGT-­3′), which
was chosen from the human GeCKOv2 CRISPR knockout pooled library,52 was cloned into the lentiviral expression vector, and the resultant vectors containing  | 5 of 16

SLC35A3-­specific gRNA were confirmed by DNA sequencing. To prepare the viruses, PEI MAX was used
to transfect the resultant vector into 293T cells withpackaging plasmids. HeLa cells were then infected by
the obtained viruses and selected for stable integration
with 2 µg/ml puromycin for 72 h. Stable independent clones were isolated using a limiting dilution. The
SLC35A5-­KO was confirmed by genomic sequences.
Genomic DNAs were isolated from several clones of the
KO cells, and amplified by PCR using forward primer(5′-­AAAGCTCTCCTTTGCAGTCC-­3′) and reverse
primer (5′-­GACAAGAGCATTGCCAGATATT-­3′).

2.12 | Enzymatic assays for GnT-­IVb and
GnT-­V
To confirm whether the C-­terminally tagged GnT-­IVb and
GnT-­V were still active, enzymatic activities were performed as described previously,53 with minor modification. In brief, 50 μg of enzyme sauces extract from 293T
cells transfected with or without (con) expression vector of
GnT-­IVb or GnT-­V, were incubated at 37°C for indicatedtimes with different buffers as described53 containing 20-­
μM pyridylamine-­labeled GlcNAc-­terminated biantennary glycan (GnGnbi-­PA) and 20 mM UDP-­GlcNAc in the
total volume of 10 μl. 30 μl of 10 mM EDTA (pH 7.0) orMilliQ water was added after the incubation, and the enzyme reactions were stopped by boiling for 2 min. Thenthe reaction mixtures were centrifuged at 20 000× g for
15 min at 4°C. We injected 20 μl of the resulting supernatant into an HPLC system equipped with an ODS-­80TM
column (250 × 4.6 mm; Tosoh) to separate the products
and substrates. The products of GnT-­IVb were isocratically eluted at a flow rate of 1.2 ml/min at 50°C using
50-­mM ammonium acetate buffer (pH 4.0) containing
0.12% 1-­butanol. The products of GnT-­V were isocratically eluted at a flow rate of 1 ml/min at 55°C using a 20-­mM ammonium acetate buffer (pH 4.0) containing 0.1%
1-­butanol. Fluorescence was monitored using excitationand emission wavelengths of 320 and 400 nm.

2.13| Wound-­healing assay

A wound was scratched into a confluent cell layer on 6-­cm
dishes using a 200-­μl pipette tip, and after 24 h incubation,
the floating cells were gently removed. The cells were further cultured in the medium supplemented with 5% FBS for
24 h. The wound gaps were photographed under a phase-­
contrast microscope (Olympus, Tokyo, Japan) at 0 and 24 h.
The migration distance was evaluated, and then the cell migration rate was quantified relative to the WT (100%).

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SONG et al.

2.14

|   

| Cell proliferation

Cells were cultured at a rate of 2000 cells per 100 µl of
culture medium per well of 96-­well plates and stopped
at different times by adding 10 µl of 5 mg/ml 3-­[4,5-­dimethylthiazol-­2-­yl]-­2,5-­diphenyltetrazolium bromide(MTT; Dojindo, Kumamoto, Japan) with further incubation for 4 h at 37°C. After the removal of the medium,
100 µl of DMSO (Millipore Sigma) was added to dissolve
the resultant formazan crystals within the cells. The optical absorbance was assessed at 490 nm using a microplate reader (SpectraMax® iD5; Molecular Devices). Cell
proliferation rates were determined by relative increases(folds) compared with corresponding cells at the replating time (0 h).

2.15| Statistical analysis

Each experiment included at least three independent
procedures. GraphPad Prism® 5.0 software (GraphPad
Software, Inc., La Jolla, CA) was used for statisticalanalysis, and p < .05 was considered statistically significant (*p < .05; **p < .01; ***p < .001; n.s, no significanceby two-­tail unpaired t-­test or the analysis of variance(ANOVA) tests).

3|

R E S U LTS

3.1 | Changes in the expression levels of
GlcNAc-­branched N-­glycans in OGT-­KD
Cells
First, we used the OGT knockdown (OGT-­KD) HeLa
cells to investigate N-­glycan expression via lectin blot.
These cells were previously established by the DOX-­
dependent inducible shRNA KD system in our lab.54
O-­GlcNAcylation is highly dynamic and often occurs
transiently in response to diverse environmental andphysiological cues. Hence, lectin blots were performedat several different time points after cell replating. As
shown in Figure 1A, the O-­GlcNAcylation levels in thewild-­type cells (WT) gradually increased at the indicated times, although these levels were persistently suppressed in the OGT-­KD cells in the presence of DOX.
We believed that a decrease in OGT would increase theproduction of GlcNAc-­branched N-­glycans in OGT-­KD
cells. Surprisingly, lectin blot analysis using DSA, which
preferentially recognizes β1,4-­linked GlcNAc glycans,showed that the reactive abilities with DSA were decreased in the OGT-­KD cells compared with that in WT(Figure 1B). The underlying molecular mechanism for

SONG et al.

the increase in O-­GlcNAc and DSA staining 48–­72 hafter replating in the WT cells requires further study. On
the other hand, the reactive abilities with ConA lectin(Figure 1C) and SNA lectin (Figure 1D) did not seemto cause significant changes between OGT-­KD and WT
cells. These results prompted us to investigate whether
OGT-­KD alters the expression levels of the glycotransferase genes that are involved in the biosynthetic pathways of GlcNAc-­branched N-­glycans. Results obtained
from a semi-­quantitative RT-­PCR using the primers
listed in Table S1 showed no significant differences between the WT and OGT-­KD cells after each cell replating
at indicated times (Figure S1).

3.2 | OGT-­KD specifically decreased β1,​
NAc-­branched tri-­and tetra-­antennaryN-­glycans
To further identify and characterize the GlcNAc-­branchedN-­glycan structures, N-­glycans were released by the treatment with PNGase F and labeled with 2-­aminobenzamide(2-­AB), and further treated with sialidase and galactosidase, as described in the section for “Experimental
procedures.” The N-­glycans then were analyzed via
normal-­phase HPLC, and each peak was subjected tomass spectrometry (MS) analysis. The N-­glycan structuresin each peak were verified, as shown in Figure S2A, andglycan assignments, precursor m/z, and charges observed
were shown in Table S2.
To distinguish the products of GlcNAc-­  branched N-­
glycans catalyzed by GnT-­IV or by GnT-­V, each corresponding peak separated in normal-­phase HPLC was
further subjected to reversed-­phase HPLC, and peaks 3, 4,
and 5 were further confirmed by MS (Table S3). Attempts
to verify the proposed glycan structures after reversed-­phase HPLC were pursued via annotation using a fragmentation mass-­matching approach based on the MS/
MS data. Although, the tri-­antennary glycans were of low
abundance precluding acquiring MS/MS spectra of sufficient quality, the elution patterns of two-­dimensional
2-­pyridylaminated N-­glycans on normal and reversed-­phase HPLC,48,49 verified that peaks 4, 5, and 6 containedtri-­, tri'-­, and tetra-­antennary N-­glycans, respectively, as
shown in Figure S2B. To directly compare these complex
types of N-­glycans, samples were purified by reversed-­phase HPLC to remove the high-­mannose types andthe remaining types of N-­glycans were then analyzed
via normal-­phase HPLC. As shown in Figure 2A, theamounts of β1,4-­GlcNAc-­branched (tri-­antennary) andtetra-­antennary N-­glycans were lower in OGT-­KD cells
than in the WT cells. The ratios of tri-­antennary or tetra-­antennary N-­glycans versus the sum of the four species

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6 of 16  | 7 of 16

F I G U R E 1 Comparison of glycan changes after cell replating at different times between WT and OGT-­KD cells. HeLa cells pretreatedwith (OGT-­KD) or without (WT) DOX at 0.1 μg/ml for 24 h, were seeded on culture dishes and then harvested at different times as
indicated. The same amounts of cell lysates were subjected to 7.5% SDS-­PAGE gel and stained either with the indicated antibody or withlectins. (A) A representative western blot analyzed by the anti-­O-­GlcNAc antibody. The relative intensities were obtained by quantifying
the intensities of the bands that fell between 75~150 kDa from 3 independent experiments and then normalized to α-­tubulin. And the valueof WT cells at 1 h was set as 1. Values represent the mean ± SE. *p < .05; **p < .01; ***p < .001; n.s, no significance by two-­way analysis
of variance (ANOVA). (B) Lectin blotting with DSA lectin, which preferentially recognizes β1-­4 GlcNAc-­branched glycans. The relative
intensities were obtained by quantifying the intensities of the bands that fell between 75~100 kDa from three independent experiments
and then normalized to α-­tubulin. And the value of WT cells at 1 h was set as 1. Lectin blot analysis with ConA (C) and SNA (D), which
preferentially recognizes high mannose types of glycans and α2,6 sialylated N-­glycans, respectively

areas for N-­glycans (bi-­, tri-­, tri'-­, and tetra-­antennary)consistently showed a significant reduction in OGT-­KD
cells compared with that in the WT cells (Figure 2B). By
contrast, the ratios of bi-­antennary and β1,6-­GlcNAc-­
branched (tri'-­antennary) N-­glycans in OGT-­KD cells
showed a slight increase compared with that in WT cells.
The data suggest that O-­GlcNAcylation specifically regulates the biosynthesis of tri-­antennary N-­glycans catalyzed by GnT-­IV.

3.3 | SLC35A3, a UDP-­GlcNAc
transporter, was modified by O-­GlcNAc
To explore the underlying molecular mechanisms responsible for the effect of O-­GlcNAcylation on the expression of tri-­  and tetra-­antennary N-­glycans, we focusedon the UDP-­GlcNAc transporter. Previous studies had
revealed that deficient UDP-­GlcNAc transporter activity
caused a decrease in the production of GlcNAc-­branched

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SONG et al.

|   

SONG et al.

F I G U R E 2 Analysis of GlcNAc-­branched N-­glycans isolated from WT and OGT-­KD cells. The N-­glycans labeled with 2-­AB wereprepared as described in the “Experimental procedures” section. To avoid complexity from sialylation and galactosylation, these N-­glycans
were treated with neuraminidase A and β1-­4 galactosidase and were further purified by reversed-­phase HPLC to remove the high mannose
types of N-­glycans. (A) A representative elution pattern for the complex types of major N-­glycans on a normal-­phase HPLC in WT and
OGT-­KD cells. Based on analysis using mass spectrometry combined with the two-­dimensional elution patterns of N-­glycans on normal
and reversed-­phase HPLC as described in the “Experimental procedures” section, the major peaks could be assigned as putative N-­glycan
structures. (B) Relative expression of GlcNAc-­branched N-­glycans. The ratio represents each peak area against the sum of the 4 peaks areas(bi-­, tri-­, tri'-­, and tetra-­antennary N-­glycans), which was set as 1. The data were obtained from three independent experiments. Values
represent the mean ± SE. *p < .05; **p < .01 by two-­way analysis of variance (ANOVA)N-­glycans.55 In addition, SLC35A3 has been considered one of the main UDP-­GlcNAc transporters thatplay important roles in the biosynthesis of complex
N-­glycans.56–­58
Based on the results of those studies, we speculated
that the UDP-­GlcNAc transporter might be modified by
OGT, thereby affecting the biosynthesis of branched N-­
glycans. To test this hypothesis, we constructed an expression vector containing human SLC35A3 tagged with
VSV (vesicular stomatitis virus glycoprotein) or FLAG
(Figure S3) and overexpressed them in the Hela, 293T, or
DOX-­induced OGT-­KD 293T cells.54 As expected, western

blot analysis showed that SLC35A3 containing either a
VSV tag (Figure 3A) or a FLAG tag (Figure S4) containing neither serine nor threonine residue, was detected by
the anti-­O-­GlcNAc antibody. O-­GlcNAcylation levels for
SLC35A3 were significantly decreased in OGT-­KD cells,
compared with those in WT cells (Figure 3A). To further
confirm the existence of O-­GlcNAcylation in SLC35A3,
we conducted a chemoenzymatic-­labeling assay using an
azido-­GalNAc sugar, as described in the “Experimental
procedures” section, and SLC35A3 was clearly labeled.
These data proved that SLC35A3 is an O-­GlcNAcylated
protein (Figure 3B).

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8 of 16  | 9 of 16

F I G U R E 3 Determination of O-­GlcNAcylation on SLC35A3. (A) 293T cells transfected without (Con) or with VSV-­SLC35A3, wereincubated without (WT) or with DOX (OGT-­KD). The cell lysates and immunoprecipitates (IP) with anti-­VSV antibodies were subjected
to 15% SDS-­PAGE gel and western blotting either with anti-­O-­GlcNAc or the indicated antibodies, respectively. The experiments wereindependently repeated at least three times. The relative intensity refers to O-­GlcNAcylated SLC35A3 against total SLC35A3. Values
represent the mean ± SE. *p < .05 (two-­tail unpaired t-­test). (B) Confirmation of O-­GlcNAcylation on SLC35A3. Cell lysates of 293T cells
transfected with SLC35A3 were immunoprecipitated (IP) with anti-­VSV antibodies, followed by click chemistry labeling of O-­GlcNAc
residues with (+) or without (−) GalT and UDP-­GalNAz, and were detected using an ABC kit as described in the “Experimental procedures”
section. The experiments were independently repeated at least three times

3.4 | Suppression of O-­GlcNAcylation in
SLC35A3 reduced its stability
Moreover, we found that the O-­GlcNAcylation of
SLC35A3 contributed to its stability. When cells weretreated with cycloheximide, a protein synthesis inhibitor, the decay of SLC35A3 in the OGT-­KD cells was significantly faster than that in WT cells (Figure 4), whichsuggests that O-­GlcNAcylation regulates the synthesisof branched N-­glycans most likely via the modulation of
SLC35A3 stability.

3.5 | Decrease in the expression levels oftri-­and tetra-­antennary N-­glycans in
SLC35A3 knockout (SLC35A3-­KO) cells
To confirm whether regulation of SLC35A3 is a key factor for the decreased expression of tri-­ and tetra-­antennaryN-­glycans in the OGT-­KD cells as described above, weestablished SLC35A3-­KO HeLa cells, which were confirmed by DNA sequencing (Figure S5). A representative
elution pattern for the four species of N-­glycans (bi-­, tri-­,tri'-­, and tetra-­antennary) on a normal-­phase HPLC appears in Figure 5A. Interestingly, the relative expression
levels of tri-­  and tetra-­antennary N-­glycans, but not thetri'-­antennary N-­glycans, were significantly decreasedin the SLC35A3-­KO cells compared with those in WT
cells (Figure 5B), which is similar to that in OGT-­KD
cells. By contrast, the ratios of bi-­antennary N-­glycans in

SLC35A3-­KO cells showed an increase compared withthat in the WT cells. These data further suggest that O-­
GlcNAcylation affects the synthesis of tri-­  and tetra-­antennary N-­glycans via SLC35A3.

3.6 | SLC35A3 is specifically associatedwith GnT-­IV
To further understand why SLC35A3-­KO resulted in a
specific decrease in tri-­antennary N-­glycans, but not in
tri'-­antennary N-­glycans, we speculated that there could
be an interaction between SLC35A3 and GnT-­IV. For
this purpose, 293T were transiently transfected withplasmids enabling the expressions of SLC35A3 taggedwith VSV and GnT-­IVa, GnT-­IVb, or GnT-­V tagged with
FLAG. Intriguingly, the immunoprecipitates with anti-­
VSV antibody contained FLAG-­GnT-­IVb (Figure 6A) or
FLAG-­GnT-­IVa (Figure S6A), but not FLAG-­GnT-­V. Theinteraction between GnT-­IVb and SLC35A3 was also confirmed in Hela cells (Figure S6B). Since GnT-­IVa expression is known to be tissue-­specific, whereas GnT-­IVb ismore ubiquitously expressed,59,60 so we selected GnT-­IVb
for further study. The C-­terminally tagged GnT-­IVb and
GnT-­V displayed the expected enzymatic activities using
pyridylamine-­labeled GlcNAc-­terminated bi-­antennaryglycan (GnGnbi-­PA) as a substrate (Figure S7).
Furthermore, we found that OGT-­KD decreased thecomplex formation between SLC35A3 and GnT-­IVb(Figure 6B). Thus, we concluded that OGT regulates

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SONG et al.

|   

F I G U R E 4 Effect of O-­GlcNAcylation on SLC35A3 stability.
(A) 293T cells were transfected with SLC35A3 and were treated
without (WT) or with DOX (OGT-­KD). After transfection for 48 h,these cells were treated with cycloheximide (CHX), a protein
synthesis inhibitor, for indicated times. Total cell lysates wereused for western blotting with an anti-­VSV antibody. α-­tubulin
was used as load control. (B) Quantitative analysis of the turn-­overfor SLC35A3. The percentage of SLC35A3 at each indicated time
point was normalized by comparing the density with that at time
0, and the density at 0 h was set as 1. The data were obtained fromthree independent experiments. Values represent the mean ± SE.
**p < .01; ***p < .001 by two-­way analysis of variance (ANOVA)

SLC35A3 stability and SLC35A3-­GnT-­IVb complex formation, which plays a crucial role in the biosynthesis oftri-­antennary N-­glycans.

3.7 | OGT-­KD and SLC35A3-­KO alter cellmorphology and inhibit cell migration andproliferation
To gain insight into the roles of OGT in SLC35A3-­
mediated functions, we further compared the cellularbiologic functions between OGT-­KD and SLC35A3-­KO
cells. Our previous study demonstrated that OGT-­KD
led to an increase in cell spreading and an alteration in
the cell shape from elongated spindle shapes to a morerounded morphology.54 A similar phenomenon was also
observed in the SLC35A3-­KO cells (Figure 7A). We further used a wound-­healing assay to compare the cell migration and found that the wound closure capabilities
of both OGT-­KD and SLC35A3-­KO cells were significantly suppressed, compared with those in the WT cells

SONG et al.

F I G U R E 5 Analysis of GlcNAc-­branched N-­glycans isolated
from WT and SLC35A3-­KO cells. The methods for the analysis
of GlcNAc-­branched N-­glycans are described in Figure 2. (A) A
representative elution pattern for the complex types of major N-­
glycans on a normal-­phase HPLC in WT and SLC35A3-­KO cells.
(B) Relative expression of GlcNAc-­branched N-­glycans. Values
represent the mean ± SE. *p < .05; ***p < .001; n.s, no significanceby two-­way analysis of variance (ANOVA)(Figure 7B). Moreover, cell proliferation was also significantly decreased in both OGT-­KD and SLC35A3-­KO cells
compared with that in the WT cells (Figure 7C). Taken
together, these data functionally support the existence ofa novel regulatory mechanism of O-­GlcNAcylation that
impacts the biosynthesis of tri-­antennary N-­glycans via
the OGT-­SLC35A3-­GnT-­IV axis.

4|

DISC USSION

In the present study, we clearly showed that O-­
GlcNAcylation interacts with N-­glycosylation. OGT-­KD
specifically decreased the biosynthesis of β1,4-­GlcNAc-­
branched tri-­  and tetra-­antennary N-­glycans via
SLC35A3, a UDP-­GlcNAc transporter, and served as acentral hub that specifically interacted with GnT-­IV.
The suppression of O-­GlcNAcylation on SLC35A3 affects its stability and the specific complex formation with
GnT-­IV, which specifically decreases the biosynthesis

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F I G U R E 6 Complex formation between SLC35A3 and GnT-­IV. (A) 293T cells co-­transfected without (−) or with (+) expression
vectors containing SLC35A3 and either GnT-­IVb or GnT-­V, were immunoprecipitated (IP) by the indicated antibodies. The cell lysates andimmunoprecipitates were subjected to 7.5% SDS-­PAGE gel and blotted with the indicated antibodies, respectively. (B) Effects of OGT-­
KD on the interaction between SLC35A3 and GnT-­IVb. Cell lysates obtained from the WT and OGT-­KD 293T cells that were transfectedwithout (Con) or with expression plasmids of SLC35A3 and GnT-­IVb were immunoprecipitated (IP) with an anti-­FLAG antibody. The
immunoprecipitates were blotted with the indicated antibodies. Ratios of SLC35A3 in those immunoprecipitates were quantitatively
calculated from three independent experiments. Values represent the mean ± SE. *p < .05 (two-­tail unpaired t-­test)of β1,4-­GlcNAc-­branched N-­glycans (Figure 8). The
identification and specific functional analysis of O-­
GlcNAcylated sites on SLC35A3 require further study.
The specificity for the biosynthesis of these N-­glycans
in OGT-­KD cells could also be confirmed using
SLC35A3-­KO. Furthermore, the effect of SLC35A3-­KO
on cell adhesion, migration, and proliferation was similar to that observed in the OGT-­KD cells. Collectively,
these results clearly show a novel crosstalk pathway between O-­GlcNAcylation and N-­glycosylation via the axisof OGT/SLC35A3/GnT-­IV, which may help us to further
understand the functions of O-­GlcNAcylation and theregulation of N-­glycan biosynthesis.
Generally, different PTMs often coordinate with eachother, and O-­GlcNAcylation is no exception. Given theimportance of O-­GlcNAcylation in multiple fundamental cellular processes, it is reasonable to assume that O-­
GlcNAcylation is involved in the pathological conditionsof many diseases such as diabetes, neurological disorders,cardiovascular disease, and tumor progression.25,32,33
UDP-­GlcNAc is an essential common donor substrate
for O-­GlcNAcylation as well as GlcNAc-­branched N-­
glycosylation. Complex types of N-­glycans cannot be
fully synthesized without an efficient delivery of UDP-­
GlcNAc from the cytosol to the Golgi lumen. For example, SLC35A3-­KD resulted in a decrease in the amountof UDP-­GlcNAc in the Golgi lumen, which reduced the
production of tri-­  and tetra-­antennary N-­glycans.55 The
loss of OGT in C. elegans caused a more than two-­foldincrease in total UDP-­GlcNAc.44 Thus, O-­GlcNAcylation

may reciprocally interact with N-­glycosylation through ashared pool of UDP-­GlcNAc. We assumed that the production of GlcNAc-­branched N-­glycans would increase in
OGT-­KD HeLa cells. However, the results in the present
study clearly contradicted our speculation.
Why OGT-­KD specifically decreased the tri-­ and tetra-­antennary N-­glycans is intriguing. Dorota Maszczak-­Seneczko et al. previously showed that the silencing of
SLC35A3 reduces the GlcNAc-­branched N-­glycans, andthen speculated that SLC35A3 might supply UDP-­GlcNAc
as a substrate mainly for GnT-­IV and GnT-­V.55 Their assumptions provided a hint that SLC35A3 could be modified by OGT at the cytoplasmic domains since SLC35A3
is a type III multi-­transmembrane protein. As expected,
OGT did modify SLC35A3 with O-­GlcNAc. The O-­
GlcNAcylation contributed to its stability at the protein
level (Figure 4). Furthermore, SLC35A3-­KO specificallydecreased the production of tri-­  and tetra-­antennary N-­
glycans, which was partially supported by the results ofthe previous study of SLC35A3-­KD CHO and HeLa cells.55
Although we observed those changes in GlcNAc-­branchedN-­glycans in the current study, we cannot exclude changes
in other types of glycans. Moreover, we believe that O-­
GlcNAcylation may also modify other transporters besides
SLC35A3, which would result in changes in other typesof N-­glycans or other glycosylation pathways rather than
exclusively the N-­glycosylation pathway.
However, our observation could not be simply explained by a decrease in UDP-­GlcNAc concentrationsin the Golgi lumen for GnT-­IV and GnT-­V in either

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SONG et al.

|   

SONG et al.

F I G U R E 7 Comparison of effects of either OGT-­KD or SLC35A3-­KO on cell morphologies, cell migration, and proliferation. (A) Helacells treated with (OGT-­KD) or without (WT) DOX, or SLC35A3-­KO Hela cells were pre-­incubated for 24 h and then replated on culture
dishes. After incubation for 48 h, cells were photographed under a phase-­contrast microscope. An enlarged view is shown in the right panel.
Scale bars, 15 μm. (B) Cells as indicated were grown to more than 90% confluence, and the monolayers were incised with a pipette in eachwell. Photographs were taken by phase-­contrast microscopy at 0 and 24 h. The migration distance was evaluated and then the cell migration
rate was quantified (right panel). The migration rate for the WT cells was set as 100%. Experiments were independently repeated threetimes. Values represent the mean ± SE. *p < .05 by one-­way analysis of variance (ANOVA). Scale bars, 50 μm. (C) The WT, OGT-­KD, and
SLC35A3-­KO cells were supplied with DMEM containing 5% FBS seeded on 96-­well dishes. The value after incubation for 6 h was set as 0
points (0 h), cells were further incubated, and cell numbers were measured at the indicated times (24, 48, 72, or 96 h) using MTT assay. The
relative rate of cell proliferation was shown as increased folds at indicated times compared with that at 0 h, which was set as 1. Experiments
were independently repeated three times. Values represent the mean ± SE. ***p < .001 by two-­way analysis of variance (ANOVA)

SLC35A3-­KO or OGT-­KD cells because GnT-­IV has lower
Km values than those of GnT-­V with UDP-­GlcNAc asa donor substrate.61 Thus, a decrease in UDP-­GlcNAc
should have a greater impact on the production of GnT-­V
compared with that of GnT-­IV. Apparently, this was
not the case for the present study, as described above.
Interestingly, the co-­immunoprecipitation analysis revealed that SLC35A3 specifically interacts with GnT-­IV
rather than with GnT-­V, which strongly suggests the
specificity of SLC35A3 for tri-­antennary N-­glycan biosynthesis and further supports our observations. The FRET
approach in A375 and 293 cells, however, showed that
SLC35A3 and GnT-­V seemed to be close in the Golgi membrane, although the association could not be detected byco-­immunoprecipitation.55,62 Whether the difference isdue to cell lines, association strength between molecules,
or other mechanisms involved will require further study.
In addition, we also noticed that the increase in O-­GlcNAc
and DSA staining 48–­72 h after replating, but the mRNAlevels of those transferases involved in the biosynthetic
pathways of GlcNAc-­branched N-­glycans showed no significant differences at indicated times in the WT cells. We
speculated that diverse environmental cues such as celladhesion and the cell cycle during the timepoints may
influence the glycosylation and secretory pathways ofglycoproteins and their expression levels. The underlying
molecular mechanism will require further study.
This study also showed that SLC35A3 may not be oneof the main UDP-­GlcNAc transporters for N-­glycosylation
since the biosynthesis of bi-­antennary N-­glycans was not
significantly blocked in the SLC35A3-­KO cells. The recent
study by Bożena Szulc et al. also came to a similar conclusion, they also speculated that SLC35A3 is not the main
supplier of UDP-­GlcNAc for N-­glycosylation and speculated there is a more efficient multi-­protein complex thatfunctions as a single transporter of UDP-­GlcNAc for N-­
glycosylation.63 In addition, SLC35A3 has been reported
to either directly or indirectly interact with SLC35A2,58

15306860, 2022, 2, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202101520R by Tohoku Ika Yakka Daigaku, Wiley Online Library on [14/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

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F I G U R E 8 Schematic diagram of the proposed molecular mechanism for regulation of the biosynthesis of β1,4-­GlcNAc-­branchedN-­glycans by O-­GlcNAcylation. Biosynthesis of N-­glycans in the endoplasmic reticulum (ER) and the Golgi apparatus involves complex
machinery that incorporates both enzymatic reactions and transportation. Once a glycoprotein with the high mannose type of N-­glycan istransported from the ER to the Golgi lumen, it is sequentially modified first by GnT-­I and GnT-­II, and then by GnT-­III, GnT-­IV, or GnT-­V
to produce a diverse GlcNAc-­branched functional glycoprotein. In the present study, we found that OGT could modify SLC35A3, a UDP-­
GlcNAc transporter. Suppression of O-­GlcNAcylation on SLC35A3 affects its stability and the specific complex formation with GnT-­IV,
which specifically decreases the biosynthesis of β1,4-­GlcNAc-­branched N-­glycans. These data are the first to demonstrate a novel crosstalk
pathway between O-­GlcNAcylation and N-­glycosylation via the axis of OGT-­SLC35A3-­GnT-­IV

SLC35A4,64 GnT-­IV,62 and GnT-­V,55,62,64 and the interactions among glycosyltransferases and nucleotide sugartransporters to form multi-­enzyme/multi-­transportercomplexes in the Golgi membranes may facilitate the efficient synthesis of complex N-­glycans.64 It is also possible,
however, that some Golgi-­resident nucleotide sugar transporters may specifically serve corresponding glycosyltransferases. For example, SLC35A2 interacts with B4GalT1,
which is widely expressed in all tissues and is consideredquite important in the galactosylation of N-­glycans. Also,a deficiency in either SLC35A2 or B4GalT1 results in a significant lack of galactose moieties on N-­glycans.65–­67
Emerging data have clearly shown that O-­
GlcNAcylation plays critical role in the progress of humandiseases, particularly diseases such as cancers, diabetes, and Alzheimer's disease.25,32–­34 The regulation of O-­
GlcNAcylation may be directly involved in diabetes. OGT
and O-­GlcNAc-­modified protein levels are increased in
the pancreatic islets, as well as in the tissues of the sciatic
nerve, kidney, and liver in diabetic rats.68 Curiously, a decrease in O-­GlcNAcylation caused by the overexpressionof OGA in the pancreatic β cells of transgenic mice led
to a decrease in insulin secretion.69 On contrary, it is wellknown that a loss of GnT-­IV inhibits GlcNAc-­branching
formation on Glut-­2, which decreases Glut-­2 stabilityon the cell surface and promotes its endocytosis, which

decreases insulin secretion and results in the pathogenesis of type 2 diabetes in mice.12 Based on these observations together with the results of this study, it would bevery interesting to investigate the relationship between O-­
GlcNAcylation and Glut-­2 expression on the cell surface.
Cell migration is a central process in the development and maintenance of multicellular organisms.70 In
the previous study, we found that the suppression of O-­
GlcNAcylation inhibited integrin-­mediated cell migration due to an aberrant increase in cell-­ECM adhesion
via an enhancement of focal-­adhesion plaque formation.54 The present study also revealed another potential
mechanism whereby O-­GlcNAcylation could regulate
the modification of GlcNAc-­branched N-­glycans onmembrane proteins such as integrins to govern cell migration. Therefore, it will be very important for glycobiology to examine the effects of both O-­GlcNAcylation
and N-­glycosylation rather than focusing on one or theother.
ACKNOWLEDGMENTS
This work was supported in part by a Grant-­in-­Aid for
Scientific Research (19H03184 to J.G. and 19K07049
to T.I.) from the Japan Society for the Promotion of
Science; and by a Grant-­in-­Aid for Scientific Research on
Innovative Areas (20H04909 to J.G.) from the Ministry15306860, 2022, 2, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202101520R by Tohoku Ika Yakka Daigaku, Wiley Online Library on [14/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

SONG et al.

|   of Education, Culture, Sports, Science, and Technologyof Japan.
DISCLOSURESThe authors declare no conflicts of interest and no competing financial interests.
AUTHOR CONTRIBUTIONS
Wanli Song, Tomoya Isaji and Jianguo Gu designed theresearch; Wanli Song performed all experiments. Wanli
Song, Tomoya Isaji, and Caixia Liang performed the PCR
analysis. Miyako Nakano performed the mass spectrometry analysis. Wanli Song, Tomoya Isaji, Caixia Liang, andTomohiko Fukuda assisted with the experiments. Tomoya
Isaji, Tomohiko Fukuda, and Jianguo Gu analyzed andinterpreted the data. Wanli Song and Jianguo Gu wrote
and revised the manuscript. All authors approved the final
version of the manuscript.
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59. Oguri S, Yoshida A, Minowa MT, Takeuchi M. Kinetic properties and substrate specificities of two recombinant humanN-­acetylglucosaminyltransferase-­IV isozymes. Glycoconj J.
2006;23:473-­480.
60. Takamatsu S, Antonopoulos A, Ohtsubo K, et al. Physiological andglycomic characterization of N-­acetylglucosaminyltransferase-­
IVa and -­IVb double deficient mice. Glycobiology. 2010;20:​485-­497.
61. Lau KS, Dennis JW. N-­Glycans in cancer progression.
Glycobiology. 2008;18:750-­760.
62. Maszczak-­Seneczko D, Sosicka P, Kaczmarek B, et al. UDP-­
galactose (SLC35A2) and UDP-­N-­acetylglucosamine (SLC35A3)
Transporters Form Glycosylation-­related Complexes with
Mannoside Acetylglucosaminyltransferases (Mgats). J Biol
Chem. 2015;290:15475-­15486.
63. Szulc B, Sosicka P, Maszczak-­Seneczko D, et al. Biosynthesis of
GlcNAc-­rich N-­ and O-­glycans in the Golgi apparatus does not
require the nucleotide sugar transporter SLC35A3. J Biol Chem.
2020;295:16445-­16463.
64. Khoder-­Agha F, Sosicka P, Escriva Conde M, et al. N-­
acetylglucosaminyltransferases and nucleotide sugar transporters form multi-­enzyme-­multi-­transporter assemblies in Golgi
membranes in vivo. Cell Mol Life Sci. 2019;76:1821-­1832.
65. Wiertelak W, Sosicka P, Olczak M, Maszczak-­Seneczko D.
Analysis of homologous and heterologous interactions between
UDP-­galactose transporter and beta-­1,4-­galactosyltransferase 1
using NanoBiT. Anal Biochem. 2020;593:113599.

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online version of the article at the publisher’s website.
How to cite this article: Song W, Isaji T, Nakano
M, Liang C, Fukuda T, Gu J. O-­GlcNAcylation
regulates β1,4-­GlcNAc-­branched N-­glycan
biosynthesis via the OGT/SLC35A3/GnT-­IV axis.
FASEB J. 2022;36:e22149. doi:10.1096/fj.20210​
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16 of 16

5.25 N-Linked Glycans in the Epithelial-Mesenchymal Transition: Implications
for Cancer Metastasis
Jianguo Gu, Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical
University, Sendai, Japan© 2021 Elsevier B.V. All rights reserved.

Glossary
EMT (epithelial-mesenchymal transition) EMT is a biologic process that allows polarized epithelial cells, which normallyinteract with basement membrane, to undergo multiple biochemical changes that enable them to acquire a mesenchymal cell
phenotype, which includes enhanced migratory capacity, invasiveness, and greatly increased production of extracellular matrix
as well as EMT biomarkers such as fibronectin, N-cadherin and vimentin.
MET (mesenchymal-epithelial transition) MET is a reversible biological process of EMT, which is also shown to occur innormal development and cancer metastasis.

Epithelial-mesenchymal transition (EMT) is a biological process that allows polarized epithelial cells to undergo multiple
biochemical changes that enable them to acquire a mesenchymal phenotype, and migrate to secondary sites.1 EMT is characterized
by a loss of cell-cell adhesion and acquisition of cell motility, and has been featured by a decreased expression of cell-cell adhesion
molecules and epithelial markers such as E-cadherin, and an increased expression of intermediate filament proteins and mesenchymal cell markers including N-cadherin and integrins (Fig. 1). Therefore, at a cell adhesion view, EMT could be easily believed
as a decreased cell-cell adhesion and an increased integrin-mediated cell-ECM (extracellular matrix) adhesion. EMT has evolved
as a critical developmental program, is essential for gastrulation, somitogenesis and neural crest delamination during embryonicdevelopment. However, this process is also recapitulated under pathological conditions, prominently in invasion and cancer
metastasis, and also associated with tumor stemness and resistance to therapy in cancer.2 To understand the molecular mechanisms
involved in EMT is critical for developing effective treatments for tumor growth and cancer metastasis.
E-cadherin is the core transmembrane protein of the adherens junction and is required for binding and localization of a numberof important cytoplasmic proteins, termed catenins, that connect the cadherin complex to the actin cytoskeleton and several
signaling pathways.3 The catenin family comprises a-catenin, b-catenin and g-catenin (plakoglobin). The regulation of cadherinmediated adhesion and associated adherence junctions is thought to underlie the dynamics of inter-cellular adhesive interactions,which are regulated during tissue development and homeostasis, as well as during tumor cell progression. In epithelial derived
tumors, loss of cell–cell adhesion is correlated with down-regulation of E-cadherin as well as increased proliferation and tumor
invasiveness. Therefore, most studies have focused on the identification and characterization of transcriptional repressors ofE-cadherin expression in epithelial tumor cells. The most prominent factors identified in these studies included the related factors,
Slug, Snail, SIP1 and Twist, which are best known for their roles in early embryogenesis and tumor progression4 (Fig. 1).
On the other hand, E-cadherin can be post-translationally modified by phosphorylation, O-linked and N-linked glycosylation.
Casein kinase II, a serine-threonine kinase, phosphorylates the cytosolic tail of E-cadherin and enhances binding to a-catenin.5
Cytoplasmic O-GlcNAcylation of the E-cadherin cytosolic tail has been shown to occur in response to endoplasmic reticulum stressand inactivate E-cadherin-mediated intercellular adhesion by preventing its transport to the cell membrane.6 N-Glycosylation ofE-cadherin has a great impact on its mediate cell adhesion. Enhanced expression of the first enzyme of N-glycosylation, DPAGT1, isactivated by the Wnt/b-catenin pathway which results in strong N-glycosylation of E-cadherin and reduction of cell-cell adhesion in
adherens junctions.7 In normal epithelial cells, E-cadherin has been shown to harbor mainly bisecting N-glycans due to the activityof N-acetylglucosaminyltransferase III (GnT-III) enzyme (Fig. 2). In carcinoma cells, GnT-III is frequently downregulated bypromoter methylation, and its counterpart N-acetylglucosaminyltransferase V (GnT-V) is up-regulated leading to the formationof tri- and tetra-antennary complex N-glycans on cadherins and other proteins. This results in E-cadherin internalization to thecytoplasm and disruption of cell-cell contacts, which compromises the associated signaling pathways, resulting in EMT, invasion
and metastasis.8 E-cadherin-mediated cell adhesion can be regulated by GnT-III. Overexpression of GnT-III increased the retentionof E-cadherin at the cell border, which might result in an enhancement of E-cadherin-mediated homotypic adhesion.9,10 In contrast,
inhibition of GnT-V expression in fibrosarcoma cells resulted in decreased levels of b1,6 GlcNAc branched N-glycans on N-cadherin,
an antagonist of E-cadherin in EMT, increased cell-cell contacts, and decreased migration and invasion.11 Thus, expression ofE-cadherin is regulated, not only by transcriptional factors, but also by post-transcriptional modifications such as N-glycosylation(Fig. 1).
During the TGF-b-induced EMT, the expression of GnT-III was significantly suppressed, whiles GnT-V was promoted.12,13 GnTIII is usually believed as an important glycosyltransferase in N-glycan biosynthetic pathways, since GnT-V cannot use the bisectedoligosaccharide as a substrate.14 Introduction of a bisecting GlcNAc blocks further branching and elongation of N-glycans catalyzed
by GnT-V. Conversely, overexpression of GnT-III suppressed the TGF-b-induced EMT, and inhibited the metastasis of multiple typesof carcinomas. Inhibition of GnT-V expression prevented liver fibrosis and suppressed TGF-b1-induced EMT in hepatocytes by440

Comprehensive Glycoscience, 2nd edition

https://doi.org/10.1016/B978-0-12-819475-1.00027-4

N-Linked Glycans in the Epithelial-Mesenchymal Transition: Implications for Cancer Metastasis

441

Fig. 1 Proposed functions of expression of N-glycans during EMT. It has been proposed that EMT occurs through distinct intermediate states with different
invasive and metastatic characteristics, that are associated with different tumor functions. During EMT process in different tumors, the function and expression ofthese N-glycans related glycogenes could be dynamic changes, implications for cancer metastasis.

Fig. 2 N-Glycan structures and its related glycosyltransferases. N-Glycans are covalently linked to asparagine residues in the Asn-X-Ser/Thr motifs (X is any aminoacid except proline) of proteins. The representative N-glycan structures of bisecting GlcNAc, b1,6 GlcNAc branch, a1,6-fucosylation and sialylation, are catalyzed byglycosyltransferase GnT-III, GnT-V, FUT8, and ST6GAL1 or ST3GAL4, respectively.

reversal of EMT markers.15 In contrast, overexpression of GnT-V promoted EMT and keratinocyte migration in GnT-V-transgenic
mouse.16 FUT8, which catalyzes a1,6-fucosylation in mammals (Fig. 2), also affects the biological functions of E-cadherin, and was
up-regulated during EMT through transactivation of b-catenin pathway.17 In addition, fucosyltransferase IV (FUT4) could activate
PI3K/Akt and NF-kB signaling systems and facilitated acquisition of a mesenchymal phenotype during EMT.18 These changes ofN-glycosylation in tumor progression and EMT, were further confirmed by glycomic analysis including mass spectrometry,glycogene microarray, and lectin microarray analysis.19
However, in fact, there also exist some inconsistent results. It has been reported for decades that GnT-III expression was enhanced
in human hepatomas, and in the ovarian cancer,20 and an ovarian cancer cell line, which carries an abundance of bisecting GlcNAc
glycans, displays highly metastatic property.21 These raised the question as to the mechanistic roles of GnT-III in regulating themetastatic cell behaviors.
Increasing evidence demonstrates that sialylation plays fundamental roles in normal biological processes, such as cell proliferation, cell differentiation, apoptosis and cell signaling.22,23 The role of sialylation in TGF-b-induced EMT had also been investigatedusing the EMT and MET (mesenchymal-epithelial transition, the converse process of EMT) models. The b-galactoside a2,6
sialyltranferase 1 (ST6GAL1), an enzyme that primarily creates terminal a2,6 sialic acid linkages on N-glycans (Fig. 2), is specificallyupregulated during TGF-b induced EMT. Knockdown of ST6GAL1 clearly inhibited EMT with the concomitant increase inE-cadherin. Interestingly, overexpression of ST6GAL1, but not b-galactoside a2,3 sialyltranferase 4 (ST3GAL4) promoted EMT,
indicating the specific role of ST6GAL1 during the EMT process.24 It had been demonstrated that the effects of GnT-III on cell
migration are dependent on the cell sialylation pattern on integrins.25 GnT-III plays an anti-migratory role under an a2,6
hyposialylated context. The overexpression of GnT-III dramatically suppresses a2,3 sialylation, while increases a2,6 sialylation on
integrins. High expression of a2,6 sialylation could antagonize the inhibitory effects of GnT-III and endow the cells with high442

N-Linked Glycans in the Epithelial-Mesenchymal Transition: Implications for Cancer Metastasis

migratory ability. So, it is reasonable to consider that the decrease in a2,3 sialylation is, at least partially, due to the reduced branchesof N-glycans. Also, the down-regulated a2,3 sialylation after GnT-III overexpression could be also attributed to the branch
specificities of sialyltransferases. In fact, previous reports showed that a low degree of branching favors a2,6 sialylation, but not
favor a2,3 sialylation.26–28
Since a2,3- and a2,6-sialyltranfserases may compete with common substrates to a certain extent, and this competition is
manifested in specific glycoproteins such as b1 integrin. Very recently, interestingly, among the three a2,3-sialyltransferases(ST3GAL3, ST3GAL4, and ST3GAL6) involved in the synthesis of N-glycans, ST3GAL4 deficiency (KO) alone reciprocally increased
the levels of a2,6-sialylation, while the overexpression of ST3GAL4 conversely decreased the a2,6-sialylation levels of b1 integrin.29
The ST3GAL3 KO and ST3GAL6 KO cells expressed more E-cadherin on the cell surface, compared to the ST3GAL4 KO cells.
Therefore, when evaluating the effect of a2,3-sialylation, the influences of a2,6-sialylation could not be neglected, since the a2,6sialylation of b1 plays important roles in cell adhesion and EMT as described above. Although the detailed molecular mechanism
for the roles of different sialylation remain unclear, one could be explained that the a2,6-sialylation but not a2,3-sialylation of
N-glycan is able to inhibit its binding to galectin-3.30 Tumor cells exhibit significant changes in cell surface glycosylation.31 Certain
glycan structures are closely associated with cell invasion and cell survival, which provide important sources of markers for tumor
progression. It has been reported that the ST6GAL1 is highly expressed in induced pluripotent stem (iPS) cells and cancer stem cells(CSCs).32 Taking together, EMT program may relate with CSCs, or cells with stem cell-like properties, systematic analysis offunctions of N-glycans, especially roles of N-glycosylation in glycoproteins expressed on the cell surface will provide insight into
EMT as well as the development of promising therapeutic methods for cancers.
Recently, EMT has recently been proposed that, rather than being a binary process, it occurs through distinct intermediate stateswith different invasive, metastatic and differentiation characteristics, that are associated with different tumor functions33 (Fig. 1).
The diversity and heterogeneity of N-glycans in different kinds of tumors or/and different states even in the same tumor cells, may
have important implications for our understanding of tumor heterogeneity, growth, invasion, metastasis and resistance to therapy.

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＜糖鎖構造生物学部門＞
教授

山口 芳樹

講師

真鍋 法義

助手

大野 詩歩
糖鎖構造解析のための技術基盤の構築

生体分子としての糖鎖の機能を明らかにするためには，糖鎖の立体構造の特徴を理解し，レクチンや抗体など糖鎖結合分子による認識の構造基盤を解明することが重要であるとの立場で研究を進めている．本年度は昨年度に引き続き，糖鎖構造解析（実験および計算）の技術基盤の構築，および糖鎖に関連するタンパク質の大量発現シス
テムの構築を目指した．
I．糖鎖の動的構造解析
リビトール（C5H12O5）は非環状の糖アルコールであり，分子中央に対称面をもつメソ化合物である(図 1)．生体内の構成成分としてはグラム陽性菌の細胞壁中に見られるタイコ酸の構成成分として報告されており，哺乳動物ではジストログリカン上の O-マンノース
糖鎖中にも見出されている．リビトールリン酸
トランスフェラーゼの遺伝的欠乏は筋ジストロ
フィーを引き起こすことから，骨格筋の機能においてリビトールは重要な役割を示すことが分

図 1. リビトールの化学構造と二面角の定義

かる．ラミニンは，リビトールを含有している
O-マンノース糖鎖と相互作用することが知られており，リビトールはその非環状構造
からラミニンと O-マンノース糖鎖を架橋する際のヒンジとして機能している可能性がある．したがって，リビトールの構造とコンホメーションダイナミクスを明らかにすることは，O-マンノース糖鎖の機能解明に貢献することができる．しかし，環状糖と比較して，非環状糖アルコールの構造特性とコンホメーションダイナミクスに関する知見は限られている．我々は NMR と計算化学的手法により，リビトールの安定構造および動的構造の解析を行った(1)．
結晶中でのリビトールのコンホメーションを解析するため，データベース（PDB お
よび Cambridge Crystallographic Data Centre）からリビトール主鎖二面角を調べたところ，リビトール主鎖二面角は 180°, 60°, −60°を中心に分布していた．しかし二面角 φ とψ は独立しておらず，相手の二面角の値を避けるような相反性が存在した．また，固105

体および溶液中のリビトールの構造に関する情報を得るため，固体および溶液 13CNMR を測定し，両 NMR スペクトルを比較したところ，リビトールの固体 13C-NMR
において，C1 および C5 に由来するシグナルは異なる化学シフトを与えた．一方，溶液 13C-NMR を測定したところ，C1 と C5，C2 と C4 は同じ化学シフトで観測された．
よって，リビトールは非対称なコンホメーションを安定構造とし得るが，溶液中では
各コンホメーション間をすばやく遷移していることが示唆された．さらに，溶液 NMR
において溶液中でのリビトールのコンホメーションの割合を評価するため，スピン結合定数（3J）を求めカープラス型の式から二面角の情報を得ることを試みた．しかし，リビトールの 1H-NMR スペクトルにおいてカップリングに高次の項が観察されたため，270 MHz，400 MHz，600 MHz の各共鳴周波数において 1H-NMR スペクトルを測定し，NMR スペクトルの反復シミュレーションを行うことで 3J 値を求めた．その結果，リビトール主鎖二面角 φ および ψ の占有数の合計の比は，180° : 60° : −60° = 55 :7 : 38 と見積もられた．この比から水溶液中においても φ と ψ は相反性を持つことが示唆された．さらにリビトールは分子中央に対称面をもつため，二面角 φ と ψ を区別して議論するために分子動力学計算を行い，コンホメーションの時間変化を観察した．その結果，データベース解析の結果で見られたように，φ と ψ の一方が 180°付近の場合，もう一方は±60°付近を占有する折れ曲がり構造であることが示唆された．同様にリビトールの 2 位と 4 位の水酸基が水素に置換されている 1, 3, 5-ペンタノールの分子動力学計算を行ったところ．リビトールよりも柔軟性が高く，直線構造を優先的に占有する傾向がみられた．これは，2 位と 4 位の水酸基がなくなることで直線構造を占有するようになったことを示していた．
以上から，リビトールは水溶液中で二面角 φ と ψ が相反性を持つ複数の非対称なコ
ンホメーションの安定配座間を遷移していることが明らかになった(1)．さらにリビトールは，環状糖よりも柔軟な二面角を有するが，ある程度決まった二面角(180°, 60°,−60°)をとり，2 位と 4 位の水酸基間の反発により，非対称の折れ曲がり構造を安定構造とする傾向がみられた．以上の知見は，リビトールの動的構造と，O-マンノース糖鎖の機能解明のための基礎となる．
II．糖タンパク質の構造機能相関の解析と応用
免疫グロブリン G（IgG）の Fc 領域と Fcγ 受容体および補体成分との相互作用は，
Fc 領域に結合している糖鎖に依存している．溶液 NMR 法により Fc 領域に結合している糖鎖に着目して構造解析を行った．その結果，糖鎖のガラクトース残基の欠損が，Fc 領域の構造的なゆらぎを引き起こすことが示された(2)．
免疫グロブリン A（IgA）および免疫グロブリン M（IgM）は，通常，J 鎖の寄与に106

より 2 量体または 5 量体になり，高分子 Ig 受容体（pIgR）の分泌成分（SC）に結合して粘膜表面に輸送される．IgA と IgM は，様々な免疫反応，特に粘膜免疫において極めて重要な役割を担っている．J 鎖と SC を含む IgA と IgM の立体構造やこれらの分子のジスルフィド結合や N 型糖鎖修飾について概説した(3)．
抗体薬物複合体（ADC）は，細胞障害性薬剤をモノクローナル抗体に結合させたバ
イオ医薬品の一種であり，標的薬物送達を可能にする．近年，部位特異的結合により，薬物と抗体の比率を制御した ADC 製剤が様々な糖鎖工学的手法により開発されており，ADC 製剤の治療域，有効性，製造バッチ間の一貫性が向上している．抗体-薬物複合化のための様々な糖鎖工学的アプローチを要約し，次世代の ADC 開発における利点についてまとめた(4)．
III．レクチン受容体の構造と機能に関する研究
糖鎖は一般に分岐構造を有しており，そこから生み出される多様性は細胞接着や細胞内シグナリングなど，様々な生理機能を調節していることが明らかとなりつつある．最近になり，レクチンや糖転移酵素が糖鎖の分岐構造を特異的に認識する現象が見出されており，これまでのレクチンの認識の概念では単純に証明できないような新しい知見が数多く報告されている．この糖鎖を特徴づける分岐構造に着目し，核磁気共鳴（NMR）法や X 線結晶構造解析法などの手法により，糖鎖の各分岐それぞれに個性がもたらされる構造的な要因や，分岐型糖鎖に作用するレクチン・糖転移酵素の特異な性質の一端を明らかにした(5)．
β(1,3)-グルカンは，真菌や植物の細胞壁の構成成分である．病原体の β グルカン
は，宿主の防御システムにおいて非自己成分として認識されている．長鎖の β グルカンは三重らせん構造を形成することができ，その三次構造は β グルカン結合タンパク質との相互作用に深く影響すると考えられている．β グルカンの 3 次元的な構造特性と β グルカン結合タンパク質との相互作用の様式について総説としてまとめた(6)．また，レクチン受容体の立体構造情報の蓄積により，微生物糖鎖の認識様式はいくつかのグループに分類されることがわかってきた．代表的な哺乳類レクチン受容体による病原体の認識機構について，知見をまとめた(7)．
SHA は，ヒト B 型赤血球を凝集させる L-ラムノースおよび D-ガラクトース結合型
レクチン受容体である．ゲノム解析とタンパク質生化学的解析を用いて，SHA を分泌する Streptomyces 株を同定し，これらの推定タンパク質の発現と結合活性を検討した
(8)．

107

IV．その他
本年度も内外の共同研究を進め，主に生体分子の構造・モデル解析を行うことによって成果発表に貢献した．脳内に蓄積する蛋白質線維を感度よく検出することは，神経変性疾患の発症を早期に捉えることにつながるため，様々な PET プローブが世界中で開発されている．しかしながら，前頭側頭葉変性症（FTLD）のタウ封入体の高感度検出は困難であった．PM-PBB3 というイメージングプローブが開発され，様々なタウ沈着物を検出することに成功した(9)．
自然発症の頭蓋内圧亢進症（SIH）は，脳脊髄液（CSF）の漏出によって引き起こされる．SIH の診断に 2 つのアイソフォームを持つトランスフェリンが有用なバイオマ
ーカーとして利用できることを示した(10)．また，アルツハイマー病患者において，トランスフェリン上の糖鎖が高くマンノシル化していることを示した(11)．これは，アルツハイマー病の新しいバイオマーカーになり得る．
腸管運動異常はいくつかのミトコンドリア脳筋症の特徴であり，TYMP や POLG などの遺伝子の変異が，これらの希少疾患に関連していることが判明している．新たにリガーゼ III（LIG3）遺伝子に腸管運動障害，脳症，神経筋異常を特徴とするミトコン
ドリア脳筋症を引き起こす変異を同定した(12)．

＜発表論文＞
1.

Ohno, S., Manabe, N., Yamaguchi, T., Uzawa, J., and Yamaguchi, Y. (2021) Ribitol insolution is an equilibrium of asymmetric conformations. Molecules 26, 5471

2.

Yanaka, S., Yamaguchi, Y., Takizawa, T., Miyanoiri, Y., Yogo, R., Shimada, I., andKato, K. (2021) NMR assignments of the N-glycans of the Fc fragment of mouse
immunoglobulin G2b glycoprotein. Biomol NMR Assign 15, 187-192

3.

Pan, S., Manabe, N., and Yamaguchi, Y. (2021) 3D structures of IgA, IgM, andcomponents. Int J Mol Sci 22, 12776

4.

Manabe, S., and Yamaguchi, Y. (2021) Antibody glycoengineering and homogeneous
antibody-drug conjugate preparation. Chem Rec 21, 3005-3014

5.

山口芳樹. (2021) 分岐型糖鎖の高次構造と相互作用原理の解明. BIO Clinica 36,
79-84

6.

Manabe, N., and Yamaguchi, Y. (2021) 3D structural insights into β-glucans and theirbinding proteins. Int J Mol Sci 22, 1578

7.

Manabe, N., and Yamaguchi, Y. (2021) 3D structural view of pathogen recognition bymammalian lectin receptors. Front Mol Biosci 8, 670780

108

8.

Fujita-Yamaguchi, Y., Muramatsu, H., Tapia, A., Bagramyan, K., Desai, M., Takehana,Y., Igarashi, M., Yamaguchi, Y., and Kalkum, M. (2021) Proteolytic processing,maturation, and unique synteny of the Streptomyces hemagglutinin SHA. Microbiol
Spectr 9, e0076621

9.

Tagai, K., Ono, M., Kubota, M., Kitamura, S., Takahata, K., Seki, C., Takado, Y.,
Shinotoh, H., Sano, Y., Yamamoto, Y., Matsuoka, K., Takuwa, H., Shimojo, M.,
Takahashi, M., Kawamura, K., Kikuchi, T., Okada, M., Akiyama, H., Suzuki, H.,
Onaya, M., Takeda, T., Arai, K., Arai, N., Araki, N., Saito, Y., Trojanowski, J. Q., Lee,V. M. Y., Mishra, S. K., Yamaguchi, Y., Kimura, Y., Ichise, M., Tomita, Y., Zhang, M.
R., Suhara, T., Shigeta, M., Sahara, N., Higuchi, M., and Shimada, H. (2021) Highcontrast in vivo imaging of tau pathologies in Alzheimer's and non-Alzheimer's diseasetauopathies. Neuron 109, 42-58 e48

10.

Iijima, J., Hoshi, K., Ito, H., Kanno, M., Murakami, Y., Takahashi, K., Matsumoto, K.,
Yamaguchi, Y., Nakajima, M., Miyajima, M., Arai, H., Kanai, M., Kitazume, S., Honda,
T., and Hashimoto, Y. (2021) Total transferrin in cerebrospinal fluid is a novel
biomarker for spontaneous intracranial hypotension. Fukushima J Med Sci 67, 64-70

11.

Hoshi, K., Ito, H., Abe, E., Fuwa, T. J., Kanno, M., Murakami, Y., Abe, M., Murakami,T., Yoshihara, A., Ugawa, Y., Saito, T., Saido, T. C., Matsumoto, K., Yamaguchi, Y.,
Furukawa, K., Arai, H., Kanai, M., Miyajima, M., Arai, H., Ogawa, N., Akatsu, H.,
Hashizume, Y., Tateno, H., Honda, T., and Hashimoto, Y. (2021) Transferrin
biosynthesized in the brain is a novel biomarker for Alzheimer's disease. Metabolites
11, 616

12.

Bonora, E., Chakrabarty, S., Kellaris, G., Tsutsumi, M., Bianco, F., Bergamini, C.,
Ullah, F., Isidori, F., Liparulo, I., Diquigiovanni, C., Masin, L., Rizzardi, N., Cratere, M.
G., Boschetti, E., Papa, V., Maresca, A., Cenacchi, G., Casadio, R., Martelli, P., Matera,
I., Ceccherini, I., Fato, R., Raiola, G., Arrigo, S., Signa, S., Sementa, A. R., Severino,
M., Striano, P., Fiorillo, C., Goto, T., Uchino, S., Oyazato, Y., Nakamura, H., Mishra, S.
K., Yeh, Y. S., Kato, T., Nozu, K., Tanboon, J., Morioka, I., Nishino, I., Toda, T., Goto,
Y. I., Ohtake, A., Kosaki, K., Yamaguchi, Y., Nonaka, I., Iijima, K., Mimaki, M.,
Kurahashi, H., Raams, A., MacInnes, A., Alders, M., Engelen, M., Linthorst, G., deKoning, T., den Dunnen, W., Dijkstra, G., van Spaendonck, K., van Gent, D. C.,
Aronica, E. M., Picco, P., Carelli, V., Seri, M., Katsanis, N., Duijkers, F. A. M.,
Taniguchi-Ikeda, M., and De Giorgio, R. (2021) Biallelic variants in LIG3 cause a novel
mitochondrial neurogastrointestinal encephalomyopathy. Brain 144, 1451-1466

109

＜学会発表＞・液体クロマトグラフィー/イオンモビリティー質量分析法を利用した 57 種類の N-結合型糖鎖の保持時間・衝突断面積・m/z のデータセット
真鍋法義，大野詩歩，川瀬泰司 a，廣瀬賢治 a，山口芳樹（日本ウォーターズ a）
第 33 回バイオメディカル分析科学シンポジウム（BMAS2021），京都，2021 年 9月，演題番号 P23
・MUC1 糖ペプチドと抗 MUC1 抗体 MY.1E12 の NMR 相互作用解析とドッキングシ
ミュレーション
國府 涼香，倉谷 太豪，高橋 優花，大野 詩歩，真鍋 法義，清水 弘樹 a，
千葉 靖典 a，伝田 香里 b，築地 信 c，入村 達郎 b，山口 芳樹（産総研細胞分子工学 a，順天堂大院医 b，星薬科大薬 c）
第 15 回東北糖鎖研究会，岩手，2021 年 9 月，演題番号 O-5・非環状糖鎖成分リビトールリン酸の構造解析
大野 詩歩，真鍋 法義，田村 純一 a，山口 芳樹（鳥取大農 a）
第 15 回東北糖鎖研究会，岩手，2021 年 9 月，演題番号 P-7
・Structural role of N-glycosylation on human IgA and J chain
Pan Shunli，Noriyoshi Manabe，Yoshiki Yamaguchi
第 15 回東北糖鎖研究会，岩手，2021 年 9 月，演題番号 P-8
・新規ミスセンス変異を認めた本邦初の B4GALNT1 関連神経変性症の 1 例
中村 勝哉 a,b，土田 奈緒美 c，井ノ口 仁一，小島 朋美 a，池田 淳司 b，小平 農 b，
稲森 啓一郎，永福 正和，新田 昂大，真鍋 法義，大野 詩歩，山口 芳樹，下畑 享
良 d，松本 直通 c，古庄 知己 a,e,f，関島 良樹 b
（信州大学医学部附属病院 遺伝子医療研究センターa，信州大学医学部 脳神経内科
リウマチ・膠原病内科 b，横浜市立大学大学院医学研究科 遺伝学 c，岐阜大学大学院
医学系研究科 脳神経内科学分野 d，信州大学医学部 遺伝医学教室 e，信州大学医学
クリニカル・シークエンス学講座 f）
日本人類遺伝学会第 66 回大会第 28 回日本遺伝子診療学会大会合同開催
横浜，2021 年 10 月，演題番号 P15-16
・タンデムリビトールリン酸含有 O-マンノシルグリカンの系統的合成とその糖鎖
田村 純一 a，田村 敬裕 a，真鍋 法義，大野 詩歩，山口 芳樹，萬谷 博 b，遠藤 玉夫b（鳥取大院連大農 a，都健康長寿研 b）

110

第 40 回日本糖質学会年会，鹿児島，2021 年 10 月，演題番号 1B-01(A)・NMR 法による GlcA/Xyl リピートとラミニンの相互作用様式の解析
真鍋 法義，大野 詩歩，田村 敬裕 a，田村 純一 a，萬谷 博 b，遠藤 玉夫 b，山口芳樹
（鳥取大院連大農 a，都健康長寿研 b）
第 40 回日本糖質学会年会，鹿児島，2021 年 10 月，演題番号 1C-03(B)・抗 iPS 細胞抗体 R-17F と lacto-N-fucopentaose I の相互作用解析
大野 詩歩，齋藤 祐希，真鍋 法義，湯浅 徳行 a，松崎 祐二 a，築地 信 b，川嵜 敏
祐 c，山口 芳樹（東京化成工業 a，星薬大薬 b，立命館大 c）
第 40 回日本糖質学会年会，鹿児島，2021 年 10 月，演題番号 P-127
・全ヒト糖鎖関連遺伝子 Variant の網羅的抽出と解析
西原 祥子 a，木下 聖子 a，栂谷 内晶 a，細田 正恵 a，伊藤 和義 a，山口 芳樹，真鍋 法義，大野 詩歩，稲森 啓一郎，井ノ口 仁一，灘中 里美 b，北川 裕之 b，城田
松之 c，木下 賢吾 c
（創価大糖鎖生命システム融合研究所 a，神戸薬科大 b，東北大東北メディカル・メ
ガバンク機構 c）
第 40 回日本糖質学会年会，鹿児島，2021 年 10 月，演題番号 3C-02(A)・3D structural view of β-glucan and its receptors
Yoshiki Yamaguchi and Noriyoshi Manabe
第 65 回日本医真菌学会総会・学術総会/真菌症フォーラム 2021, Tokyo, Japan, Oct.
2021, 16:10-17:50
・水溶性・不溶性 β-glucan と Dectin-1 受容体との相互作用解析
真鍋 法義
第 5 回東北医真菌研究会, 仙台, 2021 年 12 月
・Structural role of N-glycosylation on human IgA and J-chain
Shunli Pan，Noriyoshi Manabe，Yoshiki Yamaguchi
日本薬学会第 142 年会，オンライン開催，2022 年 3 月，演題番号 26P06-am1-10S
・MUC1 糖ペプチドと抗 MUC1 抗体 MY.1E12 の相互作用解析
國府 涼香，倉谷 太豪，高橋 優花，大野 詩歩，真鍋 法義，清水 弘樹 a，千葉 靖
典 a，伝田 香里 b，築地 信 c，入村 達郎 b，山口 芳樹（産総研細胞分子工学 a，順天堂大院医 b，星薬科大薬 c）
日本薬学会第 142 年会，オンライン開催，2022 年 3 月，演題番号 26P01-am1-46S

111

・Lacto-N-fucopentaose I の立体構造および抗 iPS 細胞抗体 R-17F との相互作用の解析
大野 詩歩，大内 陽翔，齋藤 祐希，真鍋 法義，湯浅 徳行 a，松崎 祐二 a，築地信 b，川嵜 敏祐 c，山口 芳樹（東京化成工業 a，星薬科大薬 b，立命館大 c）
日本薬学会第 142 年会，オンライン開催，2022 年 3 月，演題番号 26L-pm09
・NMR 法による αXyl-βGlcA の繰り返し構造とラミニンの相互作用解析
真鍋 法義，大野 詩歩，伊藤 良汰，田村 敬裕 a，田村 純一 a，萬谷 博 b，遠藤 玉夫 b，山口 芳樹（鳥取大院連大農 a，都健康長寿研 b）
日本薬学会第 142 年会，オンライン開催，2022 年 3 月，演題番号 27C-pm05
・Dectin-1 と不溶性 β-グルカン・Euglena gracilis EOD-1 由来 paramylon との相互作用解析
千葉 花子，真鍋 法義，内藤 淳子 a，西田 典永 a，髙橋 円 a，大野 尚仁 b，山口芳樹
（神鋼環境ソリューション a，東京薬大薬 b）
日本薬学会第 142 年会，オンライン開催，2022 年 3 月，演題番号 28N-pm11S

112

International Journal of

Molecular Sciences
Review

3D Structures of IgA, IgM, and Components
Shunli Pan, Noriyoshi Manabe

and Yoshiki Yamaguchi *Division of Structural Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical andPharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Miyagi, Japan;
22052501@is.tohoku-mpu.ac.jp (S.P.); manabe@tohoku-mpu.ac.jp (N.M.)* Correspondence: yyoshiki@tohoku-mpu.ac.jp; Tel.: +81-22-727-0208

Abstract: Immunoglobulin G (IgG) is currently the most studied immunoglobin class and is frequently used in antibody therapeutics in which its beneficial effector functions are exploited. IgG is
composed of two heavy chains and two light chains, forming the basic antibody monomeric unit. In
contrast, immunoglobulin A (IgA) and immunoglobulin M (IgM) are usually assembled into dimers
or pentamers with the contribution of joining (J)-chains, which bind to the secretory component (SC)of the polymeric Ig receptor (pIgR) and are transported to the mucosal surface. IgA and IgM play apivotal role in various immune responses, especially in mucosal immunity. Due to their structuralcomplexity, 3D structural study of these molecules at atomic scale has been slow. With the emergenceof cryo-EM and X-ray crystallographic techniques and the growing interest in the structure-function
relationships of IgA and IgM, atomic-scale structural information on IgA-Fc and IgM-Fc has been
accumulating. Here, we examine the 3D structures of IgA and IgM, including the J-chain and SC.
Disulfide bridging and N-glycosylation on these molecules are also summarized. With the increasing
information of structure–function relationships, IgA- and IgM-based monoclonal antibodies will bean effective option in the therapeutic field.


Citation: Pan, S.; Manabe, N.;

Keywords: IgA; IgM; J-chain; secretory component; glycosylation; molecular assembly; disulfide
bridge; 3D structure

Yamaguchi, Y. 3D Structures of IgA,
IgM, and Components. Int. J. Mol. Sci.
2021, 22, 12776. https://doi.org/
10.3390/ijms222312776
Academic Editor: Manlio Ferrarini
Received: 6 November 2021
Accepted: 23 November 2021
Published: 26 November 2021

Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/

1. Introduction
Immunoglobulins (Igs) are categorized into several classes (IgM, IgD, IgA, IgG, andIgE), and all play an invaluable role in the immune response to pathogens. Natural
IgG is a monomer composed of two heavy chains and two light chains. This simple
structure allows relatively easy expression and purification. IgG is the most studied
immunoglobin class and is frequently used in antibody therapeutics. In contrast, IgA wasinitially considered to be a non-inflammatory antibody for the maintenance of mucosal
homeostasis [1]. It has since been found that the different assembly forms of IgA and IgM
and their interaction with different receptors allow them to actively or passively inhibit or
initiate inflammatory responses, thus forming the basis of mucosal immunity [2–4]. Unlike
IgG, IgA and IgM are assembled into dimers or pentamers in the presence of J-chains.
Moreover, they are transported to the mucosal surfaces by a secretory component (SC) as
the first-line defense against infections [5]. Due to this structural complexity and inherent
flexibility, 3D structural study at atomic scale has only recently commenced. Until now,
structure–function relationships could only be discussed in crude terms based on lowresolution images and homology models. Without detailed knowledge of the 3D atomic
structure and species differences, the development of this class of antibody therapeutics
has been impeded. This situation is improving rapidly, and now atomic-scale structural
information on IgA and IgM molecules is available due to the use of cryogenic electron
microscopy (cryo-EM) and X-ray crystallographic techniques, which reveal the mode ofmolecular assembly. Electron densities originating from attached N-linked glycans have4.0/).

Int. J. Mol. Sci. 2021, 22, 12776. https://doi.org/10.3390/ijms222312776

https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 12776

2 of 13been partially solved, and these should help gain a greater understanding of the role ofN-glycan modifications in the functions of IgM and IgA.
2. Structure of IgAIgA plays an important role in mucosal immunity and is the most abundant immunoglobulin, with even more being produced than the sum of the other immunoglobulins [6]. Unlike other human immunoglobulins, IgA exists either as a monomer or as
soluble polymers. Only polymeric IgA can bind to polymeric immunoglobulin receptors(pIgR) for transcytosis, with the presence of J-chain being required for binding [7–9]. In
common with other immunoglobulins, the IgA monomer consists of two identical heavy
chains and two identical light chains forming a Y-shape structure (Figure 1). Each chain
extends a variable region from its N-terminal, followed by a constant region. The heavy
chains of IgA are composed of four domains (VH , Cα 1, Cα 2, and Cα 3), while the lightchains are divided into two domains (VL and CL ) [10]. The hinge region of IgA lies between
the Cα 1 and Cα 2 structural domains of each heavy chain. The hinge affords the flexibility
to the whole IgA molecule. This is critical for its activity, and it is this region that differs
most between the two subclasses IgA1 and IgA2 (being particularly extensive in humanIgA1 but absent in human IgA2). Unlike IgG, there is an 18-residue tailpiece extension at
the C-terminus of the IgA and IgM heavy chains, which is essential for immunoglobulin
assembly [11]. Fab is composed of VH , Cα 1, VL , and CL domains and engaged in antigen
binding. Fc comprises two Cα 2 and two Cα 3 domains, responsible for triggering effector
functions through interaction with complements or Fcα receptors [3]. Fc is stabilized
by several intra-molecular disulfide bonds, Cys266-Cys323 (Cα 2), Cys369-Cys432 (Cα 3),and every Cys242 forms an inter-chain disulfide bond with the Cys299 of another chain(Figure 1). The major differences between IgA1 and IgA2 are the hinge region (an octapeptide sequence Pro-Ser-Thr-Pro-Pro-Thr-Pro-Ser is repeated in IgA1) and the number andlocation of O- and N-glycans [7,12,13]. The Cys133 of IgA1 Cα 1 forms a disulfide bondwith the light chain, which is absent in IgA2 [14]. There are three IgA2 allotypic variants:
m1, m2, and mn. These allotypes differ in disulfide bridging between a heavy chain andlight chain so that Cys220 forms a covalent linkage when followed by Arg221 (IgA2 m2
and IgA2 mn) but not by Pro221 (IgA2 m1). In IgA2 m1, the replacement of Arg221 with
Pro221 introduces a kink in the peptide backbone, which precludes the formation of heavy
chain-light chain disulfide bridges but instead induces the formation of light chain-lightchain disulfide bonds [13–16]. In contrast to the previous speculation that IgA2 mn could
form heavy chain-light chain disulfide bonds in the absence of Cys220, mutation of Cys241
or Cys242 interferes with such disulfide bond formation [17]. Serum IgA molecules aremostly monomeric, while secretory IgA molecules are found as dimers and, to a lesser
extent, as trimers and tetramers. In the dimer, two IgA molecules are linked together
by another polypeptide designated as J-chain. Dimeric IgA is bound by the pIgR on3 oftheInt. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW
14
basolateral surface of epithelial cells, followed by the secretory component (SC) of thereceptor covalently binding to dIgA to form secretory IgA (sIgA) [18,19].

Figure1.1. Schematic
Schematicdrawings
drawingsof
ofhumanhumanIgA.
IgA.Disulfide
Disulfidebonding
bondingand
andN-glycosylation
N-glycosylationsites
sitesareareindicated.
indicated.
FigureCrystallization is often difficult due to the inherent flexibility of intact IgA monomers. Earlier electron microscopy (EM) studies showed that sIgA has a double-Y-likeshape [20]. With the significant advances in cryo-EM in recent years, three-dimensional

Int. J. Mol. Sci. 2021, 22, 12776

3 of 13

Crystallization is often difficult due to the inherent flexibility of intact IgA monomers.
Earlier electron microscopy (EM) studies showed that sIgA has a double-Y-like shape [20].
With the significant advances in cryo-EM in recent years, three-dimensional structures ofsIgA-Fc dimers have been determined [21]. The two Fcα are arranged in a boomeranglike shape with four tailpiece β-strands bundled together to mediate the interactions
between the two Fcα molecules (Figure 2) [21,22]. The dIgA is further stabilized throughdisulfide bridges between the Fc tail (Cys471) and the J-chain (Cys15 or Cys69), in which
two parallel β-strands of Fc1 and Fc2 extend to the bottom and top sheets of the twisted
J-chain β-sandwich. In the sIgA structure, interactions between SC domains and dIgA
(disulfide bonding between Cys468 of SC and Cys311 in the Cα 2 domain) stabilize a bent
and tilted relationship (an angle of 110◦ caudally) between two IgA monomers. Two orInt. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW
4 of 14three additional Fcs are added to the plane of the original dimer to form a higher-ordermultimer [21–25].

Figure 2.
2. 3D
3D structure
structure ofof human
human IgA-Fc
IgA-Fc dimer
dimer (gray)(gray) inin complex
complex with
with J-chain
J-chain (sky(sky blue)blue) andand SCSC
Figure(pink)determined
determinedby
bycryo-EM
cryo-EManalysis
analysis(PDB(PDBID:
ID:6UE7).
6UE7). Glycan
Glycanresidues
residuesare
areshown
shownin
inred
redwithwithstickstick(pink)
representation.
representation.

3. Structureof IgM
Amino acidsequences of IgA show significant species differences in mammals andbirds;Immunoglobulin
however, N-linked
sitesabundantof the tailpiece
are wellconserved
Both
M glycosylation(IgM) is anotherantibody
subclass
active [26].
in humanisotypes
of
IgA
have
two
conserved
N-linkedglycosylationsites,
Asn263inC
2αmucosal immunity, providing the first line of defense against pathogens [18,37]. The andpreAsn459
in thetail
has mostly
biantennaryglycanα2dominant
form
ofpiece.
mouseAsn263
and human
IgM antibodies
is complex-typepentameric, buttherewithare also
6-linkedsialicacids,
while
Asn459isdominatedbytriantennaryglycan
with
α2-6
and
small amounts of hexamers and monomers [38]. The multiple antigen-binding sites give
α2-3-linked
sialicacids [27,28].
glycans
Asn263in Cαis2 composed
are locatedof
ontwo
the heavyouter
IgM a higher
apparent
affinity N-linked
for antigen.
Each at
IgM
monomersurfaceof Fcfiveto avoid
potential
instability
caused by considerable surface exposure of thechains withdomains(VH, C
μ1, Cμ2, Cμ3, and Cμ4) and two light chains with two dounpairedC2
structural
domain[23,29].
Recentsuggestthat N-glycan
attachedonαlinked withan inter-chain
disulfidebondmains (VL, CL). The heavy chains are covalently studies
Asn263does
notimpactIgA
binding
to
Fc
R1
[30],
consistentwithearlierreports[31,32].
αCys337–Cys337, and each light chain is bonded to Cys136 in the heavy chain (Figure 3).
Asn459IgA2 tailpiececomplex- andhigh mannose-typeglycans but
Every Fcinisthestabilized
withtwo hasintra-moleculardisulfidebonds: Cys367–Cys426(Cμis3) notandfully
glycosylated
[33].
The
glycancontrols
theformationof
IgA
polymers,andsialicCys474–Cys536 (Cμ4). According to the sequence alignment, Cμ1, Cμ3, and Cμ4 of IgMacid
areresidues
foundin theN-linkedglycanactivity [30,33].
The
sialicequivalent
to theCγ1,complex
Cγ2, andCγ3 of IgG.
Cμmediate
2 of IgMantiviralis an additional
constant
domainacid
residues directlywith certain
seemingly
actingnecessary
as bait tofor
neutralizethat corresponds
to theinteract
hinge regionof IgG,viruses,
providingthe flexibility
bindingthem [30]. Human IgA2 has additional N-glycosylation sites (Asn166 in the Cα 1 andto antigens on cell surfaces. Cys414 of the Cμ3 domain is essential for IgM polymerization
Asn337 in the Cα 2), and IgA2m(2) has an additional N-linked glycan at Asn211 in the Cα 1
and can form an inter-monomeric disulfide bond with the Cys414 of an adjacent monodomain. These additional sites attach more neutralizing glycans, which creates a greater
mer. Like IgA, there is a tailpiece with a short 18 amino acid peptide sequence at the Cproinflammatory response of neutrophils and macrophages [31,34–36]. However, details ofterminus of the heavy chain. The Cys575–Cys575 disulfide bond in the tailpieces is criticalthe mechanism are not known.
for IgM polymerization. Five IgM monomers are linked by disulfide bonds to each otherand to the J-chain to form a pentamer, while the J-chain is absent in the IgM hexamer [38–43].

Int. J. Mol. Sci. 2021, 22, 12776

4 of 13

3. Structure of IgM
Immunoglobulin M (IgM) is another abundant antibody subclass active in humanmucosal immunity, providing the first line of defense against pathogens [18,37]. The
predominant form of mouse and human IgM antibodies is pentameric, but there arealso small amounts of hexamers and monomers [38]. The multiple antigen-binding sites
give IgM a higher apparent affinity for antigen. Each IgM monomer is composed of
two heavy chains with five domains (VH , Cµ 1, Cµ 2, Cµ 3, and Cµ 4) and two light chainswith two domains (VL , CL ). The heavy chains are covalently linked with an inter-chain
disulfide bond Cys337–Cys337, and each light chain is bonded to Cys136 in the heavy chain(Figure 3). Every Fc is stabilized with two intra-molecular disulfide bonds: Cys367–Cys426(Cµ 3) and Cys474–Cys536 (Cµ 4). According to the sequence alignment, Cµ 1, Cµ 3, andCµ 4 of IgM are equivalent to the Cγ 1, Cγ 2, and Cγ 3 of IgG. Cµ 2 of IgM is an additional
constant domain that corresponds to the hinge region of IgG, providing the flexibility
necessary for binding to antigens on cell surfaces. Cys414 of the Cµ 3 domain is essential
for IgM polymerization and can form an inter-monomeric disulfide bond with the Cys414
of an adjacent monomer. Like IgA, there is a tailpiece with a short 18 amino acid peptide
sequence at the C-terminus of the heavy chain. The Cys575–Cys575 disulfide bond in the
tailpieces is critical for IgM polymerization. Five IgM monomers are linked by disulfide
to each other and to the J-chain to form a pentamer, while the J-chain is absent in5 theInt. J. Mol. Sci. 2021, 22, x FOR PEERbonds
REVIEWof 14
IgM hexamer [38–43].

FigureFigure3.3.Schematic
Schematicdrawings
drawingsofofhuman
humanIgM
IgMpentamer.
pentamer.Disulfide
Disulfidebonding
bondingand
andN-glycosylation
N-glycosylationsites
sites
are
indicated.
are indicated.

The
Themolecularmolecularsize
sizeofofaafully
fullyassembled
assembledIgM
IgMcomplex
complexisisnearly
nearlyaamillion
millionDaltons;
Daltons;hence,
determinationof
itsdetailed
structureischallenging.
Earlierstudiessuggested
hence, determination of its detailed structure is challenging. Earlier studies suggested
that
the pentamer in the presence of the J-chain has a star-like appearance with five-fold symmetry [44,45]. However, a recent cryo-EM study proves that five Fcμ units are arranged inalmost perfect hexagonal symmetry with a 61° gap occupied by the J-chain, and a triangular SC perpendicularly docked to the Fcμ-J near-planar structure to form secretory IgMInt. J. Mol. Sci. 2021, 22, 12776

5 of 13that the pentamer in the presence of the J-chain has a star-like appearance with fivefold symmetry [44,45]. However, a recent cryo-EM study proves that five Fcµ units arearranged in almost perfect hexagonal symmetry with a 61◦ gap occupied by the J-chain,
and a triangular SC perpendicularly docked to the Fcµ -J near-planar structure to form
secretory IgM (sIgM) [46] (Figure 4). Cµ 3–Cµ 4 and the tailpiece play important roles in
the formation of IgM pentamers. Two neighboring Cµ 3–Cys414 form interchain disulfide
bonds, and two neighboring IgM-Cµ 4 domains interact with each other by their FG loops (aloop between F and G strands). Ten tailpieces are arranged into two five-stranded parallel
β-sheets. The two β-sheets stack together in an antiparallel fashion, which is critical for
IgM pentamer stability. Cys575 residues of two adjacent Fcµ form a disulfide bond andInt. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW
6 of 14
Cys575 on the side of the hexagonal gap is linked to the J-chain, which is essential for IgM
oligomerization [44,46,47].

Figure 4. 3D structure of human IgM-Fc pentamer (gray) in complex with J-chain (sky blue) and SC
disulfide bonds
bonds andand FG
FG loops
loops inin Fc
(pink) determined by cryo-EM analysis (PDB ID: 6KXS). Cys414 disulfide
arehighlighted inyellow andand magenta,
magenta, respectively.
respectively. Glycan
shown inin redred withwith stickstick
are highlighted
in yellow
Glycan residues
residues areare shownrepresentation.
representation.

4. Structureof five
J-chain
There areconserved N-glycosylation sites: Asn171 (Cµ 1), Asn332 (Cµ 2), Asn395(Cµ 3),
Asn402(C
3),
and Asn563(tailpiece):
Asn171,
Asn332,
andsIgM,
Asn395µ identified
The J-chain was
in 1971
in fractions
fromsIgA andbut arenotexposedin thosetosolvent
withcomplex-typeglycans(differencesinglycanbranchingandsialylation)from IgG, IgE, and IgD [51]. It is highly conserved among different species, andthere isattached.
These glycans
are vulnerable
to J-chain
enzymatic
andpolypeptide
involved incomthenoother homologous
protein[52]. Humanis a processing
15-kDa acidic
binding
IgM
to T-cellsurfacereceptors(asialo
IgM remains
while sialylated
IgM
posed
ofof
137
amino
acids,
including
six Cysresidues
involvedbound,in intramoleculardisulfideis internalized)[48]. TheCys71–Cys91,
glycosylation andof Asn402(as with Asn297
of IgG)is crucial for
bridges(Cys12–Cys100,
Cys108–Cys133)
and twoin intermolecularcomplement
activation
with highmannose-typeglycans.
Asn563
theAlthoughtailpiece, various
located(Cys15and Cys69)
disulfides
to theFc of IgM andIgA (Figure
5) of
[53].
in
the
centerofthe
IgM
polymer,possesses
high-mannose-typeglycanswhichfacilitate
predictions of the structure of the J-chain have been made based on the pattern of disulfide
assembly
the IgMhas
monomerare necessary
to prevent
IgM
aggregation[49,50].
bonds,
its ofstructure
not beenand
resolveduntil recently.
Now,
with
the structuresof sIgAand sIgM determined, the three-dimensional structure of the J-chain is resolved in their
4. Structure of J-chain
polymers. As predicted, the J-chain consists almost entirely of β-sheets and loops in the
The J-chain
wasidentified
in [21,46,53].
1971 in fractions
fromdimer,sIgA andsIgM, but
not inIgA dimer
and theIgM
pentamerIn the IgA
the central
regionof those
the Jfrom
IgG,
IgE,
and
IgD
[51].
It
is
highlyconserved
among
different
species,and
there
is asnochain contains four β-strands (β1–β4) and three hairpins (Figure 5). β1–β3 strands areotherhomologous
protein[52].
HumanJ-chainisa15-kDaacidicpolypeptide
composed
sembled into a β-sheet together with two tailpiece strands of Fcα2. Strand β4 is packed
onto the tailpieces of Fcα1 to form another β-sheet. These two β-sheets fold into a single βsandwich-like domain through strong hydrophobic interactions. β-hairpins 1 and 2 interact with the top surface of Fc2, while the bottom surface of Fc1 interacts with β-hairpin 3.
β-hairpin 1, β3–β4 loop, β-hairpin 2, and C-terminal β-hairpin 3 form four lassos to further

Int. J. Mol. Sci. 2021, 22, 12776

6 of 13of 137 amino acids, including six Cys residues involved in intramolecular disulfide bridges(Cys12–Cys100, Cys71–Cys91, and Cys108–Cys133) and two in intermolecular (Cys15 andCys69) disulfides to the Fc of IgM and IgA (Figure 5) [53]. Although various predictionsof the structure of the J-chain have been made based on the pattern of disulfide bonds, its
structure has not been resolved until recently. Now, with the structures of sIgA and sIgM
determined, the three-dimensional structure of the J-chain is resolved in their polymers. As
predicted, the J-chain consists almost entirely of β-sheets and loops in the IgA dimer andthe IgM pentamer [21,46,53]. In the IgA dimer, the central region of the J-chain contains
four β-strands (β1–β4) and three hairpins (Figure 5). β1–β3 strands are assembled into aβ-sheet together with two tailpiece strands of Fcα 2. Strand β4 is packed onto the tailpiecesof Fcα 1 to form another β-sheet. These two β-sheets fold into a single β-sandwich-likedomain through strong hydrophobic interactions. β-hairpins 1 and 2 interact with the
top surface of Fc2, while the bottom surface of Fc1 interacts with β-hairpin 3. β-hairpin 1,
β3–β4 loop, β-hairpin 2, and C-terminal β-hairpin 3 form four lassos to further interact
with the Fcα . Cys69 and Cys15 of the J-chain form disulfide bonds with the penultimate
cysteine residues Cys471 of Fcα 1 and Fcα 2, which are the foundation for the locking of two
IgA monomers to the J-chain [21,22,25]. As in the IgA dimer, the J-chain forms a radiallycentered β sandwich-like structure with the tailpieces of five Fcs in the IgM pentamer. The
β3 and β4 strands are packed on the tailpieces of Fc5 and Fc1 through a disulfide bondMol. Sci. 2021, 22, x FOR PEER REVIEW
of 14of Fc5 throughCys15 (J-chain)-Cys575 (Fc1), while the β2–β3 loop interacts with the 7basea disulfide bond C69 (J-chain)-Cys575 (Fc5). The hairpin of the C-terminal wing forms
extensive hydrophobic interactions with Fc1 and also interplays with pIgR/SC, allowing
IgMstructures
to attach to[46].
differenceof the sIgAof the sIgA andthesIgM
is thethatreceptor
β-hairpin
2 inThethebiggest
IgA dimer
is not between
observedthein J-chains
IgM
and
sIgM
structuresisthat
β-hairpin
2
in
the
IgA
dimer
is
not
observedinIgM[54]. The[54]. The structure of the J-chain without IgA/IgM has not been reported yet. There is a
structureoftheJ-chainwithout
IgA/IgM
has
not
been
reported
yet.
There
is
a
conservedconserved N-glycosylation site at Asn49 of the human J-chain. The attached oligosacchaN-glycosylation site at Asn49 of the human J-chain. The attached oligosaccharides are
rides are represented in three forms, each differing in the amount of sialic acid, 30% conrepresented in three forms, each differing in the amount of sialic acid, 30% contain two
tain two sialic acid residues, 55% contain a sialic acid residue on the α l,3-linked mannose
sialic acid residues, 55% contain a sialic acid residue on the α l,3-linked mannose branch,branch, and 15% have no sialic acid but two terminal galactose residues [55]. The glycoand 15% have no sialic acid but two terminal galactose residues [55]. The glycosylated
sylated murine J-chain mutant with an Asn48 (corresponding to Asn49 of human J-chain)murine J-chain mutant with an Asn48 (corresponding to Asn49 of human J-chain) to Ala
to Ala substitution in human IgA1 has reduced dimer assembly [56]. The N-glycosylation
substitution in human IgA1 has reduced dimer assembly [56]. The N-glycosylation site
site of the J-chain is distant from any SC-pIgA interaction interface in sIgA, which may beof the J-chain is distant from any SC-pIgA interaction interface in sIgA, which may be
relevant for promoting host and pathogen lectin binding [15,22,25]. Likewise, the glycan
relevant for promoting host and pathogen lectin binding [15,22,25]. Likewise, the glycan
attached Asn49 is completely exposed to the surface of the human sIgM molecule without
attached Asn49 is completely exposed to the surface of the human sIgM molecule without
any contact with protein [46].
any contact with protein [46].

Figure 5. Schematic drawings of human J-chain. Disulfide bonding and N-glycosylation site are indicated. 3D structure ofhuman J-chain bound to IgA-Fc is shown on the right (PDB ID: 6UE7). IgA-Fc and SC are omitted for clarity.

Figure 5. Schematic drawings of human J-chain. Disulfide bonding and N-glycosylation
5. Structureof Secretorysite are indicated.
3D structureof humanComponent
J-chain bound to IgA-Fc is shown on the right(PDB ID: 6UE7). IgA-Fc
andpIgR
SC areomittedfor clarity.
Human
plays
a dominantrole in mucosal immunity. The extracellular ligandbinding portion (known as SC) of pIgR is proteolytically cleaved and transported into
5. Structure of mucosal
Secretory
Component
secretions
along with dimeric IgA (dIgA) or pentameric IgM (pIgM) [5]. SCs bindandexcludepathogens
as in
part
of the immunity.
immune response[5,57,58]. ligandHuman SC contains
Human pIgR plays a dominant rolemucosalThe extracellularbinding portion (known as SC) of pIgR is proteolytically cleaved and transported into
mucosal secretions along with dimeric IgA (dIgA) or pentameric IgM (pIgM) [5]. SCs bindand exclude pathogens as part of the immune response [5,57,58]. Human SC contains 620
amino acids, forming five Ig-like domains named D1-D5 from the N-terminus [59]. ThereInt. J. Mol. Sci. 2021, 22, 12776

7 of 13620 amino acids, forming five Ig-like domains named D1-D5 from the N-terminus [59].
There is a short non-immunoglobulin-like sequence between the D5 terminus and themembrane [60]. The three-dimensional structure of SC in a free form has been determinedby X-ray crystallography, and the five domains (D1–D5) are organized into a compact, platelike isosceles triangle (Figure 6). D2–D3 and D4–D5 form two edges, and D1 comes into
contact with D2 and D4–D5 to form a third edge [19]. There are seven N-glycosylation sites(Asn65, 72, 117, 168, 403, 451, and 481), with fucosylated and sialylated N-glycans [61]. The
ordered glycans are observed at four N-linked glycosylation sites (Asn65, Asn72, Asn168,
and Asn481) and are mostly exposed on surfaces of the molecule. [19,60] They help the SC
bind to pIg ligands and facilitate interactions between lectin and free SC, in addition toprotecting SC from proteolytic degradation [5,22]. Each domain has a basic folded topology
consisting of two β-sheets connected by a conserved disulfide bond (Cys22–Cys92 in D1,
Cys134–Cys202 in D2, Cys239–Cys307 in D3, Cys353–Cys423 in D4, and Cys468–Cys526 in
D5) [19,60,61]. With the determination of the sIgA and sIgM structures, the 3D structureof SC bound to IgA and IgM was also reported. A drastic conformational change of
SC is observed in sIgA and sIgM (Figure 6). The D4–D5 side rotates 120◦ to be colinear
Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW
8 of to14
with unchanged D2–D3, and D1 is packed on D2–D3 by rotating 84◦ to allow binding
dIgA and pIgM [21,46]. Noncovalent interactions with three complementary determinants(CDRs) of D1 play an essential role in the assembly of sIgA or sIgM in the presence ofthetheJ-chain,
andofthere
is a broadly
consistent
D1 interaction
in sIgAinterface
and sIgM.
inpresence
the J-chain,and there
is a broadly
consistentinterface
D1 interaction
in
D2–D4
providethe correct
spacing
D1 andD5, thereby
assistinginteraction
with interdIgA
sIgA
andsIgM. D2–D4
providethe ofcorrectspacingof D1 andD5, thereby
assistingand pIgM
D5 interacts
withD5
Fc interacts
through thebond
Cys468
actionwith[21,25,46,54].
dIgA and pIgM
[21,25,46,54].
withdisulfide
Fc throughthebetween
disulfidebond(SC D5) and Cys311 (IgA Fc2) or Cys414 (IgM Fc2) [62,63].
between Cys468 (SC D5) and Cys311 (IgA Fc2) or Cys414 (IgM Fc2) [62,63].

Figure 6.
6. 3D
3D structures
structures of SC in free form
andSC
SCboundbound toto IgA-Fc
IgA-Fc dimer
dimer andFigureform (PDB(PDB ID:
ID: 5D4K,
5D4K,(left))left) andJ-chainright). Glycan
J-chain (PDB ID: 6UE7, (right)).
Glycanresidues
residuesare
areshown
shownin
inred
redwithwith stick
stick representation.
representation. On
On therightpanel,
IgA-Fcwasomittedforclarity.
right panel, IgA-Fc was omitted for clarity.

6. Interaction
Interaction with
with Fc
Fc Receptors
Receptors andand Others
Others6.
IgAandandIgM
IgMFcsFcsareareknown
knowntotointeract
interact with
with aavariety
variety ofofreceptors
receptors toto mediate
mediate multiple
multiple
IgA
effector functions,functions, such
suchasaspIgR,
pIgR,Fc
Fcαα/μ/R,µ R,
complement
receptor,
Fcµetc.
R, etc.
Under
α RI,
effector
FcαFc
RI,complement
receptor,
FcμR,
Under
hohomeostatic
conditions,
IgA
complexed
withpIgR
inhibits
bacterial
adherencetothemeostatic conditions, IgA complexed with pIgR inhibits bacterial adherence to the mucomucosal
wall,
which
functions
as
the
first
line
of
defense
of
the
body.
Interactions
withsal wall, which functions as the first line of defense of the body. Interactions with pIgR
pIgR been
have discussed
been discussed
SC section.
Once pathogens
enter
the bloodstream,
havein the in
SCthe
section.
Once pathogens
enter thebloodstream,
serumserum
monmonomeric
IgA
initiates
the
third
line
of
immunedefenseasaproinflammatory
antibody
omeric IgA initiates the third line of immune defense as a proinflammatory antibody
ininteracting
with
IgA-specificreceptor
FcαHere,RI. Here,
we elaborate
oninteractions
the interactions
teractingwiththetheIgA-specificreceptor
FcαRI.
we elaborateon thewithwith human
R and
the IgA-specificFcαIgM
RI, andwith the apoptosis
α /µthehuman
Fcα/μRFcand
IgA-specificreceptorreceptor
FcαRI, andwithIgM
the apoptosis
inhibitor
inhibitorofmacrophages(AIM).
IgA
interacts
with
Fc
RI
to
triggerclearanceof IgA-boundαof macrophages (AIM). IgA interacts with FcαRI to trigger clearance of IgA-boundaggresaggressive
pathogens.
Most IgA-dependent
are mediatedsivepathogens.
Most IgA-dependent
immune immune
responsesresponses
are mediatedthrough FcthroughαRI, inFcα RI, inducing
phagocytosis,
antibody-dependentcell-mediatedcytotoxicity,
respiratory
ducingphagocytosis,
antibody-dependentcell-mediatedcytotoxicity,
respiratory
burst,and cytokine release [64]. Crystal structures of the FcαRI complex with IgA1 Fc havegreatly facilitated our understanding of their interactions with IgA. Like most Ig receptors,
FcαRI is a transmembrane glycoprotein comprising two extracellular Ig-like structural domains (EC1 and EC2), a transmembrane region, and a short cytoplasmic tail. The two ex-

Int. J. Mol. Sci. 2021, 22, 12776

8 of 13burst, and cytokine release [64]. Crystal structures of the Fcα RI complex with IgA1 Fc
have greatly facilitated our understanding of their interactions with IgA. Like most Ig
receptors, Fcα RI is a transmembrane glycoprotein comprising two extracellular Ig-likestructural domains (EC1 and EC2), a transmembrane region, and a short cytoplasmic tail.
The two extracellular structural domains are folded together at an angle of approximately
90◦ [23,32,65]. In each extracellular domain, several β-strands are assembled into two
β-sheets, where the first β-strand is shared between the two β-sheets and with one strand
absent in EC2. Both domains include three short helices, and there is an extra polyproline
type-II helix in EC2 [23]. There are six potential N-glycosylation sites (Asn44, Asn58,
Asn120, Asn156, Asn165, and Asn177) and several O-glycosylation sites in free Fcα RI.
A recent study shows that Asn177 may not be glycosylated [66,67]. IgA monomer andpolymer can all bind Fcα RI. The crystal structure of the Fcα RI/Fcα complex shows that asingle Fc dimer binds two Fcα RI receptors. The interaction between Fcα RI and IgA occurs
at the Cα 2-Cα 3 interface, consisting of a central hydrophobic core and two sides of charged
residues [23,66,68,69]. Free Fcα RI receptor binds to Fcα with a conformational change in
two loops and a strand in EC1 [23]. Asn58 of Fcα RI is closest to the IgA interaction surface,and deglycosylation at Asn58 results in increased IgA binding [67]. There is an increase inaffinity upon desialylation at Asn58, and Asn58 modified with a single GlcNAc residue
has the highest receptor affinity [70]. The glycans attached at the other sites exert little orno influence on IgA binding [67,70].
Another novel Fc receptor Fcα /µ R, the only IgM Fc receptor on hematopoietic cells,
plays a significant role in triggering IgA- and IgM-mediated immune responses [71–73].
The Fcα /µ R gene is located adjacent to pIgR on chromosome 1. Amino acid sequencealignment shows that there is a conserved motif in SC D1 that is crucial for interactingwith sIgA and pIgM [74,75]. This type-1 transmembrane protein has a unique extracellularloop containing three domains, a short EC1, an Ig variable region-like EC2 that shares 43%
homology with SC D1, and a stalk-like EC3 [76]. Studies using an antibody against Fcα /µ R
and 3D homology modeling suggest the presence of a conserved peptide sequence (alsopresent in SC D1) that interacts with IgA or IgM [73,77,78]. Like pIgR, Fcα /µ R has three
CDR-like loops in EC2, and the CDR1-like loop lies exactly at the position of the conserved
peptide sequence (TIHCHYAPSSVNRHQRKYW). Substitution experiments indicate that
the CDR1- and CDR2-like loops together play a more essential role than the CDR3-likeloop in binding to IgA and IgM, where positively charged Arg31 and the hydrophobic side
chain of Val29 of the CDR1-like loop and Asn54-Gln55 of the CDR2-like loop contribute toligand binding, while the CDR3-like loop assists in stabilizing the structure. Fcα /µ R has ahigher affinity for IgM than IgA and only binds to IgM and IgA polymers [78–80]. There
are two potential N-glycosylation sites at Asn167 and Asn276, but the function of putative
N-glycans in binding to IgA and IgM is unclear [74,81].
Apoptosis inhibitor of macrophages (AIM) is a member of a scavenger receptor
cysteine-rich (SRCR) superfamily and is expressed on the surface of macrophages [82]. Recent studies show that the pentameric IgM is a transporter for the effector protein AIM andthat the trapping by pIgM inactivates AIM and increases its level in the blood by decreasing
renal excretion [83,84]. AIM is released from an AIM–IgM complex to inhibit the apoptosisof thymocytes, especially during acute kidney injury, and this enhances the clearance ofexcess fat, bacteria, cancer cells, and dead cell debris [85,86]. sIgM is able to bind to thecomplement receptor and Fcα /µ R on antigen-presenting cells, including macrophages andfollicular dendritic cells (FDC) [87]. AIM-containing IgM-antigen complexes are ligated to
the FDC surface by complement receptors. In contrast, cells lacking AIM bind to Fcα /µ R
and are processed and present on MHC class II after internalization [88].
Single-particle negative-stain EM of a pIgM-AIM complex has shown how pIgM
associates with AIM [47]. There are three conserved SRCR domains in the AIM molecule.
SRCR2 and SRCR3 are essential for the formation of the pIgM-AIM complex, especially
Cys194 of SRCR2. SRCR1 does not appear to bind pIgM according to a homology-based
structural model and mutational analysis. AIM assumes a broad bean-like structure, locatedInt. J. Mol. Sci. 2021, 22, 12776

9 of 13in a 50◦ gap in pIgM with the J-chain. The Cys194 of SRCR2 forms an intermoleculardisulfide bond with the Cys414 of the Fc-Cµ 3 domain at one edge, and the positively
charged amino acid cluster (H294, K298, R300, K301, K340) of SRCR3 interacts with thenegatively charged cluster of the Fc-Cµ 4 domain on the opposite edge [47,89,90]. There
are three potential N-glycosylation sites (Asn99 of SRCR1, Asn229 of SRCR2, and Asn316
of SRCR3) in murine AIM and these N-glycans affect the secretion and lipolytic functions.
It is unclear whether the N-glycans play a role in IgM interactions [91]. Potential Nglycosylation sites of human AIM do not appear to possess any glycans.
7. Therapeutic Potential of IgA and IgM
Although IgA and IgM play an essential role in human mucosal immunity, conventional vaccinations usually do not induce mucosal immunity unless administrated to themucosal surface. The progress in determining their structure and interaction with receptors suggests that IgA and IgM-based immunotherapy is an important field for thedevelopment of vaccinations and therapeutics. More than 90 therapeutic IgG antibody
products have FDA approval, whereas almost no IgA and IgM antibodies have beentested in humans [92]. The superiority of IgA over IgG for recruiting polymorphonuclear
cells and the development of bispecific antibodies (BsAb) have opened up prospects for
IgA antibody applications [93–95]. For instance, anti-HER2 × Fcα RI (against both HER2
and Fcα RI) effectively clears breast cancer cells through neutrophil accumulation, but
anti-HER2 × Fcγ RI does not [96]. Another BsAb, anti-HLA II × Fcα RI is also a powerful
candidate in recruiting polymorphonuclear cells against human B cell malignancies [97].
In addition, bispecific IgM with ten or twelve binding sites potentially allows very highaffinity binding to difficult or rare tumor antigens, enabling selective contact with T cells
resulting in tumor cell death. Anti-CD20 × CD3 IgM is currently in clinical trials against
refractory or resistant non-Hodgkin’s lymphoma [98,99].
While there are many possible advantages of IgA and IgM in antibody therapy, there
are several issues that need to be overcome. For example, how does IgA mediate both proand anti-inflammatory functions via FcαRI in order to exert a therapeutic effect. The shorthalf-life makes use of IgA potentially expensive, and the need for the high frequency ofadministration is inconvenient for patients. Another issue is the efficiency of expression,
production, purification, and complete assembly of recombinant IgA and IgM monoclonal
antibodies with appropriate homogeneity. A more critical limitation is the lack of suitableanimal models, one example being the differences in the polymerization state of serum
IgA and the lack of Fcα RI in the mouse. Many of these issues are now being addressed.
For instance, modification of terminal N-glycans successfully lengthened the half-lifeof asialoglycoprotein receptor-mediated clearance [100,101]. Systems for increasing Igs
expression and rational assembly are being gradually developed [33,101,102]. Successful
establishment of human CD89 and IgA transgenic mice now enables in vivo studies of
IgA [103,104].
The recently resolved three-dimensional structures of IgM Fc pentamers and IgAFc multimers have provided us with an excellent base to help understand and modify
these complex macromolecules. We hope that this review provides some ideas for theengineering of bispecific IgA and IgM antibodies and the development of more effective
therapeutics.
Author Contributions: Conceptualization, Y.Y.; writing—original draft preparation, S.P.; writing—review and editing, S.P., N.M. and Y.Y.; visualization, N.M.; supervision, Y.Y. All authors have readand agreed to the published version of the manuscript.
Funding: This work was supported in part by JSPS KAKENHI Grant Number 19H03362.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

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Acknowledgments: S.P. is a recipient of the special scholarship from Tohoku Medical and Pharmaceutical University and the scholarship from the Tohoku Kaihatsu Memorial Foundation.
Conflicts of Interest: The authors declare no conflict of interest.

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ArticleRibitol in Solution Is an Equilibrium of Asymmetric
Conformations
Shiho Ohno 1 , Noriyoshi Manabe 1 , Takumi Yamaguchi 2 , Jun Uzawa 3 and Yoshiki Yamaguchi 1,3, *
1

2

3

*



Citation: Ohno, S.; Manabe, N.;
Yamaguchi, T.; Uzawa, J.; Yamaguchi,
Y. Ribitol in Solution Is an
Equilibrium of Asymmetric
Conformations. Molecules 2021, 26,5471. https://doi.org/10.3390/

Division of Structural Glycobiology, Institute of Molecular Biomembrane and Glycobiology,
Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku,
Sendai 981-8558, Miyagi, Japan; s.ohno@tohoku-mpu.ac.jp (S.O.); manabe@tohoku-mpu.ac.jp (N.M.)
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai,
Nomi 923-1292, Ishikawa, Japan; takumi@jaist.ac.jp
Structural Glycobiology Team, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa,
Wako 351-0198, Saitama, Japan; uzawa_fe79@ck9.so-net.ne.jp
Correspondence: yyoshiki@tohoku-mpu.ac.jp; Tel.: +81-22-727-0208

Abstract: Ribitol (C5 H12 O5 ), an acyclic sugar alcohol, is present on mammalian α-dystroglycan as acomponent of O-mannose glycan. In this study, we examine the conformation and dynamics of ribitolby database analysis, experiments, and computational methods. Database analysis reveals that theanti-conformation (180◦ ) is populated at the C3–C4 dihedral angle, while the gauche conformation(±60◦ ) is seen at the C2–C3 dihedral angle. Such conformational asymmetry was born out in asolid-state 13 C-NMR spectrum of crystalline ribitol, where C1 and C5 signals are unequal. On
the other hand, solution 13 C-NMR has identical chemical shifts for C1 and C5. NMR 3 J coupling
constants and OH exchange rates suggest that ribitol is an equilibrium of conformations, under
the influence of hydrogen bonds and/or steric hinderance. Molecular dynamics (MD) simulations
allowed us to discuss such a chemically symmetric molecule, pinpointing the presence of asymmetricconformations evidenced by the presence of correlations between C2–C3 and C3–C4 dihedral angles.
These findings provide a basis for understanding the dynamic structure of ribitol and the function ofribitol-binding enzymes.
Keywords: ribitol; conformation; dynamics; NMR; MD simulation; hydrogen bondmolecules26185471
Academic Editor: Hideyuki Takeuchi

1. Introduction
Received: 9 August 2021
Accepted: 6 September 2021
Published: 8 September 2021

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with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Ribitol (C5 H12 O5 ) is an acyclic sugar alcohol and a component of teichoic acid foundin Gram-positive bacteria [1] (Figure 1). Ribitol is also found in riboflavin (vitamin B2) andflavin mononucleotide (riboflavin 50 -phosphate). Ribitol phosphate (D-ribitol 5-phosphate)is a component of O-mannose glycans on mammalian α-dystroglycan [2]. A genetic
deficiency of ribitol phosphate transferase leads to muscular dystrophy, highlighting theessential role of ribitol phosphate in the development of skeletal muscle [3]. Laminin is
known to interact with ribitol-containing O-mannose glycans, and the ribitol residues arelikely acting as a hinge in bridging laminin and α-dystroglycan. Hence, the structure anddynamics of ribitol are now receiving much attention. However, compared with the cyclic
hemiacetal sugars, our knowledge of the structural properties and dynamics of such an
acyclic sugar alcohol is rather limited.
Jeffrey et al. investigated the crystal structures of nine alditols, including ribitol [4].
In the crystalline state, the carbon chain of alditols tends to adopt an extended, planar
zigzag conformation when the configurations at alternate carbons are different (e.g., Cn
and Cn+2 will be D and L or L and D). When the configurations of alternate carbons are thesame (D and D or L and L), the Cn -O and Cn+2 -O bonds align in parallel. This arrangement
causes steric hindrance such that the carbon chain changes its conformation to bent andnon-planar. Ribitol adopts the same configuration at C2 and C4 (D and D), and the steric

Molecules 2021, 26, 5471. https://doi.org/10.3390/molecules26185471

https://www.mdpi.com/journal/molecules

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Molecules 2021, 26, 5471

hindrance brought by O2 and O4 is avoided by rotating the C2–C3 or C3–C4 bonds by120◦ . In fact, the crystal structure of ribitol adopts a bent conformation, avoiding the steric
2 of 11hindrance induced by a stretched planar zigzag structure. Likewise, the ribitol moiety ofriboflavin exhibits a bent conformation in crystals [5,6].

Figure 1. Chemical
Chemicalstructures
structuresofof
compounds
examinedin thisstudy.
(a) Ribitol,(b) ribitol
thethecompounds
examinedin thisstudy.
(a) Ribitol,(b) ribitol
phosphosphate(D-ribitol
5-phosphate
is analyzed
this study,
is thefoundform infound
in nature),phate (D-ribitol
5-phosphate
is analyzed
in thisinstudy,
which whichis the form
nature),and (c)
1,3,5-pentanol(referencecompound).
Carbon andoxygennumberingis indicatedthe figure.
and(c) 1,3,5-pentanol(referencecompound).
Carbonandoxygen numbering
isin
indicatedin Thethemain-chain
dihedral angles
are defined
as φ1(O1–C1–C2–C3),
φ2 (C1–C2–C3–C4),
φ3 (C2–C3–C4–
figure.
The main-chain
dihedralangles aredefinedas ϕ1 (O1–C1–C2–C3),
ϕ2 (C1–C2–C3–C4),
ϕ3
C5), and φ4 (C3–C4–C5–O5)
throughout thismanuscript.
(C2–C3–C4–C5),
and ϕ4 (C3–C4–C5–O5)
throughout
this manuscript.

Jeffrey etetal.al.investigatedthe crystal
structures
alditols,
including[4].
Hawkesperformed solution
NMR
analysisoftonine
determinethestructureribitolof ribitol
3 J(H,H) values
In theoncrystalline
state, [7].
the Ribitol
carbonequilibrates
chain of alditols
tends
to adopt
an andextended,planarbased
betweena twistat C2–C3
one at C3–C4,
zigzag conformation
when the configurations
at alternate
carbonsbearedifferent(e.g., Cnremoving
the O2/O4 interactionin the planar chain
form. It should
notedthat 3 J(H2,H3)
n+2 will be
and L areor Lindistinguishable
and D). When theinconfigurations
alternatecarbons
are theand 3CJ(H3,H4)
ofDribitol
solution NMR.ofTherelationshipbetween
same
(
D
and
D
or
L
and
L
),
the
C
n
-O
and
C
n+2
-O
bonds
align
in
parallel.
This
arrangement
C2–C3 and C3–C4 torsion angles is not definitive solely from solution NMR analysis.
13 C-labeledcausessteric andhindrance
suchthat the carbon
chain changes
its conformation
bent13andGarrett
Serianni
synthesizedsugar alcohols,
includingtoD-[1C]3
ribitol,
for NMR
analysisis 1.7 Hz, whichintermediate≈1.3
Hz
non-planar.
Ribitol
adopts[8].
theJ(C1,C4)
same configuration
at C2isand
C4 (D andbetweenD), and thesteric(gaucheconformations)
2.3 Hz(anti conformation),
indicating
the C2–C3
orhindrance
brought by O2andand≈O4is avoided
by rotating theC2–C3 orthat
C3–C4
bonds by
3 J(H,H)C3–C4
is crystal
a mixtureof extended
andadopts
bent forms.
et al. foundthat thethe
120°. Inlinkage
fact, thestructureof ribitola bentFranks
conformation,
avoiding
stericvaluesare significantly
inplanarwater andorganic
solvents,suggestingthat hydration
hindrance
induced by adifferent
stretchedzigzagstructure.
Likewise,
the ribitol
moiety ofandhydrogenbonding
affecttheconformationofribitol[9].
riboflavin exhibits a bent conformation in crystals [5,6].
Klein
et al.
MDsolution
and Monte
Carlo(MC)tosimulations
tetrasaccharideHawkes
et performed
al. performed
NMR
analysis
determinefor
thethestructureof ribitol
3J(H,H)ribitolunit
in teichoic
acid
Theyequilibrates
found thatbetween
the GalNAc-ribitol
linkageis onemore
based onvalues[7].[9].
Ribitola twist at C2–C3
andat flexC3–
ible
than otherthe
glycosidic
linkages, but
is It
not
completely
flexible.
C4, removing
O2/O4 interactionin thethe ribitol
planarchain
chainitself
form.
shouldbe notedthat
3J(H2,H3)
3J(H3,H4) of ribitol
Hatcheretand
al. parameterized
the CHARMm
force field foracyclic sugar
and perare indistinguishable
in solution
NMR.alcohols
The relationshipformedMD
calculations
for
sugar
alcohols,
includingribitol
[10].
Other
than
these,
between C2–C3 and C3–C4 torsion angles is not definitive solely from solution NMR thereanalareysis.few studies on the conformational dynamics of ribitol.
Our
present
study aims
to build13on
previous
knowledge
the conformationofGarrett
and Serianni
synthesized
C-labeledsugar
alcohols,on
includingD-[1-13C] ribiribitol
to deepen
our[8].
understanding
dynamic
structures betweenof ribitol through
3J(C1,C4) is oftol, forand
NMR
analysis
1.7static
Hz, andwhich
is intermediate≈1.3 Hz
databaseanalysis,experimental
NMR
analysis,andMD
simulation.
(gauche conformations) and ≈2.3 Hz (anti conformation), indicating that the C2–C3 or C3–C4 linkage is a mixture of extended and bent forms. Franks et al. found that the 3J(H,H)
2. Results and Discussion
values are significantly different in water and organic solvents, suggesting that hydration
2.1. Database Analysis of Ribitol and Ribitol Phosphateand hydrogen bonding affect the conformation of ribitol [9].
Crystalof oligosaccharides
boundto proteinsprovidegood
insight into
Klein et structures
al. performed
MD and Monte Carlo(MC)
simulations
for thetetrasaccharidethemorestableconformationsof
each
glycosidic
linkage[11].
Following
this flexible
notion,ribitol unit in teichoic acid [9]. They found that the GalNAc-ribitol linkage is more
weextracted
theconformation(s)ofribitolandribitol
phosphatefromtheCambridge
than other glycosidic linkages, but the ribitol chain itself is not completely flexible.
Crystallographic
Data Centre the(CCDC)and theforce
ProteinBank (PDB),
total, six
ribitol
Hatcher et al. parameterized
CHARMmfieldData
for acyclic
sugar inalcohols
andperandfour
ribitol-phosphate
structures(Figure2).
formed MD calculations for sugar alcohols, including ribitol [10]. Other than these, there
are few studies on the conformational dynamics of ribitol.
Our present study aims to build on previous knowledge on the conformation of ribitol and to deepen our understanding of static and dynamic structures of ribitol throughdatabase analysis, experimental NMR analysis, and MD simulation.

Molecules 2021, 26, 5471

Crystal structures of oligosaccharides bound to proteins provide good insight into
the more stable conformations of each glycosidic linkage [11]. Following this notion, we
extracted the conformation(s) of ribitol and ribitol phosphate from the Cambridge Crystallographic Data Centre (CCDC) and the Protein Data Bank (PDB), in total, six ribitol and3 of 10four ribitol-phosphate structures (Figure 2).

Figure 2. Representative 3D structure of ribitol and ribitol phosphate extracted from the database.
(a) Crystal
structure of 3D
ribitol(PDB of
ID:ribitol
5IAI).and(b)ribitol
Ribitolphosphate
phosphate(PDB ID:
6H4F).
(c) Ribitol
Figure2. Representative
structure
extracted
fromthe database.
with watermolecules(PDB
4A0S).
The structuresare shownin stick
representation.
(a)interacting
Crystal structureof ribitol(PDB
ID: ID:
5IAI).
(b) Ribitol
phosphate(PDB ID:
6H4F).
(c) Ribitol in-In
teractingwatermolecules
ID: 4A0S).
Themeshstructuresare shownstickProteinsrepresentation.
In(a,b), thewith
electrondensitymap (PDB
is depicted
in gray
contouredat 2.0 σinlevel.
are omitted(a,b),
the electron
map is depicted
in gray
meshwithcontouredσ level.
are omitted
for clarity.
In (c),density
water molecules
hydrogenbondedribitol atare2.0shown
in Proteinscyan sphere.
for clarity. In (c), water molecules hydrogen bonded with ribitol are shown in cyan sphere.

The dihedral angles of the carbon main chain and the C1–C5 distance of extracted
The dihedral
anglesof the 1.
carbonmain chain
andarethelimited,
C1–C5 distance
extracted
structures
are listed
in Table
Althoughthe data
one can of
examinethestructures
are listed infor
Table
Althoughthe data
are structure,
limited, one
examinethe suggest
stablestable conformations
each1.dihedral
angle.
In eachthecandihedralanglesconformations
for each dihedral
In example
each structure,thedihedralanglesangles
suggeststagstaggered conformations.
Exceptangle.
for one(6H4M),
thefour dihedral(ϕ1–ϕ4)
gered
conformations.
Exceptfor oneexample (6H4M), somethe fourdihedralangles (φ1–φ4)
do notall simultaneously
assumeanti-conformations;preferbent conformations.
do not all simultaneously assume anti-conformations; some prefer bent conformations.
(◦ ),φ2
C1–C5
distance (Å), andresolution(Å) of ribitol and ribitol
phosphate extracted
Table 1. Main chain dihedral angles
When(C1–C2–C3–C4)
is in
the anti-conformation,
φ3 (C2–C3–C4–C5)
adoptsfrom the CCDC and the PDB.
NA; notgaucheandavailable.
vice versa. These results indicate that φ2 and φ3 correlate with each other.
C1–C5 distances were also calculated to ascertain the degree of carbon chain stretch. The
CCDC
ϕ1
ϕ2
ϕ3
ϕ4
Deposition
Distance
Resolution
C1–C5
distance of the(C2–C3–C4–C5)
fully extended(C3–C4–C5–O5)
ribitol measuredC1–C5
5.0 Å.
The averagevalue ofReference
the C1–(O1–C1–C2–C3)(C1–C2–C3–C4)
Number/DB ID
C5 distance of 10 extracted ribitol and ribitol phosphate structures is 4.6 Å, suggesting
ribitolthat ribitol−62
and ribitol phosphate
are not completely
extended,possibly tocon1249410
171
−172
71
4.5
- avoid the[12]662559
171
−61
−171
73
4.6
[13]
tinuousanti-conformations
that
induceO2/O4
repulsion.
Inhigh-resolution
structuresof1015979
171
−61
−170
73
4.6
[14]
5IAI
173
166 ID; 5IAI and 4Q0S),
60
170bonding with surrounding
4.5
1.6
NA is
ribitol (PDB
hydrogenwatermolecules
4Q0S
−176
−62
−167
176
4.5
1.9
[15]
2c). This77suggests that −intermolecularinteraction
with2.3
water molecules
4F2D−150 observed−(Figure
174
147
4.5
NA
Ribitol phosphatemaybeinvolvedinthevariousconformationsofribitol.
It
seems
that
the
phosphorylation
6H4F
149
94
83
156
4.4
2.2
[16]
6H4M
131
136 not significantly−143 change the−158
5.0
2.7it needs furtherof ribitol −doesribitol conformation,although
[16]
6HNQ
166
160
69
155
4.6
2.4
[16]
detailedstudy.
In
this
paper,
wemainlyfocus
onribitol.
6KAM
−75
164
96
−168
4.8
2.5
[17]
Table 1. Main chain dihedral angles (°), C1–C5 distance (Å), and resolution (Å) of ribitol and ribitol phosphate extracted
ϕ2 (C1–C2–C3–C4) is in the anti-conformation, ϕ3 (C2–C3–C4–C5) adopts
from the CCDC and the PDB. NA; When
not available.

gauche and vice versa. These results indicate that ϕ2 and ϕ3 correlate with each other.
φ3 calculated to ascertain the degree of carbon chain stretch. The
CCDC
φ1
C1–C5 distanceswere also
φ4
C1–C5
φ2
Deposition(O1–C1–C2–C1–C5 distance of the(C2–C3–
fully extended ribitol measured 5.0 Å. The Resolution
average value Referenceof the C1–C5(C3–C4–C5–O5) Distance(C1–C2–C3–C4)
C4–C5)
Number/DB ID
C3)
distance of 10 extracted
ribitol and ribitol phosphate structures is 4.6 Å, suggesting that
ribitol and ribitol phosphate are not completely extended, possibly to avoid the continuous
ribitolanti-conformations
that
induce O2/O4
high-resolutionof ribitol
1249410
171
−62
−172
71repulsion. In4.5- structures[12]
(PDB
ID;
5IAI
and
4Q0S),hydrogenbondingwithsurroundingwater
molecules
isobserved
662559
171
−61
−171
73
4.6
[13]
(Figure 2c).
interaction
may be1015979
171−61 This suggests−170that intermolecular73
4.6 with water- molecules[14]
involvedinthevariousconformationsofribitol.
It
seems
that
the
phosphorylation
of
5IAI
173
166
60
170
4.5
1.6
NAribitol
does not significantly change the ribitol conformation, although it needs further detailed
4Q0S
−176
−62
−167
176
4.5
1.9
[15]
study. In this paper, we mainly focus on ribitol.
4F2D
−150
−174
77
−147
4.5
2.3
NA
2.2. NMR Analysis
2.2.1. Solid-State 13 C-NMR Analysis of Crystalline Ribitol
We can conclude from the database analysis that stable conformations of ribitol are
likely asymmetric. To investigate the conformation of crystalline ribitol from another aspect,solid-state 13 C-NMR was performed. When the conformation of ribitol is asymmetric,carbon signals will be observed separately in the NMR spectra. Indeed, a 13 C CP-MAS
NMR spectrum of crystalline ribitol shows four separate signals (Figure 3a). By comparison
with the solution 13 C-NMR spectrum of ribitol (Figure 3b), partial assignments of 13 C

2.2.1. Solid-State C-NMR Analysis of Crystalline Ribitol

Molecules 2021, 26, 5471

We can conclude from the database analysis that stable conformations of ribitol are
likely asymmetric. To investigate the conformation of crystalline ribitol from another aspect, solid-state 13C-NMR was performed. When the conformation of ribitol is asymmetric,carbon signals will be observed separately in the NMR spectra. Indeed, a 13C CP-MAS
4 of 10
NMR spectrum of crystalline ribitol shows four separate signals (Figure 3a). By comparison with the solution 13C-NMR spectrum of ribitol (Figure 3b), partial assignments of 13C
signals were
were possible
possible forfor 7272 and
and73
73ppm
ppmto
toC2/C3/C4
C2/C3/C4 andand 6262 ppmppm totosignals
and thethe peaks
peaks at
at 60
60 and
C1/C5.
Interestingly,
C1/C5
shows
two
separatepeaks.
This
observation
may
indicatethat
C1/C5. Interestingly, C1/C5 shows two separate peaks. This observation may indicate that
C1 andand C5C5 ofofribitol
ribitolare
areinindifferent
differentmicroenvironments.
microenvironments.
seen
above,
ribitols
deposited
AsAs
seen
above,
ribitols
deposited
in
in
CCDC
have
an
asymmetricstructure(Table1).
It
isplausiblethatribitolhas
an asymCCDC have an asymmetric structure (Table 1). It is plausible that ribitol has an asymmetric
metric structurein thesolidwell,gives
which
gives different
shifts. C2/C3/C4
structurein the solidstate
as statewell, aswhich
different
chemicalchemical
shifts. C2/C3/C4
signals
signals aroundappear thearoundsame frequency;
interpretation
here is impossible
appearsamethefrequency;
therefore,therefore,
interpretation
here is impossible
without a
without aassignment.
complete assignment.
complete

Figure 3. Comparison of solid-state and solution 13 C-NMR spectra of ribitol. (a) Solid-state 13 C
13C-NMR spectra of ribitol. (a) Solid-state 13C CPFigure 3. -NMR
Comparisonof solid-state
andribitol.
solution
13 C-NMR spectrum of ribitol dissolved
CP-MAS
spectrumof crystalline(b) Solution
MAS -NMR spectrum of crystalline ribitol. (b) Solution 13C-NMR spectrum of ribitol dissolved inin D2 O.
D2O.

In contrast, the solution 13 C-NMR of ribitol showed that C1 and C5, C2 and C4 have
13C-NMR of ribitol showed that C1 and C5, C2 and C4 have
In contrast,
theshifts
solution
the samechemical(Figure3b). These results suggest that ribitol transits rapidly inthe samebetween
chemicalstable
shiftsasymmetric(Figure 3b).conformations,
These results suggest
thattoribitol
transits
rapidly insolutiongiving riseaveragedsignals.
solution between stable asymmetric conformations, giving rise to averaged signals.
2.2.2. Solution NMR Analysis of Ribitol Based on Coupling Constant
2.2.2.Then,
Solution
AnalysisBasedon Coupling
the NMRdihedralangle ofofRibitol
the ribitol
main
chain wasConstantanalyzed from the coupling

Molecules 2021, 26, 5471

3 J(H,H)from
Then,obtained
the dihedral
angle ofthe Weribitol
maintried
chaintowas
analyzed
constants
by solution
NMR.
initially
obtainfromthethe coupling
splitting3
constants
obtainedby solution
NMR. We
initially triedHowever,to obtain the
J(H,H)
from the splittingofthe signals,
assuming
a first-orderapproximation.
assumption
was notof theduesignals,
assuming
a first-order
However, theassumption
was
not
valid
to strong
coupling
in ribitol,approximation.
even at the 1 H observation
frequencyof 600MHz.
5 of 11valid due to3 Jstrong
in ribitol,at the 1H
observationof 600MHz.
Therefore,
valuescoupling
were estimatedbyevencomparing
simulated
andfrequency
experimental
spectra1 H NMR measurements were performed at the 1 H
iteratively.
improve
theestimatedaccuracy, 1DTherefore, 3To
J values
wereby comparing
simulated and experimental spectra it1H
observation
ofaccuracy,
270, 400, and
600NMR
MHz,measurements
and the iterative
comparison
done
eratively. Tofrequencies
improve the
1D-1H
were
performedwas
at the33J(C,H) was estimated from the HR-HMBC method [18]. Theateach
frequency(Figure4).
observation
frequencies
of4).
270, 400, and 600
MHz, andthetheiterative
comparison
at each frequency(Figureestimatedfrom
HR-HMBC
methodwas[18].doneTheobserved
observed chemicalchemical shifts
shifts andand coupling
coupling constants
constants areare summarizedsummarizedin
inTableTable2.2.

1H-NMRspectra
Figureribitol(upper)at 1atH 1observation
frequencies
of 600400 (middle),
and 270
MHz
Figure4.4.Solution
Solution1 H-NMR
spectraofof
ribitol(upper)H observation
frequenciesof (left),
600 (left),
400 (middle),
and
270
1
1
H frequency
are shownthe lower
MHz (right).
Simulated
NMR spectra
each(right).
Simulated
NMR spectra
at eachat H
frequency
are shownin the inlowerpanel. panel.

Table 2. 1H and 13C chemical shift and coupling constant of ribitol determined in this study.

H1R(pro-R)
H1S(pro-S)
H2

Chemical Shift (ppm)
3.79
3.64
3.81

Molecules 2021, 26, 5471

5 of 10

Table 2. 1 H and 13 C chemical shift and coupling constant of ribitol determined in this study.
Chemical Shift (ppm)
H1R(pro-R)
H1S(pro-S)
H2
H3
C1
C2
C3
3 J(H1R,H2)(pro-R)
3 J(H1S,H2)(pro-S)
3 J(H2,H3)
2 J(H1R,H1S)
3 J(C1,H3)
3 J(C3,H1S)

3.79
3.64
3.81
3.68
65.1
74.8
74.9
Coupling constant (Hz)
3.00
7.20
6.50
−12.703.8 *2.9 ** 3 J(C,H) may include errors due to the presence of strong 3 J(H,H) coupling.

Chemical shifts and 2,3 J(H,H) coupling constants are in substantial agreement withthose of previous studies [7,8,10,19]. 3 J(C1,H3) was measured as 3.8 Hz in this study, similar
to a previous report (3.7 Hz) [8]. Then, these coupling constants are used for estimating
the distribution of each conformer using the Haasnoot equation, which includes a setof empirical constants [20]. Regarding the O1–C1 dihedral angle (ϕ1 = O1–C1–C2–C3),
the population of three conformers (ϕ1 = 180◦ , −60◦ , and +60◦ ) was calculated using
3 J(H1R,H2)(pro-R) and 3 J(H1S,H2)(pro-S) with reference to the method of Hawkes [7]. The
ϕ1 ratio was estimated as 180◦ :−60◦ :+60◦ = 64:36:0. This closely conforms to the result ofHawkes [7]. For the C1–C2 dihedral angle ϕ2 (ϕ2 = C1–C2–C3–C4), the conformations
can be estimated from 3 J(H2,H3) and 3 J(C1,H3). The population of each ϕ2 conformation
was calculated as 180◦ :−60◦ :+60◦ = 2:46:52. These data suggest the presence of a rapidtransition (>>10 Hz in terms of coupling constant) around the C1–C2 axis and a relatively
small population of the anti-conformation. It should be noted that ϕ1 and ϕ4 or ϕ2 andϕ3 cannot be discussed separately because their NMR signals (e.g., H1 and H5, H2 andH4) overlap in solution.
2.2.3. NMR Analysis of Hydroxy Protons
Franks et al. found that the 3 J(H,H) values of ribitol differ significantly in water andorganic solvents [19], and this suggests that hydration and hydrogen bonding affect theconformation of ribitol. In this study, we analyzed the hydroxyl group of ribitol by directly
observing the hydroxyl proton by 1 H-NMR. The assignments of OH signals are shownin Figure 5. At 0 ◦ C, two sharp peaks are observed at 5.9 ppm and 5.8 ppm, and these
correspond to OH3 and OH2/OH4 (Figure 5a). The OH3 signal gives a relatively sharp
peak at 0 ◦ C, suggesting that OH3 is stabilized by hydrogen bonding. To validate theexchange of the hydroxyl proton, the temperature coefficient (ppb K−1 ) was estimatedas 11 ppb K−1 for ribitol OH3. According to Sandströ et al., the temperature coefficientis greater than 11 ppb K− 1 when the OH proton is fully hydrated [8]. This suggests that
OH3 is mostly exposed to solvent, but some transient hydrogen bond may exist. The
exchange rate constant of OH3 was also estimated from 2D EXSY. From the build-up curveof exchange peak volume, it was estimated as kex = 193 s−1 . To estimate the effect of an
intramolecular hydrogen bond on the temperature coefficient and OH exchange rate, thesame experiment was performed using the model compound 1,3,5-pentanol (Figure 1),
which does not have hydroxyl groups at the 2 and 4 positions. 1,3,5-Pentanol gives a broad
OH peak around 5.7 ppm at 0 ◦ C. This peak can be assigned to OH1/OH3/OH5, and thepeak almost disappears at 25 ◦ C (Figure 5b). We reasoned that this is due to the lack of ahydrogen bond, and hence, OH3 shows faster proton exchange.

Molecules 2021, 26, 5471

gen bond on the temperature coefficient and OH exchange rate, the same experiment was
performed using the model compound 1,3,5-pentanol (Figure 1), which does not have hydroxyl groups at the 2 and 4 positions. 1,3,5-Pentanol gives a broad OH peak around 5.7
ppm at 0 °C. This peak can be assigned to OH1/OH3/OH5, and the peak almost disappears
at 25 °C (Figure 5b). We reasoned that this is due to the lack of a hydrogen bond, 6andof 10
hence, OH3 shows faster proton exchange.

1 H-NMR (OH region) spectra of ribitol and 1,3,5-pentanol measured at 0, 5,
Figure5.5.PartPart
1D1H-NMR
Figureof of
1D(OH region) spectra of ribitol and 1,3,5-pentanol measured at 0, 5, 10,◦ C in 10 mM sodium acetate buffer, pH 6.0 (H O:D O = 1:1). (a) Ribitol.
10,20,15,
25,30and
3010
15,
25,20,
and°C in
mM sodium acetate buffer, pH 6.0 (H2O:D2O =21:1).2(a) Ribitol. (b) 1,3,5(b) 1,3,5-Pentanol.
x = impurity.
Pentanol.
x = impurity.

Forcomparison,comparison,thethetemperature
coefficientof 1,3,5-pentanol
calculatedFor
temperature
coefficientof 1,3,5-pentanol
waswascalculated
as 13as−1 for OH1/3/5. Similarly, the exchange rate of OH1/3/5 was estimated from
−1
13
ppb
K
ppb K for OH1/3/5. Similarly, the exchange rate of OH1/3/5 was estimated from EXSY toto be
s−1 . Thesesuggest
observations
suggestribitol
OH3
tends to form
ex = 650
beEXSYkex = 650
s−1.kTheseobservations
that ribitolOH3thattends
to form
water-mediatedwater-mediatedor
intramolecularhydrogenbonds.
or intramolecular hydrogen bonds.
2.3. MD Simulations
2.3. MD Simulations
Distribution of Main Chain Dihedral Angles
Distribution of Main Chain Dihedral Angles
Since solution NMR does not distinguish between 3 J(H2,H3) and 3 J(H3,H4), MD
3J(H2,H3) and 3J(H3,H4), MD simSince
solution
NMR does tonotsheddistinguishsimulations
were performed
light on between
the dihedral
angles of ϕ2 and ϕ3 separately.
ulations
were
performed
to
shed
light
on
the
dihedralanglesof4F2D)
φ2 andasφ3
We
We used three ribitol coordinates in PDB (PDB ID: 5IAI, 4Q0S,
anseparately.
initial structureusedthree ribitol coordinates
in PDB (PDB
ID:simulations
5IAI, 4Q0S,(referred
4F2D) astoanasinitialstructure(Supplementary
Table S1). Independent
MD
Run #1,#2, and(Supplementary
Table
S1).
IndependentMDsimulations(referred
to
as
Run
#1,
#2, and#3, respectively) were performed for 100 ns for each initial structure. The MD simulation#3,
respectively)
were performedfor 100 nsthe
forfollowing
each initialstructure.isThe
MDonsimulation
resultsare essentiallythe same; therefore,
discussion
based
the results
resultsareessentially
the
same;
therefore,
thefollowing
discussion
is
based
onRun
the resultsof Runs #1–#3. Figure 6 shows the results of MD simulations of ribitol (left,#1) andof1,3,5-pentanol
Runs #1–#3. Figureshows
the resultsof MDof ribitol(left, results
Run #1)ofand(right) 6foreach dihedralangle
ϕ1 simulations
to ϕ4. The MD
simulation
Run
1,3,5-pentanoleach dihedral angle
φ1S1.
to φ4. The MD simulation results of Run#2 and #3 are (right)
shownforin Supplementary
Figure#2 and #3 are shown in Supplementary Figure S1.
Table 3. Comparison of calculated and experimental coupling constants of ribitol. Calculated J values
are obtained from the average of three independent MD simulations (mean ± SD).

3 J(H1R,H2)(pro-R)
3 J(H1S,H2)(pro-S)
3 J(H2,H3)
3 J(C1,H3)
3 J(C3,H1S)

Calculated J Value (Hz)Experimental J Value (Hz)3.11 ± 0.05
7.57 ± 1.02
6.41 ± 0.09
4.20 ± 0.36
2.67 ± 0.64

3.00
7.20
6.50
3.9
3.1

It seems that ϕ2 and ϕ3 tend to share the same dihedral angle, i.e., (ϕ2, ϕ3) = (+60◦ , +60◦ ) or (−60◦ , −60◦ ) during
the MD trajectory. This differs from the database analysis, in which (ϕ2, ϕ3) mostly adopts (anti, gauche) or(gauche, anti). This discrepancy may come from the intermolecular contacts in the crystal lattice. The combinationof ϕ2 and ϕ3 (+60◦ , +60◦ or −60◦ , −60◦ ) can avoid steric hindrance between O2 and O4, and this arrangement
seems reasonable.

MD simulations of 1,3,5-pentanol were also performed to investigate the role ofOH2 and OH4 in ribitol (Figure 5, right). In 1,3,5-pentanol, ϕ1 and ϕ4 assume all three
conformations (180◦ , +60◦ , −60◦ ) almost equally (Supplementary Table S2). ϕ2 and ϕ3also assume all three conformations, but −60◦ is slightly less populated for ϕ2 and +60◦ for
ϕ3. Overall, transitions are frequent among conformers in 1,3,5-pentanol, while they areless so in ribitol. These results suggest that the presence of OH groups at positions 2 and
4 affects the frequency of conformational transitions. It is likely that this is because ribitol

Molecules 2021, 26, 5471

Molecules 2021, 26, 5471

7 of 10

7 of 11

OH2 and OH4 form water-mediated or intramolecular hydrogen bonds that restructure
the molecule.

Figure 6. Dihedral angle distribution of ribitol (left, Run#1) and 1,3,5-pentanol (right) in MD simulaFigure
6. Dihedralangle
of ribitol(left, Run#1)
1,3,5-pentanol(right)arein defined
MD simutions. The
results of
eachdistribution
dihedral angle
are shown
after 10andns. Thedihedral angles
aslations.
The results ofϕ2
eachdihedral angleϕ3
are(C2–C3–C4–C5),
shown after 10 and
ns. Thedihedral angles are
ϕ1 (O1–C1–C2–C3),(C1–C2–C3–C4),
ϕ4 (C3–C4–C5–O5).
ϕ1definedand ϕ4 asof
φ1(O1–C1–C2–C3),
φ2 (C1–C2–C3–C4),(C2–C3–C4–C5),
and(C3–C4–C5–O5).
φ1 and φ4 ofribitol
are mostly distributedin anti (180◦ )φ3
andgauche (−60◦ for
ϕ1φ4
and+60◦ for ϕ4) (Supplementary
ribitol
are mostly
distributedwith
in antigauche(−60°theforpopulations
φ1 and +60° of
fordihedralφ4) (Supplementary
Table S2).
This is consistentthe(180°)
NMRandresult,in whichangle ϕ1 are
Table S2). This is ◦consistent
with the NMR result, in which the populations of dihedral angle φ1
are◦◦estimated as 180 :−60 :60 = 64:36:0. For ϕ2 and ϕ3 of ribitol, gauche conformations (−60◦ andestimated
as 180°:−60°:60° = 64:36:0. For φ2 and φ3 of ribitol, gauche conformations (−60° and +60°)◦+60 ) are dominant, but the anti-conformation is also populated. This is consistent with the NMR
are dominant, but the anti-conformation is also populated. This is consistent with the NMR result3
result (180◦ :−60=◦2:46:52).
:+60◦ = 2:46:52).
To validate
MD results
quantitatively,
and 3 J(C,H)
are
3J(H,H) J(H,H)
and 3J(C,H)
are calcu(180°:−60°:+60°
To validate
the MDthe
results
quantitatively,
calculated
the population
antigauche
and gauche
conformations,andthe calculated
J values
arelatedfrom fromthe populationof antiofand
conformations,and thecalculated
J values
are comcomparedexperimental
values(TableThecalculated
J valuesare
aremostly
mostlyconsistentconsistent with
with thepared
withwith
experimental
values(Table3). 3).
The
calculated
J values
experimental
experimental values,
values, suggestingsuggesting thatthat MDMD simulation
simulation quantitatively
quantitatively reflects the energy level of eachconformer
conformer based
based on the Boltzmann distribution.

Molecules 2021, 26, 5471

8 of 10

3. Materials and Methods
3.1. Database Analysis of Ribitol and Ribitol Phosphate
Three-dimensional (3D) crystal structures of ribitol and ribitol phosphate (resolution
less than 2.7 Å) were extracted from the Cambridge Crystallographic Data Centre (CCDC,
as of May, 2020) and the Protein Data Bank (PDB, as of April 2020). Coordinates of ribitol
phosphate were extracted that had at least one terminal CH2 OH group. When analyzing
tandem ribitol phosphate, the terminal ribitol with the CH2 OH group was chosen. The
dihedral angles of ribitol and ribitol phosphate are defined as ϕ1 (O1–C1–C2–C3), ϕ2(C1–C2–C3–C4), ϕ3 (C2–C3–C4–C5), and ϕ4 (C3–C4–C5–O5).
3.2. Solution NMR Analysis
Ribitol was purchased from Tokyo Chemical Industry (Tokyo, Japan), and 1,3,5pentanol was purchased from ChemCruz. Solution NMR analyses were performed using
JNM-ECZ600R/S1, JNM-ECZ400S/L1 and EX270 spectrometers (JEOL, Tokyo, Japan).
The probe temperature was set from 273 to 303 K. A sample (18–30 mg) was dissolved in
650 uL of D2 O for signal assignment or 10 mM sodium acetate buffer, pH 6.0 (H2 O:D2 O= 1:1) for detection of OH chemical shifts. 1 H and 13 C chemical shits were reportedly
related to the internal standard of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS, 0 ppm).
NMR chemical shifts of ribitol were assigned by analyzing 1D-1 H, 1D-13 C, 2D-DQF-COSY,NOESY, TOCSY, and 1 H-13 C HSQC spectra. NMR chemical shifts of ribitol phosphate
were assigned by analyzing 1D-1 H, 1D-13 C, 2D-1 H-13 C HSQC, and 2D-1 H-13 C HMBC
spectra. Stereospecific assignment of pro-R and pro-S protons in ribitol was conductedwith the aid of 1D-1 H-NMR spectral simulation by Mnova 14.1.1 (Mestrelab Research,Santiago, Spain). Due to the presence of a second-order term, the 3 J(H,H) and 2 J(H,H)
coupling constants of ribitol were obtained by iterative comparison of simulated NMR
spectra with observed NMR spectra obtained at 1 H observation frequencies of 270, 400,
and 600 MHz. 3 J(C,H) coupling constants were obtained from HR-HMBC spectra [18].
A scaling factor (n) of 25 was used, and the digital resolutions for f1 (13 C) and f2 (1 H)
were 4.4 and 0.7 Hz/point, respectively. The conversion from coupling constant 3 J(H,H)
to dihedral angle was done using the Haasnoot equation, which includes a correction
for electronegativity of substituents and the orientation of each substituent relative to thecoupled protons [20]. 3 J(C,H) is also applied to an empirical prediction equation [21,22].
The exchange rate of hydroxyl protons with water was calculated from 2D chemical
exchange spectroscopy [23,24]. Mixing times of 3 to 24 ms with increments of 3 ms wereused. The exchange rate constant was calculated from the initial build-up rate of the
diagonal peak volume over the exchange cross-peak. The temperature coefficient (ppb K–1 )of hydroxyl protons in ribitol and 1,3,5-pentanol was measured by collecting a series of
1D-1 H-NMR spectra at different temperatures (273, 278, 283, 288, 293, 298, and 303 K).
NMR data processing was performed using Delta5. 3. 1 (JEOL, Tokyo, Japan), and NMR
spectral analyses were performed using Mnova.
3.3. Solid-State 13 C-CP-MAS NMR
13 C CP-MAS spectra of crystalline ribitol (Tokyo Chemical Industry, Tokyo, Japan)

were obtained using a Bruker Avance III 500 spectrometer at a 1 H frequency of 500 MHz.
A VPN probe (4 mm) was used at the spinning rate of 15,000 Hz or 8000 Hz with a spectral
width of 300 ppm or 200 ppm, respectively. The data point was set to 2k. 13 C chemical
shifts were referenced to carboxyl carbon of glycine (176.03 ppm).
3.4. MD Simulation
MD simulations were performed using Discovery Studio 2019 [25]. The coordinates ofribitol (PDB ID: 5IAI, 4Q0S and 4F2D) were used as the initial structure (Supplementary Table S1),and the three simulations were performed independently. CHARMm36 was assigned as theforce field [10]. Hydrogens were generated using the “Add Hydrogens” protocol in Discovery
Studio. The simulation time was set to 100 ns. An explicit periodic boundary was used asMolecules 2021, 26, 5471

9 of 10the solvation model. An orthorhombic cell shape was used in the explicit periodic boundary
solvation model. The minimum distance from the periodic boundary was set to 7.0 Å. For
each ribitol coordinate, 182–184 water molecules were explicitly placed based on the protocol in
Discovery Studio 2019. CHARMm36 was optimized using the TIP3P water model [26], which
was used as the force field template. Explicit waters were 190 for 1,3,5-pentanol. Minimization of
the initial coordinate was done in two steps. The first step is to eliminate distortion of the entire
structure with the steepest descent algorithm. In the second step, minimization was performed
with the adopted basis Newton–Raphson (NR) algorithm. Heating was done at 500 K. Afterequilibration, the time step was set to 2 fs, and production was done under nPT ensemble. MD
simulations were also performed for 1,3,5-pentanol under the same protocol. The initial structureof 1,3,5-pentanol is a zigzag form with each dihedral angle of the carbon main chain being 180◦ .
4. Conclusions
Until the present study, it has been assumed that the C-C bonds of alditols are rather
unstructured, and the molecule exhibits little or no stable folded structure. Here, NMR
and MD simulation of ribitol reveal that ribitol is a dynamic conformational equilibriumof staggered conformations. Then, ribitol, is rather structured compared with model compound 1,3,5-pentanol. Conformational asymmetry may prevail in ribitol due to repulsion
between OH2 and OH4 and to transient weak hydrogen bonds involving OH3. Thesefindings provide a basis for understanding the dynamic structure of ribitol and the functionof ribitol-related enzymes involved in ribitol biosynthesis and glycan chain elongation.
Supplementary Materials: The following are available online, Figure S1: Dihedral angle distributionof ribitol in MD simulations Run #2 and #3, Table S1: The initial XYZ coordinates for MD simulations,and Table S2: The population (%) of dihedral angles in MD simulations of ribitol (Runs #1–#3) and
1,3,5-pentanol.
Author Contributions: Manuscript conception, writing and original draft preparation, S.O., Y.Y.;
NMR analysis, S.O., T.Y., Y.Y.; MD simulations, S.O.; editing, data analysis, and interpretation, S.O.,
N.M., J.U., Y.Y. All authors have read and agreed to the published version of the manuscript.
Funding: This work was supported in part by grants-in-aid of KAKENHI 19H05648 and 19H03362.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We acknowledge Ohgi Takahashi (Shonan University of Medical Sciences)
for technical advices on computational calculations, and Hiroshi Manya and Tamao Endo (Tokyo
Metropolitan Geriatric Hospital and Institute of Gerontology) and Jun-ichi Tamura (Tottori University)
for intriguing discussions and encouragement. We also express our thanks to Tomoyuki Matsuki
and Shin-ichi Sato (Tohoku Medical and Pharmaceutical University) for NMR measurements. This
work was partly supported by Nanotechnology Platform Program (Molecule and Material Synthesis)of MEXT.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.

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