!

( '( )+,34+5347(3/(*.643027-3,132307(Prof. Akira Kurosaka, Ph. D.Ħ
( (Assist. Prof. Yoshiaki Nakayama Ph. D.
ED!(

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ŹŲŸǖűƏƂǕ\^Ƅ0ųƦƾƽƗǆǌǒƂWŴƑ

ŘŘĽŋŔŃķōĪňĴǕĪňĮĴ ƃÝìƂµoŲŸǖĦ
Fig. 1 A phylogenetic tree of humanppGalNAc-TsǙǔŶƃƃńĪƢǌƤƨƵ>ÉēƃÝì
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ơǏƼƸƦƚƴǒƨƩÏÿƕøƂ#eŲŽŧƑŰſĩĦ ħœœœĨŶT18a KDƃôÆǈƞƶƪǇƃ¸ÓƄßƮǐƺơýƃăĆª[ƂƎƑǌƭƭǒǇ¬ƃ­£ƕ`ŭþŰŴſŧŨ|ŸƁ¶êƕ

WTT18a KDdŸīĦ ŰƒƏƃÐƄ ŘŘĽŋŔŃķōĪňĮĳ ƂƎƐblűƒƑǇưǐ>ÉēŬĩĦ ǅơǏƼƸƦƚƴǒƨƩƕćųŸÍÜC!Iƃ*ƐĄƊƕ#eŴƑŰſƂƎƐĩĦ ÍÜĠ£kƃǂǈƝƩƮƨƩƃÑ s ƂĖ  ŴƑŰſƕ¹ 7 ŲŽŧƑǓļœőīĦįǔīĦ Ħ ĦĦFig. 3 Suppression of ppGalNAc-T18a in zebrafish
embryos gave rise to the altered tail formation during thedevelopment.
ĦǙǔŶƃƃńĪƢǌƤƨƵ>ÉēƃÝìĦ ÍÜýǕŪƎƆÍÜCƂêŧŹűƒƑ ńĪĽŔōŃķō >ÉēƃÝƕƬƾǋƽƙƱƨǊƕ¨ŧŽôƈŸǖŰƃÉēĈƃ/lƂĖƔƑÉĂ¾ĎËƕƬƾǋƽƙƱƨǊž0O
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Research projects and annual reports
O-Glycosylation is an important post-translational
modification of proteins, and is classified into several

Fig. 2 Hypothetical roles of ppGalNAc-T17
in membrane trafficking

types

basedstructures.

on

the

carbohydrate-protein

linkage

We have been investigating roles of sugar

chains with the linkage structures, GalNAcα1→Ser(Thr),ĦManα1→Ser(Thr), or GlcNAcβ1→Ser(Thr).

įĨĦ ƬƾǋƽƙƱƨǊƕ¨ ŧŸ ŘŘĽŋŔŃķōĪň ƽƗǆǌǒthem, GalNAcα1→Ser(Thr) is the most frequentlyƃÝìĦ

observed linkage, and O-glycans with this linkage areĦ ƬƾǋƽƙƱƨǊƕ¨ŧŽ ŘŘĽŋŔŃķōĪňĴĩĦ ĪňĮĴ ƃÝcalled the mucin carbohydrates since they are highlyìƕäŻŸǖƳǒƮǀǒƩÌƂƎƐǕƬƾǋƽƙƱƨǊ

expressed in mucins secreted from epithelial cells.

žƄàƃĐèƂƎƐǕŘŘĽŋŔŃķōĪňĴ ƂżŧŽƄǛż

mucin carbohydrate biosynthesis is initiated by a groupħŘŘĽŋŔŃķōĪňĴŋ  ŏĨ Ǖ ŘŘĽŋŔŃķōĪňĮĴ Ƃ ż ŧ Ž Ƅ ǘ żofpolypeptide

N-acetyl-ħŘŘĽŋŔŃķōĪňĮĴŋĩĦ ŌĨƃƺǋǏƢċIŬJ<ŲŽŧƑŰ

galactosaminyltransferases (ppGalNAc-Ts).

ppGalNAcſƕêŧŹŲŸǖ¤<ƄǕŰƒƏƘƚƭƧƚǇƃơǏǒƶǐ

-Ts consist of a large gene family with 20 isozymes inƢǕ®¤!ZìǕƸƱơƯƛǐPģƕĊƋŽŧƑŬǕ

humans (Fig. 1).

\^ƄǕŘŘĽŋŔŃķōĪňĮĴŋĩĦ ŌĦ ƃ ōĺŃķ ơǏǒƶǐƢƕM

-T17, and -T18 are catalytically inactive when assayedŲŸǖŰƒƏƃƘƚƭƧƚǇƂżŧŽǕņňĪŅĹņ ƂƎƐƀwith classical assay methods, while ppGalNAc-T9 andźƏƌ+Èc ĮĴ ĕūƏ®¤ŲŽŪƐǕűƏƂ œŖĦ ŚœśŜĦ-T17, which are cloned by us, are brain-specificŒŝŌřœŎœŞŋśœŗŖ ƂƎƐǕ+Èc įı ĕƃƬƾǋƽƙƱƨǊÛ

isozymes and are biologically important for the neuralƂŪŧŽǕŘŘĽŋŔŃķōĪňĮĴŋĦ ŕņŃķ ŬāǕ¢ƂÞƍVdifferentiation.

ƂaŮ®¤ŲŽŧƑŰſƕêŧŹŲŸǖƉŸǕ

focused on the functions of glycosyltransferases to makeŘŘĽŋŔŃķōĪňĮĴŋ ƂSŴƑǉǍǂǌƸƘǐưƫǐƩƝǌƥƕO-glycan carbohydrate-linkage structures, and obtained¨ŧŸƸƱơƯƛǐPģūƏǕŰƃƘƚƭƧƚǇŬƬƾǋƽ

the following findings.

enzymes,

UDP-GalNAc:

AmongThe

Interestingly, ppGalNAc-T8, -T9,

Based on these backgrounds, we have1) Roles of mucin-type carbohydrates in intracellular

We found that the suppression of O-GlcNAcylation inmembrane trafficking

zebrafish resulted in the abnormal development of

Recent genome-scale analysis of HEK293T cells

embryos.

treated with a high GlcNAc concentration demonstratedĦ

that ppGalNAc-T17 is one of the genes upregulated,

GD

whichphase

A. Miyake, S. Nihno, Y. Murakoshi, A. Satsuka, Y. Nakayama,

To assess its roles, we first biochemicallyand N. Itoh: Neucrin, a novel secreted antagonist of canonicalarepossibly

endocytosis.

involvedinthefluid$(characterized recombinant ppGalNAc-T17 in COS7 cells,Wnt signaling, plays roles in developing neural tissues inand demonstrated that it was N-glycosylated, andzebrafish. Mech. Dev. in press.

localized mainly in the Golgi apparatus.

We thenĦ

suppressed the expression of endogenous ppGalNAc-T17HD9=<#(in HEK293T cells using siRNA.

The suppression led toS. Sasaki, Y. Nakayama, M. Konishi, A. Miyake, N. Itoh: The

phenotypiccellschangesofreduced

FGF Family in Humans, Mice, and Zebrafish: Development,lamellipodia formation, altered O-glycan profiles, andPhysiology, and Pathophysiology. Human Genetic Diseases

unusual accumulation of glycoconjugates in the late

37-56 (2011)

endosomes and lysosomes.

thewithAnalysis of endocytotic

Ħ

pathways revealed that macropinocytosis, but neitherID

clathrin-

ƁŲĦ

nor

caveolin-dependentendocytosis,elevated in the knockdown cells.

wasThis was further%8?CA@>B(Ħsupported by the findings that recombinant ppGalNAc-JD

T17 overexpressed in HEK293T cells inhibited macro-Y. Nakayama, N. Nakamura, and A. Kurosaka: The BiologicalĦpinocytosis, and rescued the influences observed for theRoles

knockdown cells.

WBSCR17. Glyco21, Vienne (Austria), 2011. 8. 21-26

Our data provide the first implicationofa

putative

Polypeptide

GalNAc-transferase/that a subset of mucin-type O-glycosylation produced by

N. Nakamura, E. Kaneda, Y. Nakayama, and A. Kurosaka:

ppGalNAc-T17 is involved in the control of dynamic

Developmental Roles of Putative Polypeptide GalNAc-

membrane trafficking probably between the cell surface

transferases in Zebrafish.

and the late endosomes through macro- pinocytosis, inSociety for Glycobiology, Seattle, Washington (USA) 2011.

response to the nutrient concentration as exemplified by
the environmentally available GlcNAc (Fig. 2).

11. 11
Y. Nakayama, A. Wada, N. Nakamura, and A. Kurosaka: A2) Analysis of ppGalNAc-T family using zebrafish

putative

We have been investigating ppGalNAc-T8 and -T18

polypeptide

GalNAc-transferase,

WBSCR17,

regulates endocytosis in HEK293T cells. Ã 34 :~!Ithat are catalytically inert under the conventional assay
conditions.

2011 Annual Conference of the¦¡K\, Y, 2011.12.13-16

To characterize these isozymes, we first

H. Fujiwara, T. Satoh, Y. Tsuji, Y. Nakayama, N. Nakamura,screened the database, and found that zebrafish have 5

and A. Kurosaka: Characterization of a brain-specificand 2 paralogue genes for ppGalNAc-T8, and -T18,

polypeptide N-acetylgalactosaminyltransferase. Ã 34 :~respectively.

For ppGalNAc-T18, we cloned bothparalogue genes, and showed that they were expressed
throughout

theearly

stronger

Functional analysis of N-acetylgalactosaminyltransferase

expression in the brain and the tail.

Suppression of-like 4 in zebrafish. Ã 34 :~!I¦¡K\, ppGalNAc-T18awithinembryos
thewithembryos

the!I¦¡K\, Y, 2011.12.13-16
N. Nakamura, E. Kaneda, Y. Nakayama, and A. Kurosaka:specificmorpholino oligos demonstrated the abnormal tailY, 2011.12.13-16
T. Kawai, K. Ohshita, A. Kakii, N. Nakamura, Y. Nakayama,

formation (Fig. 3).

and A. Kurosaka: Functional analysis of N-acetylgalactos-3) Analysis of other O-glycosylation

aminyltransferase 8 in zebrafish. Ã 34 :~!I¦¡K

We investigated the expression and roles of glycosyltransferases that are involved in the O-GlcNAcylation.

\, Y, 2011.12.13-16
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