November 28
PROGRAM07

Glycoscience : diversity and integration

Dual roles of gastric gland mucin-specificO-glycan in prevention of gastric cancer
Jun Nakayama, M.D., Ph.D.
Professor
Department of Molecular Pathology
Shinshu University Graduate School of Medicine, JAPANJun Nakayama
ProfileJun Nakayama is a pathologist, who is interested in gastrointestinal pathology. Heearned his M. D. at Shinshu University
School of Medicine, Japan in 1983 and hisPh. D. in medicine at Shinshu University
Graduate School of Medicine, Japan in1987. In the graduate school, he studied
the histochemical expression of bloodgroup-related carbohydrate antigens inhuman colorectal mucosa supervised byProfessor Emeritus (Associate Professor atthat time) Tsutomu Katsuyama. He started
his professional carrier on pathology at
the Central Clinical Laboratories, Shinshu University Hospital, Japan from 1987.
From 1993 to 1995, he received researchtraining on glycobiology in the laboratoryof Professor Minoru Fukuda at La JollaCancer Research Foundation (currentlySanford-Burnham Medical Research Institute) in San Diego, U.S.A. as a Visiting Scientist. Here, he worked on the expression
cloning of α2,8-sialyltransfereases, andidentified two distinct cDNAs encoding
polysialyltransferase (ST8SiaIV) and GT3/GD3 synthase (ST8SiaII), respectively. After returning to Shinshu University, he succeeded in the expression cloning of α1,4N-acetylglucosaminyltransferase (α4GnT).
Currently, his research group is studying
pathogenesis and prevention of gastriccancer focusing on α4GnT. He appointed
Professor in Department of MolecularPathology, Shinshu University Graduate School of Medicine in 2002, and from2011 he is a Vice Dean of Shinshu Universty School of Medicine. Dr. Nakayama isa recipient of a Bergmeyer-Kawai Award(1999) from Japanese Society of Laboratory Medicine and serves as an Editorial
Board Member for Journal of Histochemistry & Cytochemistry and Acta Histochemica et Cytochemica.

Gastric mucins are classified into two subtypes; i.e., surface mucin secreted fromsurface mucous cells lining the gastricmucosa, and gland mucin secreted fromgland mucous cells such as pyloric glandcells and mucous neck cells located inlower layer of the mucosa 1). The glandmucin characteristically contains O-glycans having terminal α1,4-linked GlcNAc
residues (αGlcNAc) attached to its scaffold
MUC6, and the expression of αGlcNAc isexclusively limited to the gland mucouscells and duodenal Brunner’s glands 2).
Previously we isolated cDNA encoding
α1,4- N -acetylglucosaminyltransferese(α4GnT), which is responsible for the biosynthesis of αGlcNAc by expression cloning 3), and showed that α4GnT is expressedin the gland mucous cells, where αGlcNAc
is secreted 4).

Helicobacter pylori (H. pylori), a causative microbe for gastric cancer, largely
colonizes the surface mucin, while thismicrobe is barely found in the glandmucin 5), suggesting that αGlcNAc plays
a protective role in H. pylori infection.
To test the hypothesis, we incubated H.
pylori with recombinant soluble CD43(sCD43) having αGlcNAc 6). We foundthat the growth and motility of H. pylori
were significantly suppressed. The abnormal morphology such as elongation andfolding were also found. By contrast, thecontrol sCD43 without αGlcNAc had noeffects on the bacteria. Hirai et al. demonstrated that the cell wall of H. pylori characteristically contains a unique glycolipid,cholesteryl-α-D-glucopyranoside (CGL) 7).
We then demonstrated that αGlcNAc sup-adenocarcinoma, H. pylori,
Keywords knockout mouse,mucin, stomach

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the 20th Anniversary of the Mizutani Foundation for Glycoscience

pressed cholesterol α-glucosyltransferase(CHLαGcT) that forms CGL in vitro 8),
and that the active form of CHLαGcT waspresent in the membrane fraction of thebacteria 9). H. pylori requires exogenous
cholesterol for the biosynthesis of CGL.
Thus, we cultured H. pylori in the absenceof cholesterol, and showed that H. pylori
lacked CGL, exhibited reduced growthand motility, and died off completely upon
prolonged incubation up to 21 days, indicating that CGL is indispensable for H.
pylori survival 6). These results show that
αGlcNAc functions as a natural antibiotic
against H. pylori by inhibiting the biosynthesis of CGL, thus protecting the gastricmucosa from the infection.
Recently, we generated α4GnT-deficient
mice by disrupting the A4gnt gene that
encodes α4GnT in mice 10). Immunoistochemistry using HIK1083 antibody
specific for αGlcNAc 2) and MALDI-TOFMS analysis revealed that A4gnt–/– mice
showed complete lack of αGlcNAc expression in gastric gland mucin, indicatingthat α4GnT is a sole enzyme responsible
for the biosynthesis of the O-glycans invivo. Surprisingly, all the mutant micedeveloped gastric differentiated-type adenocarcinoma through a hyperplasia-dysplasia-carcinoma sequence in the absenceof H. pylori infection, indicating thatαGlcNAc serves as a tumor suppressor forthe gastric adenocarcinoma. In fact, significant reduction of αGlcNAc compared
to MUC6 was found in human gastrictumors including early differentiated-typeadenocarcinoma and its potentially premalignant lesion tubular adenoma. To elu-Figure 1. Dual roles of αGlcNAc in prevention of gastric cancer.
O -glycans having αGlcNAc are characteristically contained in the gland mucin secreted from lower layer of the gastricmucosa, and α4GnT is a sole enzyme that forms αGlcNAc in vivo . These particular O -glycans exert anti-microbial effecton H. pylori . In addition, they also suppress gastric tumorigenesis. Thus, αGlcNA in the gland mucin plays dual roles inprevention of gastric cancer.

cidate pathways linking αGlcNAc to tumor
suppression, microarray and quantitative
RT-PCR analyses were carried out. We
found that genes encoding inflammatory
chemokine ligands such as Ccl2, Cxcl1,and Cxcl5, proinflammatory cytokines
such as Il-11 and Il-1β, and growth factors
such as Hgf and Fgf7 were upregulated in

the gastric mucosa of A4gnt–/– mice. On
the other hand, genes encoding Amh, Egf,and Pthlh were downregulated. In addition, inflammatory cell infiltrations suchas mononuclear cells and neutrophils, andangiogenesis were progressively increased
as they aged. These results demonstratethat the absence of αGlcNAc triggers gas-tric carcinogenesis through inflammationassociated pathways in vivo.
Taken together, the gastric gland mucinspecific αGlcNAc plays dual roles inpreventing gastric cancer by inhibiting
H. pylori infection and also suppressing
tumor-promoting inflammation.

References
MN, Fukuda M, Katsuyama T, Nakayama J. Natural antibiotic function of1) 	Ota H, Katsuyama T, Ishii K, Nakayama J, Shiozawa T, Tsukahara Y. A dual
staining method for identifying mucins of different gastric epithelial mucousa human gastric mucin against Helicobacter pylori infection. Science (305):cells. Histochem. J. (23): 22-28, 1991.
1003-1006, 2004.
2) 	Ishihara K, Kurihara M, Goso Y, Urata T, Ota H, Katsuyama T, Hotta K. Pe- 7) 	Hirai Y, Haque M, Yoshida T, Yokota K, Yasuda T, Oguma K. Unique cholesripheral α-linked N-acetylglucosamine on the carbohydrate moiety of mucinteryl glucosides in Helicobacter pylori: composition and structural analysis.
derived from mammalian gastric gland mucous cells: epitope recognized by aJ. Bacteriol. (177): 5327-5333, 1995.
newly characterized monoclonal antibody. Biochem. J. (318 (Pt 2)): 409-416, 8) 	Lee H, Kobayashi M, Wang P, Nakayama J, Seeberger PH, Fukuda M. Ex1996.
pression cloning of cholesterol α-glucosyltransferase, a unique enzyme that3) 	Nakayama J, Yeh JC, Misra AK, Ito S, Katsuyama T, Fukuda M. Exprescan be inhibited by natural antibiotic gastric mucin O-glycans, from Helicobacter pylori. Biochem. Biophys. Res. Commun. (349): 1235-1241, 2006.
sion cloning of a human α1,4-N-acetylglucosaminyltransferase that forms
GlcNAcα1→4Galβ→R, a glycan specifically expressed in the gastric gland mu- 9) 	Hoshino H, Tsuchida A, Kametani K, Mori M, Nishizawa T, Suzuki T, Nakamura H, Lee H, Ito Y, Kobayashi M, Masumoto J, Fujita M, Fukuda M, Nakayacous cell-type mucin. Proc. Natl. Acad. Sci. USA (96): 8991-8996, 1999.
ma J. Membrane-associated activation of cholesterol α-glucosyltransferase,4) 	Zhang MX, Nakayama J, Hidaka E, Kubota S, Yan J, Ota H, Fukuda M. Immunohistochemical demonstration of α1,4-N-acetylglucosaminyltransferase
an enzyme responsible for biosynthesis of cholesteryl-α-D-glucopyranosidethat forms GlcNAcα1,4Galβ residues in human gastrointestinal mucosa. J.
in Helicobacter pylori critical for its survival. J. Histochem. Cytochem. (59):Histochem. Cytochem. (49): 587-596, 2001.
98-105, 2011.
5) 	Hidaka E, Ota H, Hidaka H, Hayama M, Matsuzawa K, Akamatsu T, Na- 10) 	Karasawa F, Shiota A, Goso Y, Kobayashi M, Sato Y, Masumoto J, Fujiwara
kayama J, Katsuyama T. Helicobacter pylori and two ultrastructurally disM, Yokosawa S, Muraki T, Miyagawa S, Ueda M, Fukuda MN, Fukuda M,
tinct layers of gastric mucous cell mucins in the surface mucous gel layer. GutIshihara K, Nakayama J. Essential role of gastric gland mucin in preventing(49): 474-480, 2001.
gastric cancer in mice. J. Clin. Invest. (122): 923-934, 2012.
6) 	Kawakubo M, Ito Y, Okimura Y, Kobayashi M, Sakura K, Kasama S, Fukuda

Commemorative Symposium33