ANNIVERSARYCOMM
25thI

nvited LectureSystematic Clinical, Biochemical, andMolecular Elucidation ofGenetic Disorders due toDefective Synthesis ofO-Mannose Type Sugar Chains,
including Fukuyama-type Muscular
Dystrophy, and Discovery ofSugar Chains of Novel Structures

Tamao Endo

Vice-director, Tokyo Metropolitan Geriatric Hospitaland Institute of Gerontology, JAPANTatsushi Toda

Professor and Chairman,
Department of Neurology Graduate School of Medicine,
The University of Tokyo, JAPANDr. Tamao Endo graduated from the Faculty of Pharmaceutical Sciences, Universityof Tokyo in 1977 and obtained his Ph.D. at the same institution in 1982 (under Prof.
Shoshichi Nojima). He was a postdoctoral fellow at the Baylor College of Medicine(Prof. Donald M. Marcus), and a research associate at the Institute of Medical Science, University of Tokyo (Prof. Akira Kobata). Since 1994, he has been head of theDepartment of Glycobiology, Tokyo Metropolitan Institute of Gerontology (TMIG). In
2012, TMIG was reestablished as a new foundation and he was appointed as a vicedirector. He served as president of the Japanese Society of Carbohydrate Researchfrom 2011 to 2013 and has served on the editorial board of Glycobiology since 2006.
He has received the Tokyo Metropolitan Governor’s Award, the Pharmaceutical Society of Japan Award for Divisional Scientific Promotion, the Asahi Award, and theJapan Academy Prize. His current research interests are glycobiology in aging, dementia, and muscular functions.

Dr. Tatsushi Toda is Professor and Chairman of Neurology, Kobe University Graduate
School of Medicine. He graduated from the University of Tokyo in 1985 and joined
its Department of Neurology. In 1996, he was appointed as an associate professorof the Institute of Medical Science, University of Tokyo. In 2000, he was appointed
Professor of Clinical Genetics, Osaka University. He holds his current position since2009. Dr. Toda has identified genes for Fukuyama muscular dystrophy, muscle-eyebrain disease, and susceptibility to Parkinson’s disease. His research interests include the genetics of Parkinson’s disease, neuromuscular disorders, higher brain
functions, and antisense or sugar therapy for Fukuyama muscular dystrophy. He hasreceived the Japanese Society of Human Genetics Award, the Japan Foundation forAging and Health Award, the Japanese Society of Neurology Award, the Tokizane
Memorial Award, the Asahi Award, Award from the Japanese Minister of Education,Culture, Sports, Science and Technology, and the Japan Academy Prize.

24the 25th Anniversary of Mizutani Foundation for Glycoscience

MEMORATIVE SYMPOSIUM
Fukuyama congenital muscular dystrophy (FCMD) is an autosomalrecessive genetic disorder, found almost exclusively in Japan, which
is characterized by congenital muscular dystrophy, cobblestone lissencephaly, and eye anomalies. The affected individuals are neverable to stand or walk. There was less awareness about this disease,with no known cause or cure. In contrast, studies of glycosylation
have a long history, and the significance of glycosylation can bedemonstrated by its role in ABO blood group subtype specification.
Furthermore, glycosylation is an active field in the progression ofvarious areas of research, including cancer and other disorders, viral
infection, and antibody medicine.
Dr. Tatsushi Toda identified the gene responsible for FCMD and laid
the foundation for its genetic diagnosis, prenatal diagnosis, and accurate disease-type classification. FCMD is the first human disease
known to be caused by an ancient insertion of a “selfish” jumping
gene, the SVA retrotransposon, into a functional gene. This SVA
insertion occurred in a single ancestor, who lived approximately
2,000 years ago. Upon its discovery, the gene product, fukutin, wasa completely new protein of unknown function. In subsequentstudies, Dr. Toda discovered that aberrant splicing underlies themolecular pathogenesis of FCMD. SVA insertion activates a rare,alternative donor site in the last exon and creates a new splice-acceptor site in the retrotransposon sequence (exon trapping). This
causes incorrect splicing of the mRNA and abnormal “cutting off”
of the gene. Assuming that the prevention of this aberrant splicing would lead to the treatment of the disease, Dr. Toda appliedan “antisense therapeutic strategy” in which he designed antisense
oligonucleotides that bind to the targeted region at the pre-mRNAlevel to suppress exon trapping. This strategy corrected the abnormal splicing and restored normal fukutin expression in a mouse

FCMD model. Dr. Toda aims to conduct clinical trials for future drug
approval, thus paving the way for the development of a definitive
treatment for FCMD. However, the function of fukutin remained unknown until it was elucidated through collaborative research withDr. Tamao Endo.
In the late 1990s, Dr. Endo reported that α-dystroglycan, a muscle
protein, contains sugar chains linked by mannosyl-serine/threonine groups. This was the first time that the structure of the mammalian O-mannosyl glycan was described. A series of enzymaticstudies performed by Dr. Endo to elucidate the biosynthetic mechanism of O-mannosyl glycans in mammals found that the protein
O-mannose β1,2-N-acetylglucosaminyltransferase (POMGnT1) is
involved in the formation of O-mannosyl glycan. Dr. Endo and Dr.
Toda collaborated and found that muscle-eye-brain disease (MEB),a related disease to FCMD, is inherited as a functional loss of thePOMGnT1 gene. Additionally, a selective deficiency of glycosylated
α-dystroglycan was found in MEB patients. These findings indicatedthat α-dystroglycan is a potential target of POMGnT1, and that hypoglycosylation of α-dystroglycan is the pathological basis of MEB.
Therefore, the abnormal muscle and brain phenotypes in MEB canbe explained by abnormal glycosylation of α-dystroglycan. This led
the researchers to propose a new disease concept, dystroglycanopathy. Dr. Endo also found that the proteins O-mannosyltransferase
1 (POMT1) and POMT2 are the initiation enzymes of O-mannosylglycan, and discovered a defect in POMT1 or POMT2 genes in Walker–Warburg syndrome (WWS) patients. WWS is another form ofcongenital muscular dystrophy that is related to MEB. These studies suggested that FCMD may be a glycosylation defect disease because FCMD is related to MEB and WWS.

Figure 1
An SVA retrotransposal insertion induces abnormal splicing in FCMD. FCMD is the first human disease knownto be caused by an ancient insertion of a “selfish” jumping gene, the SVA retrotransposon, into a functionalgene.

Commemorative Symposium

25

ANNIVERSARY
COMM
25thFigure 2
Proposed structure of O-mannosyl glycan and its processing glycosyltransferases. The processing enzymes
marked in red have been characterized by us. GalNAc,N-acetylgalactosamine; GlcA, glucuronic acid; GlcNAc,N-acetylglucosamine; Man, mannose; Rbo, ribitol; Xyl, xylose; B3GALNT2, β1,3-N-acetylgalactosaminyltransferase
2; B4GAT1, β1,4-glucuronosyltransferase 1; FKRP, fukutinrelated protein; ISPD, isoprenoid synthase domain-containing protein; LARGE, acetylglucosaminyltransferaselike; POMGnT1, protein O -linked mannose β-1,2-Nacetylglucosaminyltransferase 1; POMGnT2, protein Olinked mannose β-1,4-N-acetylglucosaminyltransferase
2; POMK, protein O-mannose kinase; POMT1/2, protein
O-mannosyltransferase 1/2; TMEM5, transmembrane protein 5.

To address the functional role of fukutin on the processing of Omannosyl glycan, we continued to collaborate to elucidate the Omannosyl glycan structure. Recently, we proposed the entire structure of O-mannosyl glycan, [3GlcAβ1,3Xylα1,] n 3GlcAβ1,4Xylβ1,4Rbo5P,1Rbo5P,3GalNAcβ1,3GlcNAcβ1,4(phosphate-6)Man, whichis required for the binding of α-dystroglycan to extracellular matrix proteins. The tandem Rbo5P, a phosphoric ester of pentitol, isunique. We determined three enzymes to be involved in the synthesis of tandem Rbo5P: isoprenoid synthase domain-containing
protein (ISPD), a CDP-Rbo synthetase, and fukutin and fukutin-related protein (FKRP), Rbo5P transferases that utilize CDP-Rbo.
POMGnT1 is causative for MEB, as described above. Although
POMGnT1 does not catalyze any reactions involved in the aboveproposed O-mannosyl glycan structure, POMGnT1 KO mice and
MEB patients lack the GlcA-Xyl repeat. This suggests that POMGnT1
plays an important role in the processing of GalNAcβ1,3GlcNAcβ1,4(phosphate-6)Man. Fukutin forms a complex with POMGnT1, andthe POMGnT1 crystal structure reveals that the stem domain ofPOMGnT1 recognizes GalNAcβ1,3GlcNAc. Since fukutin is requiredfor the first Rbo5P modification of the GalNAcβ1,3GlcNAcβ1,4(phosphate-6)Man glycan, the POMGnT1-fukutin complex is important informing a platform that requires further glycosylation. These findings expand our knowledge on glycosylation machinery.
Our joint research between genomic medicine/clinical genetics
and carbohydrate biochemistry has established that the elusive
cause of FCMD and other related muscular dystrophies is the defective synthesis of O-mannose sugar chains on α-dystroglycan. Furthermore, the discovery of a new sugar chain and its significancefurther contributed to the recognition of sugar chains as an important biological material. We anticipate that our findings will lead to
new treatments for these diseases.

References1)	Toda, T., Segawa, M., Nomura, Y., Nonaka, I., Masuda, K., Ishihara, T., Sakai, M., Tomita, I., Origuchi, Y., Ohno, K., Misugi, N., Sasaki, Y., Takada, K., Kawai, M.,
Otani, K., Murakami, T., Saito, K., Fukuyama, Y., Shimizu, T., Kanazawa, I., and Nakamura, Y. Localization of a gene for Fukuyama type congenital muscular dystrophy to chromosome 9q31-33. Nature Genet. (5): 283-286, 1993.
2)	Chiba, A., Matsumura, K., Yamada, H., Inazu, T., Shimizu, T., Kusunoki, S., Kanazawa, I., Kobata, A., and Endo, T. Structures of sialylated O-linked oligosaccharidesof bovine peripheral nerve α-dystroglycan: The role of a novel O-mannosyl-type oligosaccharide in the binding of α-dystroglycan with laminin. J. Biol. Chem. (272):
2156-2162, 1997.
3)	Kobayashi, K., Nakahori, Y., Miyake, M., Matsumura, K., Kondo-Iida, E., Nomura, Y., Segawa, M., Yoshioka, M., Saito, K., Osawa, M., Hamano, K., Sakakihara, Y.,
Nonaka, I., Nakagome, Y., Kanazawa, I., Nakamura, Y., Tokunaga, K., and Toda T. An ancient retrotransposal insertion causes Fukuyama-type congenital muscular
dystrophy. Nature (394): 388-392, 1998.
4)	Yoshida, A., Kobayashi, K., Manya, H., Taniguchi, K., Kano, H., Mizuno, M., Inazu, T., Mitsuhashi, H., Takahashi, S., Takeuchi, M., Herrmann, R., Straub, V., Talim,B., Voit, T., Topaloglu, H., Toda, T., and Endo, T. Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev.
Cell (1): 717-724, 2001.
5)	Manya, H., Chiba, A., Yoshida, A., Wang, X., Chiba, Y., Jigami, Y., Margolis, RU., and Endo, T. Demonstration of mammalian protein O-mannosyltransferase activity:
Coexpression of POMT1 and POMT2 required for enzymatic activity. Proc. Natl. Acad. Sci. USA. (101): 500-505, 2004.
6)	Manya, H., Suzuki, T., Akasaka-Manya, K., Ishida, H., Mizuno, M., Suzuki Y., Inazu, T., Dohmae, N., and Endo, T. Regulation of mammalian protein O-mannosylation: Preferential amino acid sequence for O-mannose modification. J. Biol. Chem. (282): 20200-20206, 2007.
7)	Taniguchi-Ikeda, M., Kobayashi, K., Kanagawa, M., Yu, CC., Mori, K., Oda, T., Kuga, A., Kurahashi, H., Akman, HO., DiMauro, S., Kaji, R., Yokota, T., Takeda, S.,and Toda, T. Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy. Nature (478): 127-131, 2011.
8)	Kanagawa, M., Kobayashi, K., Tajiri, M., Manya, H., Kuga, A., Yamaguchi, Y., Akasaka-Manya, K., Furukawa, J., Mizuno, M., Kawakami, H., Shinohara, Y., Wada, Y.,
Endo, T., and Toda, T. Identification of a post-translational modification with ribitol-phosphate and its defect in muscular dystrophy. Cell Rep. (14): 2209-2223, 2016.
9)	Kuwabara, N., Manya, H., Yamada, T., Tateno, H., Kanagawa, M., Kobayashi, K., Akasaka-Manya, K., Hirose, Y., Mizuno, M., Ikeguchi, M., Toda, T., Hirabayashi, J.,
Senda, T., Endo, T., and Kato, R. Carbohydrate-binding domain of the POMGnT1 stem region modulates O-mannosylation sites of α-dystroglycan. Proc. Natl. Acad.
Sci. USA. (113): 9280-9285, 2016.
10)	Manya, H., Yamaguchi, Y., Kanagawa, M., Kobayashi, K., Tajiri, M., Akasaka-Manya, K., Kawakami, H., Mizuno, M., Wada, Y., Toda, T., and Endo, T. The muscular
dystrophy gene TMEM5 encodes a ribitol β1,4-xylosyltransferase required for the functional glycosylation of dystroglycan. J. Biol. Chem. (291): 24618-24627, 2016.

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the 25th Anniversary of Mizutani Foundation for Glycoscience