Akira Kurosaka

Brain-specific expression of a novel human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T9).

We isolated cDNA coding for the ninth of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-T9) from human brain by the polymerase chain reaction. The polypeptide encoded by GalNAc-T9 contained the structural features characteristic of GalNAc transferases, such as a GT1 motif, a Gal/GalNAc transferase motif, (QXW)(3) repeats, and conserved His, Cys, and acidic amino acid residues. Northern blot analysis revealed the mRNA expression of the enzyme to be confined to the brain. The brain-specific expression of GalNAc-T9 suggested that this isozyme catalyzes O-glycosylation in the brain.

Mucin-carbohydrate directed monoclonal antibody

To raise monoclonal antibodies recognizing cancer‐associated alterations of the carbohydrate structure of glycoproteins, Balb/c mice were immunized with human colonic cancer cells (LS 180 from ATCC). One of the generated hybridomas produced a monoclonal antibody that bound to the carbohydrate moiety of mucin‐type glycoproteins from LS 180. The antibody did not bind to glycoproteins from another colonic cancer cell line, SW 1116, or to glycolipids from any of the colonic cancer cell lines. The antibody bound to ovine and bovine submaxillary mucins (OSM and BSM). NeuAcα2→6Ga1NAc seemed to be involved in the epitope.

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker-Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.

Function of the lectin domain of polypeptide N-acetylgalactosaminyltransferase 1.

All UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases cloned to date contain a lectin domain at the C-terminus, consisting of three tandem repeat sequences (alpha,beta, and gamma). We previously reported that the alpha repeat of one of the most ubiquitous isozymes, GalNAc-T1, is a functional lectin that recognizes O-linked GalNAc residues on the acceptor polypeptides with multiple acceptor sites; the domain appears not to be involved in the glycosylation of acceptors with a single acceptor site. In this report, we studied the function of the beta and gamma repeats in the GalNAc-T1 lectin domain, by site-directed mutagenesis and analysis of the catalytic properties of mutant enzymes. We found that the beta repeat recognizes GalNAc and is involved in glycosylation of acceptors with multiple glycosylation sites. The gamma repeat, on the other hand, showed no significant GalNAc-binding activity. These results indicate that the lectin domain of GalNAc-T1 has at least two functional repeats, allowing the possibility of multivalent interactions with GalNAc residues on the acceptor polypeptide during glycosylation.

Radical fringe negatively modulates Notch signaling in postmitotic neurons of the rat brain.

Fringe was originally identified as a novel secreted signaling protein with a key role in wing formation of Drosophila. Three vertebrate fringe homologues, Radical, Lunatic and Manic fringe, were also identified, and have been shown to play major roles in neurogenesis during development. However, the expression and roles of vertebrate fringe homologues in the adult brain remain to be elucidated. We isolated the cDNA encoding rat Radical fringe (334 amino acids) from rat embryos, and found its mRNA to be most abundantly expressed in the adult rat brain by Northern blotting analysis. The localization of Radical fringe mRNA in the adult rat brain was also examined by in situ hybridization. The mRNA was abundantly expressed in most neurons, but not glial cells, throughout the brain. Notch signaling was shown to negatively modulate the stability of neurites and connections in postmitotic primary neurons. Furthermore, genetic evidence indicated that fringe modulated the Notch signaling pathway. Therefore, we examined the effects of Radical fringe on the Notch signaling pathway in primary rat neurons of the cerebral cortex using recombinant rat Radical fringe protein. Radical fringe protein significantly inhibited expression of the Notch effector Hes1 mRNA in primary neurons. These results indicated that Radical fringe functions by inhibiting Notch signaling in postmitotic neurons of the brain.

Production of Monoclonal Antibodies Directed against Carbohydrate Moieties of Cell Surface Glycoproteins

Through the use of a technique for raising monoclonal antibodies, coupled with a solid‐phase radioimmunoassay utilizing immobilized glyeopeptides prepared from the surface membranes of the colorectal cancer cells (LS 180) used for the immunization, carbohydrate‐directed monoclonal antibodies were obtained. One of the monoclonal antibodies, MLS 102, reacted immunohistochemically intensely with the colorectal cancer cell surface and the mutinous glycoproteins secreted by the cancer cells, but only weakly with normal colon tissue. The antigenic determinant recognized by MLS 102 was the carbohydrate moiety of glycoproteins with terminal sialic acid. The antigens defined by other monoclonal antibodies, MLS 103 and 104, were immunohistochemically detected in both normal colonic epithelial and cancer cells. These antibodies seemed to recognize the carbohydrate moieties of both glycoproteins and glycolipids. The method described in this report can be generally applied to raise cell surface carbohydrate‐directed antibodies.

UDP-GalNAc : GalNAc-mucin α-N-acetylgalactosamine transferase activity in human intestinal cancerous tissues

GalNAc transferase Human intestinal cancerous tissue Bovine submaxillary gland mucin O‐Glycosidically linked sugar chain

A putative polypeptide N-acetylgalactosaminyltransferase/Williams-Beuren syndrome chromosome region 17 (WBSCR17) regulates lamellipodium formation and macropinocytosis

Background: WBSCR17 is a potential polypeptide N-acetylgalactosaminyltransferase with unknown function. Results: WBSCR17, induced with N-acetylglucosamine, regulated O-glycosylation, lamellipodium formation, and macropinocytosis. Conclusion: Mucin-type O-glycosylation may be involved in lamellipodium formation and membrane trafficking through macropinocytosis. Significance: The data suggest that mucin-type O-glycosylation modulates the dynamic membrane transport of the cell and may be involved in the control of nutrient uptake. We previously identified a novel polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) gene, which is designated Williams-Beuren syndrome chromosome region 17 (WBSCR17) because it is located in the chromosomal flanking region of the Williams-Beuren syndrome deletion. Recent genome-scale analysis of HEK293T cells treated with a high concentration of N-acetylglucosamine (GlcNAc) demonstrated that WBSCR17 was one of the up-regulated genes possibly involved in endocytosis (Lau, K. S., Khan, S., and Dennis, J. W. (2008) Genome-scale identification of UDP-GlcNAc-dependent pathways. Proteomics 8, 3294–3302). To assess its roles, we first expressed recombinant WBSCR17 in COS7 cells and demonstrated that it was N-glycosylated and localized mainly in the Golgi apparatus, as is the case for the other GalNAc-Ts. Assay of recombinant WBSCR17 expressed in insect cells showed very low activity toward typical mucin peptide substrates. We then suppressed the expression of endogenous WBSCR17 in HEK293T cells using siRNAs and observed phenotypic changes of the knockdown cells with reduced lamellipodium formation, altered O-glycan profiles, and unusual accumulation of glycoconjugates in the late endosomes/lysosomes. Analyses of endocytic pathways revealed that macropinocytosis, but neither clathrin- nor caveolin-dependent endocytosis, was elevated in the knockdown cells. This was further supported by the findings that the overexpression of recombinant WBSCR17 stimulated lamellipodium formation, altered O-glycosylation, and inhibited macropinocytosis. WBSCR17 therefore plays important roles in lamellipodium formation and the regulation of macropinocytosis as well as lysosomes. Our study suggests that a subset of O-glycosylation produced by WBSCR17 controls dynamic membrane trafficking, probably between the cell surface and the late endosomes through macropinocytosis, in response to the nutrient concentration as exemplified by environmental GlcNAc.

Function of conserved aromatic residues in the Gal/GalNAc-glycosyltransferase motif of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1.

UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferases (GalNAc transferases), which initiate mucin‐type O‐glycan biosynthesis, have broad acceptor substrate specificities, and it is still unclear how they recognize peptides with different sequences. To increase our understanding of the catalytic mechanism of GalNAc‐T1, one of the most ubiquitous isozymes, we studied the effect of substituting six conserved aromatic residues in the highly conserved Gal/GalNAc‐glycosyltransferase motif with leucine on the catalytic properties of the enzyme. Our results indicate that substitutions of Trp302 and Phe325 have little impact on enzyme function and that substitutions of Phe303 and Tyr309 could be made with only limited impact on the interaction(s) with donor and/or acceptor substrates. By contrast, Trp328 and Trp316 are essential residues for enzyme functions, as substitution with leucine, at either site, led to complete inactivation of the enzymes. The roles of these tryptophan residues were further analyzed by evaluating the impact of substitutions with additional amino acids. All evaluated substitutions at Trp328 resulted in enzymes that were completely inactive, suggesting that the invariant Trp328 is essential for enzymatic activity. Trp316 mutant enzymes with nonaromatic replacements were again completely inactive, whereas two mutant enzymes containing a different aromatic amino acid, at position 316, showed low catalytic activity. Somewhat surprisingly, a kinetic analysis revealed that these two amino acid substitutions had a moderate impact on the enzymes affinity for the donor substrate. By contrast, the drastically reduced affinity of the Trp316 mutant enzymes for the acceptor substrates suggests that Trp316 is important for this interaction.

Expression and function of glycogen synthase kinase-3 in human hair follicles

Abstractβ-Catenin is involved in the hair follicle morphogenesis and stem cell differentiation, and inhibition of glycogen synthase kinase-3 (GSK-3) increases β-catenin concentration in the cytoplasm. To examine the effects of GSK-3 inhibition on the hair follicle epithelium, we first examined the expression of GSK-3 in plucked human hair follicles by RT-PCR and found GSK-3 expression in hair follicles. Western blotting with a GSK-3β-specific antibody, Y174, also demonstrated GSK-3β expression in the follicles. Moreover, GSK-3β immunostaining with Y174 showed that GSK-3β colocalized with hair follicle bulge markers. Contrary to GSK-3β, GSK-3α was widely expressed throughout the follicles when immunostained with a specific antibody, EP793Y. We then investigated the influence of GSK-3 inhibition. A GSK-3 inhibitor, BIO, promoted the growth of human outer root sheath cells, which could be cultured for up to four passages. The BIO-treated cells exhibited smaller and more undifferentiated morphology than control cells. Moreover, in organ culture of plucked human hair, outer root sheath cells in the middle of a hair follicle proliferated when cultured with BIO. These results indicate that GSK-3β is expressed in hair bulge stem cells and BIO promotes the growth of ORS cells, possibly by regulating the GSK-3 signaling pathway.