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Toru Uyama

Publications and source records attributed to Toru Uyama.

9 recordsLinked to original sources
PubMed2006

Chondroitin 4-O-sulfotransferase-1 regulates E disaccharide expression of chondroitin sulfate required for herpes simplex virus infectivity.

We have demonstrated a defect in expression of chondroitin 4-O-sulfotransferase-1 (C4ST-1) in murine sog9 cells, which are poorly sensitive to infection by herpes simplex virus type 1 (HSV-1). Sog9 cells were previously isolated as CS-deficient cells from gro2C cells, which were partially resistant to HSV-1 infection and defective in the expression of heparan sulfate (HS) because of a splice site mutation in the EXT1 gene encoding the HS-synthesizing enzyme. Here we detected a small amount of CS chains in sog9 cells with a drastic decrease in 4-O-sulfation compared with the parental gro2C cells. RT-PCR revealed that sog9 cells had a defect in the expression of C4ST-1 in addition to EXT1. Gel filtration analysis showed that the decrease in the amount of CS in sog9 cells was the result of a reduction in the length of CS chains. Transfer of C4ST-1 cDNA into sog9 cells (sog9-C4ST-1) restored 4-O-sulfation and amount of CS, verifying that sog9 cells had a specific defect in C4ST-1. Furthermore, the expression of C4ST-1 rendered sog9 cells significantly more susceptible to HSV-1 infection, suggesting that CS modified by C4ST-1 is sufficient for the binding and infectivity of HSV-1. Analysis of CS chains of gro2C and sog9-C4ST-1 cells revealed a considerable proportion of the E disaccharide unit, consistent with our recent finding that this unit is an essential component of the HSV receptor. These results suggest that C4ST-1 regulates the expression of the E disaccharide unit and the length of CS chains, the features that facilitate infection of cells by HSV-1.

Animals↗
PubMed2005

Chondroitin sulfate characterized by the E-disaccharide unit is a potent inhibitor of herpes simplex virus infectivity and provides the virus binding sites on gro2C cells.

Although cell surface chondroitin sulfate (CS) is regarded as an auxiliary receptor for binding of herpes simplex virus to cells, and purified CS chain types A, B, and C are known to interfere poorly or not at all with the virus infection of cells, we have found that CS type E (CS-E), derived from squid cartilage, exhibited potent antiviral activity. The IC(50) values ranged from 0.06 to 0.2 mug/ml and substantially exceeded the antiviral potency of heparin, the known inhibitor of virus binding to cells. Furthermore, in mutant gro2C cells that express CS but not heparan sulfate, CS-E showed unusually high anti-herpes virus activity with IC(50) values of <1 ng/ml. Enzymatic degradation of CS-E with chondroitinase ABC abolished its antiviral activity. CS-E inhibited the binding to cells of the purified virus attachment protein gC. A direct interaction of gC with immobilized CS-E and inhibition of this binding by CS-E oligosaccharide fragments greater than octasaccharide were demonstrated. Likewise, the gro2C-specific CS chains interfered with the binding of viral gC to these cells and were found to contain a considerable proportion (13%) of the E-disaccharide unit, suggesting that this unit is an essential component of the CS receptor for herpes simplex virus on gro2C cells and that the antiviral activity of CS-E was due to interference with the binding of viral gC to a CS-E-like receptor on the cell surface. Knowledge of the determinants of antiviral properties of CS-E will help in the development of inhibitors of herpes simplex virus infections in humans.

Animals↗
PubMed2003

Chondroitin proteoglycans are involved in cell division of Caenorhabditis elegans.

Glycosaminoglycans such as heparan sulphate and chondroitin sulphate are extracellular sugar chains involved in intercellular signalling. Disruptions of genes encoding enzymes that mediate glycosaminoglycan biosynthesis have severe consequences in Drosophila and mice. Mutations in the Drosophila gene sugarless, which encodes a UDP-glucose dehydrogenase, impairs developmental signalling through the Wnt family member Wingless, and signalling by the fibroblast growth factor and Hedgehog pathways. Heparan sulphate is involved in these pathways, but little is known about the involvement of chondroitin. Undersulphated and oversulphated chondroitin sulphate chains have been implicated in other biological processes, however, including adhesion of erythrocytes infected with malaria parasite to human placenta and regulation of neural development. To investigate chondroitin functions, we cloned a chondroitin synthase homologue of Caenorhabditis elegans and depleted expression of its product by RNA-mediated interference and deletion mutagenesis. Here we report that blocking chondroitin synthesis results in cytokinesis defects in early embryogenesis. Reversion of cytokinesis is often observed in chondroitin-depleted embryos, and cell division eventually stops, resulting in early embryonic death. Our findings show that chondroitin is required for embryonic cytokinesis and cell division.

Animals↗
PubMed2003

Molecular cloning of a chondroitin polymerizing factor that cooperates with chondroitin synthase for chondroitin polymerization.

We recently cloned human chondroitin synthase (ChSy) exhibiting the glucuronyltransferase-II (GlcATII) and N-acetylgalactosaminyltransferase-II (GalNAcTII) activities responsible for the biosynthesis of repeating disaccharide units of chondroitin sulfate, but chondroitin polymerization was not demonstrated in vitro using the recombinant ChSy. We report here that the chondroitin polymerizing activity requires concomitant expression of a novel protein designated chondroitin polymerizing factor (ChPF) with ChSy. The human ChPF consists of 775 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 23% identity to that of human ChSy. The expression of a soluble recombinant form of the protein in COS-1 cells produced a protein with little GlcAT-II or GalNAcT-II activity. In contrast, coexpression of the ChPF and ChSy yielded markedly augmented glycosyltransferase activities, whereas simple mixing of the two separately expressed proteins did not. Moreover, using both UDP-glucuronic acid (GlcUA) and UDP-N-acetylgalactosamine (GalNAc) as sugar donors, chondroitin polymerization was demonstrated on the so-called glycosaminoglycan-protein linkage region tetrasaccharide sequence of alpha-thrombomodulin. These results suggested that the ChPF acts as a specific activating factor for ChSy in chondroitin polymerization. The coding region of the ChPF was divided into four discrete exons and localized to chromosome 2q35-q36. Northern blot analysis revealed that the ChPF gene exhibited a markedly different expression pattern among various human tissues, which was similar to that of ChSy. Thus, the ChPF is required for chondroitin polymerizing activity of mammalian ChSy.

Amino Acid Sequence↗
PubMed2003

Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate.

Recent glycobiology studies have suggested fundamental biological functions for chondroitin, chondroitin sulfate and dermatan sulfate, which are widely distributed as glycosaminoglycan sidechains of proteoglycans in the extracellular matrix and at cell surfaces. They have been implicated in the signaling functions of various heparin-binding growth factors and chemokines, and play critical roles in the development of the central nervous system. They also function as receptors for various pathogens. These functions are closely associated with the sulfation patterns of the glycosaminoglycan chains. Surprisingly, nonsulfated chondroitin is indispensable in the morphogenesis and cell division of Caenorhabditis elegans, as revealed by RNA interference experiments of the recently cloned chondroitin synthase gene and by the analysis of mutants of squashed vulva genes.

Animals↗
PubMed2002

Molecular cloning and expression of a second chondroitin N-acetylgalactosaminyltransferase involved in the initiation and elongation of chondroitin/dermatan sulfate.

We identified a novel human chondroitin N-acetylgalactosaminyltransferase, designated chondroitin GalNAcT-2 after a BLAST analysis of the GenBank(TM) data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GalNAcT-1. Like chondroitin GalNAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUA beta 1-3Gal beta 1-O-C(2)H(4)NHCbz, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GlcUA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GalNAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains.

Amino Acid Sequence↗
PubMed2002

Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase: the key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate.

Based on sequence homology with the recently cloned human chondroitin synthase, we identified a novel beta1,4-N-acetylgalactosaminyltransferase, which consisted of 532 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 27% identity to that of human chondroitin synthase. The expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc not only to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUAbeta1--3Galbeta1-O-C(2)H(4)NH-benzyloxycarbonyl, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein linkage region of chondroitin sulfate. Hence, the enzyme is involved in the biosynthetic initiation and elongation of chondroitin sulfate and is the key enzyme responsible for the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate or heparin/heparan sulfate chains. The coding region of this enzyme was divided into seven discrete exons and localized to chromosome 8. Northern blot analysis revealed that the chondroitin GalNAc transferase gene exhibited a ubiquitous but markedly differential expression in human tissues and that the expression pattern was similar to that of chondroitin synthase. Thus, more than two distinct enzymes forming the novel gene family are required for chain initiation and elongation in chondroitin/dermatan sulfate as in the biosynthesis of heparin/heparan sulfate.

Acetylgalactosamine↗