Studies on Biosynthetic Mechanism of the Sulfated Glycosaminoglycans

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Sulfated glycosaminoglycans including heparin/heparan sulfate and chondroitin/dermatan sulfate have been implicated in numerous pathophysiological phenomena of vertebrates and invertebrates. To facilitate analyses of the functions of these glycosaminoglycans through gene manipulation in various model animals, we have cloned several genes encoding the glycosyltransferases and sulfotransferase required for the biosynthesis of the glycosaminoglycans. In this study, we cloned additional six genes encoding the glycosyltransferases and a related protein involved in glycosaminoglycan biosynthesis and found novel functions of chondroitin in Caenorhabditis elegans as follows.1)Molecular cloning of human chondroitin N-acetylgalactosaminyltransferase-2(GalNAcT-2) :We identified a novel human chondroitin GalNAcT, designated chondroitin GalNAcT-2. GalNAcT-2 transferred β1,4-N-acetylgalactosamine(GalNAc) from UDP-[^3H]GalNAc not only to a polymer chondroitin representing growing chondroitin, but als … More o to GlcUAβ1-3Galβ1-O-C2H4NHCbz, a synthetic substrate for β-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate.2)Molecular cloning of three Drosophila glucuronyltransferases :We cloned and characterized three Drosophila β1,3-glucuronytransferases. The results showed that all of them are likely involved in the synthesis of the glycosaminoglycans-protein linkage region in Drosophila.3)cDNA cloning of C.elegans chondroitin synthase and demonstration of novel functions :We cloned a chondroitin synthase homologue of C.elegans and depleted expression of its product by RNA-mediated interference. We found that blocking chondroitin synthesis results in cytokinesis defects in early embryogenesis.4)Molecular cloning of a chondroitin polymerizing factor :We previously cloned human chondroitin synthase, but chondroitin polymerization was not demonstrated in vitro using the recombinant chondroitin synthase. We reported that the chondroitin polymerizing activity requires concomitant expression of a novel protein designated chondroitin polymerizing factor with chondroitin synthase.5)In vitro heparan sulfate polymerization :We found that the core protein moieties in addition to the interaction between EXT1 and EXT2 are required for heparan sulfate polymerization. Less