Molecular cloning and expression of a human chondroitin synthase.
We have identified a human chondroitin synthase from the HUGE (human unidentified gene-encoded large proteins) protein data base by screening with two keywords: "one transmembrane domain" and "galactosyltransferase family." The identified protein consists of 802 amino acids with a type II transmembrane protein topology. The protein showed weak homology to the beta1,3-galactosyltransferase family on the amino-terminal side and to the beta1,4-galactosyltransferase family on the carboxyl-terminal side. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active enzyme, which transferred not only the glucuronic acid (GlcUA) from UDP-[(14)C]GlcUA but also N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to the polymer chondroitin. Identification of the reaction products demonstrated that the enzyme was chondroitin synthase, with both beta1,3-GlcUA transferase and beta1,4-GalNAc transferase activities. The coding region of the chondroitin synthase was divided into three discrete exons and localized to chromosome 15. Northern blot analysis revealed that the chondroitin synthase gene exhibited ubiquitous but markedly differential expression in the human tissues examined. Thus, we demonstrated that analogous to human heparan sulfate polymerases, the single polypeptide chondroitin synthase possesses two glycosyltransferase activities required for chain polymerization.
Kitagawa
H
H
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan.
Uyama
T
T
Sugahara
K
K
D000116:Acetylgalactosamine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D019556:COS Cells; D002807:Chondroitin / Q000378:metabolism; D002874:Chromosome Mapping; D002884:Chromosomes, Human, Pair 15; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D016678:Genome; D020723:Glucuronic Acid / Q000378:metabolism; D014453:Glucuronosyltransferase; D016695:Glycosyltransferases / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D064251:Multifunctional Enzymes; D017350:N-Acetylgalactosaminyltransferases; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D018076:DNA, Complementary; D064251:Multifunctional Enzymes; D020723:Glucuronic Acid; D002807:Chondroitin; D016695:Glycosyltransferases; D017350:N-Acetylgalactosaminyltransferases; D014453:Glucuronosyltransferase; C412725:chondroitin synthase; C498800:CHSY1 protein, human; D000116:Acetylgalactosamine