Chondroitin sulfate synthase-2. Molecular cloning and characterization of a novel human glycosyltransferase homologous to chondroitin sulfate glucuronyltransferase, which has dual enzymatic activities.
Chondroitin sulfate is found in a variety of tissues as proteoglycans and consists of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues with sulfate residues at various places. We found a novel human gene (GenBank accession number AB086063) that possesses a sequence homologous with the human chondroitin sulfate glucuronyltransferase gene which we recently cloned and characterized. The full-length open reading frame encodes a typical type II membrane protein comprising 775 amino acids. The protein had a domain containing beta 3-glycosyltransferase motif but lacked a typical beta 4-glycosyltransferase motif, which is the same as chondroitin sulfate glucuronyltransferase, whereas chondroitin synthase had both domains. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Surprisingly, both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, chondroitin sulfate, and their oligosaccharides were used as the acceptor substrates. The reaction products were identified to have the linkage of GlcUA beta 1-3GalNAc and GalNAc beta 1-4GlcUA at the non-reducing terminus of chondroitin for glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcripts were ubiquitously expressed in various human tissues but highly expressed in the pancreas, ovary, placenta, small intestine, and stomach. These results indicate that this enzyme could synthesize chondroitin sulfate chains as a chondroitin sulfate synthase that has both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. Sequence analysis based on three-dimensional structure revealed the presence of not typical but significant beta 4-glycosyltransferase architecture.
Yada
Toshikazu
T
Institute for Molecular Science of Medicine, Aichi Medical University, Nagakute, Aichi 480-1195, Japan.
Gotoh
Masanori
M
Sato
Takashi
T
Shionyu
Masafumi
M
Go
Mitiko
M
Kaseyama
Hiromi
H
Iwasaki
Hiroko
H
Kikuchi
Norihiro
N
Kwon
Yeon-Dae
YD
Togayachi
Akira
A
Kudo
Takashi
T
Watanabe
Hideto
H
Narimatsu
Hisashi
H
Kimata
Koji
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D019556:COS Cells; D002412:Cations; D002417:Cattle; D002455:Cell Division; D002807:Chondroitin / Q000737:chemistry; D002809:Chondroitin Sulfates / Q000737:chemistry*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004187:Disaccharides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D005822:Genetic Vectors; D020723:Glucuronic Acid / Q000737:chemistry; D014453:Glucuronosyltransferase / Q000378:metabolism; D016695:Glycosyltransferases / Q000378:metabolism; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009005:Monosaccharides; D009844:Oligosaccharides / Q000737:chemistry; D016366:Open Reading Frames; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution; D014530:Uridine Diphosphate / Q000494:pharmacology
D000596:Amino Acids; D002412:Cations; D018076:DNA, Complementary; D004187:Disaccharides; D000939:Epitopes; D009005:Monosaccharides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014530:Uridine Diphosphate; D020723:Glucuronic Acid; D002807:Chondroitin; D002809:Chondroitin Sulfates; D016695:Glycosyltransferases; D006602:Hexosyltransferases; C477294:chondroitin sulfate synthase-2; D014453:Glucuronosyltransferase; C468136:chondroitin sulfate glucuronyltransferase; D008274:Magnesium