Molecular cloning and characterization of a novel chondroitin sulfate glucuronyltransferase that transfers glucuronic acid to N-acetylgalactosamine.
We found a novel human gene (GenBank accession number, Kazusa DNA Research Institute KIAA1402) that possesses homology with chondroitin synthase. The full-length open reading frame consists of 772 amino acids and encodes a typical type II membrane protein. This enzyme had a domain containing beta 3-glycosyltransferase motifs, which might be a beta3-glucuronyltransferase domain, but no domain with beta 4-glycosyltransferase motifs, although both are found in chondroitin synthase. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Its glucuronyltransferase activity was observed when chondroitin and chondroitin sulfate polysaccharides and oligosaccharides were used as acceptor substrates. However, it was not detected when dermatan sulfate, hyaluronan, heparan sulfate, heparin, N-acetylheparosan, lactosamine tetrasaccharide, and linkage tri- and tetrasaccharide acceptors were employed. The reaction product, which was speculated to exhibit a GlcA beta 1-3GalNAc linkage structure at its non-reducing terminus, showed the following characteristics. 1) It was catabolized by beta-glucuronidase. 2) It was an acceptor for Escherichia coli K4 chondroitin polymerase (K4 chondroitin polymerase). 3) The product of K4 chondroitin polymerase was cleaved by chondroitinase ACII. On the other hand, no N-acetylgalactosaminyltransferase activity was detected toward any acceptors. Quantitative real time PCR analysis revealed that its transcripts were highly expressed in the placenta, small intestine, and pancreas, although they were ubiquitously expressed in various tissues and cell lines. This enzyme could play a role in the synthesis of chondroitin sulfate as a glucuronyltransferase.
Gotoh
Masanori
M
Glycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology, Open Space Laboratory, C-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
Yada
Toshikazu
T
Sato
Takashi
T
Akashima
Tomohiro
T
Iwasaki
Hiroko
H
Mochizuki
Hideo
H
Inaba
Niro
N
Togayachi
Akira
A
Kudo
Takashi
T
Watanabe
Hideto
H
Kimata
Koji
K
Narimatsu
Hisashi
H
D000116:Acetylgalactosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002809:Chondroitin Sulfates / Q000378:metabolism*; D003001:Cloning, Molecular; D017930:Genes, Reporter; D020723:Glucuronic Acid / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D020723:Glucuronic Acid; D002809:Chondroitin Sulfates; D014453:Glucuronosyltransferase; C468136:chondroitin sulfate glucuronyltransferase; D000116:Acetylgalactosamine