Molecular cloning and expression of glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans.
We isolated a cDNA encoding a novel glucuronyltransferase from human placenta cDNA with the use of the degenerate reverse transcriptase-polymerase chain reaction method. Degenerate primers were designed based upon the amino acid sequence alignment of rat glucuronyltransferase (GlcAT-P) involved in the biosynthesis of the carbohydrate epitope HNK-1 with putative proteins in Caenorhabditis elegans and Schistosoma mansoni. The new cDNA sequence revealed an open reading frame coding for a protein of 335 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 43% identity to the rat GlcAT-P, and the highest sequence identity was found in the COOH-terminal catalytic domain. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active glucuronyltransferase with marked specificity for a glycoserine Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. In contrast, asialoorosomucoid, which contains the Galbeta1-4GlcNAc sequence and is a good acceptor substrate for the GlcAT-P, did not serve as an acceptor. The reaction product was sensitive to beta-glucuronidase digestion and co-chromatographed with authentic GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser in high-performance liquid chromatography, suggesting that the enzyme is a beta1, 3-glucuronyltransferase. These results indicate that this new member of the glucuronyltransferase gene family is the enzyme previously described as glucuronyltransferase I that forms the glycosaminoglycan-protein linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans.
Kitagawa
H
H
Department of Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658, Japan.
Tone
Y
Y
Tamura
J
J
Neumann
K W
KW
Ogawa
T
T
Oka
S
S
Kawasaki
T
T
Sugahara
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary; D014453:Glucuronosyltransferase; D006025:Glycosaminoglycans / Q000378:metabolism*; D006602:Hexosyltransferases / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000096:biosynthesis* / Q000737:chemistry; D051381:Rats; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D006025:Glycosaminoglycans; D011509:Proteoglycans; D006602:Hexosyltransferases; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase