Molecular cloning and expression of human UDP-D-xylose : proteoglycan coreprotein beta-D-xylosyltransferase and its first isoform XT-II

Human UDP-D-xylose:proteoglycan core protein beta -D-xylosyltransferase (EC 2.4.2.26, XT-I) initiates the biosynthesis of glycosaminoglycan chains in proteoglycans by transferring xylose from UDP-xylose to specific serine res idues of the core protein. Based on the partial amino acid sequence of the purified enzyme from human JAR choriocarcinoma cell culture supernatant we isolated a cDNA encoding XT-I using the degenerate reverse transcriptase-po lymerase chain reaction method. This enzyme, which is involved in chondroit in sulfate, heparan sulfate, heparin and dermatan sulfate biosynthesis, bel ongs to a novel family of glycosyltransferases with no homology to proteins known so far. 5' and 3'-RACE were performed to isolate a novel cDNA fragme nt of 3726 bp with a single open reading frame encoding at least 827 amino acid residues with a molecular mass of 91 kDa. The human XT-I gene was loca ted on chromosome 16p13.1 using radiation hybrid mapping, and extracts from CHO-K1 cells transfected with the XT-I cDNA in an expression vector exhibi ted marked XT activity. A new 3608 bp cDNA fragment encoding a protein of 8 65 amino acid residues was also isolated by PCR using degenerate primers ba sed on the amino acid sequence of human XT-I. The amino acid sequence of th is XT-II isoform displayed 55 % identity to the human XT-I. The XT-II gene was located on chromosome 17q21.3-17q22, and the exon/intron structure of t he 15 kb gene was determined. RT-PCR analyses of XT-I and XT-II mRNA from v arious tissues confirmed that both XT-I and XT-II transcripts are ubiquitou sly expressed in the human tissues, although with different levels of trans cription. Furthermore, the cDNAs encoding XT-I and XT-II from rat were clon ed. The deduced amino acid sequences of rat xylosyltransferases displayed 9 4% identity to the corresponding human enzyme. (C) 2000 Academic Press.