Molecular cloning and expression of human UDP-d-Xylose:proteoglycan core protein 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 residues 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-polymerase chain reaction method. This enzyme, which is involved in chondroitin sulfate, heparan sulfate, heparin and dermatan sulfate biosynthesis, belongs 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 fragment 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 located on chromosome 16p13.1 using radiation hybrid mapping, and extracts from CHO-K1 cells transfected with the XT-I cDNA in an expression vector exhibited marked XT activity. A new 3608 bp cDNA fragment encoding a protein of 865 amino acid residues was also isolated by PCR using degenerate primers based on the amino acid sequence of human XT-I. The amino acid sequence of this 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 the 15 kb gene was determined. RT-PCR analyses of XT-I and XT-II mRNA from various tissues confirmed that both XT-I and XT-II transcripts are ubiquitously expressed in the human tissues, although with different levels of transcription. Furthermore, the cDNAs encoding XT-I and XT-II from rat were cloned. The deduced amino acid sequences of rat xylosyltransferases displayed 94% identity to the corresponding human enzyme.
Götting
C
C
Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstrasse 11, Bad Oeynhausen, 32545, Germany.
Kuhn
J
J
Zahn
R
R
Brinkmann
T
T
Kleesiek
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D019902:Chondrocytes / Q000378:metabolism; D002885:Chromosomes, Human, Pair 16 / Q000235:genetics; D002886:Chromosomes, Human, Pair 17 / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D005091:Exons / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D009928:Organ Specificity; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D021101:Radiation Hybrid Mapping; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D014407:Tumor Cells, Cultured; D000097608:UDP Xylose-Protein Xylosyltransferase
D007527:Isoenzymes; D010430:Pentosyltransferases; D011994:Recombinant Proteins; D000097608:UDP Xylose-Protein Xylosyltransferase