Human Xylosyltransferase II Is Involved in the Biosynthesis of the Uniform Tetrasaccharide Linkage Region in Chondroitin Sulfate and Heparan Sulfate Proteoglycans

Abstract

Human xylosyltransferase I (XT-I) initiates the biosynthesis of the glycosaminoglycan (GAG) linkage tetrasaccharide in proteoglycans. Xylosyltransferase II (XT-II) is a protein homologous to XT-I but with hitherto unknown activity or physiological function. Here, we report the enzymatic activity of XT-II and provide evidence that XT-II initiates the biosynthesis of both heparan sulfate and chondroitin sulfate GAGs. Transfection of the xylosyltransferase-deficient Chinese hamster ovary mutant pgsA-745 with XT-I or XT-II coding cDNA completely restored GAG biosynthesis. GAG disaccharide analysis revealed that XT-I- and XT-II-transfected pgsA-745 cells produced similar amounts of chondroitin sulfate and heparan sulfate. Furthermore, a high xylosyltransferase activity was measured after transfection with cDNAs encoding either isozyme. Analysis of the enzyme activity revealed that XT-II catalyzes the transfer of xylose to similar peptide acceptors as XT-I but with different efficiency. The optimal XT-II acceptor was observed using a bikunin-related peptide (Km 5.2 μm). Analysis of XT-I and XT-II mRNA expression in murine tissues showed a differential expression pattern for both enzymes. In particular, XT-II is highly expressed in liver tissue, where XT-I transcripts were not detected. This is the first report on the enzyme activity of XT-II and its involvement in chondroitin sulfate and heparan sulfate biosynthesis. Human xylosyltransferase I (XT-I) initiates the biosynthesis of the glycosaminoglycan (GAG) linkage tetrasaccharide in proteoglycans. Xylosyltransferase II (XT-II) is a protein homologous to XT-I but with hitherto unknown activity or physiological function. Here, we report the enzymatic activity of XT-II and provide evidence that XT-II initiates the biosynthesis of both heparan sulfate and chondroitin sulfate GAGs. Transfection of the xylosyltransferase-deficient Chinese hamster ovary mutant pgsA-745 with XT-I or XT-II coding cDNA completely restored GAG biosynthesis. GAG disaccharide analysis revealed that XT-I- and XT-II-transfected pgsA-745 cells produced similar amounts of chondroitin sulfate and heparan sulfate. Furthermore, a high xylosyltransferase activity was measured after transfection with cDNAs encoding either isozyme. Analysis of the enzyme activity revealed that XT-II catalyzes the transfer of xylose to similar peptide acceptors as XT-I but with different efficiency. The optimal XT-II acceptor was observed using a bikunin-related peptide (Km 5.2 μm). Analysis of XT-I and XT-II mRNA expression in murine tissues showed a differential expression pattern for both enzymes. In particular, XT-II is highly expressed in liver tissue, where XT-I transcripts were not detected. This is the first report on the enzyme activity of XT-II and its involvement in chondroitin sulfate and heparan sulfate biosynthesis. Proteoglycans are a large group of polyanionic glycoproteins expressed in virtually every animal cell on the cell surface and in the extracellular matrix (1Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1349) Google Scholar). These abundant molecules serve a wide range of functions in many biological processes, including extracellular matrix deposition, cell-cell interactions, tumor cell growth, viral infections, and neurite outgrowth. They consist of a core protein to which varying numbers and types of glycosaminoglycan (GAG) 2The abbreviations used are: GAG, glycosaminoglycan; CHO, Chinese hamster ovary; FGF-2, fibroblast growth factor 2; RT, reverse transcription; XT, xylosyltransferase; XT-I, xylosyltransferase I; XT-II, xylosyltransferase II; HPLC, high pressure liquid chromatography; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. chains are covalently attached (2Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar). The biological roles of proteoglycans are closely related to the presence and biosynthesis of the GAG chains, which contain binding sites for various growth factors and matrix proteins. The glycosaminoglycans chondroitin sulfate, dermatan sulfate, heparan sulfate, and heparin are bound to the proteoglycan core protein by a common linkage tetrasaccharide composed of d-GlcAβ1,3-Galβ1,3Galβ1,4Xyl-d-glucuronic acid-β1,3-galactose-β1,3-galactose-β1,4-xylose- (2Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar). The initial transfer of d-xylose from UDP-d-xylose to specific serine residues of the core protein is catalyzed by one or more xylosyltransferases. Xylosyltransferase I (UDP-d-xylose:proteoglycan core protein β-d-xylosyltransferase I, EC 2.4.2.26, XT-I) was shown to be the rate-limiting step enzyme in the biosynthesis of GAG chains (3Seo N.S. Hocking A.M. Hook M. McQuillan D.J. J. Biol. Chem. 2005; 280: 42774-42784Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Furthermore, serum xylosyltransferase activity was found to be a biochemical marker for the assessment of disease activity in systemic sclerosis (4Goötting C. Sollberg S. Kuhn J. Weilke C. Huerkamp C. Brinkmann T. Krieg T. Kleesiek K. J. Investig. Dermatol. 1999; 112: 919-924Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and for the diagnosis of fibrotic remodeling processes, which are characterized by an elevated proteoglycan biosynthesis (5Goötting C. Hendig D. Adam A. Schoön S. Schulz V. Szliska C. Kuhn J. Kleesiek K. J. Mol. Med. 2005; 83: 984-992Crossref PubMed Scopus (33) Google Scholar, 6Schoöttler M. Muöller S. Schoön S. Prante C. Kuhn J. Kleesiek K. Goötting C. Clin. Biochem. 2005; 38: 486-488Crossref PubMed Scopus (8) Google Scholar). In 2000, we successfully isolated the human XT-I from 2,000 liters of JAR choriocarcinoma cell culture supernatant and determined the partial amino acid sequence of the proteolytically cleaved protein (7Kuhn J. Goötting C. Schnoölzer M. Kempf T. Brinkmann T. Kleesiek K. J. Biol. Chem. 2001; 276: 4940-4947Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 8Kuhn J. Moölle K. Brinkmann T. Goötting C. Kleesiek K. J. Biotechnol. 2003; 103: 191-196Crossref PubMed Scopus (12) Google Scholar). We cloned the cDNA of the human, mouse, and rat XT-I (9Goötting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Crossref PubMed Scopus (204) Google Scholar) and demonstrated that the recombinant human XT-I is the chain-initiating enzyme in proteoglycan biosynthesis (9Goötting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Crossref PubMed Scopus (204) Google Scholar, 10Kuhn J. Muöller S. Schnoölzer M. Kempf T. Schoön S. Brinkmann T. Schoöttler M. Goötting C. Kleesiek K. Biochem. Biophys. Res. Commun. 2003; 312: 537-544Crossref PubMed Scopus (19) Google Scholar). We also cloned a cDNA encoding a putative protein highly homologous to the human XT-I, which was termed XT-II (9Goötting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Crossref PubMed Scopus (204) Google Scholar). Both amino acid sequences have a 55% overall sequence identity. In addition, highly homologous regions with more than 80% identical amino acids are present (9Goötting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Crossref PubMed Scopus (204) Google Scholar). All higher organisms have been found to harbor two xylosyltransferase genes, whereas Drosophila melanogaster and Caenorhabditis elegans have only one xylosyltransferase (11Goötting C. Muöller S. Schoöttler M. Schoön S. Prante C. Brinkmann T. Kuhn J. Kleesiek K. J. Biol. Chem. 2004; 279: 42566-42573Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 12Wilson I.B. J. Biol. Chem. 2002; 277: 21207-21212Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 13Hwang H.Y. Olson S.K. Brown J.R. Esko J.D. Horvitz H.R. J. Biol. Chem. 2003; 278: 11735-11738Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, no enzymatic activity has been shown for XT-II and, consequently, the physiological function of this protein has remained a mystery. Nevertheless, we could already present evidence that XT-II is of biological importance since mutations in the XT-II gene are genetic risk factors for diseases that are characterized by an altered proteoglycan metabolism, such as pseudoxanthoma elasticum (14Schoön S. Schulz V. Prante C. Hendig D. Szliska C. Kuhn J. Kleesiek K. Goötting C. J. Med. Genet. 2006; 43: 745-749Crossref PubMed Scopus (41) Google Scholar), osteoarthritis (15Schoön S. Huep G. Prante C. Muöller S. Christ R. Hagena F.W. Kuhn J. Kleesiek K. Goötting C. Osteoarthritis Cartilage. 2006; 14: 442-448Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), or diabetic nephropathy (16Schoön S. Prante C. Muöller S. Schoöttler M. Tarnow L. Kuhn J. Kleesiek K. Goötting C. Kidney Int. 2005; 68: 1483-1490Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In the present study, we transfected xylosyltransferase-deficient pgsA-745 CHO cells with human XT-I and XT-II cDNA to restore proteoglycan biosynthesis and to elucidate the physiological function of XT-II. XT-II is an active xylosyltransferase that is capable of restoring the GAG chain biosynthesis in mutant CHO cells, and XT-I and XT-II are differentially expressed in animal tissues. This is the first report on the physiological function of human XT-II and its involvement in the proteoglycan biosynthesis. Radiochemical Xylosyltransferase Activity Assay—The method for determining xylosyltransferase activity is based on the incorporation of d-[14C]xylose with silk, recombinant bikunin, or bikunin peptides as the acceptor and was performed as described previously (17Campbell P. Jacobsson I. Benzing-Purdie L. Roden L. Fessler J.H. Anal. Biochem. 1984; 137: 505-516Crossref PubMed Scopus (30) Google Scholar, 18Weilke C. Brinkmann T. Kleesiek K. Clin. Chem. 1997; 43: 45-51Crossref PubMed Scopus (47) Google Scholar). For the determination of the Km values, various concentrations of the acceptor proteins were incubated with XT-II solution under assay conditions. Analysis of Xylosyltransferase Activity by HPLC Electrospray Ionization Tandem Mass Spectrometry—For the determination of xylosyltransferase activity by HPLC electrospray ionization tandem mass spectrometry, the xylosyltransferase-mediated incorporation of UDP-xylose into the synthetic peptide biotin-NH-QEEEGSGGGQKK(5-fluorescein)-CONH2 was quantified as described recently (19Kuhn J. Prante C. Schoön S. Goötting C. Kleesiek K. Clin. Chem. 2006; 52: 2243-2249Crossref PubMed Scopus (27) Google Scholar). The characterization of the XT-I and XT-II reaction products was performed as described using the biotin-NH-QEEEGSGGGQKK(5-fluorescein)-CONH2 as acceptor (9Goötting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Crossref PubMed Scopus (204) Google Scholar). Biotinylation of FGF-2—The biotinylation of FGF-2 was performed as described previously (20Bai X. Wei G. Sinha A. Esko J.D. J. Biol. Chem. 1999; 274: 13017-13024Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 21Zhang L. Lawrence R. Schwartz J.J. Bai X. Wei G. Esko J.D. Rosenberg R.D. J. Biol. Chem. 2001; 276: 28806-28813Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) with modifications mentioned in the Supplemental Data. Complementation of the Proteoglycan Biosynthesis Defect in pgsA-745 CHO Cells by Human XT-I and XT-II—Wild-type CHO cells (CHO-K1, DSMZ, Braunschweig, Germany) and xylosyltransferase-deficient pgsA-745 CHO cells (22Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (489) Google Scholar) were grown in Ham’s F12 medium supplemented with 10% fetal calf serum under standard conditions. pgsA-745 CHO cells were transfected with the plasmids pSS1 and pCMV6-XL6-XYLT2 using FuGENE 6 transfection reagent (Roche Applied Science, Mannheim, Germany), as recommended by the manufacturer. pSS1 encodes a green fluorescent protein-tagged form of the full-length human XT-I (23Schoön S. Prante C. Bahr C. Kuhn J. Kleesiek K. Goötting C. J. Biol. Chem. 2006; 281: 14224-14231Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and pCMV6-XL6-XYLT2 (GenBank™ accession number NM_022167, TrueClone, Origene, Rockville, MD) contains the complete cDNA of the full-length human XT-II. Stable transfectants were selected with 400 μg/ml Geneticin (Invitrogen), and single clones were isolated. Flow cytometry analysis of FGF-2 binding was performed as described in the supplemental data. Recombinant Expression of XT-II in the Yeast Pichia pastoris—A cDNA fragment encoding the amino acids 37–865 of the human XT-II was cloned in the pPICZαA vector, and the soluble form of XT-II was expressed in P. pastoris using standard conditions. The culture medium was harvested after 24 h, and the enzyme activity was analyzed. Mock-transfected cells were used as control. HPLC Analysis of Chondroitin Sulfate and Heparan Sulfate Disaccharides—The isolation of GAG chains was performed as described in the supplemental data. 2-Amino-acridone labeling of GAG disaccharides and HPLC analysis were carried out as described previously (24Kitagawa H. Kinoshita A. Sugahara K. Anal. Biochem. 1995; 232: 114-121Crossref PubMed Scopus (108) Google Scholar, 25Vogel K. Kuhn J. Kleesiek K. Goötting C. Electrophoresis. 2006; 27: 1363-1367Crossref PubMed Scopus (12) Google Scholar). Real-time Quantitative RT-PCR Analysis of Xylosyltransferase Expression in Murine Tissues—The mRNA expression of the mouse XT-I and XT-II genes in different mouse tissues was analyzed by a fluorogenic RT-PCR assay using the Realplex Mastercycler system (Eppendorf, Hamburg, Germany) and cDNA from different mouse tissues (Mouse MTC Panel, Clontech, Heidelberg, Germany). The primer sequences and the PCR protocols are listed in the supplemental data. Real-time Quantitative RT-PCR Analysis of Xylosyltransferase I and II Expression in Wild-type and pgsA-745 CHO Cells—mRNA expression of the XT-I and XT-II genes in wild-type and pgsA-745 CHO cells was quantified by a fluorogenic RT-PCR assay using the Realplex Mastercycler system (Eppendorf) and intron-spanning primers listed in the supplemental data. Analysis of XT-II Amino Acid Sequences—Homology analysis of the amino acid sequences of the XT-II was carried out using sequences extracted form the GenBank, Swissprot, or Ensembl databases. The sequences and the procedures are listed in the supplemental data. Homology Analysis of Xylosyltransferase II Orthologs and Identification of Highly Conserved Sequence Blocks—All higher organisms were found to possess two putative xylosyltransferase genes based on surveys of available genome sequences. Using the ClustalX alignment method, a sequence alignment of the XT-II peptide sequences from multiple organisms was performed. The XT-II was highly conserved among all species (Fig. 1). XT-II of human and apes showed more than 98% amino acid sequence identity, whereas the XT-II sequence from other organisms were 60–93% identical, with a much higher portion of amino acids with similar large conserved sequence that identical amino acids among all XT-II sequences could be (Fig. These the acids and large of the protein by the in the Furthermore, the which is in human XT-II and which was found to be for enzyme activity in XT-I (11Goötting C. Muöller S. Schoöttler M. Schoön S. Prante C. Brinkmann T. Kuhn J. Kleesiek K. J. Biol. Chem. 2004; 279: 42566-42573Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), is also in the conserved sequence The amino acids of the XT-II are highly which is in with that this was for activity or enzyme in Drosophila and C. elegans H.Y. Olson S.K. Brown J.R. Esko J.D. Horvitz H.R. J. Biol. Chem. 2003; 278: 11735-11738Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, I.B. Mol. Sci. 2004; PubMed Scopus Google Scholar). These that XT-II is an highly conserved XT-II (9Goötting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Crossref PubMed Scopus (204) Google Scholar, C. Muöller S. Schoöttler M. Schoön S. Prante C. Brinkmann T. Kuhn J. Kleesiek K. J. Biol. Chem. 2004; 279: 42566-42573Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, S. Schoöttler M. Schoön S. Prante C. Brinkmann T. Kuhn J. Goötting C. Kleesiek K. Biochem. J. 2005; PubMed Scopus Google Scholar). Analysis of the gene and of the XT-II genes in different organisms revealed that consist of with highly conserved (Fig. mRNA Expression of the expression of XT-II and XT-I was analyzed in various murine tissues using a method with The mRNA of the genes and was determined to the genes encoding GAPDH, and The expression of XT-II was found in liver tissue, where the mRNA was more than higher than the XT-II expression in all (Fig. XT-II mRNA could also be in the and the the other XT-I was highly expressed in mouse and no XT-I mRNA was in liver (Fig. The expression of both genes in the mouse tissues to These the that XT-I and XT-II have a expression Expression of XT-I and XT-II mRNA in pgsA-745 CHO is a xylosyltransferase-deficient mutant CHO cell that was to have than of the activity found in wild-type cells (22Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (489) Google Scholar). However, the genetic of the proteoglycan biosynthesis in pgsA-745 is hitherto the Chinese hamster was to harbor two xylosyltransferase genes, we analyzed the expression of the genes is in pgsA-745 RT-PCR with from the mouse XT-I and XT-II mRNA sequence and cDNA was to the hamster XT-I and XT-II the putative of the XT-I gene and the of the XT-II gene were and used for the of XT-I and XT-II cDNA The sequence the mouse and hamster XT-I and XT-II cDNA sequences was and of XT-I and XT-II mRNA showed that XT-II is the present in CHO wild-type cells of of The of XT-I and XT-II mRNA in wild-type and pgsA-745 CHO cells was XT-I transcripts were in wild-type cells but not in pgsA-745 Furthermore, XT-II expression was with wild-type cells, that both genes are in the cell These to the that multiple mutations in both genes are for the of mutations or to J. Clin. Investig. 2002; PubMed Scopus Google Scholar) and a mRNA are for the of XT-I mRNA and the of XT-II mRNA in pgsA-745 However, hitherto mutations that in an XT-II enzyme are to be present in of GAG by Human XT-I and XT-II cDNA in CHO xylosyltransferase-deficient mutant CHO cell pgsA-745 is not capable of chondroitin or heparan sulfate (22Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (489) Google Scholar). has been recently shown that the GAG biosynthesis of pgsA-745 be restored by recombinant C. elegans H.Y. Olson S.K. Brown J.R. Esko J.D. Horvitz H.R. J. Biol. Chem. 2003; 278: 11735-11738Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). we transfected pgsA-745 CHO cells with human XT-I and XT-II cDNA and analyzed the binding of FGF-2 to the cell cell XT-I or XT-II were and showed a of FGF-2 binding to heparan sulfate on the cell surface (Fig. XT-I- and clones were FGF-2 binding to the cell surface could be completely by the of heparin in wild-type and and cells identical to the the of Furthermore, and cells amounts and types of GAG chains (Fig. These that both in the biosynthesis of heparan sulfate and chondroitin sulfate chains and that are capable of a of either one xylosyltransferase or both activity of XT-II for peptide and protein and GAG of transfected CHO enzyme activity of XT-II expressed in CHO Xylosyltransferase activity in the cell culture supernatant of CHO cells was measured after using the assay with different acceptors for recombinant The acceptor in the assay was for acceptor with the of and are of chondroitin sulfate and heparan sulfate in XT-I- and XT-II-transfected pgsA-745 CHO cells were into and grown for and the GAG was analyzed in the cell XT-I- and XT-II-transfected pgsA-745 cells amounts of heparan sulfate and chondroitin sulfate The GAG in pgsA-745 cells transfected with the was than of the GAG present in xylosyltransferase activity of a soluble form of human XT-II expressed in the P. supernatant of P. and the cells P. was harvested after 24 h, and the xylosyltransferase activity was determined using the peptide biotin-NH-QEEEGSGGGQKK(5-fluorescein)-CONH2 as and are not Expression of human XT-I and human XT-II in pgsA-745 cells also in of xylosyltransferase activity in the cell culture as measured by the transfer of xylose to the peptide biotin-NH-QEEEGSGGGQKK(5-fluorescein)-CONH2 in the liquid mass was determined to be in and cells in 1). The observed activity in the XT-I- and XT-II-transfected cells is higher than that in cell culture supernatant of wild-type cells, where the activity was determined to be pgsA-745 cells and pgsA-745 cells virtually no xylosyltransferase activity In the cell were for for for and than cells for pgsA-745 and mutant in CHO cell in a These that both were 1). has been shown previously that XT-I is from the surface and is into the extracellular with large proteoglycans (4Goötting C. Sollberg S. Kuhn J. Weilke C. Huerkamp C. Brinkmann T. Krieg T. Kleesiek K. J. Investig. Dermatol. 1999; 112: 919-924Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, S. Prante C. Bahr C. Kuhn J. Kleesiek K. Goötting C. J. Biol. Chem. 2006; 281: 14224-14231Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). present that XT-II is also into the culture a in proteoglycan has been the biological of this to be In addition, expression of soluble XT-II the in the P. pastoris in a high activity in the culture supernatant In the cells, no activity could be that XT-II has xylosyltransferase analysis of the human XT-II expressed in cells revealed that XT-II catalyzes the of similar acceptor peptides as human XT-I but with different acceptor The peptide biotin-NH-QEEEGSGGGQKK(5-fluorescein)-CONH2 (19Kuhn J. Prante C. Schoön S. Goötting C. Kleesiek K. Clin. Chem. 2006; 52: 2243-2249Crossref PubMed Scopus (27) Google Scholar) is the for (Km 5.2 μm). The for recombinant bikunin was determined to be The which is homologous to the 24 amino acids of FGF-2 and which has been shown to be a XT-I acceptor J. Schnoölzer M. Schoön S. Muöller S. Prante C. Goötting C. Kleesiek K. Biochem. Biophys. Res. Commun. 2005; PubMed Scopus Google Scholar), is also by XT-II (Km μm). Furthermore, is a acceptor for XT-II (Km μm). The activity of XT-II for different peptide and protein is shown in the reaction the peptide was by either recombinant XT-I or recombinant XT-II and to the of the bound xylose with and In all the xylose could be from the peptide by and but not by with These that the xylose was to the acid serine of the peptide a and that both XT-I and XT-II are core protein the XT-II gene was already described by in (9Goötting C. Kuhn J. Zahn R. Brinkmann T. Kleesiek K. J. Mol. Biol. 2000; 304: 517-528Crossref PubMed Scopus (204) Google Scholar), the XT-II enzymatic activity remained unknown for a only an of was which a transfer of xylose to a peptide A. D. J. K. I.B. J. 2006; PubMed Scopus Google Scholar). we present for the first evidence that XT-II is a xylosyltransferase that is in the biosynthesis of heparan sulfate and chondroitin sulfate proteoglycans. that transfection of either XT-I or XT-II cDNA to a complete of the of pgsA-745 CHO Furthermore, initial that both similar acceptors for but with different acceptor in the expression pattern of XT-I and XT-II were for XT-II is the only xylosyltransferase present in liver tissues. that XT-I and XT-II are highly similar with expression and that both are in the biosynthesis of the tetrasaccharide linkage in proteoglycans. The presence of two xylosyltransferase genes in all higher organisms to the importance of for In addition, no human disease is that is by a of of the xylosyltransferases. However, mutations in the XT-II gene have been shown to be genetic risk factors for diseases that are with an altered proteoglycan (14Schoön S. Schulz V. Prante C. Hendig D. Szliska C. Kuhn J. Kleesiek K. Goötting C. J. Med. Genet. 2006; 43: 745-749Crossref PubMed Scopus (41) Google Scholar, S. Huep G. Prante C. Muöller S. Christ R. Hagena F.W. Kuhn J. Kleesiek K. Goötting C. Osteoarthritis Cartilage. 2006; 14: 442-448Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, S. Prante C. Muöller S. Schoöttler M. Tarnow L. Kuhn J. Kleesiek K. Goötting C. Kidney Int. 2005; 68: 1483-1490Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). For we could that the XT-II gene to a more than risk of a disease and multiple in pseudoxanthoma elasticum (14Schoön S. Schulz V. Prante C. Hendig D. Szliska C. Kuhn J. Kleesiek K. Goötting C. J. Med. Genet. 2006; 43: 745-749Crossref PubMed Scopus (41) Google Scholar). The Adam and for for with the and Hendig for with the RT-PCR and for with