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 (K(m) 5.2 microM). 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.
Highlights
Proteoglycans are a large group of polyanionic glycoproteins expressed in virtually every animal cell on the cell surface and in the extracellular matrix [1]
The carboxyl-terminal DXD motif, which is located at positions 651– 653 in human Xylosyltransferase II (XT-II) and which was found to be important for enzyme activity in xylosyltransferase I (XT-I) [11], is situated in the fourth conserved sequence block
The carboxyl-terminal 25 amino acids of the XT-II are highly conserved, which is in accord with previous findings, indicating that this region was necessary for catalytic activity or enzyme stability in Drosophila and C. elegans xylosyltransferases [13, 26]
Summary
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 [17, 18]. HPLC Analysis of Chondroitin Sulfate and Heparan Sulfate Disaccharides—The isolation of GAG chains was performed as described in the Supplemental Data. 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). 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 wildtype 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. The sequences and the procedures are listed in the Supplemental Data
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