Abstract
Xylosyltransferase (XylT) catalyzes the initial enzymatic reaction in the glycosaminoglycan assembly pathway for proteoglycan biosynthesis. Its activity is thought to be rate-limiting. Two xylosyltransferases have been found using genomic analyses, and one of these, XylT1, has been shown to have xylosyltransferase activity. On the other hand, the less studied XylT2 in recombinant form lacks xylosyltransferase activity and has no known function. Wild-type Chinese hamster ovary cells express abundant Xylt2 mRNA levels and lack detectable Xylt1 mRNA levels. Analysis of a previously described Chinese hamster ovary cell xylosyltransferase mutant (psgA-745) shows that it harbors an Xylt2 nonsense mutation and fails to assemble glycosaminoglycans onto recombinant biglycan. Transfection of this cell line with a murine Xylt2 minigene results in the production of recombinant chondroitin sulfate-modified biglycan core protein and restoration of fibroblast growth factor binding to cell surface-associated heparan sulfate. Expression analyses on 10 different human transformed cell lines detect exclusive XYLT2 expression in two and co-expression of XYLT1 and XYLT2 in the others but at disparate ratios where XYLT2 expression is greater than XYLT1 in most cell lines. These results indicate that XylT2 has a significant role in proteoglycan biosynthesis and that cell type may control which family member is utilized.
Highlights
We discovered that the pgsA-745 cell line harbors an inactivating genetic defect in Xylt2 and that overexpression of Xylt2 in these cells complements their inability to add chondroitin sulfate to a glycosaminoglycan-free biglycan core protein and to bind basic fibroblast growth factor to surface associated heparan sulfate proteoglycans
The main finding of this study is that XylT2 has an important role in chondroitin and heparan sulfate proteoglycan biosynthesis as shown by expression and complementation analyses
We have demonstrated a correlation between expression of Xylt2 in wild-type CHO cells and chondroitin sulfate assembly on biglycan and heparan sulfate on cell surface proteoglycans and between loss of Xylt2 mRNA expression and absence of chondroitin sulfate modification and cell surface binding of fibroblast growth factor 2 (FGF-2) in the xylosyltransferase-deficient CHO cells
Summary
Northern Blot, RT-PCR, and Real-time RT-PCR—Poly(A) RNA was isolated as per the manufacturer’s instructions (Invitrogen MicroFast Track). Human cDNA probe template for XYLT1 (nucleotides 2455–3009 according to GenBank accession number NM_022166) was a fragment that spanned the last 555 bp of the FEBRUARY 23, 2007 VOLUME 282 NUMBER 8. Five overlapping genomic fragments spanning 13 kb in length were generated that included the sequence down to the provisional poly(A) addition site as identified by comparison with mouse cDNA sequence (according to GenBank accession number NM_145828). Production of Cell Lines—The coding portion of the Bgn cDNA, encoding mouse biglycan, was cloned into the expression vector pcDNA3.1 (Invitrogen) that contains a neomycinselectable marker. An Xylt minigene containing the coding portion of exon 1 spliced to the remaining portion of the locus spanning exon 2 to the translational stop codon in exon 11 was cloned into the expression vector pBudCE4.1 (Invitrogen) with the Zeocin cassette replaced with puromycinand neomycin-selectable cassettes to create the expression vector XYIISCI-44. After three washes in PBS with saponin, the slides were incubated with peroxidaseconjugated goat antimouse IgG/IgM (Jackson ImmunoResearch Laboratories) antibody for 1 h at room temperature followed by NovaRED (Vector Laboratories) incubation, counterstaining, dehydration, and coverslip mounting
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