Silk—A new substrate for UDP-d-xylose: Proteoglycan core protein β-d-xylosyltransferase

UDP-D-Xylose : Proteoglycan Core Protein β-D-Xylosyltransferase: A New Marker of Cartilage Destruction in Chronic Joint Diseases

“…It should be emphasized that the presence of a Ser-Gly pair is not in itself sufficient to convey acceptor activity to a protein, since Collagen which contains several such groups is not an acceptor (27). The K m values which have been reported for silk (182 mg/1) (13) and protein core of Sw/YA-degraded proteoglycan (143-240 mg/1) (13,15) are similar, when expressed on a weight basis. Since precise Information regarding the relative proportions of reactive and unreactive serine residues is not available for either Substrate, the more appropriate comparison based on the molar concentrations of reactive serine residues cannot be made at the present time.…”

“…The only constant feature of the Substrate structure discovered so far is the presence of a glycine residue at the C-terminal side of the xylose-accepting serine residue (26). Thus, the search for appropriate Substrates of the Xylosyltransferase was focused on proteins containing the Ser-Gly pair and it led to silk from Bombyx mori, which contains a large number of Ser-Gly sequences in the repeating hexapeptide Ser-Gly-Ala-Gly-Ala-Gly (13). It should be emphasized that the presence of a Ser-Gly pair is not in itself sufficient to convey acceptor activity to a protein, since Collagen which contains several such groups is not an acceptor (27).…”

“…Silk from Bombyx mori (l g) was dissolved in 10 ml of 600 g/l lithium thiocyanate (13,14) with stirring and gentle heating. After dilution with deionized water to about 80 ml, the solution was filtered, dialysed for 48 h against deionized water until free of thiocyanale, and equilibrated with tris buffer (0.1 mol/1, pH 7.0).…”

Decorin Core Protein Secretion Is Regulated by N-Linked Oligosaccharide and Glycosaminoglycan Additions

“…We investigated this possibility by establishing an assay of XT activity, utilizing XT present in crude cell extracts of HT1080 cells. Other investigators have utilized a variety of putative substrates to measure XT activity, including chemically deglycosylated core protein (45)(46)(47), synthetic peptides (25,48,49), silk (26,50,51), and recombinant bikunin (52). Our result showed that the recombinant decorin core protein isolated from mammalian cells (either HT1080 or the pgsA-745 cells) under native conditions was a significantly better substrate for XT than decorin produced by bacteria or a simple peptide spanning the GAG attachment domain.…”

“…Determination of XT Activity-XT activity assays were performed following a protocol previously described with some modifications (25,26). In brief, HT1080 and pgsA-745 cells were cultured in medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO 2 at 37°C until confluent.…”