Biosynthesis of Chondroitin Sulfate Proteoglycan

Abstract

Abstract The chain-initiating reaction in chondroitin sulfate synthesis is catalyzed by a xylosyltransferase, which transfers xylose from UDP-xylose to a polypeptide acceptor. The enzyme has previously been detected in several cell-free tissue preparations, which also contain endogenous xylose acceptors. However, reliable quantitation of enzyme levels has not been possible, since such crude systems contain two unknown dependent variables, enzyme concentration and acceptor concentration. The purpose of the present work was (a) to develop exogenous acceptors which would permit accurate determination of enzyme levels and (b) at the same time gain some insight into the substrate specificity of the enzyme. Many small molecular weight compounds were tested as potential acceptors, including free serine, serine derivatives, and serine-containing peptides. One of the peptides had some acceptor activity, i.e. the tripeptide, Ser-Gly-Gly, although it had a rather high Km value of 20.0 mm. Considerably higher acceptor activity was observed with peptides derived from the chondroitin sulfate-protein linkage region by Smith degradation of a chondroitin sulfate peptidoglycan preparation, which had been isolated after trypsin digestion of the proteoglycan. Smith degradation of the entire proteoglycan molecule yielded the best acceptor obtained so far; this material had a Km of 0.064 mm, expressed in terms of concentration of serine residues. The intact proteoglycan was not an acceptor, nor were the products obtained by digestion with testicular hyaluronidase or bacterial chondroitinase, although about half of the serine residues in these three compounds are unsubstituted. Characterization of the [14C]xylosyl-labeled Smith-degraded proteoglycan showed that (a) xylose had been incorporated into the protein in an alkali-labile linkage from which it could be released as xylitol on treatment with alkaline borohydride, and that (b) exhaustive proteolysis yielded a compound with electrophoretic and chromatographic properties of xylosylserine. It is therefore concluded that xylose had been transferred to the serine hydroxyl groups which had been liberated by Smith degradation. Furthermore, the failure of the intact proteoglycan and the enzymatic digestion products to serve as acceptors suggests that glycosylation occurs specifically at certain serine sites of the proteoglycan molecule.