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Biosynthesis of dermatan sulphate proteoglycans. The effect of beta-D-xyloside addition on the polymer-modification process in fibroblast cultures.

ABSTRACT: Incubation of cultured fibroblasts with p-nitrophenyl beta-D-xyloside resulted in a concentration-dependent increase in galactosaminoglycan synthesis. At low concentration of added xyloside large and small radiolabelled proteoglycans and xyloside-bound polysaccharides were recovered from the medium, whereas at high concentrations only xyloside-bound polysaccharides were found. In the cell layer proteoglycans and xyloside-bound polysaccharides were found at all concentrations tested. Only galactosaminoglycan chains were polymerized on the xyloside primer. At low concentrations of added xyloside the structure of the galactosaminoglycans formed on the xyloside was similar to that of the small dermatan sulphate proteoglycan, i.e. mainly composed of L-iduronic acid-containing 4-sulphated disaccharides. With increasing concentration of added xyloside the co-polymeric structure of the small dermatan sulphate proteoglycan and the xyloside-bound polysaccharide was changed to contain a larger proportion of D-glucuronosyl residues with only slight changes in the sulphation pattern. No structural change in the polysaccharide chains of the large glucuronic acid-rich proteoglycans occurred. At 1 mM-xyloside, where no proteoglycans were formed, the polysaccharide was shorter and composed mainly of D-glucuronosyl-containing disaccharides with a ratio of 4-sulphate to 6-sulphate substituents of 1:2. This is similar to the structure of the large glucuronic acid-rich proteoglycan synthesized by these cells. Thus the main difference induced by the xyloside treatment was changed polymer modification at high xyloside concentrations. The specific activities of the polymer-modifying enzymes, uronosyl C-5-epimerase and 4-sulphotransferase, were therefore measured and found to be decreased by 30-50% in fibroblasts treated with high xyloside concentrations. It is suggested that the protein core is of importance for regulating the activity of the polymer-modifying enzymes.

SUBMITTER: Coster L

PROVIDER: S-EPMC1151124 | biostudies-other | 1991 Jun

REPOSITORIES: biostudies-other

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Project description:1. Proteoglycans were extracted from sclera with 4 M-guanidine hydrochloride in the presence of proteinase inhibitors and purified by ion-exchange chromatography and density-gradient centrifugation. 2. The entire proteoglycan pool was characterized by compositional analyses and by specific chemical (periodate oxidation) and enzymic (chondroitinases) degradations. The glycan moieties of the molecules were exclusively galactosaminoglycans (dermatan sulphate-chondroitin sulphate co-polymers). In addition, the preparations contained small amounts of oligosaccharides. 3. The scleral proteodermatan sulphates were fractionated into one larger (I) and one smaller (II) component by gel chromatography. Proteoglycan I was eluted in a more excluded position on gel chromatography in 0.5 M-sodium acetate than in 4.0 M-guanidine hydrochloride. Reduced and alkylated proteoglycan I was eluted in the same position (in 0.5 M-sodium acetate) as was the starting material (in 4.0 M-guanidine hydrochloride). The elution position of proteoglycan II was the same in both solvents. Proteoglycans I and II had s0 20,w values of 2.8 x 10(-13) and 2.2 x 10(-13) s respectively in 6.0 M-guanidine hydrochloride. 4. The two proteoglycans differed with respect to the nature of the protein core and the co-polymeric structure of their side chains. Also proteoglycan I contained more side chains than did proteoglycan II. The dermatan sulphate side chains of proteoglycan I were D-glucuronic acid-rich (80%), whereas those of proteoglycan II contained equal amounts of D-glucuronic acid and L-iduronic acid. Furthermore, the co-polymeric features of the side chains of proteoglycans I and II were different. The protein core of proteoglycan I was of larger size than that of proteoglycan II. The latter had an apparent molecular weight of 46 000 (estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis), whereas the former was greater than 100 000. In addition, the amino-acid composition of the two core preparations was different. 5. As proteoglycan I altered its elution position on gel chromatography in 4 M-guanidine hydrochloride compared with 0.5 M-sodium acetate it is proposed that a change in conformation or a disaggregation took place. If the latter hypothesis is favoured, aggregation may be due to self-association or mediated by an extrinsic molecule, e.g. hyaluronic acid.

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Biosynthesis of dermatan sulphate proteoglycans. The effect of beta-D-xyloside addition on the polymer-modification process in fibroblast cultures.

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