Effects of primer-concentration on uronosyl-epimerization and sulfation patterns in p-hydroxyphenyl-O-β-D-xylopyranoside-primed galactosaminoglycans produced by skin fibroblasts

Abstract

By supplying skin fibroblasts with different concentrations of the galactosaminoglycan chain-primer p-hydroxyphenyl-O-β-D-xylopyranoside we have produced and recovered glycan-chains that were subsequently radio-iodinated in the hydroxyphenyl group and subjected to sequence analysis by using graded enzymic treatment followed by a combination of gel chromatography and electrophoresis. Fragments extending from the tagged reducing end to the cleavage-point were identified and quantified. Degradation by chondroitin B lyase of chains primed at 0.1 or 0.5 mM xyloside gave profiles indicating a periodic and wave-like distribution of iduronate-containing repeats, with high incidence around positions 2, 5 and onwards, whereas in chains produced at 1.0 mM xyloside the incidence of iduronate was similar in positions 1–4 and then declined. Degradation by chondroitin AC lyase indicated a high incidence of glucuronate in or near the linkage-region. There was a relatively uniform degree of sulfation in chains primed at low xyloside concentration, whereas chains primed at 1.0 mM xyloside gave very heterogeneous charge-patterns in all segments of the chain, including the linkage-region, giving the impression that adequate sulfation, probably at C-4 and at the first opportunity, is necessary to obtain an ordered and periodic epimerization pattern. Abbreviations:CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; Gal, D-galactose, GaINAc, N-acetyl-D-galactosamine; GlcUA, D-glucuronic acid; GlyUA, glycuronic acid; ΔGlyUA, 4,5-unsaturated glycuronic acid; IdoUA, L-iduronic acid; Xyl-Phe-OH, p-hydroxyphenyl-O-β-D-xylopyranoside; Xyl, D-xylose