Isolation and Partial Characterization of Dermatan Sulfate Proteoglycans from Human Post-Burn Scar Tissues

Abstract

Dermatan sulfate (DS) proteoglycans (PGs) were extracted from human post-burnscar (Sc) tissues with 4M guanidinium chloride and isolated from the extracts by DEAE-cellulose chromatography and by differential ethanol precipitation. The DS.PGs were further purified by Sepharose CL-6B column chromatography. The average molecular weight (M_r) of hypertrophic scar (HSc) tissue DS.PGs was 39,000 based on sedimentation equilibrium measurements. Alkaline borohydride treatment of DS.PGs liberated glycosaminoglycan (GAG) chains and the presence of xylitol indicated that these chains were attached to protein core by xylosyl residues. The average M_r of the DS.GAG chain from HSc and normal scar (NSc) samples were 23,500 and 20,000 respectively. After digestion of the HSc and NSc, DS.PGs with chondroitinase ABC in the presence of proteinase inhibitors, two peptide components with M_r values of 21,500 and 17,000 were detected by SDS-polyacrylamide gel electrophoresis using reducing conditions. Analysis of the protein core fractions derived from NSc and HSc DS.PGs by Sepharose CL-6B column chromatography showed the presence of a single NH₂-terminal amino acid (aspartic acid) and also that the fractions with different K_AV values had an identical NH₂-terminal sequence (A₁-A_S). The A₁-A₂₃ sequence of NSc DS.PG (major fraction, C) : NH₂Asp-Glu-Ala-O-Gly-Ile-Gly-Pro-Glu-Val-Pro-Asp-Asp-Arg-Asp-Phe-Glu-Pro-Ser-Leu-Gly-Pro-Val was the same as reported for a DS.PG isolated from human fetal membrane (HFM) tissue (Brennan et al., 1984).

ELISA inhibition assay using monoclonal antibodies raised in rabbit against the NH₂-terminal peptide (containing 15 amino acids) of human fetal membrane tissue were found to cross-react with HSc and NSc DS.PGs. Monoclonal antibodies to bovine skin DS.PGs protein core (Pearson et al., 1983) did not show any cross-reactivity with scar DS.PGs. These results show that the scar DS.PGs described here are different from normal bovine skin DS.PGs in the size and type of the protein core, and that in all the samples, the peptide components have the same NH₂-terminal amino acid sequence.