IDENTITY OF DERMATAN AND CHONDROITIN SEQUENCES IN DERMATAN SULFATE CHAINS DETERMINED BY USING FRAGMENTATION WITH CHONDROITINASES AND ION-PAIR HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The structure of a dermatan sulfate fraction from porcine skin has been analyzed by using various chondro/dermatolyases (chondoitinases ABC, AC, and B) and a highly sensitive HPLC method. Chondroitinase AC degrades the chondroitin sulfate portions of the chain and releases the dermatan sulfate fragments as Delta-saccharides, whereas chondroitinase B releases the chondroitin sulfate portions as Delta-saccharides by degrading the dermatan sulfate sections of the chain. The variously sized, sulfated Delta-saccharides have been completely separated by ion-pair reversed-phase HPLC and the type of repeating disaccharide units in each fragment was determined by digestion with chondroitinase ABC and anion-suppression HPLC or reversed-polarity HPCE. The combined use of the various chondro/dermatolyases and the HPLC/HPCE analyses has enabled us to elucidate the disaccharide sequence of portions of dermatan sulfate and quantitate the linkage region binding the galactosaminoglycans to the protein backbone. The findings suggest that the dermatan sulfate chain can be defined as a copolymer that contains short chondroitin sulfate and long dermatan sulfate regions which are distributed periodically and nonrandomly, Two glucuronic acid-containing repeats were identified-one repeat, located next to the -Gal-Gal-Xyl linkage trisaccharide, contains three glucuronic acid residues, and the other repeat contains two monosulfated disaccharides. A third non-random sequence consists of two chondroitin disaccharides separated by one non-sulfated dermatan-type disaccharide. Each of these repeats is followed by iduronic acid clusters composed of sequences with more than four sulfated disaccharides. Nonsulfated iduronic acid-containing disaccharides were identified in the nonreducing terminal in a cluster of three disaccharides, followed by a sulfated glucuronic acid-containing disaccharide. By the proposed method, the linkage region that contains fragments produced by each one of the three chondroitinases can be easily determined and the presence of phosphorylated xylose in the linkage trisaccharide is also readily identified. The dermatan sulfate fraction from pig skin that was studied contains no phosphorylated xylose, but a significant proportion is present in rat chondrosarcoma proteoglycans. (C) 1995 Academic Press, Inc.