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 bee n analyzed by using various chondro/dermatolyases (chondoitinases ABC, AC, and B) and a highly sensitive HPLC method. Chondroitinase AC degr ades the chondroitin sulfate portions of the chain and releases the de rmatan sulfate fragments as Delta-saccharides, whereas chondroitinase B releases the chondroitin sulfate portions as Delta-saccharides by de grading the dermatan sulfate sections of the chain. The variously size d, sulfated Delta-saccharides have been completely separated by ion-pa ir 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 enab led us to elucidate the disaccharide sequence of portions of dermatan sulfate and quantitate the linkage region binding the galactosaminogly cans to the protein backbone. The findings suggest that the dermatan s ulfate chain can be defined as a copolymer that contains short chondro itin sulfate and long dermatan sulfate regions which are distributed p eriodically and nonrandomly, Two glucuronic acid-containing repeats we re identified-one repeat, located next to the -Gal-Gal-Xyl linkage tri saccharide, contains three glucuronic acid residues, and the other rep eat contains two monosulfated disaccharides. A third non-random sequen ce consists of two chondroitin disaccharides separated by one non-sulf ated dermatan-type disaccharide. Each of these repeats is followed by iduronic acid clusters composed of sequences with more than four sulfa ted disaccharides. Nonsulfated iduronic acid-containing disaccharides were identified in the nonreducing terminal in a cluster of three disa ccharides, followed by a sulfated glucuronic acid-containing disacchar ide. By the proposed method, the linkage region that contains fragment s produced by each one of the three chondroitinases can be easily dete rmined and the presence of phosphorylated xylose in the linkage trisac charide is also readily identified. The dermatan sulfate fraction from pig skin that was studied contains no phosphorylated xylose, but a si gnificant proportion is present in rat chondrosarcoma proteoglycans. ( C) 1995 Academic Press, Inc.