Determination of phosphorylated and sulfated linkage-region oligosaccharides in chondroitin/dermatan and heparan sulfate proteoglycans by high performance liquid chromatography

The linkage-region oligosaccharides of chondroitin sulfate and dermatan sul fate, produced by the action of various chondroitinases, as well as of hepa ran sulfate, produced by the combined action of heparin-lyases I, II, and I II, have been separated and characterized according to their size, number o f sulfate residues, and the presence of phosphorylated xylose by HPLC. Glyc osaminoglycans and/or proteoglycans were treated by tritiated borohydride a nd the [H-3]-labeled xylitole-containing glycan chains were degraded by the various chondro/dermatolyases (chondroitinases ABC, AC and B) and/or hepar in-lyases. The produced linkage-region Delta-oligosaccharides have been com pletely separated by ion-pair reversed phase HPLC, using tetrabutylammonium as ion-pairing reagent, and detected by a radiochemical detector. Application of the method to chondroitin sulfate and dermatan sulfate revea led that the first uronic acid after the -Gal-Gal-Xyl linkage trisaccharide is always glucuronic acid and the next galactosamine residue is sulfated. In porcine skin dermatan sulfate studied, a three glucuronic acid-containin g cluster following the linkage-region oligosaccharide was found. None of c hondroitin sulfate and dermatan sulfate studied contains phosphorylated xyl ose. This phosphorylation was, however, a dominating feature in xylose link age-region of the glycans derived from rat chondrosarcoma proteoglycans. The linkage region oligosaccharide fragment of heparan sulfate had the same structure and identical retention time with that obtained from dermatan su lfate with chondroitinase AC.