STRUCTURAL-ANALYSIS OF THE LINKAGE REGION OLIGOSACCHARIDES AND UNSATURATED DISACCHARIDES FROM CHONDROITIN SULFATE USING CARBOPAC PA1

Swarm rat chondrosarcoma cell cultures were metabolically labeled with [S-35]sulfate, [H-3]glucose, or [H-3]glucosamine. Chondroitin sulfate chains were isolated from purified aggrecan using alkaline borohydrid e treatment and Superose 6 chromatography. Various linkage region olig osaccharide alditols were derived from these chains using sequential c hondroitinase digestions (ABC lyase followed by ACII lyase). They were then further processed by mercuric acetate treatment, which removed t he 4,5-unsaturated uronosyl residue from the nonreducing end of the li nkage, and then beta-galactosidase digestion which liberated the 2 gal actose residues from the xylitol reducing terminus. Alkaline phosphata se digestions were performed to verify the presence of phosphate ester s. All linkage region structures were isolated and identified using a combination of Progel-TSK G2500 and CarboPac PA1 chromatography steps in conjunction with monosaccharide analyses. This study revealed that chondroitin sulfate chains from aggrecan synthesized by rat chondrosar coma cells in vitro have the following properties: 1) three out of eve ry four of their linkage regions carry a phosphate ester on xylose, 2) nearly three out of every five chains begin the repeating disaccharid e region with an unsulfated first disaccharide unit, 3) nearly twice a s many nonphosphorylated chains have a sulfated first disaccharide tha n their phosphorylated counterparts, and 4) the vast majority of these chains do not contain sulfated galactose in their linkage regions. Th is report also describes a borohydride reduction procedure to confer a lkali stability to the 3-substituted, unsaturated disaccharides derive d from chondroitinase digests of chondroitin sulfate. Furthermore, a C arboPac PA1 method is demonstrated that separates these reduced disacc harides with exceptional resolution.