ANALYSIS OF HEPARAN-SULFATE CHAINS AND OLIGOSACCHARIDES FROM PROLIFERATING AND QUIESCENT FIBROBLASTS - A PROPOSED MODEL FOR ENDOHEPARANASE ACTIVITY

Human skin fibroblasts in different growth states were incubated with [H-3]glucosamine and/or (Na2SO4)-S-35 and extracted with Triton X-100 for various periods of time. Free heparan-sulphate oligosaccharides an d protein-bound heparan-sulphate chains were separated by chromatograp hy on octyl-Sepharose and analyzed. A pool of endogenously produced ol igosaccharides, present in the cultured cells and isolated after brief extraction, contained fragments of uniform size (approximately 7-10 k Da corresponding to approximately 14-20 disaccharides). Analysis by he parinase I and heparinase III degradations followed by electrophoretic separation (oligosaccharide mapping) showed that the oligosaccharides were rich in glucuronic acid but had a few sulphated iduronic acid re sidues at the periphery of each molecule. These results indicated that endoheparanase cleavage points were located close to linkages betweee n N-sulphated glucosamine and sulphated iduronic acid, generating frag ments that comprise a major portion of the unmodified segments and a m inor portion of the highly modified segments. Prolonged extraction (24 -48 h) of cells with Triton X-100 at 4 degrees C in the presence of pr oteinase inhibitors resulted in further degradation. There was an incr ease in the amount of heparan-sulphate oligosaccharides and a concomit ant decrease in the amount of protein-bound heparan-sulphate chains pr esent in the same extract. The heparan-sulphate oligosaccharides obtai ned after prolonged extraction were more heterogeneous in size compris ing, in addition to the major species of approximately 7-10 kDa, inter mediate and larger fragments of approximately 17 kDa and 30-40 kDa. Th is observation suggests that endoheparanase acted at periodically appe aring, specific regions in the intact heparan-sulphate chain. Furtherm ore, the enzyme and substrate should remain closely associated during cold Triton X-100 extraction. To determine if the endogenously produce d heparan-sulphate oligosaccharides were derived from a particular hep aran-sulphate species degraded during the growth phase, proteoglycan-d erived heparan-sulphate chains obtained from proliferating or quiescen t fibroblasts were also examined. These chains showed similar oligosac charide maps, except for a small increase in the amount of glucuronic acid as cell growth was arrested. Hence, an endoheparanase with restri cted specificity may generate slightly different oligosaccharides in t he various growth states.