Analysis of heparan-sulphate chains and oligosaccharides from proliferating and quiescent fibroblasts. A proposed model for endoheparanase activity.
Human skin fibroblasts in different growth states were incubated with [3H]glucosamine and/or Na(2)35SO4 and extracted with Triton X-100 for various periods of time. Free heparan-sulphate oligosaccharides and protein-bound heparan-sulphate chains were separated by chromatography on octyl-Sepharose and analyzed. A pool of endogenously produced oligosaccharides, present in the cultured cells and isolated after brief extraction, contained fragments of uniform size (approximately 7-10 kDa corresponding to approximately 14-20 disaccharides). Analysis by heparinase 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 residues at the periphery of each molecule. These results indicated that endoheparanase cleavage points were located close to linkages between N-sulphated glucosamine and sulphated iduronic acid, generating fragments that comprise a major portion of the unmodified segments and a minor 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 proteinase inhibitors resulted in further degradation. There was an increase in the amount of heparan-sulphate oligosaccharides and a concomitant decrease in the amount of protein-bound heparan-sulphate chains present in the same extract. The heparan-sulphate oligosaccharides obtained after prolonged extraction were more heterogeneous in size comprising, in addition to the major species of approximately 7-10 kDa, intermediate and larger fragments of approximately 17 kDa and 30-40 kDa. This observation suggests that endoheparanase acted at periodically appearing, specific regions in the intact heparan-sulphate chain. Furthermore, the enzyme and substrate should remain closely associated during cold Triton X-100 extraction. To determine if the endogenously produced heparan-sulphate oligosaccharides were derived from a particular heparan-sulphate species degraded during the growth phase, proteoglycan-derived heparan-sulphate chains obtained from proliferating or quiescent fibroblasts were also examined. These chains showed similar oligosaccharide maps, except for a small increase in the amount of glucuronic acid as cell growth was arrested. Hence, an endoheparanase with restricted specificity may generate slightly different oligosaccharides in the various growth states.
Department of Physiological Chemistry, University of Lund, Sweden.
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Schmidtchen
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Fransson
D002455:Cell Division; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology; D005966:Glucuronidase; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006497:Heparan Sulfate / Q000032:analysis*; D006801:Humans; D006868:Hydrolysis; D009844:Oligosaccharides / Q000032:analysis*; D012867:Skin / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D000099175:Heparanase
D005966:Glucuronidase; D006026:Glycoside Hydrolases; D006497:Heparan Sulfate; D009844:Oligosaccharides; D000099175:Heparanase