Interaction of heparin with rat mast cell protease 1.
Heparin is a sulfated glycosaminoglycan, synthesized by connective tissue-type mast cells. Rat mast cell protease 1 (RMCP-1), a chymotrypsin-like serine protease expressed specifically by connective tissue-type mast cells, is recovered in a macromolecular complex with heparin proteoglycan. The heparin.RMCP-1 complexes are stored in the secretory granules of the cells and are released following mast cell activation. We showed previously that dissociation of RMCP-1 from heparin resulted in loss of protease activity, as measured by its ability to inactivate thrombin. In the present report the binding of heparin to RMCP-1 was characterized. Affinity chromatography on heparin-Sepharose showed that RMCP-1 displayed high affinity for heparin, with approximately 1.2 M NaCl being required for elution of RMCP-1 from the affinity matrix. The structural requirements for the binding of heparin to RMCP-1 were investigated. Heparan sulfate, chondroitin sulfate, and dermatan sulfate, three glycosaminoglycans structurally related to heparin, were > or = 80-fold less effective in binding to RMCP-1 than heparin. The 2-O-sulfate, 6-O-sulfate, and N-sulfate groups in heparin were all shown to contribute in the binding. The minimal heparin sequence required for binding to RMCP-1 was found in a 14-saccharide fraction. 14-Saccharide species, obtained after separation by anion exchange chromatography, showed continuously increased binding with increasing anionic charge densities. The 16-18-saccharides were the smallest heparin oligosaccharides capable of accelerating the inactivation of thrombin by RMCP-1.
Pejler
G
G
Department of Veterinary Medical Chemistry, Biomedical Center, Uppsala, Sweden.
Maccarana
M
M
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D053818:Chymases; D004187:Disaccharides / Q000737:chemistry / Q000302:isolation & purification; D006025:Glycosaminoglycans / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007413:Intestinal Mucosa; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D013917:Thrombin / Q000378:metabolism
D004187:Disaccharides; D006025:Glycosaminoglycans; D009844:Oligosaccharides; D006493:Heparin; D012697:Serine Endopeptidases; D053818:Chymases; C509231:Mcpt1 protein, rat; D013917:Thrombin