SERGLYCIN-BINDING PROTEINS IN ACTIVATED MACROPHAGES AND PLATELETS

The major proteoglycan in macrophages and platelets is the chondroitin sulfate proteoglycan serglycin. To study the biological role of sergl ycin, its binding to secreted and cell-associated proteins from macrop hages and blood platelets was examined. Affinity chromatography with s erglycin-Sepharose and chondroitin sulfate-Sepharose was used to isola te proteoglycan-binding proteins from macrophages and platelets. Antib odies against human macrophage inflammatory protein-1 alpha (MIP-1 alp ha) precipitated a 14-kDa S-35, methionine-labeled protein among the c hondroitin sulfate binding proteins secreted from the macrophage-like U937 cells after stimulation. Two proteins from murine macrophage J774 cells with molecular masses of similar to 10 and 14 kDa were precipit ated by an antiserum against the murine MIP-1 alpha. Protein sequencin g of fragments obtained by trypsin digestion of a 14-kDa chondroitin s ulfate-binding protein from cell extracts of stimulated U937 cells rev ealed 100% homology with lysozyme, a bacteriolytic enzyme. Fragment of one other protein with approximate molecular mass of 8 kDa showed hig h homology with bone morphogenetic protein. Inhibition studies showed that chondroitin 6-sulfate inhibited the bacteriolytic activity of lys ozyme in a competitive manner more efficiently than heparin and chondr oitin 4-sulfate. Amino-terminal sequencing of two proteins from platel et extracts that bound to serglycin-Sepharose revealed that they corre sponded to multimeric forms of human platelet factor 4 (PF4). Chondroi tin sulfate-Sepharose was shown to be equally efficient in retaining P F4 from platelet extracts as serglycin-Sepharose, indicating that the glycosaminoglycan chains mediate the binding to PF4 in the intact prot eoglycan molecule. Competition experiments showed that serglycin was a s efficient as heparan sulfate in blocking the binding of [H-3]chondro itin sulfate to PF4, whereas heparin was one order of magnitude more e fficient. Affinity measurements using fluoresceinamine-labeled glycosa minoglycans showed that the affinity of heparin for PF4 is on the orde r of 30 nM, whereas chondroitin sulfate has an affinity of 260 nM. Bot h PF4, MIP-1 alpha, and lysozyme play important roles in different typ es of inflammatory reactions. The interaction with serglycin may indic ate that this proteoglycan is involved in the regulation of the inflam matory response.