Secretion, cleavage and binding of complement component C3 by the human monocytic cell line U937. |
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| Authors: | C M Maison C L Villiers M G Colomb |
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| Affiliation: | I.N.S.E.R.M. Unité 238, Laboratoire d'Immunochimie du D.R.F.-Grenoble, Alliée au C.N.R.S., France. |
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| Abstract: | Secretion of complement component C3 by U937 cells was studied. Preliminary evidence for a cell-associated proteolytic activity specific for C3 is given, as well as for a covalent-like binding of C3 fragments to the cell membranes. Secretion of C3, in the presence of 10 ng of phorbol 12-myristate 13-acetate/ml, is 120-140 ng/10(6) cells per 24 h on the third day after addition of the activator. As shown by SDS/polyacrylamide-gel electrophoresis, the intracellular pro-C3 (200 kDa) and the extracellular secreted C3 (alpha-chain 110 kDa and beta-chain 75 kDa) are identical with the forms of C3 previously characterized from human serum. Incubation of U937 cells in the presence of exogenous radiolabelled C3 shows that membrane-bound proteinase(s), not related to the classical-pathway or the alternative-pathway C3 convertases, is (are) able to cleave C3; this cleavage leads to the binding of the resulting C3 fragments to the cell membrane through reaction of membrane acceptors with the carbonyl group of C3 revealed after disruption of the intramolecular thioester bond. The proteolysis appears to be fairly specific to C3, as C4, which also possesses an intramolecular thioester bond, is not cleaved and does not bind to the cells. p-Nitrophenyl p'-guanidinobenzoate (1 mM) and di-isopropyl phosphorofluoridate (2 mM) are potent inhibitors of the proteolysis, whereas soya-bean trypsin inhibitor (1 mM), leupeptin (0.1 mg/ml) and 1,10-phenanthroline (1 mM) were ineffective. Immunological characterization of the cell-bound C3 fragments with monoclonal antibodies shows an evolution of the proteolysis of the fragments from iC3b to C3dg epitopes. Extraction of membrane-bound fragments by detergent, followed by SDS/polyacrylamide-gel electrophoresis, shows two fragments, of 43 kDa and 46 kDa, with C3dg-like characteristics. |
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