Lack of binding of human C3, in its native state, to C3b receptors.

Abstract

Abstract To understand better the role of C3 in opsonization, we investigated the binding of purified C3 and C3b to C3b receptors. To ensure that only physiologically relevant binding would be detected, we assayed the ability of isolated proteins to inhibit formation of rosettes between EA(lgM)C4b3b and normal, viable human polymorphonuclear leukocytes. Freshly prepared C3, with specific hemolytic activity equal to that of serum, was capable of only 30% inhibition at 7.5 mg/ml, 5 times the concentration in serum. After cleavage to C3b, 50% inhibition was achieved at 12 µg/ml. These experiments were performed in the presence of the protease inhibitor, phenylmethyl sulfonyl fluoride (PMSF). When PMSF was omitted, the intact, native C3 became much more effective at blocking the receptor, giving 50% inhibition at 43 µg/ml, which suggested cleavage of the C3 by neutrophil proteases. We also investigated a preparation of C3 that had lost 60% of its hemolytic activity on storage. Although this material had intact protein chains, it was much more effective at blocking the receptor than was the fresh preparation, giving 50% inhibition at 1.4 mg/ml. The stored material was resolved into hemolytically active and inactive components by chromatography on QAE-Sephadex. The hemolytically active material had less affinity for the C3b receptor than did the unresolved mixture whereas the hemolytically inactive material had enhanced affinity. Chaotrope and ammonium-treated C3 preparations were also studied. These preparations also lost hemolytic activity without protein chain cleavage and had increased affinity for the receptor. Previous observations of binding of uncleaved C3 to C3b receptors may thus be due to the presence of C3b-like, hemolytically inactive molecules and/or cleavage of native molecules by cell-associated proteases. Our results suggest that native C3 does not bind to the C3b receptor, whereas C3b binds very effectively at 6 × 10−8 M. The lack of competition by fluid phase C3 in the interaction of particle-bound C3b with C3b receptors may partly explain the importance of C3b in opsonization even in the presence of antibody.