Membrane cofactor protein (MCP; CD46), a widely distributed regulator of complement activation, is a cofactor for the factor I-mediated degradation of C3b and C4b deposited on host cells. MCP possesses four extracellular, contiguous complement control protein modules (CCPs) important for this inhibitory activity. The goal of the present study was to delineate functional sites within these modules. We employed multiple approaches including mutagenesis, epitope mapping, and comparisons to primate MCP to make the following observations. First, functional sites were located to each of the four CCPs. Second, some residues were important for both C3b and C4b interactions while others were specific for one or the other. Third, while a reduction in ligand binding was invariably accompanied by a parallel reduction in cofactor activity (CA), other mutants lost or had reduced CA but retained ligand binding. Fourth, two C4b-regulatory domains overlapped measles virus interactive regions, indicating that the hemagglutinin docks to a site important for complement inhibition. Fifth, several MCP regulatory areas corresponded to functionally critical, homologous positions in other CCP-bearing C3b/C4b-binding proteins. Based on these data and the recently derived crystal structure of repeats one and two, computer modeling was employed to predict MCP structure and examine active sites.

Membrane cofactor protein (MCP) of human complement is an iC3/C3b-binding glycoprotein with a characteristic two-band (63 kDa and 55 kDa) pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using affinity chromatography, it has been found on human mononuclear cells and platelets. MCP has been purified and shown to be a cofactor for the I-mediated cleavage of C3b. A rabbit polyclonal antibody was produced to the purified protein and this reagent employed to analyze the distribution of MCP on human peripheral blood cells. Flow cytometric analysis indicated that MCP is unimodally present on all platelets, granulocytes, T helper lymphocytes, T suppressor/cytotoxic lymphocytes, B lymphocytes, natural killer cells and monocytes but not erythrocytes. The presence of MCP on granulocytes was unexpected. To evaluate this, MCP was isolated by immunoprecipitation and analyzed by SDS-PAGE followed by autoradiography. The Mr of granulocyte MCP was that of a single broad band in which the typical two-band pattern could not be distinguished. Alterations in the conditions of the affinity column procedure increased the efficiency of the isolation of monocyte MCP and led to the reproducible isolation of granulocyte MCP. These results indicate that MCP of granulocytes has both structural and functional differences compared to MCP of plateletes and mononuclear cells. The wide distribution of MCP among peripheral blood cells supports the concept that MCP is important in the protection of host cells from complement-mediated damage.

Immune adherence is the attachment of C-bearing immune complexes via the major activation fragment of the third component of C(C3b) to C3b binding membrane proteins. On primate E, the C3b-R, termed CR1, mediates immune adherence. In nonprimates, immune adherence involves platelets instead of E. However, these functional data have not been corroborated by the identification of the binding protein. In this work, we have identified a C3b/iC3 binding protein of rabbit platelets and characterized it as a single chain structure with a Mr of 150 kDa (nonreducing) or 175 kDa (reducing). This protein binds to rabbit iC3 or C3b but not C3d. This specificity of binding and the ability to rebind to a second column of iC3- or C3b-thiol-Sepharose are comparable to human CR1. Also, a molecule with the identical Mr as well as other structural and binding characteristics is present on rabbit PBMC. No such protein was isolated from rabbit E. Our data strongly suggest that the C3b/iC3 binding protein of rabbit platelets is the homologue of human CR1. If so, this represents an interesting evolutionary switch in the tissue specific expression of the immune adherence R from platelets in the nonprimate to E in the primate.