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Functional properties of membrane cofactor protein of complement.

ABSTRACT: Membrane cofactor protein (MCP or gp45-70) of the complement system is a cofactor for factor I-mediated cleavage of fluid-phase C3b and C3b-like C3, which opens the thioester bond. In the present study the activity of MCP was further characterized. Unexpectedly, in the absence of factor I, MCP stabilized the alternative- and, to a lesser extent, the classical-pathway cell-bound C3 convertases and thereby enhanced C3b deposition. Soluble MCP, if added exogenously, hardly functioned as cofactor for the cleavage of erythrocyte-bound C3b to iC3b; i.e. its activity, compared with the cofactor activity of factor H, was inefficient, since less than 10% of the bound C3b was MCP-sensitive. Further, exogenously added soluble MCP was also a weak cofactor for the cleavage of C3b bound to zymosan. Likewise, factor I, in the presence of cells bearing MCP, cleaved fluid-phase C3b inefficiently. These results imply that MCP has very little extrinsic cofactor activity for factor I. In contrast, exogenously added MCP and factor I mediated efficient cleavage of erythrocyte-bound C3b if the concentration of Nonidet P40 was sufficient to solubilize the cells. Interestingly, soluble MCP and factor I degraded C3b attached to certain solubilized acceptor membrane molecules more readily than others. The cleavage reaction of fluid-phase and cell-bound C3b by soluble MCP and factor I produced iC3b, but no C3c and C3dg. These and prior data indicate that soluble MCP has potent cofactor activity for fluid-phase C3b or C3b bound to solubilized molecules, but acts inefficiently towards C3b on other cells. This functional profile is unique for a C3b/C4b binding protein and, taken together with its wide tissue distribution, suggests an important role for MCP in the regulation of the complement system.

SUBMITTER: Seya T

PROVIDER: S-EPMC1133618 | biostudies-other | 1989 Dec

REPOSITORIES: biostudies-other

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Project description:Human granulocytes (polymorphonuclear leucocytes, PMN) possess a membrane cofactor protein (MCP, CD46), which is structurally and functionally distinct from the MCPs of other cell types: it shows a single broad band of 56-80 kDa (without the doublet pattern characteristic of MCP) on SDS/PAGE and has less affinity for complement component C3b. We purified PMN MCP using monoclonal antibodies in order to study the molecular differences between it and other MCPs. Several forms of PMN MCP with size heterogeneity were noted on SDS/PAGE and by immunoblotting. O-Glycanase treatment decreased this heterogeneity, yielding a fast-migrating component identical in position on SDS/PAGE to the O-glycanase-treated MCP of other cells. The cell-specific variation of MCP, therefore, arises from post-translational glycosylation and not from a difference in primary structure. The Factor I cofactor activity of PMN MCP was more efficient in cleaving the methylamine-treated complement components C4/C3 than was MCP from other cells, which shared a similar potency of cofactor activity on a weight basis. Two types of small-form PMN MCP were identified during purification. These were 42 kDa and 30 kDa in size; the former was recognized by M177 (a monoclonal antibody against the active site marker), possessed N-linked sugars [located on the short consensus repeats (SCRs)] but not O-linked ones (on the Ser/Thr-rich region), and retained cofactor activity for C3b/C4b cleavage, similar in potency to that of other MCPs. The functionally active soluble form of MCP was observed specifically in PMN. Protease inhibitors did not inhibit liberation of the fragments, although the generated fragments became susceptible to serine proteases. The findings show that the SCRs are the functional domain of MCP and that the MCP proteolysis found only in PMN may modulate the properties of PMN MCP. In conclusion, the structural features of PMN MCP largely reflect a variability in the O-linked sugars, and the decreased affinity for C3b may be in part attributable to proteolysis.

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Functional properties of membrane cofactor protein of complement.

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