The consequences of complement activation can be protective or pathological. The objectives of the studies proposed in this application are to elucidate further the molecular mechanisms by which human erythrocytes modulate the functional activity of the complement system, and to discern both the normal physiology of complement regulation and the pathophysiological sequela of regulatory dysfunction. Human erythrocytes have been the paradigm for investigation of membrane regulation of complement, and the affected erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) have provided important insights into the physiological relevance of membrane proteins which regulate complement. Interactions with discrete cell-surface glycoproteins may inhibit the activity of a membrane-associated complement component in the following two ways, (1) Directly, by disrupting, non-enzymatically, the functional integrity of the component. (2) Indirectly, by acting as a cofactor for endogenous plasma regulators which proteolytically degrade the component. Recent investigations have suggested that there exists a regulatory system which inhibits the assembly of the cytolytic membrane attack complex by inactivating membrane-associated complement C6. This regulatory system appears to involve both cellular and plasma constituents, and the most complement sensitive PNH erythrocytes may lack the membrane component of this laboratory system.
Specific Aim I : To isolate and characterize the putative C6 inactivator. Standard chromatographic procedures will be employed to purify the inactivator from plasma. Antibodies will be developed, and the biochemical properties of the inactivator will be determined subsequently.
Specific Aim II : To characterize the functional properties of the inhibitor. Serum will be immunochemically depleted of the C6 inactivator, and the hemolytic activity of the inactivator deficient serum will be compared with that of normal serum. Binding of the isolated inactivator to cell- bound C6 will be analyzed. To identify and characterize the membrane constituent which acts as the cofactor for the C6 inactivator. Erythrocytes will be radiolabeled and complement will be activated on the membrane surface. A reducible cross-linker will be introduced, and the binding complex involving C6 and the putative C6 inactivator cofactor will be immunoprecipitated with anti-C6. The immunoprecipitate will be analyzed subsequently by SDS-PAGE (under reducing conditions) and autoradiography. The inactivator will be isolated from solubilized erythrocyte membrane by affinity chromatography using immobilized C6, and antibodies will be produced. The presence of the inactivator cofactor in PNH erythrocytes will be determined by antibody binding studies, immunoprecipitation and immunoblotting.
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