Clearance of immune complexes from the circulation is facilitated by activation of the clinical complement pathway which results in the covalent binding of C4b and C3b to the complexes and their uptake by the C3b/C4b receptor (CRl,CD35) on erythrocytes. Erythrocytes transport the complexes to the liver and spleen, for removal. The strong association of deficiencies of the classical pathway components with immune complex disease, particularly systemic lupus erythematosus (SLE) indicates the importance of this pathway. In addition, a high percentage of patients with SLE have complete or partial deficiency of the C4A isotype of C4. It is postulated that C4A isotype is more effective C4B in the clearance of immune complexes, and that C4A deficiency is therefore a predisposing factor in SLE. C4A and C4B are encoded by two genes within the major histocompatibility complex. C4A selectively forms amide linkages and C4B ester linkages during complement activation. A sequence of six amino acids accounts for the differences between the C4A and C4B isotypes, and these isotype-specific residues also determine their covalent binding reactivities. Greater binding of C4A than C4B to immune complexes has been observed using purified complement proteins; however, in serum the extent of binding of C4A and C4B to immune complexes is similar. Following C4b and C3b fixation, immune complexes bind to erythrocytes through the complement receptor CRl. In addition to serving as a receptor for C3b and C4b, CRl is a cofactor for the cleavage of C3b and C4b by the serum enzyme factor 1. CR1-bound immune complexes may be released from erythrocytes by this cleavage, and the resulting complexes no longer bind to erythrocytes, but can interact with B lymphocytes. Preliminary evidence indicates that immune complexes containing C4A are more efficiently bound by CR1 and are subsequently less susceptible to release by factor I than immune complexes containing C4B. A recent report describes greater binding of C4A than C4B to soluble CR1. The proposed research will examine the effect of C4 isotype on binding of immune complexes to CR1 and release by factor I. Binding constants will be determined using human erythrocytes and radiolabeled dimers of C3b, C4b, and mixed C4b-C3b dimers prepared using purified C4A and C4B. The effect of C4 isotype on binding and release of soluble immune complexes treated with human serum will also be studied. Cleavage reactions required to release the dimers and immune complexes from the cells will be analyzed by SDS-PAGE and autoradiography using labeled C4 or C3. CR1 has three binding sites for C4b and C3b. Transfected cells expressing CR1 or fragments of CR1 lacking individual binding sites will be used to study the role of these sites in the binding and cleavage of C4b and C3b. Site-directed mutagenesis of C4B at the isotype specific residues will be used to determine the effect of individual amino acids on C4b conformation an interactions with CR1. The proposed studies will analyze the effect of C4 isotype on the molecular interaction required for the clearance of soluble immune complexes by complement and CR1, and address the reason for the association of C4A deficiency with SLE.
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