application). Systemic Lupus Erythematosus (SLE) is a chronic multisystem disease that is associated with a deficiency of the C4A but not the C4B isotype of the fourth component of human complement. Pathogenesis is attributed to the inadequate clearance and processing of immune complexes. Normally, processing of immune complexes in vivo requires the activation and covalent binding of C4 (C4b) and C3 (C3b) to the immune complexes thus their aggregation and precipitation. Complexes then bind to C4b/C3b receptors (CR1) found on erythrocytes and are removed from the circulation to the mononuclear phagocytic system for normal disposal. Studies have shown that C4A-coated complexes bind better to CR1 and are less likely to precipitate than C4B-coated complexes. The overall hypothesis to be tested is that structural differences exist between the C4 isotypes that differentially influence immune precipitation and binding to CR1. The proposed research will examine the mechanism(s) responsible for these differences by first characterizing the substrate binding site(s) of C4A and C4B to immunoglobulin isotypes by reacting the cleaved thioester of bound C4 with 14C-iodoacetamide, followed by proteolytic digestion, reverse-phase HPLC and analysis of the relevant peptides by amino acid sequencing. This will determine if C4A and C4B have different possibly overlapping binding sites on immunoglobulin thus influencing immune precipitation. Second, structural studies will be done using near and far UV circular dichroism, 8-anilino-1-naphthalene sulfonate (ANS) fluorescence, and site-specific monoclonal antibodies to probe for differences in C4A and C4B that predictably would affect their interaction with CR1. The results obtained from this study may explain, in part, the genetic association between C4A deficiency and SLE and provide insight into the disease process.
| Reilly, B D (1999) Analysis of human C4A and C4B binding to an immune complex in serum. Clin Exp Immunol 117:12-8 |
