EVIDENCE SHOWING THAT THE 1105-ISOTYPIC AND 1106-ISOTYPIC RESIDUES OFTHE FOURTH COMPONENT OF HUMAN-COMPLEMENT, C4A, ARE NOT INVOLVED IN AMIDE BOND FORMATION

Human C4A and C4B have different functions that may stem from their ab ility to bind hydroxyl or free amino groups on complement activating s urfaces. Previous studies suggest that C4B binds to hydroxyl or amino groups whereas C4A binds to free amino groups on acceptor molecules. C omparison of the derived amino acid sequences of C4A and C4B has shown that differences exist between them at positions 1101, 1102, 1105 and 1106. These residues appear to be involved in the binding specificity of C4B. Less is known about the corresponding residues of C4A. It has been suggested that the aspartic acid of C4A at position 1106 is invo lved in amide bond formation by serving as a catalytic residue for the reaction or by promoting an increased interaction with amino nucleoph ilic groups. To examine the functional role of residues 1101-1106, we studied the effects of the C4A site-specific antipeptide mAb, AII-I in assays dependent on the covalent binding properties of C4A; the C4 me diated inhibition of hemolysis and the C4 mediated inhibition of immun e precipitation. This study shows that mAb AII-1 has no effect on C4-m ediated hemolysis or its ability to inhibit the rate of immune precipi tate formation. The lack of interference by AII-I in these assays coul d not be explained by low affinity interaction between antibody and C4 A showing that mAb AII-1 does not affect the covalent binding activity of C4A. Furthermore, results from epitope mapping studies show that A II-1 binds to Leu(1105) and Asp(1106) suggesting that these residues a re not critical for amide bond formation by C4A.