Covalent binding properties of the C4A and C4B isotypes of the fourth component of human complement on several C1-bearing cell surfaces.
In a previous study we demonstrated that the thioester-mediated transacylation of the human C4B isotype onto sheep erythrocytes (ES) was approximately fourfold more efficient than that of C4A. Moreover, although C4B formed predominantly ester linkages, C4A displayed a preference for amide bond formation. We therefore suggested that the relative functional activity observed for the two isotypes would be a combined reflection of their nucleophilic preference and the surface composition of the C1-bearing target. The present study tests this hypothesis. Chemical modification of amino groups on Es with ethylacetimidate produced a twofold decrease in the C1-dependent binding of C4A isotype, while having a negligible effect on C4B binding. Furthermore, with human erythrocytes and two human leukocyte cell lines, K562 and U937, the C4B to C4A deposition ratio decreased from greater than 4 with ES to between 1.5 and 2. Irrespective of the target, C4A and C4B maintained their preference for forming amide and ester bonds, respectively. Interestingly, SDS-PAGE profiles of radiolabeled C4A and C4B, which had been covalently deposited on the various cells, suggested a further degree of transacylation specificity, as the two isotypic alpha-chains sometimes bound to different membrane components. These differences were not easily accounted for by simple differences in the abundance of the preferred nucleophile for each isotype on a given surface constituent, nor were they due to the preferential binding of one isotype to the sensitizing antibody. We speculate that nascent C4B may contain a substrate binding site that facilitates productive attack on the thioester carbonyl by molecules containing the class of nucleophile preferred by each isotype.
D000215:Acylation; D000818:Animals; D000918:Antibody Specificity; D002236:Carbohydrate Conformation; D003172:Complement C1 / Q000378:metabolism*; D003181:Complement C4 / Q000302:isolation & purification / Q000378:metabolism*; D015934:Complement C4a; D015935:Complement C4b; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007096:Imidoesters; D007075:Immunoglobulin M / Q000502:physiology; D046911:Macromolecular Substances; D008970:Molecular Weight; D010447:Peptide Hydrolases; D010641:Phenotype; D012756:Sheep
D003172:Complement C1; D003181:Complement C4; D007096:Imidoesters; D007075:Immunoglobulin M; D046911:Macromolecular Substances; C014539:ethyl acetimidate; D015934:Complement C4a; D015935:Complement C4b; D010447:Peptide Hydrolases