Chemical modification of carboxyl groups in human Fc γ fragment: Structural role and effect on the complement fixation

Abstract

The role played by acidic residues of immunoglobulin molecules in complement activation by the classical pathway has been studied using the Fc fragment obtained from human IgG by papain digestion. Delta- and Epsilon-carboxyl groups of aspartic and glutamic acid were selectively modified by coupling to glycine-ethyl-ester through the mediation of ethyl-dimethylaminopropyl-carbodiimide (EDC). ‡ ‡ EDC, 1-ethyl-3(3-dimethyl-amino-propyl)-carbodiimide; SDS sodium dodecyl sulfate; PBS, phosphate-buffered saline (pH 7.2); CFD; complement fixation diluent; Cl, first component of complement; PAGE, polyacrylamide gel electrophoresis; Fc-1, 5, 10, 30-min, Fc fragment obtained at different times of modification; WSC, water-soluble carbodiimide. A rapid loss of anticomplementary activity was observed accompanied by small conformational changes in the Fc fragments. As shown by circular dichroism data, a gradual decreasing in the band at 225 nm (corresponding to the interaction Cγ2-Cγ3) was detected, as well as an increase of about 15% in the Stokes radius. No loss of anticomplementary activity was observed in the first minute of the reaction. However, after 5 min, when 7 out of 26 carboxyl groups per Fc chain had been modified, anticomplementary activity was completely abolished. In addition, using an immune serum to native Fc, the modified fragment was found to gradually lose its antigenicity as a function of the reaction time. These results point to an important involvement of some acidic residues of the immunoglobulin molecule in the activation of the complement sequence through the classical pathway.