Effect of heavy chain signal peptide mutations and NH sub(2)-terminal chain length on binding of anti-digoxin antibodies

In certain instances, antibody variable region mutations outside of the antigen-combining site influence antigen binding. We reported previously that a heavy chain mutation (Ser-94 arrow right Arg) decreased binding of the anti-digoxin antibody 40-150, whereas an additional signal peptide mutation at the -2 position (Gln arrow right Pro) causing NH sub(2)-terminal 2-residue truncation partially restored binding. To assess the combined effects on binding of two seemingly distant mutations, we constructed signal peptide mutations and NH sub(2)-terminal deletions in the presence of Ser-94 and Arg-94. Introduction of Pro at the signal peptide -3 position in 40-150 resulted in cleavage at alternative sites, with varying effects on affinity. Introduction of Pro at -2 into the anti-digoxin antibody 26-10 resulted, unexpectedly, in expression of heavy chains with 3 extra NH sub(2)-terminal residues, causing an approximately 100-fold reduction in affinity. Thus, both extensions and deletions of the heavy chain amino terminus can enhance or reduce antigen binding, depending on the structural context of specific antigen combining sites.