EFFECT OF HEAVY-CHAIN SIGNAL PEPTIDE MUTATIONS AND NH(2)-TERMINAL CHAIN-LENGTH ON BINDING OF ANTIDIGOXIN ANTIBODIES

In certain instances, antibody variable region mutations outside of th e antigen-combining site influence antigen binding. We reported previo usly that a heavy chain mutation (Ser-94 --> Arg) decreased binding of the anti-digoxin antibody 40-150, whereas an additional signal peptid e mutation at the -2 position (Gln --> Pro) causing NH2-terminal 2-res idue truncation partially restored binding. To assess the combined eff ects on binding of two seemingly distant mutations, we constructed sig nal peptide mutations and NH2-terminal deletions in the presence of Se r-94 and Arg-94. Deletions of one to three amino acids had little effe ct on binding for Ser-94 mutants, whereas 2-residue truncations produc ed directly or by signal peptide mutation increased affinity approxima tely 40-fold for Arg-94 mutants. These observations are consistent wit h the reported computer-generated model of antibody 40-150. Introducti on of Pro at the signal peptide -3 position in 40-150 resulted in clea vage at alternative sites, with varying effects on affinity. Introduct ion of Pro at -2 into the anti-digoxin antibody 26-10 resulted, unexpe ctedly, in expression of heavy chains with 3 extra NH2-terminal residu es,.causing an approximately 100-fold reduction in affinity. Thus, bot h extensions and deletions of the heavy chain amino terminus can enhan ce or reduce antigen binding, depending on the structural context of s pecific antigen combining sites.