Immunoglobulin gene joints compensate for reduced on-rates imposed by somatic mutations in a V-H gene

Affinity maturation in the B lymphocyte response to a protein epitope appea rs to be largely due to a decrease in the off-rate constant of the antibodi es (Ab) resulting from somatic mutation without a significant increase in t he on-rate constant. Here, we show by site-directed mutagenesis of a germli ne encoded single-chain Fv that somatic mutations frequently selected in th e Ab response to mouse cytochrome c (CYT) at heavy (H) chain positions 31 a nd 58 actually cause a two and three-fold decrease, respectively, in the on -rate constant as well as a two and five-fold decrease, respectively, in th e off-rate constant and together cause nearly an eight-fold decrease in the off-rate. However, additional selection for a tyrosine residue at position 96 in the V-kappa-J(kappa) joint compensates for the decreased on-rate imp osed by the somatic mutations. This allows for an increase in the affinity of Ab during the secondary response. Certain sequences at the V-H-D-J(H) jo int were also shown to maintain a normal on-rate constant in the context of the common H chain mutations and, in addition, to reduce the off-rate, thu s increasing the affinity. The results support the idea that both faster on -rates and slower off-rates for B lymphocyte antigen-specific receptors are Favored during the maturation of the Ab response to mouse CYT. (C) 2000 El sevier Science Ltd. All rights reserved.