Role of conserved amino acid residues in the complementarity determining regions on hapten-antibody interaction of anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies

Abstract

Monoclonal antibodies (mAbs) specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) were prepared at various times after immunization and the amino acid sequences of VH and Vλ1 in these mAbs were deduced from cDNA nucleotide sequences. Replacements due to somatic mutation were not found in day 7 mAbs but were found in those of days 14, 84 and 294. The affinity of day 7 mAbs to NP-glycine(NP-Gly) was in the order of 10 4 M −1 and it increased about 8000-fold with time after immunization. The extrinsic circular dichroism (CD) spectrum of the NP-ϵ-aminocaproic acid (NP-Cap)/Ab complex was unique for each mAb, although the spectra were grouped into two types, which tended to shift from one type to another with time, suggesting a variation in the micro-environments around NP-Cap in the combining sites. All these data indicate that the structure of the combining site was altered by somatic mutation; however, the fine-specificity measured by cross-reactivity with hapten analogues did not change significantly with time. We examined the amino acid residues in CDRs responsible for recognition of NP-haptens by comparing the amino acid sequences of anti-NP mAbs. Analyses revealed the presence of several conserved amino acid residues in CDRs of VH and Vλ1, such as Tyr-32H, and Tyr-60H, in addition to a core segment involving Arg-50H. It was predicted that these conserved residues determine Ab-specificity and that the amino acid residues introduced by somatic mutation may act by refining the orientation of specificity determining residues and resulting in an increase in affinity.