The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement

Abstract

The variable (V) genes of antigen-selected antibodies are known to exhibit a higher frequency of amino acid replacement mutations in the sequences encoding the antigen-contacting complementarity-determining regions (CDRs) than in those encoding the 'structural' framework regions (FRs). Here, Bernard Chang and Paolo Casali analyse the impact of regional differences in the codon composition of human germline Ig VH and VL genes on regional differences in the frequency of replacement mutations in the gene products (i.e. the antigen-binding sites of antibody molecules). This analysis reveals that CDR and FR sequences can differ significantly in their inherent susceptibility to amino acid replacement given any single nucleotide change. Thus, the CDR sequences of all the Ig VH genes analysed comprise a higher frequency of codons susceptible to replacement mutations than would be expected for a random sequence. Conversely, the FR sequences comprise codons less susceptible to replacement mutations than expected. Random accumulation of nucleotide changes throughout the coding sequence of an Ig V-gene segment containing CDRs inherently more prone to replacement mutations than the respective FRs would inevitably yield a higher rate of amino acid replacements in the CDRs than in the FRs. This would provide a fertile structural substrate of hypervariability for antigen selection while still maintaining the structural integrity of the FRs.

Fig. 1

The human germline V_HH11 gene sequence was analysed using the computer algorithm that calculates the inherent susceptibility to amino acid replacement given any single nucleotide change. The complementarity-determining regions (CDRs) and framework regions (FRs) of the gene sequence displayed extremely different inherent R:S mutation ratios. The sequence of the whole CDR1 and clusters of codons from the FR2 and FR3 are depicted. Each circle above each nucleotide represents a single viable substitution that is possible at that base (substitutions that result in a stop codon are excluded). Closed and open circles represent single-base substitutions that result in an amino acid replacement (R mutation) and no replacement (S mutation), respectively. Listed below each codon are the germline-encoded amino acid (underlined), and all possible amino acid replacements resulting from single-base substitutions at that codon. For example, the first codon in the CDR1 of H11 is AGC, which encodes Ser and has a total of nine possible single-base substitutions for its three nucleotides. Eight of these substitutions result in an amino acid replacement, and one does not. The sequence of the CDR1 of the human V_HH11 gene is identical to that of the mouse V_H186.2 gene CDR1.

Fig. 2

Inherent R mutation susceptibility profiles of human germline V_H- and V_L-gene sequences. The complementarity-determining region (CDR) and framework region (FR) sequences of the most-commonly expressed human Ig V_H and V_L genes, as listed in Tables 1 and 2, were analysed using the computer algorithm that calculates the inherent susceptibility to amino acid replacement given any single nucleotide change. Inherent R mutation susceptibility profiles were created by plotting the inherent R:S mutation ratio (ratio of total possible single-base substitutions resulting in amino acid replacement to total possible single-base substitutions not resulting in amino acid replacement) for each of the Ig V regions versus the respective V region nucleotide residue numbers. (a) The inherent R mutation susceptibility profiles of the six human germline V_H-gene sequences displaying the highest Rf in CDR1 are depicted in the upper six panels (where Rf indicates replacement frequency, or proportion of total possible point mutations that result in amino acid replacement), (b) The inherent R mutation susceptibility profiles of the three human V_L-gene sequences (two V_κ and one V_λ displaying the highest Rf in CDR1 are depicted in the lower three panels. Numbers below each region represent the inherent R:S mutation ratio for that region.