Introduction
Although DNA mutations are a cornerstone of species evolution and adaptation, somatic mutagenesis is generally suppressed in eukaryotes. An important exception is the somatic hypermutation (SHM) of antibody variable (V) region genes, which is initiated by activation-induced cytidine deaminase (AID) and provides the structural basis of affinity maturation during physiological Ab responses. However, a byproduct of SHM is the generation of B cells with autoreactive receptors. Although, autoreactive B cells are normally eliminated by immune self-tolerance mechanisms, they sometimes escape censorship to participate in systemic autoimmune diseases such as lupus erythematosus (SLE). In particular, Abs directed against nuclear antigens (ANA) are a hallmark of SLE.
In prior studies involving a spontaneous mouse model of SLE, we have shown that many ANA arise by SHM of non-autoreactive B cells and that this conversion was strongly correlated with SHM of complementarity-determining regions (CDR) AGY Ser codons to Arg codons (1, 2). Arg residues are known to contribute substantially and often decisively to the binding energy between ANA and their nuclear targets (1, 3–11). In agreement with this, D regions that are enriched with Arg residues have a profound effect in B cell development and can induce spontaneous autoantibody production in mice (12). Moreover, AGY codons are unique in their potential to mutate to an Arg codon by any one of three different single-base changes, and the AGC trinucleotide is an intrinsically preferred target of SHM (13–16).
Curiously, our analyses of the germline repertoire of IgV-region genes revealed that these seemingly dangerous AGY Ser codons are unusually abundant in sequences specifying CDRs, a phenomenon that is conserved in human and mouse repertoires (1, 2). As such, VH, Vκ, and Vλ genes appear poised to mutate in a manner that would frequently generate antinuclear activity in the specified Ab product. Moreover, AGY Ser codons are more frequent than TCN Ser codons in germline IgV-region CDRs, a bias that does not apply to αβTCRV-region genes, which favor TCN over AGY codons (3, 17, 18). Because AGY, but not TCN, is an intrinsically preferred target of SHM, it was speculated that this AGY bias evolved to enhance targeting of SHM to antibody CDRs (17).
In this study, we asked whether the AGY serine codon bias and abundance in CDRs were highly conserved from an evolutionary perspective, and if so, whether this might be explained by a selection pressure to enhance overall CDR mutability. We found these features to be conserved in the most primitive vertebrates with an acquired immune system, but not solely because they enhance CDR mutability. Notably, the AGY triplet was abundant only in the Ser reading frame. In an unexpected twist, we found that AGY codons in antiviral antibodies were frequently mutated to codons specifying most of the amino acids that were reported to be key binding-site contact residues for antigen (Ag), as determined from more than 100 crystal structures of Ag–Ab complexes (19). Because the germline codons that gave rise to somatically generated contact residues were not determined in this study, we conducted additional analyses of published Ab–Ag crystal structures to identify germline codons that mutated to codons specifying contact residues. Our independent analyses revealed that somatic mutations in AGY codons created Ag-contact residues more often than mutations in any other synonymous codon group. As such, it appears that AGY CDR codons were preserved because of their exceptional functional plasticity in the context of SHM and affinity maturation.