Preferential Use of AGY Triplets in the Ser Codon Reading Frame
Because the AGC triplet has been shown to be an intrinsically preferred target for AID-dependent SHM (13, 15, 16, 34, 35), it is plausible that high frequencies of CDR AGY codons resulted solely from an evolutionary pressure to ensure high somatic mutation frequencies in CDR sequences during immune responses. This would be consistent with the fact that αβTCR genes do not share the CDR AGY abundance and bias features with Ig genes (17, 18) (Figures S2C,D in Supplementary Material). If CDR AGY codons were preserved solely to enhance mutability, we would predict that AGY triplets would be equally frequent in all three reading frames. However, this was not the case. Even when only one AGY base was required to be contained within a CDR for inclusion in the non-coding CDR frame counts, AGY triplets in the Ser reading frame were nearly always more frequent than the combined frequencies of those in the two other reading frames (Figures 2A–C). This trend also held for AGC triplets contained within the context of the extremely mutable AGCT sequence (16, 36) (Figures S3A,B in Supplementary Material). Finally, the intrinsically mutable AGC triplet was consistently more frequent in the Ser reading frame than was the combined frequency for GCT triplets in all three reading frames (AGC on opposite strand), the only exception being the small mouse Vλ gene family (Figure S3C in Supplementary Material). These results argue that the abundance of germline CDR AGY codons was not solely due to an evolutionary selection pressure for high CDR mutability via SHM.
Figure 2  Preferential use of the AGY triplets among CDR sequences in the Ser reading frame. (A) Schematic of how AGY triplets in the different reading frames were determined at CDR boundaries. AGY triplets at CDR boundaries were counted in non-coding frames if one or two bases were located in the CDR. (B) Numbers of in-frame Ser AGC codons compared to combined numbers of AGC triplets in two non-coding frames. (C) Same analysis as in (B) applied to AGT. Box plots and whiskers were defined in Figure 1 and in Section “Materials and Methods.”