Somatic hypermutation of the new antigen receptor gene (NAR) in the nurse shark does not generate the repertoire: possible role in antigen-driven reactions in the absence of germinal centers

Abstract

The new antigen receptor (NAR) gene in the nurse shark diversifies extensively by somatic hypermutation. It is not known, however, whether NAR somatic hypermutation generates the primary repertoire (like in the sheep) or rather is used in antigen-driven immune responses. To address this issue, the sequences of NAR transmembrane (Tm) and secretory (Sec) forms, presumed to represent the primary and secondary repertoires, respectively, were examined from the peripheral blood lymphocytes of three adult nurse sharks. More than 40% of the Sec clones but fewer than 11% of Tm clones contained five mutations or more. Furthermore, more than 75% of the Tm clones had few or no mutations. Mutations in the Sec clones occurred mostly in the complementarity-determining regions (CDR) with a significant bias toward replacement substitutions in CDR1; in Tm clones there was no significant bias toward replacements and only a low level of targeting to the CDRs. Unlike the Tm clones where the replacement mutational pattern was similar to that seen for synonymous changes, Sec replacements displayed a distinct pattern of mutations. The types of mutations in NAR were similar to those found in mouse Ig genes rather than to the unusual pattern reported for shark and Xenopus Ig. Finally, an oligoclonal family of Sec clones revealed a striking trend toward acquisition of glutamic/aspartic acid, suggesting some degree of selection. These data strongly suggest that hypermutation of NAR does not generate the repertoire, but instead is involved in antigen-driven immune responses.

Figure 1

The frequency of mutations and their distribution among Tm and Sec clones. (A) Sec clones (n = 74) had a significantly higher number of mutations than the Tm clones (n = 62) (P < 0.05, one-tail t test for paired comparisons). Shared mutations in oligoclonal families were counted once. (B) The proportion of clones with a large number of mutations (>5/clones) was also highest in the Sec clones (P < 0.05, Kolmogorov-Smirnov Test).

Figure 2

Distribution of Tm and Sec mutations in a type 2 NAR gene of an individual shark. CDRs are in bold italics and underlined. A triplet insertion is underlined. The insertion precedes the triplet where indicated. The CDR3 of each sequence was unique.

Figure 3

Deviation from the intrinsic mutation pattern of replacement substitutions in Sec (n = 66), but not Tm (n = 60), clones suggests selection. Replacement analysis excluded clones with more than 15 mutations (mut). (A) Synonymous changes include data from an additional 31 clones used in a previous study (2), but excludes c to t changes generated by synonymous changes to the AGC hotspot and to a TGC codon in framework region (FR) 2 of type 1 NAR that is under stringent selection (35). (B) Even when excluding the third base of each codon, the Sec CDR replacements differ from the intrinsic pattern of mutation. Conservatively, we excluded the few examples where a third base generated a replacement, because they tend to be generated by transversions. The overall G test comparison of all groups was significant (P < 0.05), which was mostly attributed to the comparison of the replacement Sec group vs. the synonymous group.

Figure 4

An oligoclonal family of Sec clones has been selected for changes to acidic residues. The numbers adjacent to lines connecting clones are the number of mutations shared (and unique mutations when ending the lineage) and numbers in parenthesis enumerate acquired acidic residues. Shaded circles represent theoretical intermediates. The bold circle reflects the most common member of the family. Hypervariable region 4 (HV4) is modeled to be in a similar position as in T cell receptors (29).

Figure 5

Unlike shark IgM, NAR hypermutation does not target GC bases. (A) The proportion of synonymous mutations (mut) at each base in NAR is similar to expected based on the base composition at degenerate sites. (B) Mutation of shark IgM genes targets g:c bases, particularly guanines.