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 glutamic͞aspartic 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.Like the hallmark molecules of adaptive immunity (T cell receptors, Ig, and major histocompatibility complex) and the Ig gene rearrangement machinery, somatic hypermutation of Ig genes is present in the oldest group of extant jawed vertebrates, the cartilaginous fish (1-3). Similar to mutation and selection in adaptive evolution, somatic hypermutation in mice and humans is an antigen-driven process that randomly introduces mutations to Ig genes, and B cells whose receptors have affinity-enhancing mutations are selected in the germinal centers (GC) of secondary lymphoid tissues (reviewed in ref. 4). In sheep and cows, however, somatic hypermutation generates the primary repertoire in a foreign antigen-independent process during B-cell development in the Ileal Peyer's Patches (5, 6). Generally, species that use somatic hypermutation and͞or gene conversion to generate the repertoire use only one or a few variable (V) genes, whereas those using somatic hypermutation only in secondary responses express many V families (5-10). In the amphibian Xenopus, somatic hypermutation is not used to generate the repertoire (11), and the few examples where it is known to generate the repertoire occur in mammals. Such data suggest that somatic mutation first evolved for secondary immune responses and later was recruited to generate the repertoire in some species; studies of somatic hypermutation in the cartilaginous fish address this issue.In the nurse shark, Ginglymostoma cirratum...

The Nucleotide Targets of Somatic Mutation and the Role of Selection in Immunoglobulin Heavy Chains of a Teleost Fish

“…The generally observed imbalance of A compared with T mutations in mammalian systems has been a basis for proposing a strand-biased mechanism that differentially targets A:T but not G:C pairs (discussed below). Earlier studies on shark (29) and Xenopus (28) H chains had analyzed a limited number of mutations and concluded that these lower vertebrates exhibited a strong mutational bias toward G and C. However, extensive mutational analysis on shark L chains (30) and shark new Ag receptor (31) have subsequently shown that mutations in A and T generally represent Ͼ40 and Ͼ50% of the mutations, respectively. These latter studies have also observed that tandem mutations, ranging in length from two to four nucleotides, are characteristic of the mutational pattern and may represent Ͼ50% of the total mutations.…”

“…Subsequent studies on shark L chains and shark NAR have shown that mutations in A and T can account for 40 -50% of the mutations. These latter studies have also observed that tandem mutations, ranging in length from 2 to 4 nts, can represent from 25 to 50% of the total mutations, suggesting that alternative mutational and/or repair mechanisms may exist (30,31).…”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…A minority of V NAR s (type IV) bear only the single canonical disulfide linkage. As for V H Hs, most V NAR Cys residues in CDR1, FR2 and FR4 are probably encoded in the germline and non-canonical disulfide linkages are formed during primary repertoire development 4,27,28 Although the precise roles of non-canonical disulfide linkages in sdAb structure and function remain unclear, these linkages very likely influence sdAb paratope structure, since patterns of antigen-driven somatic hypermutation appear to vary depending on their presence and location. 27

10.1080/19420862.2018.1489633-F0002Fig 2.Properties of sdAb vs .

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“…27 Unlike the V H domains of IgMs and IgWs, V NAR s accrue somatic hypermutations upon encounter with antigen primarily in CDR1 and CDR3 but also in HV2 and HV4. 27,28 …”

Unprecedented Multiplicity of Ig Transmembrane and Secretory mRNA Forms in the Cartilaginous Fish

“…The IgNAR V recognizes Ag as a single domain (see Fig. 8) and its gene mutates to very high levels, suggesting that it is important for the shark adaptive immune system (12)(13)(14).…”