Shark IgW C Region Diversification through RNA Processing and Isotype Switching

Abstract

Sharks and skates represent the earliest vertebrates with an adaptive immune system based on lymphocyte antigen receptors generated by V(D)J recombination. Shark B cells express two classical immunoglobulins (Ig), IgM and IgW, encoded by an early, alternative gene organization consisting of numerous autonomous miniloci, where the individual gene cluster carries a few rearranging gene segments and one constant region, μ or ω. We have characterized eight distinct Ig miniloci encoding the nurse shark omega heavy (H) chain. Each cluster consists of VH, D and JH segments and six to eight constant (C) domain exons. Two interspersed secretory exons, in addition to the 3’-most C exon with tailpiece, provide the gene cluster with the ability to generate at least six secreted isoforms that differ as to polypeptide length and C domain combination. All clusters appear to be functional, as judged by the capability for rearrangement and absence of defects in the deduced amino acid sequence. We previously showed that IgW VDJ can perform isotype switching to μ C regions; in this study we found that switching also occurs between ω clusters. Thus C region diversification for any IgW VDJ can take place at the DNA level, by switching to other ω or μ C regions, as well as by RNA processing to generate different C isoforms. The wide array of pathogens recognized by antibodies require different disposal pathways, and our findings demonstrate complex and unique pathways for C effector function diversity that evolved independently in cartilaginous fishes.

Deiminated proteins in extracellular vesicles and serum of llama (Lama glama)—Novel insights into camelid immunity

“…Furthermore, deimination of Ig's in teleost fish was recently described (Magnadóttir et al, 2019a), as well as in shark (Criscitiello et al, 2019). Given the increased focus on understanding of Ig diversity throughout phylogeny (Smith et al, 2012;Zhang et al, 2013;de los Rios et al, 2015;Zhang et al, 2016Zhang et al, , 2017Stanfield et al, 2018) and the unique features of camelid immunoglobulins (Plasil et al, 2019), our current finding of deimination of llama Ig's highlights a novel concept that may further understanding of Ig diversity throughout evolution. Of additional interest is the finding that some deiminated Ig proteins were also found to be exported in EVs.…”

Assembly and Expression of Shark Ig Genes

“…Whereas within a cluster, recombining gene segments such as V H -D1 or V L -J L or V NAR -D1 are 250–500 bp apart (Fig. 2B), the clusters themselves are spatially distant: 120 to >200 kb between Igμ, 82 to >200 kb between Igω [18, 19]. Clusters can be chromosomally dispersed, as shown in clearnose skate by fluorescence in situ hybridization [20].…”

“…There is no information about chromatin folding during V(D)J recombination in shark. As some IgH are linked [18, 19], chromatin folding may bring the distant clusters into proximity. However, intercluster recombination is rarely observed [17, 26], and the reason could be that in any pro-B cell few (1–3) Igμ genes are accessible to RAG at the same time (see H chain exclusion).…”

“…All the nurse shark IgH clusters are distinct in their V H , J H and C H [17, 19], so that it was possible to distinguish chimeric sequences from adult spleen cDNA libraries that carried VDJ from one IgH gene and C region from another [60, 19]. Combinations were found between Igμ clusters or Igω clusters or between Igμ and Igω clusters.…”

Nurse shark T‐cell receptors employ somatic hypermutation preferentially to alter alpha/delta variable segments associated with alpha constant region

“…S6D). However, mutation in IgHV was substantially lower than would be expected during an antigen-specific response in B cells as they affinity mature in spleen [39,41,42]. Mutations to CDR were biased toward G and C nucleotides (71%) within WRCH/DGYW motifs (59%) and tended to be transitions (51%).…”