Shark IgW C Region Diversification through RNA Processing and Isotype Switching Cecilia Zhang, Louis Du Pasquier and Ellen Hsu This information is current as J Immunol 2013; 191:3410-3418; Prepublished online 9 of September 27, 2021. August 2013; doi: 10.4049/jimmunol.1301257 http://www.jimmunol.org/content/191/6/3410 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2013/08/12/jimmunol.130125 Material 7.DC1 References This article cites 43 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/191/6/3410.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Shark IgW C Region Diversification through RNA Processing and Isotype Switching Cecilia Zhang,* Louis Du Pasquier,† and Ellen Hsu* Sharks and skates represent the earliest vertebrates with an adaptive immune system based on lymphocyte Ag receptors generated by V(D)J recombination. Shark B cells express two classical Igs, 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 C region, m or v. We have characterized eight distinct Ig miniloci encoding the nurse shark v H chain. Each cluster consists of VH, D, and JH segments and six to eight C domain exons. Two interspersed secretory exons, in addition to the 39-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 m in the deduced amino acid sequence. We previously showed that IgW VDJ can perform isotype switching to C regions; in this Downloaded from study, we found that switching also occurs between v clusters. Thus, C region diversification for any IgW VDJ can take place at the DNA level by switching to other v or m C regions, as well as by RNA processing to generate different C isoforms. The wide array of pathogens recognized by Abs requires different disposal pathways, and our findings demonstrate complex and unique pathways for C effector function diversity that evolved independently in cartilaginous fishes. The Journal of Immunology, 2013, 191: 3410–3418. http://www.jimmunol.org/ he B lymphocytes in jawed vertebrates express IgM as ulocytes, suggesting conservation across species of a common a cell surface receptor and as secreted protein. Although immune function that has yet to be fully elucidated (2, 6). T the quaternary structure can vary among species, the basic Very little is known about IgW in cartilaginous fishes, where it m H chain is highly conserved in structure with its rearranged V has been variously called IgX, IgNARC, and IgW in various species region and four C region domains (1). The ubiquity and constancy of skates and sharks (7–12). Some genomic sequence obtained of IgM suggest a strongly preserved function, in contrast to a from the clearnose skate (13) confirmed that the IgW H chain was second Ig class called IgD or IgW, also present in most vertebrate encoded by multiple genes of the “cluster” type that consisted of classes although intriguingly absent in some species (for a review, genetic elements similar to the IgM H chain clusters (14). Each m see Ref. 2). The IgW H chain (v in cartilaginous fishes and gene contains VH, D, and JH gene segments all within 1–2 kb that by guest on September 27, 2021 lungfish (3) is an ortholog of the IgD H chain (d in bony fishes and can somatically recombine, and the rearranged VDJ is transcribed tetrapods (4). Delta is often characterized by its position 39 of the with a set of C region exons (15). The m clusters in nurse shark IgM H chain C exons and dependence on m transcription, which function autonomously and are as isolated from each other, .120 was also key to its classification in bony fishes (5) and the am- kb, as from the v clusters (16, 17). phibian Xenopus (4). In this study the name IgW is retained for the The multicluster organization is considered an early alternative systems such as sharks where the IgW H chain genes rearrange form evolved from a primordial Ig gene. The number of m clusters and are expressed autonomously. Thus, IgD/IgW appears to be as varies greatly among species, from 100 to 200 in horned shark old as IgM, although its function remains unclear. Mouse and (18) to 15 in nurse shark (16). Ab combining site diversity is due human IgD bind to basophils that upon crosslinking induces to junctional diversity and is generated by rearrangement. How- proinflammatory activity; catfish IgD also binds a subset of gran- ever, with apparently only three kinds of serum Ig classes (IgM, IgW, IgNAR) (8) there would appear to be a limit on shark C region effector function, compared with the eight Ig isotypes in mouse. Comparison of the five subfamilies of m sequences in *Department of Physiology and Pharmacology, State University of New York Health Science Center at Brooklyn, Brooklyn, NY 11203; and †Institute of Zoology and nurse shark showed that, whereas CH3 and CH4 were highly Evolutionary Biology, University of Basel, Basel CH-4051, Switzerland conserved among clusters, CH2 and VH were under strong posi- Received for publication May 13, 2013. Accepted for publication July 5, 2013. tive selection for amino acid diversity (16). This observation This work was supported by National Institutes of Health Grant GM068095. suggested that C region function could differ among the Ig The sequences presented in this article have been submitted to GenBank (http:// clusters, a notion reinforced when it was found that VDJ from one www.ncbi.nlm.nih.gov/genbank/) under accession numbers KC920789–KC920812, cluster could switch to the C region of another cluster through KF184389–KF184390, and KF192877–KF192884. recombination within the J-C intron (17). The frequency of Address correspondence and reprint requests to Dr. Ellen Hsu, Department of Phys- switching coincided with the expression of activation-induced cy- iology and Pharmacology, State University of New York Health Science Center at Brooklyn, 450 Clarkson Avenue, Box 31, Brooklyn, NY 11203. E-mail address: tidine deaminase, and the switch junctions mostly appeared to be [email protected] the results of double-strand DNA breakage repaired by nonho- The online version of this article contains supplemental material. mologous end-joining. Abbreviations used in this article: BAC, bacterial artificial chromosome; nC-domain, Not only could m clusters switch among themselves, but VDJ C region sequence where n = 1 to 8 domains; RSS, recombination signal sequence; from v clusters could also acquire m C regions. These observations Sec, secreted; Tm, transmembrane; TMR, transmembrane region; tp, tailpiece. demonstrated that the elasmobranch Ig cluster system was not as Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 static as originally appeared. Because the v C region is so entirely www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301257 The Journal of Immunology 3411 different from that of m (11, 12), the shark interclass switching Probes strongly suggested C effector function selection. We embarked on the Probes to IgM derived from group 2 cDNA (vh, cm1, cm2, cm3–cm4) have characterization of nurse shark v genes with the long-term view of been described elsewhere (16). Probes to IgW were derived from cDNA or clarifying their status in an IgM-expressing B lymphocyte. Eight selected BAC clones as follows: wV (333 bp, JWF1, 59- distinct v clusters have been identified; with the genomic organiza- GATTGCTCCWAATCTCKG-39 and IgWFR3R2, 59-GACACCGCCATC- 9 9 9 tion elucidated for the first time, we discovered that most v clusters TATTAC-3 ) and transmembrane (wTM) with 3 UT (472 bp, WTMF, 5 - GGTCCCTCCAGATGTGAA-39 and WTMUTR, 59-GGAATCAGTCCTC- are each capable of producing six secreted H chain isoforms. CACCC-39) were amplified from pup spleen cDNA primed with oligo(dT). Given the existence of shark immune cells (neutrophils, eosin- wCH1/2 (561 bp, IgWCH1F1, 59-AAGATGAGATCAGCCTCC-39 and ophils, monocytes, macrophages, dendritic cells) (19, 20) that in W543R, 59-GATC- other vertebrates mediate Ab-dependent cellular cytotoxicity, CTGGTACTGAAGCT-39), wCH3/4 (508 bp, WC3F, 59-CCACCCTG- GTGTGTACA-39 and WC4R, 59-GCAGGAAGGAGGTGAAGA-39), wCH5 opsonization, and cell degranulation, it is anticipated that shark (260 bp, WC5F, 59-CTGGAGGAACAGAACAGC-39 and WC5R, 59- Abs likewise recruit effector cells against pathogens. The wide GGGCTTTCCTGATGAACC-39), and wCH6 (231 bp, W1580F, 59-AACTG- array of pathogens recognized by Abs require different disposal CTCATACTGCGAC-39 and W1792R, 59-GTTGTGACTGACTCGTGT-39) pathways, and this need for a flexible array of processing was were amplified from a cloned W6 sequence (C region identical to GenBank met with by uncoupling the Ag recognition site from a fixed accession number U51450).