The Journal of Immunology Expression of IgM, IgD, and IgY in a Reptile, Anolis carolinensis1 Zhiguo Wei,2* Qian Wu,2* Liming Ren,* Xiaoxiang Hu,* Ying Guo,* Gregory W. Warr,3† Lennart Hammarström,‡ Ning Li,4* and Yaofeng Zhao4* The reptiles are the last major group of jawed vertebrates in which the organization of the IGH locus and its encoded Ig H chain isotypes have not been well characterized. In this study, we show that the green anole lizard (Anolis carolinensis) expresses three Ig H chain isotypes (IgM, IgD, and IgY) but no IgA. The presence of the ␦ gene in the lizard demonstrates an evolutionary continuity of IgD from ␦ fishes to mammals. Although the germline gene contains 11 CH exons, only the first 4 are used in the expressed IgD membrane-bound ␮ form. The chain lacks the cysteine in CH1 that forms a disulfide bond between H and L chains, suggesting that (as in IgM of some amphibians) the H and L polypeptide chains are not covalently associated. Although conventional IgM transcripts (four CH domains) encoding both secreted and membrane-bound forms were detected, alternatively spliced transcripts encoding a short membrane-bound form were also observed and shown to lack the first two CH domains (VDJ-CH3-CH4-transmembrane region). Similar to duck IgY, ␷ ⌬ lizard IgY H chain ( ) transcripts encoding both full-length and truncated (IgY Fc) forms (with two CH domains) were observed. The absence of an IgA-encoding gene in the lizard IGH locus suggests a complex evolutionary history for IgA in the saurian lineage leading to modern birds, lizards, and their relatives. The Journal of Immunology, 2009, 183: 3858–3864. he Igs are uniquely characteristic of the adaptive immune which are encoded by the ␮, ␦, ␥, ␧, and ␣ genes, respectively (1, systems of all jawed vertebrates, being found in jawed fish, 2). The evolutionary history of these genes is of interest from many T amphibia, reptiles, birds, and mammals (1, 2). A typical Ig perspectives, two of which have received particular attention. The molecule consists of two H and two L chains, with the H and L chains first is the question of how the genes encoding the H chains arose, being encoded in separate IGH5 and IGL loci (2). The H and L one from the other, and how their subsequent evolutionary diver- polypeptides are typically covalently linked by disulfide bonds gence permitted the acquisition of novel Ab functions including formed by positionally conserved cysteine residues; however, nonco- transport and secretion into external body fluids such as mucus and valent interactions alone are sufficient to hold L and H chains together, milk, or the mediation of allergic reactions. In summary, first, IgM as observed for human IgA2m (1) and Igs of Rana catesbeiana (3, 4). is the only class of Ab that is expressed in all species of jawed The Ig classes expressed in mammals are IgM, IgD, IgG, IgE, vertebrates (1, 2, 5). Second, IgD is a class of Ab that is probably and IgA. This classification is based on their H chain isotypes, as ancient as IgM, existing in elasmobranchs as a class that was originally named IgW but is now recognized as a homolog of IgD (6). IgD is an enigmatic Ab the structure of which is highly vari- *State Key Laboratory of Agrobiotechnology, College of Biological Sciences, able (which, in some cases, is due to duplication or loss of CH † China Agricultural University, Beijing, Peoples Republic of China; Medical Uni- exons; Ref. 7), the function of which is a matter of debate (8), and versity of South Carolina, Marine Biomedicine and Environmental Sciences Cen- ter, Charleston, SC 29425; and ‡Division of Clinical Immunology, Department of which has clearly been lost from certain species. In bony fish and Laboratory Medicine, Karolinska University Hospital Huddinge, Stockholm, pigs, IgD can be expressed as chimeric H chains with inclusion of Sweden the IgM CH1 domain (9, 10). Although IgD/W has been described Received for publication September 30, 2008. Accepted for publication July 13, 2009. in fish (cartilaginous and bony), amphibians, and numerous mam- The costs of publication of this article were defrayed in part by the payment of page mals (6, 9, 11, 12), it is absent in birds and rabbits (13, 14). Third, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the major class of low-m.w. Ab in the amphibia, reptiles, and birds 1 This work was supported by National Science Fund for Distinguished Young Schol- is IgY, which is thought to have been derived from IgM by a gene ars Grant 30725029, Program for New Century Excellent Talents in University of duplication event (5). Fourth, IgY played a central role in Ab evo- China, National Key Basic Research Program Grant 2006CB102100, and National Natural Science Foundation of China Grant 30671497. lution in the tetrapod lineage, giving rise to IgA (through an ap- parent recombination event with IgM; Ref. 15) and to IgG and IgE This material is based in part on work supported by the National Science Foundation. Any opinion, finding, and conclusions or recommendations expressed in this material through a gene duplication event followed by differentiation of are those of the author and do not necessarily reflect the views of the National Science structure and function (16). Overlaid on this broad scheme are Foundation. other important events in the evolutionary history of Abs where 2 Z.W. and Q.W. contributed equally to this work. certain lineages developed unique classes of Abs, such as the Ig- 3 Current address: Division of Molecular and Cellular Biosciences, National Science NAR in elasmobranchs (17), IgT/Z in some species of bony fish Foundation, 4201 Wilson Boulevard, Arlington, VA 22230. (18, 19), and IgX and IgF in amphibia (12, 20). 4 Address correspondence and reprint requests to Dr. Yaofeng Zhao, State Key Lab- oratory of Agrobiotechnology, College of Biological Sciences, China Agricultural Uni- A second important perspective on the evolution of the Ab versity, Beijing 100193, Peoples Republic of China. E-mail address: yaofengzhao@ genes is the structure of the IGH locus. Interest in this question was cau.edu.cn, or Dr. Ning Li, State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, Peoples Repub- stimulated by observations that in cartilaginous fish (phylogeneti- lic of China. E-mail address: [email protected] cally the most primitive jawed vertebrates), the IGH loci show a 5 Abbreviations used in this paper: IGH, Ig H chain gene; IGL, Ig L chain gene; unique structure. Whereas in the mammals the genes present in the BLAST, basic local alignment search tool; NCBI, National Center for Biotechnology IGH locus are arranged in a (V ) -(D ) -(J ) -(C ) order, the Information; IgSF, Ig superfamily; TM, transmembrane region; sIg, secretory Ig. H n H n H n H n IGH loci of cartilaginous fish show a multicluster organization, Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 where multiple clusters, each consisting of (V-DH-DH-JH-CH), www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803251 The Journal of Immunology 3859 are arranged in tandem (21–23). These alternative organizations and IgY cDNA. All the resulting PCR products were cloned into a T vector (translocon or multicluster) present different challenges for the or- (pMD19; Takara) and sequenced. The sequences have been deposited in derly rearrangement and clonal expression by B lymphocytes of the NCBI GenBank under the following accession numbers: EF683585 and EF690357–EF690361 (http://www.ncbi.nlm.nih.gov/sites/entrez). the Ab H chains (24). Although the IGH loci have been extensively investigated in Southern blotting fish, amphibia, birds, and mammals, our current knowledge of the The above amplified ␮ (membrane-bound form), ␦ (membrane-bound Ig genes in reptiles remains limited. Reptiles are known, on the form), and ␷ (secreted form) cDNAs were used as probes, which were all basis of studies at the protein level, to express two Ig classes, IgM labeled using a PCR digoxygenin probe synthesis kit (Roche). The hybrid- and IgY (25, 26), and an IgM encoding gene has been cloned in ization and detection were conducted by following the manufacturer’s digoxygenin hybridization instruction. turtles (27). Two types of IgY (7S, 5.7S) that differ in the lengths of their H chain have been described at the biochemical level in Tissue expression of the lizard IGH genes turtles (28, 29), but it is not known whether the genetic basis of this Tissue expression of the membrane-bound and secreted IgM were detected phenomenon is the same as that underlying the expression of two by PCR using the primers IgMCH3s (5Ј-CATCAGTGGCATCCCTCA isoforms of IgY in the duck (30). A gene encoding an IgA-like CA-3Ј) and IgMTMas3 (5Ј-ATCCCATCTAGGTAAGCCGT-3Ј) or lizard isotype was recently cloned in the leopard gecko (15), suggesting that sIgMas2. Full-length IgY expression was detected using primer IgY de- tections (5Ј-TTTCCTGATCCGGTCACAGTGC-3Ј) and IgY detections the repertoire of Ig classes in reptiles may be similar to that of birds. (5Ј-AGCCCTTCTTCACTTTCCAGAT-3Ј), whereas IgY (⌬Fc) expres- The IgD-encoding gene, despite its absence in birds, was also recently sion was detected using primer IgY detections and IgYDFc detections (5Ј- reported in the gecko (31). A paucity of knowledge of the structure of CAACAATTAGATGCCCATAC-3Ј). Expression of IgD was detected by the reptilian IGH locus and of its encoded Ig H chain isotypes has a nested PCR using primers JHs1 and IgDTMas1, JHs2 and IgDTMas2. been a barrier to a more complete understanding of the evolutionary Analysis of the expressed Ig isotypes in the small intestine history of Igs in vertebrates.