Anolis Carolinensis Expression of Igm, Igd, and Igy in a Reptile

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 apparent 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,

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 detections (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. We report here an investigation of the Small intestine total RNA was reverse-transcribed using the primer NotI- expression of the IGH genes of a reptile, the green anole lizard (Anolis Ј d(T)18 (5 -AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTT carolinensis). (While our paper was under revision, another group TTTTTTT-3Ј) and Moloney murine leukemia virus reverse transcriptase. (32) reported a computational analysis of the lizard IgH locus but One round of 3Ј-RACE PCR was conducted using a mixture of JH2s (5Ј- without providing any experimental data). CTGGGGGAAAGGCATGATCGTTGTC-3Ј, JH2 derived) and JH8s (5Ј- CTGGGGGGATGGGACATTCCTCCTCGTA-3Ј, JH8 derived) as sense Ј Ј Materials and Methods primers and R2 (5 -AAGAATTCGCGGCCGCAGGAA-3 ; this primer was designed based on the anchor sequence of the primer NotI-d(T)18)as Annotation of the green anole lizard (A. carolinensis) IGH an antisense primer. The 3Ј-RACE PCR parameters were 94°C for 5 min constant genes for 1 cycle; followed by 40 cycles of 94°C for 30 s, 66.5°C for 30 s, and 72°C for 90 s and a final extension at 72°C for 7 min. The polymerase used Basic local alignment search tool (BLAST) searches were performed against was LA-TaqDNA polymerase (Takara). The resulted ϳ1.6-kb PCR prod- the genome sequence of the lizard (AnoCar1.0 assembly) deposited in En- ucts were cloned into pMD19-T vector (Takara) to generate an Ig cDNA sembl (http://www.ensembl.org/index.html). Genomic scaffolds were retrieved minilibrary. The white clones (after blue-white screening) were subject to for further analysis. To search for putative Ig domains, the genomic DNA PCR screening using primers M13F (5Ј-AGCGGATAACAATTTCACAC sequences were translated into proteins, which were subsequently subject to AGG-3Ј) and M13R (5Ј-AGACAGGGTTTTCCCAGTCACGAC-3Ј) for protein to protein BLAST searches in the National Center for Biotechnology positively recombined clones containing correct insert size; IgM1 (5Ј-AT Information (NCBI) GenBank. Recombination signal sequences for VH, CCGTGTAGAGACTATTGC-3Ј) and IgM2 (5Ј-GGTTTACCCGTGTTCT DH, and JH gene segments were examined using the program FUZZNUC TGTC-3Ј) for IgM-positive clones; and IgY1 (5Ј-AGAAATCTGCTTGA (http://bioweb.pasteur.fr/seqanal/interfaces/fuzznuc.html). GGGG-AC-3Ј) and IgY2 (5Ј-CTTTTGGCCATCCCATTGTTG-3Ј) for Animals, RNA isolations, and reverse transcriptions IgY-positive clones. The clones containing correct insert size but negative for both IgM and IgY were sent for sequencing to identify the nature of the About 3-year-old green anole lizards were purchased from a local Beijing inserts. pet market. Total RNA from different tissues was prepared using a TRNzol kit (Tiangen Biotech). Reverse transcription was conducted using Moloney DNA and protein sequence computations murine leukemia virus reverse transcriptase following the manufacture’s DNA and protein sequence editing, alignments, and comparisons were instructions (Invitrogen). performed using the DNAStar program. Phylogenetic trees were made using MrBayes3.1.2 (33) and viewed in TreeView (34). Multiple se- Cloning of the IgM, IgD, and IgY H chain constant region quence alignments were performed using ClustalW. Accession numbers cDNAs for the sequences (http://www.ncbi.nlm.nih.gov/sites/entrez) used in phylogenetic analysis are as follows. ␣ or ␹ gene: chicken, S40610; Two JH primers, JHs1 (5Ј-GGGGCCAAGGAACATTTCTG) and JHs2 cow, AF109167; duck, AJ314754; human, J00220; mouse, J00475; (ACATTTCTGGCTGTAACTTCAG-3Ј) were designed based on con- platypus, AY055778; Xenopus laevis, BC072981. ␦ gene: catfish, served JH sequences. Membrane-bound IgM cDNA was amplified by a U67437; cow, AF411245; dog, DQ297185; fugu: AB159481; human, nested PCR using JHs1, and IgMTMas1 (5Ј-TTGATGACAGTCACGGT BC021276; horse, AY631942; mouse, J00449; pig, AF411239; rat, GGCAC-3Ј, in which TM is transmembrane region), JHs2 and IgMTMas2 AY148494; sheep, AF515673; Xenopus tropicalis, DQ350886; ze- (5Ј-TGGCACTGTAGAACAGGCTCAC-3Ј), whereas the secreted IgM brafish, BX510335. ␥ gene: human, J00228; mouse, J00453; platypus, cDNA was amplified by a nested PCR using primers JHs1 and lizard sIg- AY055781. ␧ gene: cow, AY221098; human, J00222; mouse, X01857; Mas1 (5Ј-GGTGATGGTTAGTGGCAAGTGT-3Ј, in which sIg is secre- platypus, AY055780. ␮ gene: catfish, X52617; chicken, X01613; cow, tory Ig), JHs2 and lizard sIgMas2 (5Ј-AGTGTTGCCAGTATCGGAGAG AY145128; duck, AJ314754; human, X14940; horned shark, X07781; C-3Ј). IgD constant region cDNA was amplified using JHs1 and IgDTMas1 lungfish, AF437724; mouse, V00818; nurse shark, M92851; platypus, (5Ј-GTGCAACGACAAACAGAACCAT-3Ј) and JHs2 and IgDTMas2 AY168639; skate, M29679; trout, X65261; X. laevis, BC084123; ze- (5Ј-GAAACTTTGTTCCAGACG T-3Ј). IgY full-length cDNA (secreted brafish: AF281480. ␷ gene: chicken, X07175; duck, X78273; X. laevis, form) was amplified using JHs1 and IgYas1 (5Ј-TCTGGGATAGGACA X15114. ␨/␶ gene: zebrafish, AY643752; trout, AY872256. ␻ gene: AGGAGGGC-3Ј), JHs2 and IgYas2 (5Ј-GGGCTGTGATGCTGGATAGA sandbar shark, U40560; lungfish, AF437727; nurse shark, U51450. GA-3Ј), whereas IgY (DFc) cDNA was amplified using primers JHs1 and IgYDFcas1 (5Ј-TTTATATCCTGCCCTTCTCA-3Ј) and JHs2 and IgYD- Fcas2 (5Ј-CAACAATTAGATGCCCATAC-3Ј). The IgY membrane- Results bound form cDNA was amplified using primers JHs1 and lizard IgYTMas1 The lizard IGH locus (5Ј-AAATTGCCATGCTTATAGGAGA-3Ј) and JHs2 and IgYTMas2 (5Ј- ␮ CTTGGCCAGGGACTTCAGCTTT-3Ј). The DNA polymerase used for Using the turtle gene sequence as a template (27), a BLAST amplification of IgD cDNA was LA-TaqDNA polymerase (Takara), search was performed against the green anole lizard whole-genome whereas TaqDNA polymerase (Tiangen) was used for amplification of IgM shotgun sequences deposited in the Ensemble database. A 369-kb 3860 THE LIZARD IGHC GENES

E H P E H P E H P

8 kb

6 kb

4 kb 3 kb

2 kb

FIGURE 1. Genomic organization of the lizard Ig H chain constant region gene loci. CH-encoding exons for each gene and JH gene segments are indicated using Arabic numbers. µ δ υ FIGURE 2. Southern blotting detection of the lizard Ig H chain constant genomic contig (scaffold 869) was found to contain sequences re- region genes. E, EcoRI; H, HindIII; P, PstI. lated to those of the turtle ␮. An examination of this scaffold revealed that it contained the 3Ј-region of the lizard IGH locus, with ␮ ␦ ␷ VH,DH, and JH segments and , , and genes being readily two C domains (i.e., VDJ-C 3-C 4-TM) was also observed ␮ ␦ ␷ H H H identifiable and as expected, arranged in a VH-DH-JH- - - order (Fig. 4; supplemental Figs. 4–6). However, Northern blotting 6 (Fig. 1; see supplemental Figs. 1 and 2 for deduced DH and JH suggested that this short IgM transcript was a very minor form segments). We subsequently performed Southern blottings using as it was not detectable using this approach (data not shown). the ␮, ␦, and ␷ cDNA probes, revealing that the hybridization Transcripts encoding the secreted form were detected only with bands either were consistent with the expected patterns or could be four CH exons (VDJ-CH1-CH2-CH3-CH4-s) in both PCR and readily explained by loss or gain of restriction enzymatic sites Northern experiments (Figs. 4 and 5, supplemental Fig. 4, and (polymorphisms in an outbreed species) (Fig. 2). This, to a certain data not shown). Transcripts encoding both the membrane- extent, confirmed the assembling accuracy of the IGH locus. bound and secreted forms of ␮ were detectable in multiple tissues (Fig. 5). The lizard ␮ gene Four exons encoding sequences homologous (43.8% similarity at Characterization of the rearranged VDJ-C␮ fragments the overall protein level) to the turtle ␮ C 1–4 sequences were ␮ H To analyze the expressed VDJ-C sequences, we used four VH identified downstream of the J locus. Phylogenetic analysis indi- H sense primers that matched most of the VH genes identified in the cates that the identified gene is the lizard ␮ gene (Fig. 3). Align- current genome assembly (including those not assembled to scaf- ment of the lizard ␮ sequence (inferred amino acids) with those fold 369), and an antisense primer derived from the IgM CH1to from other species revealed a distinct pattern with regard to the amplify the rearranged VDJ-C␮ fragments by RT-PCR. We se- distribution of noncanonical cysteines; i.e., those cysteines not in- quenced 92 clones, which provided 34 uniquely rearranged VDJ volved in the intradomain disulfide bond (supplemental Fig. 3). junctions (supplemental Fig. 7). Among these 34 clones, only 1 Surprisingly, the cysteine residue in the N-terminal region of C 1, H (LDA24) was expressed from the 8 VH gene segments (VH7) iden- typically involved in disulfide bonding of heavy and L chains, is tified in scaffold 369. The frequency of JH utilization was JH1 (0), absent (supplemental Fig. 3). This was confirmed in both the re- JH2 (7), JH3 (1), JH4 (3), JH5 (4), JH6 (5), JH7 (5), JH8 (7), and JH9 trieved genomic sequence and by sequencing of RT-PCR products (2); see supplemental Fig. 7. The most frequently used DH seg- generated using forward primers in the JH region and reverse ϭ ϭ ments were DH11 (or DH20, n 10) and DH2 (or DH4, n 6). primers in the 3Ј-region of the ␮ sequence. In contrast, three non- The average length of the CDR3 was 8.9 codons, which is nearly canonical cysteines were observed in the CH2 domain (supplemen- the same as that in Xenopus (8.6 codons) and mice (8.7 codons); tal Fig. 3). The cysteine residues that covalently polymerize pen- see Ref. 39). On average, the D gene plus N and P nucleotides tameric IgM in mammals (35) are also found in the CH3 and contributed 43.1% of the CDR3 length, a ratio lower than that secreted tail of the lizard ␮ (supplemental Fig. 3). Three potential observed in X. laevis (57%) and adult mice (57%) (39). N-linked glycosylation sites, one in CH1, one in CH2, and one in the secreted tail, are conserved in reptiles, birds, and Xenopus (sup- Identification of the lizard ␦ gene plemental Fig. 3). The first and third (invariant NVS) glycosylation An array of 11 C domain-encoding exons spanning ϳ12 kb was sites are also conserved between reptiles and mammals (36, 37), identified further downstream of the ␮ gene. Two typical Ig suggesting a functional importance (37, 38). transmembrane region-encoding exons were also observed 1 kb To examine the splicing pattern of the lizard ␮ gene transcript, downstream of the CH11 exon. A BLAST search using the we used primers in JH, TM, or secreted tail sequences to perform translated protein sequence from the 11 exons showed homol- nested RT-PCR amplifications. This approach revealed the pres- ogy to the Xenopus and fugu IgD H chains, supporting the no- ence of 4-CH secreted and membrane-bound forms (VDJ-CH1- tion that it is the ␦ ortholog in lizards. This conclusion was also CH2-CH3-CH4-s; VDJ-CH1-CH2-CH3-CH4-TM). Surprisingly, a supported by a phylogenetic analysis (Fig. 3 and supplemental short, membrane-bound encoding transcript, lacking the first Figs. 8 and 9). ␦ All the CH domains show C1-set Ig superfamily structure with 6 The online version of this article contains supplemental material. seven strands, and canonical cysteines at invariant positions (Ref. The Journal of Immunology 3861

FIGURE 3. Phylogenetic analysis of the lizard Ig genes. The tree was constructed using protein sequences of the last CH domains (CH8 for the lizard IgD, CH7 for the X. tropicalis IgD, and CH6 for the catﬁsh IgD that show maximal homology to the mammalian ␦CH3 were used). Except the sequences of the lizard IgM, IgD, and IgY (obtained in this study), and X. tropicalis IgF, IgM, and IgX (12), the remaining Ig sequences were obtained from the NCBI GenBank.

40 and supplemental Fig. 10). In the CH1, the second canonical IgW; Refs. 6, 12, and 41), the N-terminal region of CH1 contains tryptophan is replaced by a phenylalanine, in CH4 by a glycine, two consecutive cysteines, either of which could potentially pro- and in CH8 by a glutamine. As in Xenopus IgD (and in lungﬁsh vide the disulﬁde bond to the L chain.

FIGURE 5. Tissue expression of the lizard Ig genes. Although all other Ig genes were detected by one round of RT-PCR, IgD (transmembrane form) was detected only by two rounds (nested) of RT-PCR. TM, mem- FIGURE 4. Splicing patterns of the lizard Ig H chain genes. a, RNA brane-bound form; ␷ truncated form, IgY(⌬Fc). splicing patterns of the IgM H chains; b, RNA splicing patterns of the IgY

H chains; c, RNA splicing pattern of the IgD H chain. The CH exons are indicated in Arabic numbers. 3862 THE LIZARD IGHC GENES

ard, we searched the CH2-CH3 intron for a potential transcriptional termination signal (or polyadenylate addition signal, AATAAA).

Such signal sequences were found 464 bp downstream of the CH2 exon. To identify the putative expression of the truncated IgY H chains, RT-PCR was conducted using primers for the predicted Ј 3 -untranslated region and conserved JH sequences. The sequences of the amplicon generated showed that a truncated IgY transcript was indeed present (Fig. 4 and supplemental Figs. 15–17) but ex-

pressed much weaker than the four-CH forms in many tissues as shown by Northern blotting (data not shown). FIGURE 6. Structure of IgD H chains in different species. IgA-encoding gene is absent in the lizard An ␣ gene was also recently cloned in a reptile, the leopard gecko Domain to domain comparisons of the lizard and human ␦ se- (15). However, neither the intron between the lizard ␦ and ␷ genes quences revealed a relatively high identity between the lizard CH7 (ϳ17 kb) nor the DNA (ϳ238 kb in the retrieved genomic scaffold) and the human C 2 (30.8%), and between the lizard C 8 and ␷ H H downstream of the lizard gene contained any potential Ig CH do- human CH3 (38.3%; supplemental Table I). Two potential main-encoding sequences. We also searched the entire lizard genome N-linked glycosylation sites in the C 7 and C 8 are conserved H H and did not find any potential IgA CH encoding sequences in any across species when compared with the mammalian ␦ sequences other contigs, suggesting an absence of the IgA-encoding gene in the (supplemental Fig. 10). Strikingly, CH10 contains seven potential lizard. To further confirm this point, an Ig cDNA-specific minilibrary N-linked glycosylation sites. was constructed using total RNA derived from the small intestine ␦ ␦ The presence of 11 CH exons suggested that the lizard gene where IgA, if present, is predicted to be highly expressed. By screen- may be expressed as a long, multiple domain H chain as in Xeno- ing of the library using PCR, we obtained 302 clones containing pus and bony fish (6, 9, 12). However, a nested RT-PCR using ϳ1.6-kb inserts, of which 267 contained an IgM sequence, whereas primers derived from the conserved JH sequence and the predicted 22 were IgY. No IgD cDNA or additional Ig isotypes were found in ␦TM sequence generated a single product of ϳ1.4 kb. Sequencing this minilibrary, because the remaining clones were all shown to have ␦ of this amplicon showed that the first 4 CH exons were directly non-Ig sequences. Taken together, these data strongly suggest that ␦ spliced to the TM exon, with the remaining exons (CH5–CH11) there is no IgA encoding gene in the lizard. being spliced out (Fig. 4 and supplemental Fig. 11). We were unable to identify the 3Ј-end of a secreted form using 3Ј-RACE Putative switch regions are present upstream of the ␮ and ␷ but PCR, due, perhaps, to the overall low level of expression of ␦ not ␦ genes transcripts. The encoded lizard IgD H chain is thus more similar in Expression of H chain isotypes other than IgM (IgD) in tetrapods size to mammalian IgD than to that of bony fish and Xenopus (Fig. is accomplished through class switch recombination, in which a 6). The membrane form of the lizard ␦ gene, however, appears to functional VDJ region is translocated from upstream of an ex- be weakly expressed given that even a two-round, nested RT-PCR pressed ␮ gene to a position immediately 5Ј of the H chain gene to generated only weak amplicons in the spleen and lung (Fig. 5). be expressed (42). The recombination is mediated by switch (S) regions, which are located upstream of the ␮ gene and of the other Identification of the lizard ␷ gene H chain genes (except for ␦, which is expressed by RNA process- Approximately 17 kb downstream of the ␦TM2 exon, a third Ig ing rather than by class-switch recombination). The switch regions

gene consisting of four CH domain-encoding exons, plus two trans- are typically repeat rich (in mammals; Ref. 42) and contain short membrane region-encoding exons, was identiﬁed. A comparison repetitive sequences rich in AGCT motifs (43). Analysis by dot- of sequences, as well as phylogenetic analysis, indicated that this plot under relatively relaxed conditions (window, 30 bp; percent ␷ was the lizard gene (Fig. 3). The entire gene, including four CH- match, 70%) of the JH-␮ intron identiﬁed an ϳ3.2-kb DNA re- and two TM region-encoding exons, spans ϳ8 kb of DNA. petitive sequence block (supplemental Fig. 18a). In this 3.2-kb Alignment of the lizard IgY H chain (␷) sequence with those of DNA region, 12 CTGGG (both strands) and 18 CTGAG (both other species revealed several conserved features (supplemental strands) motifs that characterize the mammalian S␮, were found. Fig. 12). These included the three cysteines (potentially linking H The region is also rich in the AGCT motifs (supplemental Fig.

chains and L chains) in the N-terminal region of the CH1, and three 18a) that can promote isotype switching (43), suggesting that this noncanonical cysteines located in the N- and C-terminal regions of region is most likely the S␮. Using the same method, an ϳ5-kb S␷

the CH2 domain, all of which are conserved across the species was also identiﬁed (supplemental Fig. 18c). When dot-plot anal- examined. The two potential N-linked glycosylation sites in the ysis was applied to the ␮-␦ intron, some repetitive sequences

lizard CH2 and CH3 are also found in birds, but not in Xenopus. (mainly due to the presence of two (ATT)n microsatellite sites) RT-PCR using primers for the JH and secreted tail (or TM re- could be identiﬁed. This intron is, however, very much less rich in gion) followed by sequencing of the amplicons revealed that the AGCT motifs than are the putative S␮ and S␷ regions (supple- lizard IgY is expressed in secreted and membrane-bound forms mental Fig. 18b), suggesting that there is no S␦ region in the lizard. that contain all four C-region exons (Fig. 4 and supplemental Figs. The ␦ gene is thus likely to be expressed through cotranscription 13 and 14). together with the ␮ gene, followed by alternative processing of the primary transcript, as occurs in other species that express IgD. IgY(⌬Fc) is expressed in the lizard Based on serological data, a truncated IgY form has previously Discussion been reported in turtles (28). This is reminiscent of the situation in Making use of the recently released whole-genome sequence com- ducks, in which both full-length IgY and IgY(⌬Fc) lacking the Fc bined with experimental investigation of gene expression, we re-

region (CH3 and CH4), have been reported (16, 30). To conﬁrm port that the anole green lizard expresses three IgH isotypes (IgM, whether a truncated form is also expressed in the green anole liz- IgD, and IgY) but no IgA. These ﬁndings have implications for our The Journal of Immunology 3863

␮ ␷ the and genes, with the ﬁrst two CH domains being derived from ␷ ␮ the CH1–2 and the last two derived from CH3–4, a scenario that also applies to bird ␣ and Xenopus IgX H chains (15). The hypothesis that ␣ originated from a recombination between the ␮ and ␷ genes explains why only IgM and IgA can form polymeric Abs and also why IgM and IgA share the same polymeric Ig receptor (46, 47). Indeed, sequence comparisons of the gecko ␣ with the lizard ␮ and ␷, show strikingly high similarities at the inferred protein level between ␣ ␷ the gecko CH1–2 and the lizard CH1–2 (59.0% identity), and ␣ ␮ between the gecko CH3–4 and the lizard CH3–4 (59.6% identity; supplemental Table II and supplemental Fig. 19). Dot plot analysis of the lizard ␮ and ␷ with the gecko ␣ gene supports the same conclusion

(supplemental Fig. 20). Although IgA contain 4 CH domains in birds and reptiles, mammalian IgA possess only three and a hinge region.

Phylogenetic analysis suggested that the CH2 domain (of 4-CH IgA) was lost in mammals (supplemental Fig. 21). The position of IgA-encoding genes in the IGH locus differs greatly between birds and mammals. In mammals it is found at the 3Ј-end of the locus, but in birds it is found (in an inverted tran- FIGURE 7. Phylogeny of the IgH isotypes in jawed vertebrates. Ap- scriptional orientation) between the ␮ and ␷ genes. It is possible proximate times (unit, million years) of vertebrate divergence are shown in ␣ ovals. The filled circles indicate the first appearance of an Ig isotype. Ge- that, following its creation, the gene was in an unstable chro- netic events: 1, IgA was lost during speciation of reptiles, being retained in mosomal location that favored deletion and/or recombination some species such as the gecko; 2, IgD was lost during the emergence of events, thus explaining the presence of the ␣ gene in different sites the birds; 3, IgG and IgE evolved from IgY, and hinge regions for IgD, IgG in birds and mammals and its absence in the lizard. and IgA were developed. A broad overview of the Abs present in the jawed vertebrates, including the lizard, leads to two conclusions. First, certain classes of Ab (such as IgD or IgA) can be either present or absent from other- understanding of the evolutionary history of the vertebrate IGH wise related species without any apparent effects on fitness. Second, locus and the Ab classes it encodes (Fig. 7). These results, as structural variants of Abs (such as IgY) that are predicted to impair discussed below, also allow a more complete reconstruction of the important functions are also well tolerated. An example of the latter is evolutionary history of the vertebrate IGH locus and suggest con- the presence in the lizard of two forms of IgY, one of which is of clusions (some unexpected) regarding the structural and functional normal size (four-C-domain H chain), but the other is truncated. The plasticity of this locus and of the Abs that it encodes. observation in the lizard of a message that encodes a truncated form First, the demonstration of IgD in a lizard makes clear that IgD, an of IgY, termed IgY(⌬Fc) (16), mirrors findings made in ducks (30) Ab once thought to be present only in primates, shows an evolution- and in turtles (28, 29). The IgY(⌬Fc) of the lizard appears to be a ary continuity from fish, through the amphibia and reptiles to the structural equivalent of a F(abЈ) fragment, with a V -C 1-C 2do- mammals (Fig. 7). In those taxa that are lacking IgD (e.g., chickens, 2 H H H ducks, and rabbits), its absence is clearly the result of a loss of the ␦ main structure of the H chain. Such a fragment would be predicted to gene from the IGH locus. It is perhaps surprising that Ab classes can maintain virus-neutralizing functions but to be inactive in opsoniza- be lost without compromising the immune defenses of an animal, but tion and complement fixation (16, 48), although some potential se- ⌬ this theme is illustrated not only by IgD, but also by IgA, which is a lective advantages of IgY( Fc) have been proposed, such as an in- class of Ab highly expressed in mammals and which is also believed ability to initiate anaphylactic reactions (48). In this study, it was ␷ to be a critical component of mucosal immunity (44). The fact that clearly shown that the truncated chain of the lizard has a genetic IgA appears to be present in some reptiles, such as the gecko (15), but basis different from that described in ducks. In ducks, the C-terminal ␷ ⌬ not in the anole lizard suggests that it is not essential for immune sequence of the ( Fc) chain results from the use of a small additional defense in reptiles. In humans, some IgA-deficient individuals show exon that is present in the CH2-CH3 intron (supplemental Fig. 17) Ј marked susceptibility to infection, whereas others do not, which is (30). However, the C-terminal encoding sequence and 3 -untranslated ⌬ thought to reflect increased (and compensatory) IgM secretion (44). In region of the lizard IgY( Fc) transcripts are located immediately teleost fish that do not produce IgA, its function is fulfilled by IgM downstream of the CH2 exon, encoding a slightly longer tail (45). Considering that IgM shares the same transport receptor with (GKTSCGLH; supplemental Figs. 16 and 17). Thus, processing of the IgA (or IgX) in mammals and birds and Xenopus (46, 47) and that it primary ␷ transcript in the lizard involves a choice between splicing is highly expressed in the lizard intestine (Fig. 4), it is likely to play from the donor site at the end of the CH2 exon to the acceptor site of a role in mucosal immunity when IgA is absent. Thus, the fact that an the CH3 exon or cleavage/polyadenylation of the transcript at a site absence of IgA poses no apparent problem for immunity in the lizard downstream of the CH2 splice donor site. This is reminiscent of the exemplifies the plasticity and redundancy of the vertebrate Ab system. alternative RNA processing pathways associated with the expression The failure to find the ␣ gene in the lizard was unexpected, given of the secreted vs membrane bound forms of Ig H chains (49). The that IgA is present in both mammals and birds, and its evolutionary expression of the membrane form of an Ig H chain typically involves origins must therefore have predated the divergence of the synapsid splicing of the TM1 exon into a cryptic site (consensus GG/GTAAA) ␮ (mammalian) and diapsid (bird, reptile) lineages. The demonstration within the terminal secreted exon. The CH4 exons of the lizard and of a gene encoding the IgA H chain in the leopard gecko (Eulepharis ␷ genes both use cryptic splice sites of this sequence for generating macularius) (15) is compatible with this scenario; thus, the absence of messages that encode the membrane-anchored form of IgM or IgY. In ␣ Ј ␷ IgA in the lizard most likely represents the loss of the gene from the contrast, the 3 splice site of the CH2 exon of the lizard gene (AG/ IGH locus of this species. The ␣ gene recently cloned from the gecko GTAAG) conforms more closely to the canonical splice donor se- (15) was proposed to have originated from a recombination between quence (AG/GTGAG). 3864 THE LIZARD IGHC GENES

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