Expression of IgM, IgD, and IgY in a Reptile,
Anolis carolinensis
This information is current as
of June 16, 2017.
Zhiguo Wei, Qian Wu, Liming Ren, Xiaoxiang Hu, Ying
Guo, Gregory W. Warr, Lennart Hammarström, Ning Li and
Yaofeng Zhao
J Immunol 2009; 183:3858-3864; Prepublished online 28
August 2009;
doi: 10.4049/jimmunol.0803251
http://www.jimmunol.org/content/183/6/3858
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Supplementary
Material
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*
T
he Igs are uniquely characteristic of the adaptive immune
systems of all jawed vertebrates, being found in jawed fish,
amphibia, reptiles, birds, and mammals (1, 2). A typical Ig
molecule consists of two H and two L chains, with the H and L chains
being encoded in separate IGH5 and IGL loci (2). The H and L
polypeptides are typically covalently linked by disulfide bonds
formed by positionally conserved cysteine residues; however, noncovalent interactions alone are sufficient to hold L and H chains together,
as observed for human IgA2m (1) and Igs of Rana catesbeiana (3, 4).
The Ig classes expressed in mammals are IgM, IgD, IgG, IgE,
and IgA. This classification is based on their H chain isotypes,
*State Key Laboratory of Agrobiotechnology, College of Biological Sciences,
China Agricultural University, Beijing, Peoples Republic of China; †Medical University of South Carolina, Marine Biomedicine and Environmental Sciences Center, Charleston, SC 29425; and ‡Division of Clinical Immunology, Department of
Laboratory Medicine, Karolinska University Hospital Huddinge, Stockholm,
Sweden
Received for publication September 30, 2008. Accepted for publication July 13, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Science Fund for Distinguished Young Scholars Grant 30725029, Program for New Century Excellent Talents in University of
China, National Key Basic Research Program Grant 2006CB102100, and National
Natural Science Foundation of China Grant 30671497.
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
are those of the author and do not necessarily reflect the views of the National Science
Foundation.
2
Z.W. and Q.W. contributed equally to this work.
3
Current address: Division of Molecular and Cellular Biosciences, National Science
Foundation, 4201 Wilson Boulevard, Arlington, VA 22230.
4
Address correspondence and reprint requests to Dr. Yaofeng Zhao, State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, Peoples Republic of China. E-mail address: yaofengzhao@
cau.edu.cn, or Dr. Ning Li, State Key Laboratory of Agrobiotechnology, College of
Biological Sciences, China Agricultural University, Beijing 100193, Peoples Republic of China. E-mail address: [email protected]
5
Abbreviations used in this paper: IGH, Ig H chain gene; IGL, Ig L chain gene;
BLAST, basic local alignment search tool; NCBI, National Center for Biotechnology
Information; IgSF, Ig superfamily; TM, transmembrane region; sIg, secretory Ig.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803251
which are encoded by the ␮, ␦, ␥, ␧, and ␣ genes, respectively (1,
2). The evolutionary history of these genes is of interest from many
perspectives, two of which have received particular attention. The
first is the question of how the genes encoding the H chains arose,
one from the other, and how their subsequent evolutionary divergence permitted the acquisition of novel Ab functions including
transport and secretion into external body fluids such as mucus and
milk, or the mediation of allergic reactions. In summary, first, IgM
is the only class of Ab that is expressed in all species of jawed
vertebrates (1, 2, 5). Second, IgD is a class of Ab that is probably
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 variable (which, in some cases, is due to duplication or loss of CH
exons; Ref. 7), the function of which is a matter of debate (8), and
which has clearly been lost from certain species. In bony fish and
pigs, IgD can be expressed as chimeric H chains with inclusion of
the IgM CH1 domain (9, 10). Although IgD/W has been described
in fish (cartilaginous and bony), amphibians, and numerous mammals (6, 9, 11, 12), it is absent in birds and rabbits (13, 14). Third,
the major class of low-m.w. Ab in the amphibia, reptiles, and birds
is IgY, which is thought to have been derived from IgM by a gene
duplication event (5). Fourth, IgY played a central role in Ab evolution in the tetrapod lineage, giving rise to IgA (through an apparent recombination event with IgM; Ref. 15) and to IgG and IgE
through a gene duplication event followed by differentiation of
structure and function (16). Overlaid on this broad scheme are
other important events in the evolutionary history of Abs where
certain lineages developed unique classes of Abs, such as the IgNAR in elasmobranchs (17), IgT/Z in some species of bony fish
(18, 19), and IgX and IgF in amphibia (12, 20).
A second important perspective on the evolution of the Ab
genes is the structure of the IGH locus. Interest in this question was
stimulated by observations that in cartilaginous fish (phylogenetically the most primitive jawed vertebrates), the IGH loci show a
unique structure. Whereas in the mammals the genes present in the
IGH locus are arranged in a (VH)n-(DH)n-(JH)n-(CH)n order, the
IGH loci of cartilaginous fish show a multicluster organization,
where multiple clusters, each consisting of (V-DH-DH-JH-CH),
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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.
The Journal of Immunology
Materials and Methods
Annotation of the green anole lizard (A. carolinensis) IGH
constant genes
Basic local alignment search tool (BLAST) searches were performed against
the genome sequence of the lizard (AnoCar1.0 assembly) deposited in Ensembl (http://www.ensembl.org/index.html). Genomic scaffolds were retrieved
for further analysis. To search for putative Ig domains, the genomic DNA
sequences were translated into proteins, which were subsequently subject to
protein to protein BLAST searches in the National Center for Biotechnology
Information (NCBI) GenBank. Recombination signal sequences for VH,
DH, and JH gene segments were examined using the program FUZZNUC
(http://bioweb.pasteur.fr/seqanal/interfaces/fuzznuc.html).
Animals, RNA isolations, and reverse transcriptions
About 3-year-old green anole lizards were purchased from a local Beijing
pet market. Total RNA from different tissues was prepared using a TRNzol
kit (Tiangen Biotech). Reverse transcription was conducted using Moloney
murine leukemia virus reverse transcriptase following the manufacture’s
instructions (Invitrogen).
Cloning of the IgM, IgD, and IgY H chain constant region
cDNAs
Two JH primers, JHs1 (5⬘-GGGGCCAAGGAACATTTCTG) and JHs2
(ACATTTCTGGCTGTAACTTCAG-3⬘) were designed based on conserved JH sequences. Membrane-bound IgM cDNA was amplified by a
nested PCR using JHs1, and IgMTMas1 (5⬘-TTGATGACAGTCACGGT
GGCAC-3⬘, in which TM is transmembrane region), JHs2 and IgMTMas2
(5⬘-TGGCACTGTAGAACAGGCTCAC-3⬘), whereas the secreted IgM
cDNA was amplified by a nested PCR using primers JHs1 and lizard sIgMas1 (5⬘-GGTGATGGTTAGTGGCAAGTGT-3⬘, in which sIg is secretory Ig), JHs2 and lizard sIgMas2 (5⬘-AGTGTTGCCAGTATCGGAGAG
C-3⬘). IgD constant region cDNA was amplified using JHs1 and IgDTMas1
(5⬘-GTGCAACGACAAACAGAACCAT-3⬘) and JHs2 and IgDTMas2
(5⬘-GAAACTTTGTTCCAGACG T-3⬘). IgY full-length cDNA (secreted
form) was amplified using JHs1 and IgYas1 (5⬘-TCTGGGATAGGACA
AGGAGGGC-3⬘), JHs2 and IgYas2 (5⬘-GGGCTGTGATGCTGGATAGA
GA-3⬘), whereas IgY (DFc) cDNA was amplified using primers JHs1 and
IgYDFcas1 (5⬘-TTTATATCCTGCCCTTCTCA-3⬘) and JHs2 and IgYDFcas2 (5⬘-CAACAATTAGATGCCCATAC-3⬘). The IgY membranebound form cDNA was amplified using primers JHs1 and lizard IgYTMas1
(5⬘-AAATTGCCATGCTTATAGGAGA-3⬘) and JHs2 and IgYTMas2 (5⬘CTTGGCCAGGGACTTCAGCTTT-3⬘). The DNA polymerase used for
amplification of IgD cDNA was LA-TaqDNA polymerase (Takara),
whereas TaqDNA polymerase (Tiangen) was used for amplification of IgM
and IgY cDNA. All the resulting PCR products were cloned into a T vector
(pMD19; Takara) and sequenced. The sequences have been deposited in
the NCBI GenBank under the following accession numbers: EF683585 and
EF690357–EF690361 (http://www.ncbi.nlm.nih.gov/sites/entrez).
Southern blotting
The above amplified ␮ (membrane-bound form), ␦ (membrane-bound
form), and ␷ (secreted form) cDNAs were used as probes, which were all
labeled using a PCR digoxygenin probe synthesis kit (Roche). The hybridization and detection were conducted by following the manufacturer’s
digoxygenin hybridization instruction.
Tissue expression of the lizard IGH genes
Tissue expression of the membrane-bound and secreted IgM were detected
by PCR using the primers IgMCH3s (5⬘-CATCAGTGGCATCCCTCA
CA-3⬘) and IgMTMas3 (5⬘-ATCCCATCTAGGTAAGCCGT-3⬘) or lizard
sIgMas2. Full-length IgY expression was detected using primer IgY detections (5⬘-TTTCCTGATCCGGTCACAGTGC-3⬘) and IgY detections
(5⬘-AGCCCTTCTTCACTTTCCAGAT-3⬘), whereas IgY (⌬Fc) expression was detected using primer IgY detections and IgYDFc detections (5⬘CAACAATTAGATGCCCATAC-3⬘). Expression of IgD was detected by
a nested PCR using primers JHs1 and IgDTMas1, JHs2 and IgDTMas2.
Analysis of the expressed Ig isotypes in the small intestine
Small intestine total RNA was reverse-transcribed using the primer NotId(T)18 (5⬘-AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTT
TTTTTTT-3⬘) and Moloney murine leukemia virus reverse transcriptase.
One round of 3⬘-RACE PCR was conducted using a mixture of JH2s (5⬘CTGGGGGAAAGGCATGATCGTTGTC-3⬘, JH2 derived) and JH8s (5⬘CTGGGGGGATGGGACATTCCTCCTCGTA-3⬘, JH8 derived) as sense
primers and R2 (5⬘-AAGAATTCGCGGCCGCAGGAA-3⬘; this primer
was designed based on the anchor sequence of the primer NotI-d(T)18) as
an antisense primer. The 3⬘-RACE PCR parameters were 94°C for 5 min
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
was LA-TaqDNA polymerase (Takara). The resulted ⬃1.6-kb PCR products were cloned into pMD19-T vector (Takara) to generate an Ig cDNA
minilibrary. The white clones (after blue-white screening) were subject to
PCR screening using primers M13F (5⬘-AGCGGATAACAATTTCACAC
AGG-3⬘) and M13R (5⬘-AGACAGGGTTTTCCCAGTCACGAC-3⬘) for
positively recombined clones containing correct insert size; IgM1 (5⬘-AT
CCGTGTAGAGACTATTGC-3⬘) and IgM2 (5⬘-GGTTTACCCGTGTTCT
TGTC-3⬘) for IgM-positive clones; and IgY1 (5⬘-AGAAATCTGCTTGA
GGGG-AC-3⬘) and IgY2 (5⬘-CTTTTGGCCATCCCATTGTTG-3⬘) for
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
inserts.
DNA and protein sequence computations
DNA and protein sequence editing, alignments, and comparisons were
performed using the DNAStar program. Phylogenetic trees were made
using MrBayes3.1.2 (33) and viewed in TreeView (34). Multiple sequence alignments were performed using ClustalW. Accession numbers
for the sequences (http://www.ncbi.nlm.nih.gov/sites/entrez) used in
phylogenetic analysis are as follows. ␣ or ␹ gene: chicken, S40610;
cow, AF109167; duck, AJ314754; human, J00220; mouse, J00475;
platypus, AY055778; Xenopus laevis, BC072981. ␦ gene: catfish,
U67437; cow, AF411245; dog, DQ297185; fugu: AB159481; human,
BC021276; horse, AY631942; mouse, J00449; pig, AF411239; rat,
AY148494; sheep, AF515673; Xenopus tropicalis, DQ350886; zebrafish, BX510335. ␥ gene: human, J00228; mouse, J00453; platypus,
AY055781. ␧ gene: cow, AY221098; human, J00222; mouse, X01857;
platypus, AY055780. ␮ gene: catfish, X52617; chicken, X01613; cow,
AY145128; duck, AJ314754; human, X14940; horned shark, X07781;
lungfish, AF437724; mouse, V00818; nurse shark, M92851; platypus,
AY168639; skate, M29679; trout, X65261; X. laevis, BC084123; zebrafish: AF281480. ␷ gene: chicken, X07175; duck, X78273; X. laevis,
X15114. ␨/␶ gene: zebrafish, AY643752; trout, AY872256. ␻ gene:
sandbar shark, U40560; lungfish, AF437727; nurse shark, U51450.
Results
The lizard IGH locus
Using the turtle ␮ gene sequence as a template (27), a BLAST
search was performed against the green anole lizard whole-genome
shotgun sequences deposited in the Ensemble database. A 369-kb
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are arranged in tandem (21–23). These alternative organizations
(translocon or multicluster) present different challenges for the orderly rearrangement and clonal expression by B lymphocytes of
the Ab H chains (24).
Although the IGH loci have been extensively investigated in
fish, amphibia, birds, and mammals, our current knowledge of the
Ig genes in reptiles remains limited. Reptiles are known, on the
basis of studies at the protein level, to express two Ig classes, IgM
and IgY (25, 26), and an IgM encoding gene has been cloned in
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
turtles (28, 29), but it is not known whether the genetic basis of this
phenomenon is the same as that underlying the expression of two
isoforms of IgY in the duck (30). A gene encoding an IgA-like
isotype was recently cloned in the leopard gecko (15), suggesting that
the repertoire of Ig classes in reptiles may be similar to that of birds.
The IgD-encoding gene, despite its absence in birds, was also recently
reported in the gecko (31). A paucity of knowledge of the structure of
the reptilian IGH locus and of its encoded Ig H chain isotypes has
been a barrier to a more complete understanding of the evolutionary
history of Igs in vertebrates. We report here an investigation of the
expression of the IGH genes of a reptile, the green anole lizard (Anolis
carolinensis). (While our paper was under revision, another group
(32) reported a computational analysis of the lizard IgH locus but
without providing any experimental data).
3859
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
The lizard ␮ gene
Four exons encoding sequences homologous (43.8% similarity at
the overall protein level) to the turtle ␮ CH1– 4 sequences were
identified downstream of the JH locus. Phylogenetic analysis indicates that the identified gene is the lizard ␮ gene (Fig. 3). Alignment of the lizard ␮ sequence (inferred amino acids) with those
from other species revealed a distinct pattern with regard to the
distribution of noncanonical cysteines; i.e., those cysteines not involved in the intradomain disulfide bond (supplemental Fig. 3).
Surprisingly, the cysteine residue in the N-terminal region of CH1,
typically involved in disulfide bonding of heavy and L chains, is
absent (supplemental Fig. 3). This was confirmed in both the retrieved genomic sequence and by sequencing of RT-PCR products
generated using forward primers in the JH region and reverse
primers in the 3⬘-region of the ␮ sequence. In contrast, three noncanonical cysteines were observed in the CH2 domain (supplemental Fig. 3). The cysteine residues that covalently polymerize pentameric IgM in mammals (35) are also found in the CH3 and
secreted tail of the lizard ␮ (supplemental Fig. 3). Three potential
N-linked glycosylation sites, one in CH1, one in CH2, and one in
the secreted tail, are conserved in reptiles, birds, and Xenopus (supplemental Fig. 3). The first and third (invariant NVS) glycosylation
sites are also conserved between reptiles and mammals (36, 37),
suggesting a functional importance (37, 38).
To examine the splicing pattern of the lizard ␮ gene transcript,
we used primers in JH, TM, or secreted tail sequences to perform
nested RT-PCR amplifications. This approach revealed the presence of 4-CH secreted and membrane-bound forms (VDJ-CH1CH2-CH3-CH4-s; VDJ-CH1-CH2-CH3-CH4-TM). Surprisingly, a
short, membrane-bound encoding transcript, lacking the first
6
The online version of this article contains supplemental material.
υ
two CH domains (i.e., VDJ-CH3-CH4-TM) was also observed
(Fig. 4; supplemental Figs. 4 – 6). However, Northern blotting
suggested that this short IgM transcript was a very minor form
as it was not detectable using this approach (data not shown).
Transcripts encoding the secreted form were detected only with
four CH exons (VDJ-CH1-CH2-CH3-CH4-s) in both PCR and
Northern experiments (Figs. 4 and 5, supplemental Fig. 4, and
data not shown). Transcripts encoding both the membranebound and secreted forms of ␮ were detectable in multiple tissues (Fig. 5).
Characterization of the rearranged VDJ-C␮ fragments
To analyze the expressed VDJ-C␮ sequences, we used four VH
sense primers that matched most of the VH genes identified in the
current genome assembly (including those not assembled to scaffold 369), and an antisense primer derived from the IgM CH1 to
amplify the rearranged VDJ-C␮ fragments by RT-PCR. We sequenced 92 clones, which provided 34 uniquely rearranged VDJ
junctions (supplemental Fig. 7). Among these 34 clones, only 1
(LDA24) was expressed from the 8 VH gene segments (VH7) identified in scaffold 369. The frequency of JH utilization was JH1 (0),
JH2 (7), JH3 (1), JH4 (3), JH5 (4), JH6 (5), JH7 (5), JH8 (7), and JH9
(2); see supplemental Fig. 7. The most frequently used DH segments were DH11 (or DH20, n ⫽ 10) and DH2 (or DH4, n ⫽ 6).
The average length of the CDR3 was 8.9 codons, which is nearly
the same as that in Xenopus (8.6 codons) and mice (8.7 codons);
see Ref. 39). On average, the D gene plus N and P nucleotides
contributed 43.1% of the CDR3 length, a ratio lower than that
observed in X. laevis (57%) and adult mice (57%) (39).
Identification of the lizard ␦ gene
An array of 11 C domain-encoding exons spanning ⬃12 kb was
identified further downstream of the ␮ gene. Two typical Ig
transmembrane region-encoding exons were also observed 1 kb
downstream of the CH11 exon. A BLAST search using the
translated protein sequence from the 11 exons showed homology to the Xenopus and fugu IgD H chains, supporting the notion that it is the ␦ ortholog in lizards. This conclusion was also
supported by a phylogenetic analysis (Fig. 3 and supplemental
Figs. 8 and 9).
All the ␦ CH domains show C1-set Ig superfamily structure with
seven strands, and canonical cysteines at invariant positions (Ref.
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genomic contig (scaffold 869) was found to contain sequences related to those of the turtle ␮. An examination of this scaffold revealed that it contained the 3⬘-region of the lizard IGH locus, with
VH, DH, and JH segments and ␮, ␦, and ␷ genes being readily
identifiable and as expected, arranged in a VH-DH-JH-␮-␦-␷ order
(Fig. 1; see supplemental Figs. 1 and 26 for deduced DH and JH
segments). We subsequently performed Southern blottings using
the ␮, ␦, and ␷ cDNA probes, revealing that the hybridization
bands either were consistent with the expected patterns or could be
readily explained by loss or gain of restriction enzymatic sites
(polymorphisms in an outbreed species) (Fig. 2). This, to a certain
extent, confirmed the assembling accuracy of the IGH locus.
δ
FIGURE 2. Southern blotting detection of the lizard Ig H chain constant
region genes. E, EcoRI; H, HindIII; P, PstI.
The Journal of Immunology
3861
40 and supplemental Fig. 10). In the CH1, the second canonical
tryptophan is replaced by a phenylalanine, in CH4 by a glycine,
and in CH8 by a glutamine. As in Xenopus IgD (and in lungfish
FIGURE 4. Splicing patterns of the lizard Ig H chain genes. a, RNA
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.
IgW; Refs. 6, 12, and 41), the N-terminal region of CH1 contains
two consecutive cysteines, either of which could potentially provide 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, membrane-bound form; ␷ truncated form, IgY(⌬Fc).
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
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.
3862
FIGURE 6. Structure of IgD H chains in different species.
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
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 expressed much weaker than the four-CH forms in many tissues as
shown by Northern blotting (data not shown).
IgA-encoding gene is absent in the lizard
Identification of the lizard ␷ gene
Approximately 17 kb downstream of the ␦TM2 exon, a third Ig
gene consisting of four CH domain-encoding exons, plus two transmembrane region-encoding exons, was identified. A comparison
of sequences, as well as phylogenetic analysis, indicated that this
was the lizard ␷ gene (Fig. 3). The entire gene, including four CHand two TM region-encoding exons, spans ⬃8 kb of DNA.
Alignment of the lizard IgY H chain (␷) sequence with those of
other species revealed several conserved features (supplemental
Fig. 12). These included the three cysteines (potentially linking H
chains and L chains) in the N-terminal region of the CH1, and three
noncanonical cysteines located in the N- and C-terminal regions of
the CH2 domain, all of which are conserved across the species
examined. The two potential N-linked glycosylation sites in the
lizard CH2 and CH3 are also found in birds, but not in Xenopus.
RT-PCR using primers for the JH and secreted tail (or TM region) followed by sequencing of the amplicons revealed that the
lizard IgY is expressed in secreted and membrane-bound forms
that contain all four C-region exons (Fig. 4 and supplemental Figs.
13 and 14).
IgY(⌬Fc) is expressed in the lizard
Based on serological data, a truncated IgY form has previously
been reported in turtles (28). This is reminiscent of the situation in
ducks, in which both full-length IgY and IgY(⌬Fc) lacking the Fc
region (CH3 and CH4), have been reported (16, 30). To confirm
whether a truncated form is also expressed in the green anole liz-
An ␣ gene was also recently cloned in a reptile, the leopard gecko
(15). However, neither the intron between the lizard ␦ and ␷ genes
(⬃17 kb) nor the DNA (⬃238 kb in the retrieved genomic scaffold)
downstream of the lizard ␷ gene contained any potential Ig CH domain-encoding sequences. We also searched the entire lizard genome
and did not find any potential IgA CH encoding sequences in any
other contigs, suggesting an absence of the IgA-encoding gene in the
lizard. To further confirm this point, an Ig cDNA-specific minilibrary
was constructed using total RNA derived from the small intestine
where IgA, if present, is predicted to be highly expressed. By screening of the library using PCR, we obtained 302 clones containing
⬃1.6-kb inserts, of which 267 contained an IgM sequence, whereas
22 were IgY. No IgD cDNA or additional Ig isotypes were found in
this minilibrary, because the remaining clones were all shown to have
non-Ig sequences. Taken together, these data strongly suggest that
there is no IgA encoding gene in the lizard.
Putative switch regions are present upstream of the ␮ and ␷ but
not ␦ genes
Expression of H chain isotypes other than IgM (IgD) in tetrapods
is accomplished through class switch recombination, in which a
functional VDJ region is translocated from upstream of an expressed ␮ gene to a position immediately 5⬘ of the H chain gene to
be expressed (42). The recombination is mediated by switch (S)
regions, which are located upstream of the ␮ gene and of the other
H chain genes (except for ␦, which is expressed by RNA processing rather than by class-switch recombination). The switch regions
are typically repeat rich (in mammals; Ref. 42) and contain short
repetitive sequences rich in AGCT motifs (43). Analysis by dotplot under relatively relaxed conditions (window, 30 bp; percent
match, 70%) of the JH-␮ intron identified an ⬃3.2-kb DNA repetitive sequence block (supplemental Fig. 18a). In this 3.2-kb
DNA region, 12 CTGGG (both strands) and 18 CTGAG (both
strands) motifs that characterize the mammalian S␮, were found.
The region is also rich in the AGCT motifs (supplemental Fig.
18a) that can promote isotype switching (43), suggesting that this
region is most likely the S␮. Using the same method, an ⬃5-kb S␷
was also identified (supplemental Fig. 18c). When dot-plot analysis was applied to the ␮-␦ intron, some repetitive sequences
(mainly due to the presence of two (ATT)n microsatellite sites)
could be identified. This intron is, however, very much less rich in
AGCT motifs than are the putative S␮ and S␷ regions (supplemental Fig. 18b), suggesting that there is no S␦ region in the lizard.
The ␦ gene is thus likely to be expressed through cotranscription
together with the ␮ gene, followed by alternative processing of the
primary transcript, as occurs in other species that express IgD.
Discussion
Making use of the recently released whole-genome sequence combined with experimental investigation of gene expression, we report that the anole green lizard expresses three IgH isotypes (IgM,
IgD, and IgY) but no IgA. These findings have implications for our
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Domain to domain comparisons of the lizard and human ␦ sequences revealed a relatively high identity between the lizard CH7
and the human CH2 (30.8%), and between the lizard CH8 and
human CH3 (38.3%; supplemental Table I). Two potential
N-linked glycosylation sites in the CH7 and CH8 are conserved
across species when compared with the mammalian ␦ sequences
(supplemental Fig. 10). Strikingly, CH10 contains seven potential
N-linked glycosylation sites.
The presence of 11 ␦CH exons suggested that the lizard ␦ gene
may be expressed as a long, multiple domain H chain as in Xenopus and bony fish (6, 9, 12). However, a nested RT-PCR using
primers derived from the conserved JH sequence and the predicted
␦TM sequence generated a single product of ⬃1.4 kb. Sequencing
of this amplicon showed that the first 4 ␦CH exons were directly
spliced to the ␦TM exon, with the remaining exons (CH5–CH11)
being spliced out (Fig. 4 and supplemental Fig. 11). We were
unable to identify the 3⬘-end of a secreted form using 3⬘-RACE
PCR, due, perhaps, to the overall low level of expression of ␦
transcripts. The encoded lizard IgD H chain is thus more similar in
size to mammalian IgD than to that of bony fish and Xenopus (Fig.
6). The membrane form of the lizard ␦ gene, however, appears to
be weakly expressed given that even a two-round, nested RT-PCR
generated only weak amplicons in the spleen and lung (Fig. 5).
The Journal of Immunology
understanding of the evolutionary history of the vertebrate IGH
locus and the Ab classes it encodes (Fig. 7). These results, as
discussed below, also allow a more complete reconstruction of the
evolutionary history of the vertebrate IGH locus and suggest conclusions (some unexpected) regarding the structural and functional
plasticity of this locus and of the Abs that it encodes.
First, the demonstration of IgD in a lizard makes clear that IgD, an
Ab once thought to be present only in primates, shows an evolutionary continuity from fish, through the amphibia and reptiles to the
mammals (Fig. 7). In those taxa that are lacking IgD (e.g., chickens,
ducks, and rabbits), its absence is clearly the result of a loss of the ␦
gene from the IGH locus. It is perhaps surprising that Ab classes can
be lost without compromising the immune defenses of an animal, but
this theme is illustrated not only by IgD, but also by IgA, which is a
class of Ab highly expressed in mammals and which is also believed
to be a critical component of mucosal immunity (44). The fact that
IgA appears to be present in some reptiles, such as the gecko (15), but
not in the anole lizard suggests that it is not essential for immune
defense in reptiles. In humans, some IgA-deficient individuals show
marked susceptibility to infection, whereas others do not, which is
thought to reflect increased (and compensatory) IgM secretion (44). In
teleost fish that do not produce IgA, its function is fulfilled by IgM
(45). Considering that IgM shares the same transport receptor with
IgA (or IgX) in mammals and birds and Xenopus (46, 47) and that it
is highly expressed in the lizard intestine (Fig. 4), it is likely to play
a role in mucosal immunity when IgA is absent. Thus, the fact that an
absence of IgA poses no apparent problem for immunity in the lizard
exemplifies the plasticity and redundancy of the vertebrate Ab system.
The failure to find the ␣ gene in the lizard was unexpected, given
that IgA is present in both mammals and birds, and its evolutionary
origins must therefore have predated the divergence of the synapsid
(mammalian) and diapsid (bird, reptile) lineages. The demonstration
of a gene encoding the IgA H chain in the leopard gecko (Eulepharis
macularius) (15) is compatible with this scenario; thus, the absence of
IgA in the lizard most likely represents the loss of the ␣ gene from the
IGH locus of this species. The ␣ gene recently cloned from the gecko
(15) was proposed to have originated from a recombination between
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
3⬘-end of the locus, but in birds it is found (in an inverted transcriptional orientation) between the ␮ and ␷ genes. It is possible
that, following its creation, the ␣ gene was in an unstable chromosomal location that favored deletion and/or recombination
events, thus explaining the presence of the ␣ gene in different sites
in birds and mammals and its absence in the lizard.
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 otherwise related species without any apparent effects on fitness. Second,
structural variants of Abs (such as IgY) that are predicted to impair
important functions are also well tolerated. An example of the latter is
the presence in the lizard of two forms of IgY, one of which is of
normal size (four-C-domain H chain), but the other is truncated. The
observation in the lizard of a message that encodes a truncated form
of IgY, termed IgY(⌬Fc) (16), mirrors findings made in ducks (30)
and in turtles (28, 29). The IgY(⌬Fc) of the lizard appears to be a
structural equivalent of a F(ab⬘)2 fragment, with a VH-CH1-CH2 domain structure of the H chain. Such a fragment would be predicted to
maintain virus-neutralizing functions but to be inactive in opsonization and complement fixation (16, 48), although some potential selective advantages of IgY(⌬Fc) have been proposed, such as an inability to initiate anaphylactic reactions (48). In this study, it was
clearly shown that the truncated ␷ chain of the lizard has a genetic
basis different from that described in ducks. In ducks, the C-terminal
sequence of the ␷(⌬Fc) chain results from the use of a small additional
exon that is present in the CH2-CH3 intron (supplemental Fig. 17)
(30). However, the C-terminal encoding sequence and 3⬘-untranslated
region of the lizard IgY(⌬Fc) transcripts are located immediately
downstream of the CH2 exon, encoding a slightly longer tail
(GKTSCGLH; supplemental Figs. 16 and 17). Thus, processing of the
primary ␷ transcript in the lizard involves a choice between splicing
from the donor site at the end of the CH2 exon to the acceptor site of
the CH3 exon or cleavage/polyadenylation of the transcript at a site
downstream of the CH2 splice donor site. This is reminiscent of the
alternative RNA processing pathways associated with the expression
of the secreted vs membrane bound forms of Ig H chains (49). The
expression of the membrane form of an Ig H chain typically involves
splicing of the TM1 exon into a cryptic site (consensus GG/GTAAA)
within the terminal secreted exon. The CH4 exons of the lizard ␮ and
␷ genes both use cryptic splice sites of this sequence for generating
messages that encode the membrane-anchored form of IgM or IgY. In
contrast, the 3⬘ splice site of the CH2 exon of the lizard ␷ gene (AG/
GTAAG) conforms more closely to the canonical splice donor sequence (AG/GTGAG).
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 7. Phylogeny of the IgH isotypes in jawed vertebrates. Approximate times (unit, million years) of vertebrate divergence are shown in
ovals. The filled circles indicate the first appearance of an Ig isotype. Genetic events: 1, IgA was lost during speciation of reptiles, being retained in
some species such as the gecko; 2, IgD was lost during the emergence of
the birds; 3, IgG and IgE evolved from IgY, and hinge regions for IgD, IgG
and IgA were developed.
3863
3864
Disclosures
The authors have no financial conflict of interest.
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Thus, the IgY(⌬Fc) of the lizard and of the duck arise from
different genetic events and are examples of convergent evolution.
This would argue for a selective advantage for the presence
IgY(⌬Fc) in the Ab responses of lizards, sea turtles, and ducks, but
the biological significance of IgY(⌬Fc) expression in any of these
species has yet to be established.
Overall, these observations on the Ig genes of the lizard reinforce several conclusions concerning the vertebrate IGH locus and
the Abs it encodes. First, this locus shows remarkable evolutionary
flexibility in the number of classes of Ab that are expressed in
different species of jawed vertebrate. Furthermore, the patterns of
expression (Fig. 7) show little consistency, outside the universal
presence of IgM. Second, any single class of vertebrate Ab (such
as IgD, IgA, or IgY) can show substantial structural variation, in
ways that do not clearly correlate with obvious differentiated functions. Third, it is clear that Abs show both structural plasticity and
functional redundancy. Although the evolutionary history of the
Ab family is now better described, we are still far from understanding the functional significance of the remarkable diversity
that has been uncovered.
THE LIZARD IGHC GENES