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Extensive Diversification of IgD-, IgY-, and
Truncated IgY( ∆Fc)-Encoding Genes in the
Red-Eared Turtle ( Trachemys scripta elegans
)
Lingxiao Li, Tao Wang, Yi Sun, Gang Cheng, Hui Yang,
Zhiguo Wei, Ping Wang, Xiaoxiang Hu, Liming Ren,
Qingyong Meng, Ran Zhang, Ying Guo, Lennart
Hammarström, Ning Li and Yaofeng Zhao
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http://www.jimmunol.org/content/suppl/2012/09/12/jimmunol.120018
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Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 189:3995-4004; Prepublished online 12
September 2012;
doi: 10.4049/jimmunol.1200188
http://www.jimmunol.org/content/189/8/3995
The Journal of Immunology
Extensive Diversification of IgD-, IgY-, and Truncated
IgY(DFc)-Encoding Genes in the Red-Eared Turtle
(Trachemys scripta elegans)
Lingxiao Li,* Tao Wang,* Yi Sun,* Gang Cheng,* Hui Yang,* Zhiguo Wei,† Ping Wang,*
Xiaoxiang Hu,* Liming Ren,* Qingyong Meng,* Ran Zhang,* Ying Guo,*
Lennart Hammarström,‡ Ning Li,* and Yaofeng Zhao*,x
I
mmunoglobulins are important molecules of the adaptive
immune system, and they appeared early in evolution along
with the emergence of cartilaginous fish, the most primitive
jawed vertebrates (1, 2). Based on a large number of comparative
studies of Ig genes in jawed vertebrates, including cartilaginous
fish, bony fish, amphibians, reptiles, birds, and mammals, the evolutionary history of Ig genes has been partially tracked and reconstructed. Because both IgM and IgD (W) are present in cartilaginous
fish and nearly all other jawed vertebrates (only IgD is missing in
certain species) (1, 3–5), they are believed to be the most primordial
Ig classes in evolution and thus the ancestors of other classes defined
in tetrapods, such as IgY, IgA (X), IgG, and IgE (3).
*State Key Laboratory of Agrobiotechnology, College of Biological Sciences, National Engineering Laboratory for Animal Breeding, China Agricultural University,
Beijing 100193, People’s Republic of China; †College of Animal Science and Technology, Henan University of Science and Technology, Henan 471003, People’s Republic of China; ‡Division of Clinical Immunology, Department of Laboratory
Medicine, Karolinska University Hospital Huddinge, Stockholm SE-141 86, Sweden;
and xKey Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China
Received for publication January 17, 2012. Accepted for publication August 15,
2012.
This work was supported by the National Science Fund for Distinguished Young
Scholars of China (30725029), the National Basic Research Program of China (973
Program-2010CB945300), the Innovation Fund for Graduate Students of China
Agricultural University, and the Taishan Scholar Foundation of Shandong Province.
The sequences presented in this article have been deposited in the National Center for
Biotechnology Information GenBank database (http://www.ncbi.nlm.nih.gov/
nuccore) under accession numbers JQ343852–JQ343902.
Address correspondence and reprint requests to Prof. Yaofeng Zhao, State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural
University, Beijing 100193, People’s Republic of China. E-mail address: yaofengzhao@
cau.edu.cn
The online version of this article contains supplemental material.
Abbreviations used in this article: IMGT, International ImMunoGeneTics database;
TM, transmembrane region.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1200188
IgY is a low m.w. serum Ab encoded by the y gene in nonmammalian tetrapods, including birds (6–8), reptiles (9, 10), and
amphibians (11–14). The gene is thought to have developed from
m by a duplication event (2, 15) because of their sequence homology and similar gene structures. It is also thought that the y
gene gave rise to the IgA-encoding gene (a) through a recombination event with m and may thus have emerged earlier in evolution than a (16).
IgY plays important roles in the humoral immune response and
also mediates immunity to parasite infection and allergic reactions
(15). IgY possesses the functions of IgG and IgE, two Ig classes
(encoded by the g and ε genes, respectively) expressed only by
mammals. Because of the functional similarities and relatively
high levels of sequence homology between y, g and ε genes, the y
gene is thought to be the evolutionary precursor of the mammalian
g and ε genes and may have diversified through gene duplication
events to generate multiple y genes that eventually differentiated
into g and ε. It is highly likely that y evolved to ε because of their
similar genetic structures. IgG has three constant domains and
a hinge region encoded by a separate exon, which has been thought
to be condensed into CH2 of IgY (15). However, our recent work on
platypus Ig genes identified a new IgO-encoding gene and proved
that the hinge region in IgG was independently formed at the 59 end
of yCH2 and that yCH2 was lost during evolution, probably due to
a splice-site mutation (17). Considering that IgG and IgE are present
in all mammals, the gene duplication of y that led to development
of g and ε must have taken place earlier than the appearance of
mammals. However, there are no reports of the existence of multiple
y genes in nonmammalian tetrapods.
In addition to the full-length form, a truncated IgY (5.7S) variant,
termed IgY(DFc), has also been reported in some species, including
ducks and turtles (9, 15, 18, 19). In ducks, IgY(DFc) is generated
from the alternative use of a transcriptional termination site located
between the CH2 and CH3 exons of the y gene, which also produces
the full-length IgY (20). A short exon was found to be spliced into
CH2, encoding a secretory tail of IgY(DFc) (20). A similar tran-
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IgY(DFc), containing only CH1 and CH2 domains, is expressed in the serum of some birds and reptiles, such as ducks and turtles.
The duck IgY(DFc) is produced by the same y gene that expresses the intact IgY form (CH1–4) using different transcriptional
termination sites. In this study, we show that intact IgY and IgY(DFc) are encoded by distinct genes in the red-eared turtle
(Trachemys scripta elegans). At least eight IgY and five IgY(DFc) transcripts were found in a single turtle. Together with Southern
blotting, our data suggest that multiple genes encoding both IgY forms are present in the turtle genome. Both of the IgY forms
were detected in the serum using rabbit polyclonal Abs. In addition, we show that multiple copies of the turtle d gene are present
in the genome and that alternative splicing is extensively involved in the generation of both the secretory and membrane-bound
forms of the IgD H chain transcripts. Although a single m gene was identified, the a gene was not identified in this species. The
Journal of Immunology, 2012, 189: 3995–4004.
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scriptional pattern was also observed in the green anole lizard, in
which the secretory tail is not encoded by a separate exon but rather
by the sequence immediately downstream of the CH2 exon (21).
Because Igs play important roles in the adaptive immune system
of all jawed vertebrates (1, 2), they have been extensively studied
in various groups of animals, including reptiles (16, 21–23).
However, little is known about Ig-encoding genes in turtles, except
for the description of cDNA sequences (24, 25) and serological
studies (9, 10, 19). It has previously been shown that IgY(DFc) is
expressed in these species, but it is not known whether the expression of the two isoforms of IgY is mediated through the same
mechanism as in the duck. In this paper, we present a study of the
IgH genes of the red-eared turtle (Trachemys scripta elegans) and
show that IgY(DFc) is expressed from distinct genes and that both
IgY- and IgY(DFc)-encoding genes are extensively diversified.
THE TURTLE IgH GENES
Southern and Northern blotting
The probes for Southern and Northern blotting were prepared using PCR
with primers designed from the above-amplified m, d, and y cDNAs and
labeled with digoxigenin using the DIG-High Prime DNA Labeling and
Detection Starter Kit (Roche). Hybridization and detection were performed
following the manufacturer’s instructions. All Southern and Northern blots
were washed at high-stringency conditions (2 3 30 min in 0.13 SSC
containing 0.1% SDS at 68˚C).
For Southern blotting, 10 mg liver genomic DNA was digested separately with PstI, PvuII, HincII, and DraI (NEB), subjected to electrophoresis on a 0.8% agarose gel, and transferred to a positively charged nylon
membrane (Roche). For Northern blotting, total RNA was extracted from
various tissues, including the heart, liver, spleen, lung, kidney, large intestine, small intestine, testis, and pancreas. The total RNA samples were
used for further purification of mRNA with PolyATtract mRNA Isolation
System (Promega). One microgram mRNA from each tissue was separated
on an agarose/formaldehyde gel and transferred to a nylon membrane.
Western blotting for IgY and IgY(DFc) detection in the serum
Materials and Methods
Animals, isolation of RNA, and reverse transcription
Cloning of the turtle m gene
A pair of primers, JH-forward (59-GGG GAC AAG GAA CAA TGG TGA
CCG TTT CCA-39) and m-reverse (59-ACT TGA TGA AGA GGT CAC
GGG-39), was designed based on conserved sequences among species to
amplify a segment of the turtle m gene. The obtained 1.0-kb PCR product was
cloned into the pMD19-T vector (TaKaRa, Dalian, China) and sequenced.
Amplification of the turtle VH sequences
For 59 RACE, we designed three IgM primers based on the amplified m
gene segment: R1 (59-TGG AGG CAG GGA CTG ATA G-39), R2 (59CCA AGC AAC CCA GTG CGA TAG T-39), and R5 (59-AGG GTA GAG
TGG GAA AAG AGA CG-39). cDNA was synthesized from turtle spleen
and amplified using a 59 RACE kit according to the manufacturer’s
instructions (Invitrogen).
Construction and screening of the mini-Ig–specific cDNA
libraries
A JH-specific primer, JH-primer (59-TGG GGT CAA GGG ACC ATG
GTC ACC GTC ACT-39), was designed based on the most frequently
used JH segment to amplify the C region of turtle Ig H chains using the
39 RACE System (Invitrogen). Total RNA extracted from the spleen and
small intestine was used. PCR products were cloned into the pMD19-T
vector (TaKaRa) to generate cDNA mini-libraries containing the turtle Ig
H chains. The libraries were screened via PCR using IgM-specific primers,
IgM fw50 (59-GAC CGA CAC GAG CCA AGA TAC TA-39), and IgM
rev538 (59-GTC ATT ATC CCA ATC TTT CTC CG-39). A number of
positive and all of the negative clones containing the correct insert size
were sequenced.
Genome walking and amplifications of the d gene
To identify the d gene, genome walking was initiated using two primers,
mTM-1494 (59-AAA CTT TGC TCC ACT CTG TCA CCT GCG-39) and
mTM-1538 (59-TGC CTG TGG AAT GTT CTC AGA CGT CA-39), derived from the 39 end of the mTM2 region. Walking was performed using
the Genome Walker Universal Kit (Clontech) following the manufacturer’s
instructions. For IgD cDNA, we designed IgD-specific primers based on
the obtained genomic sequences to perform 39 RACE PCR. IgD-s17 (59TTC CCT GCT GCA CCC AGA CTG-39) was used as the sense primer
to amplify both secretory and transmembrane IgD. RT-P1 (59-AAC TGG
AAG AAT TCG CGG CC-39), designed based on the adaptor sequence
of the primer NotI-d(T)18, was used as the antisense primer to amplify
secretory IgD, whereas the primer DTM-103 (59-CGG CGC TGT ACA
ACA GGC TGA G-39), which was derived from the predicted IgD transmembrane 1 (TM1) region, was used as the antisense primer to amplify the
transmembrane-encoding IgD H chain.
Sequence and phylogenetic analyses
DNA and protein sequence editing, alignments, and comparisons were
performed using DNASTAR’s Lasergene software suite (26) and Boxshade
software (http://www.ch.embnet.org/software/BOX_form.html). The identity
of turtle VH was identified using IMGT/V-QUEST from the International
ImMunoGeneTics database (IMGT; http://www.imgt.org/IMGT_vquest/
share/textes/). The BLAST program on the National Center for Biotechnology Information Web site (http://blast.ncbi.nlm.nih.gov/Blast.
cgi) was used to identify homologous sequences. Phylogenetic trees were
constructed using MrBayes 3.1.2 (27) and viewed in TreeView (28).
Results
Analysis of the expressed H chain V region sequences
To analyze the expressed VH genes in the turtle, we first amplified
a genomic fragment of the m gene, which was subsequently used to
design primers for 59 RACE PCR with spleen RNA isolated from
one turtle as the template. The resulting PCR products of ∼500 bp
were cloned and sequenced. A total of 190 VH cDNA clones were
identified using IMGT/V-QUEST on the IMGT Web site. Among
these VH sequences, 115 were unique sequences. The phylogenetic analysis revealed the existence of seven VH families (Fig.
1A), according to the 75% identity criterion (29). The alignment
of the amino acid sequences of representatives of each family
is shown in Supplemental Fig. 1. To further ascertain how these
sequences were related to the VH genes found in other species,
a phylogenetic tree of VH genes was constructed using the framework regions, as defined by the IMGT numbering system (30) (Fig.
1B). Unlike human and mouse, in which the VH families have been
classified into three major groups or clans (31, 32), the seven turtle
VH families were clustered into the five following groups: family
3 (G34) grouped into clan I, family 4 (L36) and family 6 (P51)
grouped into clan II, and family 1 (Q4) and family 7 (L59)
grouped into clan III. However, families 2 and 5 were classified
into two additional distinct groups with the VH genes of cartilaginous and bony fish.
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Turtles were purchased from a local Beijing pet market. Total RNA was
isolated from pulverized organs using the TRIzol Kit (Tiangen Biotech,
Beijing, China). Reverse transcription was conducted using Moloney
Murine Leukemia Virus Reverse Transcriptase following the manufacturer’s instructions (Invitrogen). An oligo(dT) adapter primer NotI-d
(T)18 was used (59-AAC TGG AAG AAT TCG CGG CCG CAG GAA T18-39).
The resultant cDNA mixture was used in PCR assays.
Polyclonal Abs specific to IgY CH4 and IgY(DFc) CH2 were prepared
by CoWin Biotech (Beijing, China). Briefly, two peptides (MKNHEGENETNYIT and EQRDGNTDQPTKD) that were respectively derived from
conserved sequences in CH4 of IgY and CH2 of IgY(DFc) were synthesized and used to immunize rabbits. To confirm the specificity of polyclonal Abs, both IgY and IgY (DFc) cDNAs were cloned into pcDNA3.1
and fused with a flag-encoding sequence. The obtained constructs were
transfected into 293T cells, and the cell lysates were subject to Western
blotting. For Western blotting, serum samples were obtained from an adult
turtle and prepared by centrifugation. Serum proteins were separated by
electrophoresis on 10% SDS-PAGE gels under reducing conditions and
transferred to polyvinyl difluoride membranes (Thermo Scientific). Membranes were blocked with 5% nonfat milk and probed with our polyclonal
Abs. The HRP-conjugated secondary Ab used was a goat polyclonal
secondary Ab to rabbit IgG-H&L (Abcam).
The Journal of Immunology
3997
The 115 unique VH clones accounted for a total of 61 unique
CDR3s, which resulted from independent VDJ recombination events.
The lengths of the CDR3s (according to the IMGT numbering system)
among these clones ranged from 7 to 21 aa (Supplemental Fig. 2),
with an average length of 11.4 6 2.5 aa. Although it was difficult to
determine the number of DH segments involved based only on the
expressed CDR3s, an analysis of the FR4 regions suggested that
there were at least 15 distinct JH genes (Supplemental Fig. 3A, 3B).
To analyze whether different turtles or different tissues in one
individual could show different usage of VH and JH segments, 59
RACE reactions were performed separately on the RNA samples
isolated from the spleen and small intestine of a second turtle.
Totally, 94 and 64 clones with unique CDR3 were obtained from
the spleen and small intestine, respectively. Similar to the first
turtle, the VH genes used in the spleen clones belonged to seven
families. However, only the members of families 1–5 were found
in the clones from the small intestine. Compared with the first
turtle, some common and additional JH segments were identified
in both tissues of the new individual, showing different profiles
of JH usage (Supplemental Fig. 3A–C). Also, the JH profiles
appeared to be different in the spleen and small intestine, as apparently some JH segments were used in the spleen but not in the
small intestine (Supplemental Fig. 3B, 3C).
Analysis of the IgH isotypes in the turtle
In birds and reptiles such as lizards, IgH transcripts of all isotypes
can be detected in the spleen plus intestine (21, 33, 34). To analyze
the IgH isotypes in the turtle, we designed a sense primer based on
the sequence of the most frequently used JH segment, which was
used in 39 RACE using the RNA isolated from the spleen and
small intestine. We amplified two major bands of 1.5 and 0.9 kb
from both tissues. These bands were cloned to construct Igspecific mini-cDNA libraries. A subsequent analysis showed that
all of the amplified 0.9-kb fragments from both tissues were IgY H
chains containing only two CH domains. The analysis of 960
clones in the spleen library containing the 1.5-kb inserts revealed
915 IgM H chain, 29 IgY (with 4 CH domains), and 16 non-Ig
sequences. Additionally, 458 clones of the small intestine libraries
were analyzed and showed 446 IgM H chain, 3 IgY (with four CH
domains), and 9 non-Ig sequences. No IgD or IgA H chains were
identified in either library.
The turtle m gene
The IgH C region of turtle m encodes 451 aa and shows overall
sequence identities of 47.3 and 45.3% to the gecko and lizard
m genes, respectively. The alignment of the turtle m gene (inferred amino acids) and those of other species showed a conserved
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FIGURE 1. Phylogenetic analysis of turtle VH genes. (A) Cluster (family) analysis of the turtle VH genes. (B) Phylogenetic analysis of the VH genes in jawed
vertebrates. The trees were constructed using MrBayes. The scale bars show the genetic distance, and the credibility value of each node is shown. The clans are
labeled with Roman numerals. Human TRBV1 (L36092) is used as an outgroup. The accession numbers of the sequences used are as follows: human: V1,
L22582; V2, X62111; V3, X92206; V4, Z12367; V5, M99686; V6, X92224; V7, AB019437; mouse: V1, AF304557, V2, J00502; V3, K01569; V4, V00774; V5,
AJ851868; V6, AJ972404; V7, X03253; V8, AC073939; V9, L14368; V10, AF064445; V11, AJ851868; V12, AC073590; V13, X55935; V14, X55934; V15,
U39293; V16, AC073563; chicken: AB233003.1; pig: V1, AF064686; Xenopus laevis: V1, Y00380; V2, M30025; V3, M24675; V4, M24676; V5, M24677; V6,
M24678; V7, M24679; V8, M24680; V9, M24681; V10, M27254; V11, M27255; zebrafish: BX649502; and nurse shark: V1, M92851; V2, L38965.
3998
THE TURTLE IgH GENES
distribution of cysteine and tryptophan residues (Supplemental
Fig. 4). The turtle C domains all had the conserved Cys residues
for intra- and inter-H chain disulfide bonds and for covalent
binding of the L chain. The turtle sequence also had the conserved
Cys residues found in CH3 and the secretory tail that covalently
polymerizes pentameric IgM in mammals (35). Six putative Nlinked glycosylation sites (N-X-S/T) were present in the turtle,
and three of them are also highly conserved in mammals. The
transcripts of the membrane-bound form of the IgM H chain were
also amplified using 39 RACE (Supplemental Fig. 4).
Although RT-PCR showed that IgM (both membrane-bound and
secretory forms) was expressed in all of the tissues examined (data
not shown), Northern blotting confirmed that the gene was highly
expressed in the spleen and the large intestine and relatively weakly
in the small intestine and kidney, but barely detectable in other
tissues (Fig. 2).
We also performed Southern blotting using one probe of the
CH1–3 sequence and one probe of a single CH1 sequence, which
revealed that it is likely that there is only a single copy of m in the
turtle genome (Fig. 3).
Multiple copies of d genes are present in the genome
Despite its absence in birds, the d gene, consisting of up to 11 CH
exons, has been identified in several reptiles (21, 22). We failed
to obtain d gene transcripts from the constructed cDNA minilibraries, probably due to a low level of expression. To clone the
d gene, we performed genome walking starting from the 39 end of
the mTM2 region toward downstream regions. A dCH-encoding
exon was identified ∼6.2 kb from mTM2. BLAST searches and
phylogenetic analysis (Fig. 4) confirmed this exon to be turtle d.
Through continuous walking experiments, we obtained a d genomic sequence that spanned ∼11 kb of DNA and consisted of
six CH exons and a typical TM1 exon (Fig. 5). Although the
walking process was based on overlapping sequences, nucleotide
mismatches were often observed in the overlapping regions. This
result suggested that there could be more than one d allele or d
FIGURE 3. Southern blotting detection of the turtle IgH chain C region genes. Ten micrograms of digested liver genomic DNA were loaded in each lane.
The sequences of the probes are indicated at the bottom, whereas the restriction enzymes used are indicated on the top. A DNA marker was indicated on the
left. The blot was washed under high-stringency conditions.
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FIGURE 2. Northern blotting analysis of the turtle IgH genes. One microgram of mRNA was loaded in each lane. cDNAs of m, d, and y constant regions
were used as probes. An RNA marker was indicated on the left. The blot was washed under high stringency conditions. L-int, Large intestine; S-int, small
intestine.
The Journal of Immunology
3999
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FIGURE 4. Phylogenetic analysis of the turtle IgH constant genes. The credibility value of each node is shown. The amino acid sequences were used for
tree construction. Unless otherwise indicated, all CH domains of Ig classes other than IgD are used. For those species that express IgD (and IgW) longer
than four CH domains, only the first four CH domains were used. The accession numbers of the sequences used were as follows: m: nurse shark: M92851;
skate: M29679; rainbow trout: X65261; zebrafish: AF281480; X. laevis: X15114; X. tropicalis: AAH89670; axolotl: A46532; platypus: AY168639; duck:
AJ314754; chicken: X01613; human: X14940; mouse: V00818; opossum: AAD24482; lizard: ABV66128; gecko: ABY74509; catfish: X52617; ratfish:
AAC12892.1; cod: CAA41680.1; waltl: CAE02685.1; pig: BAI82566.1; cow: AAN60017.1; horse: AAU09792.1; rabbit: (Figure legend continues)
4000
IgY- and IgY(DFc)-encoding genes in the turtle
Previous studies examining turtle serum Igs have suggested the
presence of both intact IgY and truncated IgY (DFc) (9, 10, 19).
Four CH domain-encoding IgY cDNA transcripts were obtained
from the screening of the Ig-specific mini-libraries with 1.5-kb
inserts, whereas all 0.9-kb-amplified PCR bands were shown to be
IgY(DFc) H chain cDNAs. Surprisingly, the sequencing of more
clones also revealed extensive diversification of both IgY and IgY
(DFc) subclasses; at least eight IgY and five distinct IgY(DFc)
cDNAs could be identified in the same animal (Fig. 7). This result
is consistent with Southern blotting results, in which both multiple- and single-CH probes detected multiple bands (Fig. 3).
To our surprise, sequence comparisons of turtle IgY and IgY
(DFc) revealed that IgY(DFc) was not expressed from the IgYencoding genes because they displayed distinct sequences (Fig. 7).
This result is different from the case in ducks, in which both intact
IgY and IgY(DFc) H chain cDNAs are expressed from the same
gene using different transcriptional termination sites (15, 20).
Northern blotting was performed to further examine the expression of the turtle IgY- and IgY(DFc)-encoding genes using an
IgY CH1-CH3 cDNA probe. Because all of the cloned IgY and
IgY(DFc) genes shared a highly similar CH1 exon, this probe was
expected to detect both the intact and truncated transcripts. The
hybridization revealed that intact IgY transcripts were detected in
the spleen, kidney, and large intestine, whereas the IgY(DFc)
transcripts were mainly detected in the large intestine (Fig. 2).
We developed rabbit polyclonal Abs specific to turtle IgY and
IgY(DFc). The specificity of the rabbit polyclonal Abs was first
confirmed by Western blotting on the cell lysates of 293T cells,
which were transfected by IgY and IgY(DFc) cloned in pcDNA3.1
(Fig. 8A). The polyclonal Abs were then used in the Western
blotting detection of serum proteins. The results showed that both
IgY and IgY(DFc) were present in the serum with molecular mass
of 62 and 38 kDa, respectively, both of which were in accordance
with predicted results (Fig. 8B).
Discussion
In this paper, we performed a detailed study of IgH genes in the redeared turtle and showed that this species expressed three IgH
isotypes, including m, d, and y. Unlike the m gene, both the d and y
genes are present as multiple copies. Surprisingly, unlike in ducks,
in which the same y gene produces both IgY and IgY(DFc) H
chains, multiple distinct genes have been developed to encode
truncated IgY(DFc) in the turtle. This study thus reveals a more
complex set of Ig genes in the turtle than other reptiles and birds.
The red-eared turtle has a diploid genome, with a total of 50
chromosomes (36). Neither the genome sequence nor a bacterial
artificial chromosome library is available for this species. It is
therefore currently not possible to determine the exact copy
numbers of the d and y genes or how these genes are organized in
the genome.
The d gene is as ancient as the m gene, and it has now been
identified in nearly all jawed vertebrates except birds and a few
mammalian species (3, 5, 37–40). An exceptional feature of the d
gene compared with other isotypes appears to be its highly variable structure with regard to the number of CH exons throughout
the evolution of jawed vertebrates, which can range from 2 in
mice to 16 in zebrafish (41–43). A large number of studies have
revealed that the d gene tends to have more CH-encoding exons in
nonmammalian species [including the most primitive living
AAA64251.1; panda: AAX73309.1; echidna: AAN33013.1; and dog: AAEX02009527. d: catfish: U67437; X. tropicalis: DQ350886; human: BC021276;
platypus: ACD31540; Pleurodeles waltl: CAL25718; horse: AY631942; mouse: J00449; lizard: ABV66130; gecko: ABY67439; pig: AAN03672.1; cow:
AAN03673.1; panda: AAX73311.1; dog: XM548647; rainbow trout: AAW66977.1. g: human: J00228; mouse: J00453; platypus: AY055781; opossum:
AAC79675; horse: CAC44760; pig: AAA51294.1; cow: ABE68619.1; rabbit: AAB59265.1; panda: AAX73307.1; dog: AF354264; and echidna:
AAM61760.1. a or x: chicken: S40610; cow: AF109167; duck: AJ314754; human: J00220; mouse: J00475; opossum: AAD21190; gecko: ABG72683;
platypus: AY055778; X. laevis: BC072981; X. tropicalis: AAI57651; pig: ADD51207.1; horse: AAP80145.1; rabbit: S09264; panda: AAX73304.1; dog:
L36871; echidna: AAN33012.1; and axolotl: CAO82107.1. y: chicken: X07175; duck: X78273; X. laevis: X15114; X. tropicalis: AAH89679.1. Axolotl:
S31436; lizard: ABV66132; gecko ACF60236; waltl: CAE02686.1. ε: human: J00222; mouse: X01857; pig: AAC48776.1; horse: AAA85662.1; rabbit:
AY386696; cow: AY221098; platypus: AY055780; opossum: AAC79674; panda: AAX73306.1; dog: L36872; echidna: AAM45140.1. v: sandbar shark:
U40560; lungfish: AF437727; and nurse shark: U51450. z/t: zebrafish: AY643752 and rainbow trout: AY872256.
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gene in the turtle genome, and it is possible that the genomic d
sequence we obtained was chimeric.
We subsequently performed 39 RACE to amplify the d H chain
transcripts using primers derived from the dCH1 exon. This amplification resulted in two major bands of 2.2 and 1.0 kb. Sequencing of the 2.2-kb bands (35 clones) revealed multiple, highly
similar (sharing .96% sequence identity), but distinct IgD H
chain transcripts. These sequences all encoded secretory IgD H
chains with six CH domains in which a secretory tail was encoded
by the sequence immediately downstream of dCH6. Among the 35
cloned cDNA sequences, there were 25 distinct clones that shared
a sequence identity of ,99% (Fig. 6). It was difficult to determine
exactly how many distinct d genes were transcribed based on these
cDNA sequences amplified using PCR because the PCR process
may create chimeric sequences when more than one highly similar
sequence is present in the template. To confirm whether multiple d
genes were present in the turtle genome, we therefore performed
Southern blotting using both dCH1-3 and dCH1 cDNA as probes,
both of which, as expected, detected multiple bands (Fig. 3). This
strongly suggests the presence of multiple copies of d genes in the
turtle genome, although some distinct bands could potentially be
explained by allelic polymorphisms. To analyze the 1.0-kb amplified bands, 12 clones were sequenced, revealing three different
transcripts. All three transcripts encoded two CH domains (dCH1
and dCH3). Two of these transcripts differed from each other by
only 1 nt, but both showed 5-nt differences from the third transcript. Surprisingly, none of the three short transcripts could be
matched to any of the 35 cloned 2.2-kb transcripts because they
contained several consistent nucleotides distinct from the long
transcripts (Fig. 6).
To analyze the transcripts encoding the d membrane-bound
form, RT-PCR was performed using primers derived from dCH1
and dTM1 and spleen cDNA as the template. This amplification
generated four bands of 1.0, 1.3, 1.6, and 2.0 kb. All bands were
cloned and sequenced and showed the presence of three CH
domains, four CH domains, five CH domains, and six CH domains,
respectively (Fig. 5). Similar to the secretory IgD H chain transcripts, the transcripts of each length did not have a unique sequence and appeared to be transcribed from distinct d genes.
Northern blotting showed that the turtle d gene was only
expressed in the spleen, and in addition to a major band, some
weaker bands were also observed (Fig. 2), confirming the alternative splicing of the d gene.
THE TURTLE IgH GENES
The Journal of Immunology
4001
mammal, the duck-billed platypus, in which 10 CH-encoding exons
have been identified for its d gene (17)], but no more than 3 in
placental mammals (44).
The d gene has previously been analyzed in three reptilian
species, including the green anole lizard, the leopard gecko, and
the Chinese soft-shelled turtle (21, 22, 25). In the first two species, the d gene contains 11 CH-encoding exons; however, in the
green anole lizard, only the first 4 CH exons are included in the
IgD H chain transcripts (21). In the Chinese soft-shelled turtle,
six CH-encoding exons were observed in cloned IgD H chain
cDNA (25). Additionally, two d genes were observed in a single
IgH gene locus in the leopard gecko, whereas only a single d
gene was found in the green anole lizard (21–23). The current
study revealed the presence of multiple copies of the d gene in
the genome of the red-eared turtle, and alternative splicing was
extensively involved in gene expression, suggesting that IgD
may play a significant role in immunological defense of turtles.
The d gene has been thought to be functionally redundant and
merely act as a genetic backup for m as d-knockout mice do not
show apparent immunological defects (45). Recently, however, it
was shown that IgD regulates a surveillance system at the interface between immunity and inflammation and that this
mechanism appears to be conserved in jawed vertebrates (46). In
addition to enhancing mucosal immunity, secretory IgD is able to
bind basophils and other innate immune cells to stimulate release
of immunoactivating, proinflammatory, and antimicrobial mediators (46). Although it is not known how the multiplication of the
turtle d genes occurred, these multiple copies of d genes are most
likely located together within a common IgH locus, as only
a single m gene was observed in the turtle. It may be expected that
some of the d genes would be expressed through class-switch
recombination, which would generate IgM-IgD+ B cells in the
turtle. As multiple cDNA transcripts encoding secretory IgD were
cloned, it is reasonable to speculate that IgD would mediate immunity or inflammation in this species. More importantly, the turtle
d genes show extensive sequence variations, especially at some
functional sites (for instance, glycosylation sites), which may indicate functional divergence among these different d-encoding
genes.
This study also generated two interesting findings regarding the y
genes in the turtle. First, similar to the d gene, the y gene has also
been extensively diversified to generate multiple subclass-encoding
genes. Second, distinct y genes have been developed in the turtle for
the production of IgY(DFc). This situation is clearly different from
the case of ducks, in which the same y gene is used to produce both
IgY and IgY(DFc) H chains (20). Extensive diversification of the y
gene has significant evolutionary implications when considering the
gene to be the evolutionary precursor of mammalian g and ε,
encoding IgG and IgE, two functionally divergent Ig classes (15).
The presence of multiple y genes in the turtle suggests that the
functional divergence of IgY may have already been initiated in
nonmammalian tetrapods. Indeed, our unpublished data also
revealed the presence of multiple IgY subclasses at both the DNA
and protein level in other reptiles (such as snakes and crocodiles).
Because IgY can mediate humoral responses, it would be interesting to investigate whether some IgY subclasses are specialized
in mediating allergic response in reptiles, similar to the ability of
IgE in mammals.
IgY(DFc) is a truncated form of IgY that lacks the last two CH
domains compared with four intact CH domains in IgY. The Fc
region is missing in this truncated form, which is believed to have
only Ag-binding activity but no effector functions. IgY(DFc) is
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FIGURE 5. The gene locus and RNA splicing patterns of turtle IgD. This figure was drawn to show the splicing pattern of the turtle genes. As there are
multiple d genes in the turtle, these splicing variants are not necessarily derived from a single d gene. Solid boxes indicate exons. d, IgD-encoding gene; m,
IgM-encoding gene; mIgD, membrane IgD; sIgD, secretory IgD; SP, secretory tailpiece.
4002
THE TURTLE IgH GENES
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FIGURE 6. Amino acid sequence alignment of the cloned IgD transcripts. Conserved cysteine and tryptophan residues are shaded. Potential N-linked
glycosylation sites are underlined. Dots are used to denote identical amino acids, whereas dashes are used to adjust the sequence alignments. sIgD, Short
IgD.
produced at the protein level in various birds and reptiles, including ducks and turtles (15, 18, 39, 47). The present study
clarifies that turtles possess a different mechanism than ducks for
the production of the truncated IgY form. Nonetheless, the most
interesting question of the immunological benefits of IgY(DFc)
remains to be addressed.
The Journal of Immunology
4003
This study also adds the turtle to the list of reptilian species,
including lizards and snakes (21, 48), in which an IgA-encoding
gene could not be identified. The consistent lack of IgA in reptiles
is surprising, because IgA, as a specialized Ab class in mucosal
immunity, has been observed in all mammals and birds examined
FIGURE 8. Western blotting detection of turtle IgY and IgY(DFc) in
serum. (A) Specificity confirmation of the developed polyclonal Abs.
Fragments of IgY and IgY(DFc) were separately cloned into pcDNA3.1
and fused with a flag sequence, which were transfected into 293T cells.
The cell lysates were subject to Western blotting. 1, IgY detected by the
developed polyclonal Abs; 2, IgY detected by anti-flag Abs; 3, IgY(DFc)
detected by the developed polyclonal Abs; 4, IgY(DFc) detected by antiflag Abs. (B) Detection of serum IgY and IgY(DFc) by the developed
polyclonal Abs.
so far (1, 4). Another Ab class, IgX, has also been implicated in
mucosal immunity of amphibians (49, 50). Even in the most
primitive bony vertebrates, teleost fish, IgT, was found to be
functionally specialized in mucosal immunity (51). There are two
questions with regard to the absence of IgA in reptiles: 1) why or
how the IgA encoding gene was lost? and 2) which Ab class is
used at the mucosal sites in the absence of IgA? The answer for
the first question may be derived from a comparative analysis of Ig
genes in both reptiles and birds. Reptiles are believed to share
a direct common ancestor with modern birds, among which all
examined species show an inversion of the IgA-encoding gene and
an absence of the IgD-encoding gene (34, 39, 52, 53). It seems
that the IGHC gene locus in both reptiles and birds has experienced some genetic rearrangements during their evolutionary
process. It is not known whether these rearrangements are accidental or deliberate events but somehow lead to loss of d or a genes
or inversion of a gene in these species. Our Northern data may
provide some potential answers to the second question. As a strong
IgM expression was detected in the large intestine and also in the
small intestine, it may be speculated that IgM is able to compensate
for the absence of IgA at mucosal sites in turtles. This is a reasonable speculation, as only IgM and IgA share the same polymeric Ig
receptor, which allows transport into mucosal secretions (54).
In conclusion, this study provides a detailed analysis of IgH
genes in the red-eared turtle, and it aids in the understanding of how
flexible IgH genes can be in different species and the evolutionary
process of IgH genes in tetrapods.
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FIGURE 7. Amino acid sequence alignment of different IgY and IgY(DFc) H chain transcripts. Conserved cysteine and tryptophan residues are shaded.
Potential N-linked glycosylation sites are underlined. Dots are used to denote identical amino acids, whereas dashes are used to adjust the sequence
alignments.
4004
Disclosures
The authors have no financial conflicts of interest.
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