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Molecular characterization of the VH repertoire in Canis familiaris
Veterinary Immunology and Immunopathology 137 (2010) 64–75
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Research paper
Molecular characterization of the VH repertoire in Canis familiaris Yonghua Bao a,b , Yongchen Guo b , Shuqi Xiao c , Zhihui Zhao a,∗ a b c
College of Animal Science and Veterinary Medicine, Agricultural Division, Jilin University, 5333 of Xi’an Road, Changchun, Jilin 130062, PR China State Key Laboratories for AgroBiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangzhou, Guangdong 510006, PR China
a r t i c l e
i n f o
Article history: Received 26 November 2009 Received in revised form 19 March 2010 Accepted 16 April 2010 Keywords: Immunoglobulin VH Dog Genome Diversity
a b s t r a c t The immunoglobulin heavy chain variable region in vertebrates is composed of many genes. However, the gene number and the extent of diversity among the VH gene segments vary with the species. To gain insight into the dog VH repertoire, we analyzed its genome sequence. Eighty VH, six DH and three JH segments were mapped to a 1.28 Mb region of the dog chromosome 8. Forty-one of these VH genes were potentially functional, and 39 were pseudogenes. The sequence similarity analysis suggests that the dog IgH locus harbors three VH gene families, VH1 (including 76 members), VH2 (including 3 members) and VH3 (including one member), that are classified into mammalian clan III, I and II, respectively. Subsequently, 111 cDNA positive clones from dog 26 , 22 ␥, 23 ␣, 16 ␦ and 24 chains containing almost full-length VH, DH and JH segments were selected at random. All of the sequences, except for clone G6, which belonged to the dog VH2 family, originated from the dog VH1 family. The germline segments, VH62 (human VH3-21 homology) and VH44 (human VH3-23 homology), seemed to be preferentially used. Our results suggest that the dog VH repertoire appears to be derived from limited germline gene families, and its diversity may rely on junctional diversity and somatic hypermutation. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The immune system can produce an enormous antibody repertoire in anticipation of large and diverse groups of potential pathogens (Kabat et al., 1991). The recognition of an antigen involves dimers formed by the variable domains of the heavy and light chains (VH and VL) (de Bono et al., 2004), and in some cases only heavy chains are used
Abbreviations: IgH, immunoglobulin heavy chain (Ig: immunoglobulin and H: heavy chain); VH, heavy chain variable domain (V: variable domain and H: heavy chain); VL, light chain variable domain (V: variable domain and L: light chain); DH, heavy chain diversity domain (D: diversity domain and H: heavy chain); JH, heavy chain joining domain (J: joining domain and H: heavy chain); IGHV, immunoglobulin heavy chain variable region (IG: immunoglobulin, H: heavy chain and V: variable region). ∗ Corresponding author. Tel.: +86 431 87836156; fax: +86 431 87836156. E-mail address: [email protected] (Z. Zhao). 0165-2427/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2010.04.011
(Hamers-Casterman et al., 1993). Therefore, the variable region determines the diversity of immunoglobulins. The huge variety of immunoglobulin specificities is produced by (1) the inherited set of variable (V), diversity (D) and joining (J) gene segments (germline diversity), (2) the different rearrangements of these V, D and J gene segments (combinatorial diversity), (3) the insertion and deletion of random nucleotides (junctional diversity), and (4) the introduction of somatic mutations (Berek et al., 1991). In mammals, the gene segments of VH domains of many species have been well described. Historically, the human V region is composed of 7 germline VH gene families, including approximately 100 genes and pseudogenes, approximately 30 DH gene segments, and 6 JH gene segments (van Dijk et al., 1993). In the mouse, 4 J genes, 10–13 D genes and about 150 germline V genes forming 16 families have been identified (Chevillard et al., 2002; de Bono et al., 2004; Johnston et al., 2006; Mainville et al., 1996; Ye, 2004).
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Studies of sheep (Ovis aries), cattle (Bos taurus) and pig (Sus scrofa) suggest that these species express one family of VH and rely on somatic hypermutation and gene conversion to diversify the primary repertoire of antibodies (Berens et al., 1997; Dufour et al., 1996; Sinclair et al., 1997; Sun et al., 1994). In a more recent study, the ovine VH repertoire was found to be more complex and diverse than previously thought, being composed of at least nine VH gene families. This finding suggests that the diversity of sheep VH germline genes may play a more significant role in the diversification of the primary antibody repertoire than previously suggested (Charlton et al., 2000). Similarly, at least three VH gene families were identified in the equine IgH locus (Almagro et al., 2006), in contrast with earlier reports of a limited diversity of the VH germline gene repertoire (Butler, 1998). In this study, we examined the variable region of the dog IgH locus, in which 80 germline VH segments formed three gene families. As expected, the dog VH1 family (human VH3 family analogue) was predominant at both the genome and expression levels. Sequence analysis indicated that CDR3 variability and somatic hypermutations may contribute greatly to dog antibody diversification. 2. Materials and methods 2.1. Acquirement of dog germline V, D and J segments Available cDNA sequence (GenBank accession number XM 844149) of the dog VH gene was used to search for corresponding genomic sequences by a conventional TBLASTN approach in the Ensembl database (www.ensembl.org). A region of approximately 1.28 Mb from dog chromosome 8 was found to contain potential VH, DH and JH gene segments. Combined with the specificity of the conserved recombination signal sequence (RSS) motif adjacent to the V, D or J gene segments, FUZZNUC, an on-line software package(http://anabench.bcm.umontreal.ca/anabench/ Anabench-Jsp/Applications/fuzznuc.jsp?APPLICATIONID= 81&APPLICATIONNA ME=fuzznuc), was used to identify these gene segments. 2.2. Dogs Three healthy Chinese rural dogs were selected for this experiment. The dogs were euthanized after the experiment was complete. The study was conducted with the approval of the State Ethical Commission, and met the
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International Guiding Principles for Biomedical Research Involving Animals. 2.3. Isolation of total RNA Spleen specimens were collected and frozen immediately in liquid nitrogen. Total RNA was extracted using the TRlzol Reagent (Invitrogen, USA). Equal amounts of RNA samples from the three dogs were pooled to amplify dog cDNA sequences. 2.4. Primer design Based on the characterized canine IGHC genes (GenBank accession numbers: IgM-AAEX02009527, IgG-AF354264, IgA-L36871, IgD-XM548647, and IgE-L36872), specific primers for the amplification of five classes of the immunoglobulin V region are displayed in Table 1. 2.5. 5 RACE of IGHV genes The 5 RACE system (Invitrogen, USA) was utilized to determine the immunoglobulin V region transcripts. First-stranded cDNA was synthesized using SuperscriptTM II reverse transcriptase with the Igs-GSP1 primer (Table 1). Double-stranded DNA (dsDNA) was obtained from cDNA by PCR with the primer sets, AAP (5 -ggccacgcgtcgactagtacgggggggggg-3 ) and Igs-GSP2 (Table 1). Nested PCR was performed with the primers, UAP (5 -ggccacgcgtcgactagtac-3 ) and Igs-GSP3 (Table 1). 2.6. Sequence analysis Editing and comparison of all sequences were performed using the DNAstar program. Multiple sequence alignments were made by the Clustal W method and optimized manually. 2.7. Diversity index Referring to the methods of Kabat et al. (1991), diversity index (DI) is the number of different amino acids at a particular position divided by the frequency of the most common amino acid at that position. 2.8. VH genes family definition Immunoglobulin VH genes were divided into families based on their nucleotide similarities. Criteria have been
Table 1 Primer sequences used in the 5 RACE system. GSP1 IgM IgG IgA IgD IgE
GSP2
5 -gaaccaagacacggaaat-3 5 -agggcactgtcaccat-3 5 -tgcttccagtcttcg-3 5 -tccggtatctttagcatc-3 5 -aggtcttgttgatggc-3
GSP3
5 -gggcacgttcacagatttgtcaccat-3 5 -gcggacggacgggaaggtg-3 5 -ctggatgggttccttccctttgg-3 5 -tgtcgtgacccgcacagaat-3 5 -tggctgacgatgctgtggag-3
5 -ggaaggtcttaatgtcctggttgtt-3 5 -gccagtgggaaaaccgaggg-3 5 -ggcagaggctcagcgggaacac-3 5 -gaccaagggagcagaagtgacgat-3 5 -gcgatgttgtctttacagcaggaggc-3
GSP1 was the gene specific primer for cDNA synthesis. GSP2 and GSP3 were antisense primers for PCR amplification. Sense primers (AAP and UAP) were supplied in this system.
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established that the same family members share more than 80% nucleotide identity, those sequences with less than 70% identity belong to different families, and those having between 70% and 80% similarity are examined on a case-by-case basis (Brodeur and Riblet, 1984). 3. Results 3.1. VH genes 3.1.1. Germline VH segments The publication of dog genome information in the Ensembl database allowed us to annotate its germline VH, DH and JH minigenes. A total of 80 VH segments were identified and located in a 1.2Mb region at the end of the dog chromosome 8 (Fig. 1). No significant matches were found outside of this region. In order to clarify the relations among dog germline VH segments, all the sequences were processed for pair comparison (results not shown). Considering sequence truncations (VH5, VH42, VH49 and VH76), we placed sequences having greater than 70% identity in the same family, the dog VH1 family composed of the upper 76 sequences shown in Fig. 2. VH51, VH64 and VH66, which showed greater than 80% nucleotide similarity, were classified into the dog VH2 family. For VH80, an identity values below 70% was found with all other sequences, indicating that it belongs to a single member family called dog VH3 (Fig. 2).
3.1.2. Prediction of the functional VH genes We have defined a VH gene as potentially functional if the gene retains an open reading frame (ORF), leader exons, a downstream RSS, and essential amino acid residues such as Cys residues, even though no corresponding transcripts were identified in later experiments. The gene was considered a pseudogene if it contained frame shifts or in-frame stop codons (Lefranc, 1998). Based on the above criteria, of 80 canine germline VH segments, 41 were regarded as potentially functional genes whose formal products are shown in Fig. 3. The remaining 39 segments showed various structural defects. 3.1.3. VH transcripts We investigated the usage of germline VH gene segments. A total of 111 positive clones from 26 , 22 ␥, 23 ␣, 16 ␦, and 24 chains containing almost full-length variable regions were obtained by 5 RACE-PCR. All the sequences have been deposited in the GenBank database and assigned accession numbers FJ197707–FJ197817. Comparing these expressed VH sequences with germline segments, major differences were found in the base composition. The differences were attributed to somatic hypermutation or individual differences. It is difficult to make an accurate assessment of the use of germline VH segments, but certain segments may be used more frequently than others. For example, there are 30 VH transcripts possessing high similarity with the germline VH62 (human VH3-21 homology). Another 26
Fig. 1. Dog IGHV locus. The organization of dog IGHV genes is showed to scale. Forty-one functional VH genes are shown as filled bars; 39 VH pseudogenes are represented with open bars and attached with the letter P; DH and JH are indicated with grey boxes and C genes with a black box. These VH genes are numbered based on the family to which they belong and their position in the locus. The number before VH refers to the family, while the number after VH denotes the genomic position.
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Fig. 2. Multiple sequences alignment of all the dog germline VH segments. 80 germline VH genes in dog genome were divided into three VH families, where family 1 comprises the upper 76 sequences, family 2 includes VH51, VH64 and VH66, and family 3 contains VH80. At the end of each sequence, the serial number of the gene is given. Identifies with respect to the majority of the nucleotides are represented by dots; gaps are represented by dashes. Designation of framework regions (FR) and complementarity-determining regions (CDR) are based on the IMGT numbering system (http://imgt.cines.fr/).
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Fig. 2. (Continued )
transcripts showed similarity with germline VH44 (human VH3-23 homology). The other segments were duplicated less than 13 times. Undoubtedly, the most expressed is the dog VH1 family, i.e. the human VH3 family analogue. From 111 transcripts, only clone G6 was derived from the dog VH2 family. In addition, no transcript of dog VH3 family was found.
3.1.4. High diversity index of the expressed VH genes The diversity index (DI) in the variable region (FR1–FR3) from expressed VH genes (excluding clone G6) is shown in Fig. 4. The DI of chain ␣ is the highest at 109.25 in the position 57 amino acid, followed by 91.2 ( in position 59), 89.25 (␥ in position 57), 70.57 ( in positions 50 and 54) and 35.2 (␦ in positions 50 and 54). As expected, variation was concentrated in the CDRs.
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Fig. 2. (Continued ).
3.2. DH genes 3.2.1. Germline DH segments Six germline DH segments of the dog were acquired and shown in Fig. 5. Except for DH5 with 3 -RSS including a 13-bp spacer, other five DH segments have a 5 - and 3 RSS with a 12-bp spacer. In addition, DH2, 3, 4, 5 and 6 have conserved 3 heptamers (CACAGTG). 5 heptamers of DH3, 4, 5, 6 and the 3 heptamer of DH1 differ from the consensus (5 : CACTGTG and 3 : CACAGTG) by A, G, G, A and CA, respectively. Consistently, the 5 and 3 nonamers of each DH gene segment are T-rich and A-rich, respectively. 3.2.2. DH transcripts The cDNA transcripts of the D region were compared with genomic sequences. Clones possessing <5 bp
coincidence with germline segments were not involved. Representing clones that utilized longer and relatively definite DH1–DH6 segments are presented in Fig. 6. Only clone G102 has the full-length coding region of the germline segment DH2. Various numbers of nucleotides removal and addition were found at the junction of VH, DH and JH segments. The addition of nucleotides was prominent. N-region additions were located between V–D junctions, as well as between D–J junctions, and ranged from 1 to 23 bp in length. A strong correlation was apparent between the length of the CDR3 and the number of N nucleotides. Most of the P-region additions, one or two nucleotides in length, were also identified. Nucleotide deletions from the D segments were found at either the 5 or the 3 ends, or from both the 5 and 3 ends. In addition, the D region of clone G118 seems to be a
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Fig. 3. Alignment of deduced amino acid sequences of 41 dog functional VH genes. The amino acid sequences were deduced from the obtained germline VH genes based on the codon usage. All positions where the amino acid residues are identical to those of majority are shown as small dots. Gaps and missing data are indicated by hyphens. The Designation of framework regions (FR) and complementarity determining regions (CDR) referred to the IMGT numbering system (http://imgt.cines.fr/).
fusion of DH3 and DH1. Interestingly, two groups of transcripts involved in germline DH4, clones AA5 and AA28 and clones E7, E21, E28 and E72 showed an identical CDR3.
of somatic mutation. In addition, this panel also illustrates the predominant usages of two JH segments, 1 and 2, in the rearrangements. 4. Discussion
3.2.3. Length and amino acid composition of CDR3 CDR3 lengths of the sequences obtained in this work ranged from 6 to 26 amino acid residues (solid column in Fig. 7), with a peak of 14 residues. Most of the loops ranged from 7 to 17 amino acids. The average length was 13.1 ± 3.59 residues. Three critical amino acids of canine CDR3 loops were analyzed: cysteine, glycine and serine residues. Dog CDR3 loops had 17 cysteine residues (1.2%), 149 glycine residues (10.41%), and 103 serine residues (7.2%) out of 1431 amino acids analyzed. In cattle, sheep, pigs and horses, the proportions of cysteine and glycine residues were 0.2%, 1.2%, 4.3%, and 6.4% and 17.0%, 13.2%, 12,0%, and 20.5%, respectively (Almagro et al., 2006). 3.3. JH segments There were three germline JH gene segments found in the dog IgH locus (Fig. 8). JH1, JH2 and JH3 segments were conserved in the 3 region, whereas in V–J junctions with 4, 3 and 5 different amino acid residues before tyrosine (Y) and tryptophan (W), respectively. In addition, JH1 and JH2 segments share higher amino acid homology than JH3. All JH encoded sequences were readily assigned to germline JH1-JH3 segments (Fig. 9). The small nucleotide variations observed in these JH sequences may be the effect
In the present study, we have characterized the dog immunoglobulin VH repertoire. Chromosome 8 of the dog displayed 80 VH genes, about half of which are pseudogenes (39/80). In the pool of human immunoglobulin VH gene segments, pseudogenes amount to roughly 30% of the total number of genes (Matsuda et al., 1998; VargasMadrazo et al., 1995). Among 141 VH gene segments from the mouse strain C57BL/6J, 92 are functional(de Bono et al., 2004). Pseudogenes in the Ig V region constitute a potential donor pool for gene conversion during evolution (Almagro et al., 1996; Haino et al., 1994; Vargas-Madrazo et al., 1995). Although no gene conversions were found in the 39 dog VH pseudogenes, pseudogenes reserves during evolution should be useful for dog immunoglobulin diversity. Alignment of VH gene sequences indicates that the vast majority have a high nucleotide similarity. The largest majority of VH genes are of the dog VH1 family, and the remainder are organized into two other groups, dog VH2 and VH3 family, with a few VH genes each. The three VH gene families were interspersed throughout the dog IgH locus. Earlier publications described the VH gene families as clans (Kirkham et al., 1992; Ota and Nei, 1994). The mammalian VH genes are classified into three clans, I, II, and
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Fig. 4. Comparison of the diversity index (DI) of VH transcripts for five classes of immunoglobulins. Horizontal axis represents the amino acid residue position. Vertical axis indicates DI. The calculation of DI is described in Kabat et al. (1991). DI is defined as the number of different amino acids at one position divided by the frequency of the most common amino acid at that position. The DI at position 57 of ␣ transcripts is 109.25, followed by 91.2 ( at position 59), 89.25 (␥ at position 57), 70.57 ( at positions 50 and 54) and 35.2 (␦ at positions 50 and 54).
III. Clan III is considered the most ancient (Johansson et al., 2002; Ota and Nei, 1994). When compared with the human counterpart, the dog VH1 family corresponds to human VH3 (a member of clan III), the dog VH2 family corresponds with human VH1 (a member of clan I), and the dog VH3 family corresponds with human VH4 (a member of clan II). The three dog VH gene families are classified into three different clans, but the most majority is in clan III. It has been reported that a single family belonging to clan III is expressed in some species such as camel, swine, rabbit, and chicken (Currier et al., 1988; Nguyen et al., 2000; Parvari et al., 1988; Sun et al., 1994). Bovine also uses a single VH family, which belongs to clan II (Berens et al.,
1997; Sinclair et al., 1997). In sheep, nine VH gene families belong to three clans, but the most are homologous to clan II (Charlton et al., 2000). The horse IgH locus includes at least three VH families, two of which belong to clan II, while the other was classified into clan I. Therefore, a close homology among bovine, sheep and horse IGHV genes exists. Similarly, the most primitive mammalian, platypus VH genes are also classified into three groups, the majority of which belong to a single family (clan III) (Gambon-Deza et al., 2009). The marsupial opossum IgH locus consists of only three V gene families, all belonging to clan III (Wang et al., 2009). As far as mammals are concerned, clan III seems to be the largest group.
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Fig. 5. Dog germline DH segments. The nonamer (9-mer) and heptamer (7-mer) sequences are underlined and heptamer components different from consensus (5 : CACTGTG and 3 : CACAGTG) are boxed. The 3 -RSS of the segment DH5 was intervened with 13 bp spacer (shadowed). The predicted amino acids of three reading frames of D segments are displayed.
As expected, the dog VH1 family (human VH3 family analogue) were used prevalently at the expression level (110/111). The other two proposed VH families, 2 and 3, were rarely observed, and only one cDNA sequence (clone G6) belonging to VH2 family was detected. To exclude the influence of breed on VH usage bias, all available IGHV genes sequences in the NCBI database from other studies were analyzed. Besides clone G6 in our study, only one mRNA clone (XM 843643) from the boxer breed belongs to the dog VH2 family. All other sequences originate from the dog VH1 family. Interestingly, it was demonstrated
that the bias is focused on some gene segments of the VH1 family, VH62 and VH44, human VH3-21 and VH3-23 homology, respectively, which have been demonstrated to be expressed at a high frequency in some cases (Bertoni et al., 2004; Brezinschek et al., 1997; Walsh et al., 2003). Unexpectedly, the dog diversity index (DI) value was very high. In contrast, the DI value of sheep expressed VH genes is much lower, 17 in CDR1 and 40 in CDR2 (Dufour et al., 1996). The diversity index from 51 human functional germline VH genes in position 50 (CDR2) is 70 (Cook and Tomlinson, 1995). The results from the adult cattle chain
Fig. 6. Comparison of CDR3 sequences between cDNA clones and germline segments. CDR3 sequences of 39 representing clones are presented and compared with germline DH segments. The extent of VH and JH sequences is based on genomic information and common sequences motifs of CDR3 codons. N and P (PD and PJ) addition are displayed. The clones G118, G102, AA5, AA28, E7, E21, E28 and E72 and corresponding sequences are shadowed. The D region of clone G118 seems to be a fusion of DH3 and DH1. Clone G102 used the full-length coding region of germline segment DH2. Clones AA5 and AA28 and clones E7, E21, E28 and E72 showed an identical CDR3.
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Fig. 7. Length distribution of dog CDR3. 111 cDNA sequences were analyzed for CDR3. The y-axis represents the relative frequency of each loop length in percentage and the x-axis indicates the length of the loop.
Fig. 8. Dog germline JH segments. Nucleotide and corresponding amino acid sequences of three JH segments along with RSSs are shown.
transcript showed a DI value of 73 in position 35 (CDR1) (Berens et al., 1997). Thus, a high variability in the variable region excluding CDR3 suggests that dog species may implement antibody diversification by somatic hypermutation.
The length and amino acid composition of CDR3 are considered to be important in antigen binding (Zemlin et al., 2003). The potential coding regions of dog germline DH segments are 11–31 bp in length. In humans, it is proposed that coding regions of DH segments may be characterized
Fig. 9. Alignment of JH transcripts. All JH sequences were transcribed from three germline JH segments. JH1 and JH2 are dominantly used. The dots represent those nucleotides that are identical to majority.
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by the character of their amino acids (Corbett et al., 1997). Inspection indicates that dog DH coding regions are composed of polar/hydrophilic amino acids or a stop codon(s) (details not shown). In humans and mice, the germline DH gene segments are divided into families (Kurosawa and Tonegawa, 1982; Siebenlist et al., 1981). The members of an identical family share nucleotide homology in the coding regions, RSS elements, and flanking regions (Corbett et al., 1997). However, our results indicate that six dog DH segments belong to single member gene families. In addition, compared with human and mouse, the dog DH segments are relatively few. Immunoglobulin diversification can be enhanced by inserting or deleting nucleotides at the breakpoints during site-specific recombination (Lieber, 1991). Nucleotide variations such as replacement, deletion or/and insertion are involved in CDR3 of the listed 39 transcripts. Undoubtedly, more complex events must have taken place at the V/D/J junction of the unlisted 72 clones having a less nucleotide match with known D segments. Thus, an increase in CDR3 diversity may be more important for generating variability to compensate for a limited germline VH repertoire. The immunoglobulin heavy chain V region is one of the most complex regions in the genome. Understanding gene organization and detailed structure of the region is an important aspect for understanding dog immunglobulin diversity. The study lays the foundations for further work on dog mmunglobulin. For further studies, VH gene segment usage at specific pathogen status may help in understanding the mechanism and the biological impact underlying dog antibody diversity. Additionally, knowing of gene segment organization of the light chain is essential to comprehensive understanding dog immunoglobulin feature. 5. Conclusions The study of the V gene repertoire is critical to understanding the nature of antibody molecules. In this study, we analyzed the gene organization of the dog IGHV locus. Eighty VH segments belonging to three gene families were characterized. Compared with the human and mouse VH sequences, dog should be considered a species with limited germline VH diversity. Subsequently, we isolated and analyzed a substantial number of dog expressed VH genes from five classes of immunoglobulin molecules. A preferential usage of a few germline VH segments was indicated. Complex and diverse CDR3 may contribute to dog immunoglobulin variability. The results generated from our study will provide new elements for analysis of other species immunglobulin and evolutionary relationships. Acknowledgement This research was supported by the National Science Fund (30972085). References Almagro, J.C., Dominguez-Martinez, V., Lara-Ochoa, F., Vargas-Madrazo, E., 1996. Structural repertoire in human VL pseudogenes of immunoglob-
ulins: comparison with functional germline genes and amino acid sequences. Immunogenetics 43, 92–96. Almagro, J.C., Martinez, L., Smith, S.L., Alagon, A., Estevez, J., Paniagua, J., 2006. Analysis of the horse V(H) repertoire and comparison with the human IGHV germline genes, and sheep, cattle and pig V(H) sequences. Mol. Immunol. 43, 1836–1845. Berek, C., Berger, A., Apel, M., 1991. Maturation of the immune response in germinal centers. Cell 67, 1121–1129. Berens, S.J, Wylie, D.E., Lopez, O.J., 1997. Use of a single VH family and long CDR3s in the variable region of cattle Ig heavy chains. Int. Immunol. 9, 189–199. Bertoni, F., Conconi, A., Cogliatti, S.B., Schmitz, S.F., Ghielmini, M., Cerny, T., Fey, M., Pichert, G., Bertolini, F., Ponzoni, M., Baldini, L., Jones, C., Auer, R., Zucca, E., Cavalli, F., Cotter, F.E., 2004. Immunoglobulin heavy chain genes somatic hypermutations and chromosome 11q22–23 deletion in classic mantle cell lymphoma: a study of the Swiss Group for Clinical Cancer Research. Br. J. Haematol. 124, 289–298. Brezinschek, H.P., Foster, S.J., Brezinschek, R.I., Dorner, T., Domiati-Saad, R., Lipsky, P.E., 1997. Analysis of the human VH gene repertoire. Differential effects of selection and somatic hypermutation on human peripheral CD5(+)/IgM+ and CD5(−)/IgM+ B cells. J. Clin. Invest. 99, 2488–2501. Brodeur, P.H., Riblet, R., 1984. The immunoglobulin heavy chain variable region (Igh-V) locus in the mouse. I. One hundred Igh-V genes comprise seven families of homologous genes. Eur. J. Immunol. 14, 922–930. Butler, J.E., 1998. Immunoglobulin diversity, B-cell and antibody repertoire development in large farm animals. Rev. Sci. Technol. 17, 43–70. Charlton, K.A., Moyle, S., Porter, A.J., Harris, W.J., 2000. Analysis of the diversity of a sheep antibody repertoire as revealed from a bacteriophage display library. J. Immunol. 164, 6221–6229. Chevillard, C., Ozaki, J., Herring, C.D., Riblet, R., 2002. A three-megabase yeast artificial chromosome contig spanning the C57BL mouse Igh locus. J. Immunol. 168, 5659–5666. Cook, G.P., Tomlinson, I.M., 1995. The human immunoglobulin VH repertoire. Immunol. Today 16, 237–242. Corbett, S.J., Tomlinson, I.M., Sonnhammer, E.L., Buck, D., Winter, G., 1997. Sequence of the human immunoglobulin diversity (D) segment locus: a systematic analysis provides no evidence for the use of DIR segments, inverted D segments, “minor” D segments or D–D recombination. J. Mol. Biol. 270, 587–597. Currier, S.J., Gallarda, J.L., Knight, K.L., 1988. Partial molecular genetic map of the rabbit VH chromosomal region. J. Immunol. 140, 1651–1659. de Bono, B., Madera, M., Chothia, C., 2004. VH gene segments in the mouse and human genomes. J. Mol. Biol. 342, 131–143. Dufour, V., Malinge, S., Nau, F., 1996. The sheep Ig variable region repertoire consists of a single VH family. J. Immunol. 156, 2163–2170. Gambon-Deza, F., Sanchez-Espinel, C., Magadan-Mompo, S., 2009. The immunoglobulin heavy chain locus in the platypus (Ornithorhynchus anatinus). Mol. Immunol. 46, 2515–2523. Haino, M., Hayashida, H., Miyata, T., Shin, E.K., Matsuda, F., Nagaoka, H., Matsumura, R., Taka-ishi, S., Fukita, Y., Fujikura, J., et al., 1994. Comparison and evolution of human immunoglobulin VH segments located in the 3 0.8-megabase region. Evidence for unidirectional transfer of segmental gene sequences. J. Biol. Chem. 269, 2619–2626. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Songa, E.B., Bendahman, N., Hamers, R., 1993. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448. Johansson, J., Aveskogh, M., Munday, B., Hellman, L., 2002. Heavy chain V region diversity in the duck-billed platypus (Ornithorhynchus anatinus): long and highly variable complementarity-determining region 3 compensates for limited germline diversity. J. Immunol. 168, 5155–5162. Johnston, C.M., Wood, A.L., Bolland, D.J., Corcoran, A.E., 2006. Complete sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V region. J. Immunol. 176, 4221–4234. Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S., Foeller, C., 1991. Sequences of Proteins of Immunological Interest, 5th ed. National Institutes of Health, Bethesda, MD. Kirkham, P.M., Mortari, F., Newton, J.A., Schroeder Jr., H.W., 1992. Immunoglobulin VH clan and family identity predicts variable domain structure and may influence antigen binding. EMBO J. 11, 603–609. Kurosawa, Y., Tonegawa, S., 1982. Organization, structure, and assembly of immunoglobulin heavy chain diversity DNA segments. J. Exp. Med. 155, 201–218. Lefranc, M.P., 1998. IMGT (ImMunoGeneTics) locus on focus. A new section of experimental and clinical immunogenetics. Exp. Clin. Immunogenet. 15, 1–7.
Y. Bao et al. / Veterinary Immunology and Immunopathology 137 (2010) 64–75 Lieber, M.R., 1991. Site-specific recombination in the immune system. FASEB J. 5, 2934–2944. Mainville, C.A., Sheehan, K.M., Klaman, L.D., Giorgetti, C.A., Press, J.L., Brodeur, P.H., 1996. Deletional mapping of fifteen mouse VH gene families reveals a common organization for three Igh haplotypes. J. Immunol. 156, 1038–1046. Matsuda, F., Ishii, K., Bourvagnet, P., Kuma, K., Hayashida, H., Miyata, T., Honjo, T., 1998. The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus. J. Exp. Med. 188, 2151–2162. Nguyen, V.K., Hamers, R., Wyns, L., Muyldermans, S., 2000. Camel heavy-chain antibodies: diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire. EMBO J. 19, 921– 930. Ota, T., Nei, M., 1994. Divergent evolution and evolution by the birth-anddeath process in the immunoglobulin VH gene family. Mol. Biol. Evol. 11, 469–482. Parvari, R., Avivi, A., Lentner, F., Ziv, E., Tel-Or, S., Burstein, Y., Schechter, I., 1988. Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus. EMBO J. 7, 739–744. Siebenlist, U., Ravetch, J.V., Korsmeyer, S., Waldmann, T., Leder, P., 1981. Human immunoglobulin D segments encoded in tandem multigenic families. Nature 294, 631–635. Sinclair, M.C., Gilchrist, J., Aitken, R., 1997. Bovine IgG repertoire is dominated by a single diversified VH gene family. J. Immunol. 159, 3883–3889.
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Sun, J., Kacskovics, I., Brown, W.R., Butler, J.E., 1994. Expressed swine VH genes belong to a small VH gene family homologous to human VHIII. J. Immunol. 153, 5618–5627. van Dijk, K.W., Mortari, F., Kirkham, P.M., Schroeder Jr., H.W., Milner, E.C., 1993. The human immunoglobulin VH7 gene family consists of a small, polymorphic group of six to eight gene segments dispersed throughout the VH locus. Eur. J. Immunol. 23, 832–839. Vargas-Madrazo, E., Almagro, J.C., Lara-Ochoa, F., 1995. Structural repertoire in VH pseudogenes of immunoglobulins: comparison with human germline genes and human amino acid sequences. J. Mol. Biol. 246, 74–81. Walsh, S.H., Thorselius, M., Johnson, A., Soderberg, O., Jerkeman, M., Bjorck, E., Eriksson, I., Thunberg, U., Landgren, O., Ehinger, M., Lofvenberg, E., Wallman, K., Enblad, G., Sander, B., Porwit-MacDonald, A., Dictor, M., Olofsson, T., Sundstrom, C., Roos, G., Rosenquist, R., 2003. Mutated VH genes and preferential VH3-21 use define new subsets of mantle cell lymphoma. Blood 101, 4047–4054. Wang, X., Olp, J.J., Miller, R.D., 2009. On the genomics of immunoglobulins in the gray, short-tailed opossum Monodelphis domestica. Immunogenetics 61, 581–596. Ye, J., 2004. The immunoglobulin IGHD gene locus in C57BL/6 mice. Immunogenetics 56, 399–404. Zemlin, M., Klinger, M., Link, J., Zemlin, C., Bauer, K., Engler, J.A., Schroeder Jr., H.W., Kirkham, P.M., 2003. Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures. J. Mol. Biol. 334, 733–749.
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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
Molecular characterization of the VH repertoire in Canis familiaris Yonghua Bao a,b , Yongchen Guo b , Shuqi Xiao c , Zhihui Zhao a,∗ a b c
College of Animal Science and Veterinary Medicine, Agricultural Division, Jilin University, 5333 of Xi’an Road, Changchun, Jilin 130062, PR China State Key Laboratories for AgroBiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, PR China State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangzhou, Guangdong 510006, PR China
a r t i c l e
i n f o
Article history: Received 26 November 2009 Received in revised form 19 March 2010 Accepted 16 April 2010 Keywords: Immunoglobulin VH Dog Genome Diversity
a b s t r a c t The immunoglobulin heavy chain variable region in vertebrates is composed of many genes. However, the gene number and the extent of diversity among the VH gene segments vary with the species. To gain insight into the dog VH repertoire, we analyzed its genome sequence. Eighty VH, six DH and three JH segments were mapped to a 1.28 Mb region of the dog chromosome 8. Forty-one of these VH genes were potentially functional, and 39 were pseudogenes. The sequence similarity analysis suggests that the dog IgH locus harbors three VH gene families, VH1 (including 76 members), VH2 (including 3 members) and VH3 (including one member), that are classified into mammalian clan III, I and II, respectively. Subsequently, 111 cDNA positive clones from dog 26 , 22 ␥, 23 ␣, 16 ␦ and 24 chains containing almost full-length VH, DH and JH segments were selected at random. All of the sequences, except for clone G6, which belonged to the dog VH2 family, originated from the dog VH1 family. The germline segments, VH62 (human VH3-21 homology) and VH44 (human VH3-23 homology), seemed to be preferentially used. Our results suggest that the dog VH repertoire appears to be derived from limited germline gene families, and its diversity may rely on junctional diversity and somatic hypermutation. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The immune system can produce an enormous antibody repertoire in anticipation of large and diverse groups of potential pathogens (Kabat et al., 1991). The recognition of an antigen involves dimers formed by the variable domains of the heavy and light chains (VH and VL) (de Bono et al., 2004), and in some cases only heavy chains are used
Abbreviations: IgH, immunoglobulin heavy chain (Ig: immunoglobulin and H: heavy chain); VH, heavy chain variable domain (V: variable domain and H: heavy chain); VL, light chain variable domain (V: variable domain and L: light chain); DH, heavy chain diversity domain (D: diversity domain and H: heavy chain); JH, heavy chain joining domain (J: joining domain and H: heavy chain); IGHV, immunoglobulin heavy chain variable region (IG: immunoglobulin, H: heavy chain and V: variable region). ∗ Corresponding author. Tel.: +86 431 87836156; fax: +86 431 87836156. E-mail address: [email protected] (Z. Zhao). 0165-2427/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2010.04.011
(Hamers-Casterman et al., 1993). Therefore, the variable region determines the diversity of immunoglobulins. The huge variety of immunoglobulin specificities is produced by (1) the inherited set of variable (V), diversity (D) and joining (J) gene segments (germline diversity), (2) the different rearrangements of these V, D and J gene segments (combinatorial diversity), (3) the insertion and deletion of random nucleotides (junctional diversity), and (4) the introduction of somatic mutations (Berek et al., 1991). In mammals, the gene segments of VH domains of many species have been well described. Historically, the human V region is composed of 7 germline VH gene families, including approximately 100 genes and pseudogenes, approximately 30 DH gene segments, and 6 JH gene segments (van Dijk et al., 1993). In the mouse, 4 J genes, 10–13 D genes and about 150 germline V genes forming 16 families have been identified (Chevillard et al., 2002; de Bono et al., 2004; Johnston et al., 2006; Mainville et al., 1996; Ye, 2004).
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Studies of sheep (Ovis aries), cattle (Bos taurus) and pig (Sus scrofa) suggest that these species express one family of VH and rely on somatic hypermutation and gene conversion to diversify the primary repertoire of antibodies (Berens et al., 1997; Dufour et al., 1996; Sinclair et al., 1997; Sun et al., 1994). In a more recent study, the ovine VH repertoire was found to be more complex and diverse than previously thought, being composed of at least nine VH gene families. This finding suggests that the diversity of sheep VH germline genes may play a more significant role in the diversification of the primary antibody repertoire than previously suggested (Charlton et al., 2000). Similarly, at least three VH gene families were identified in the equine IgH locus (Almagro et al., 2006), in contrast with earlier reports of a limited diversity of the VH germline gene repertoire (Butler, 1998). In this study, we examined the variable region of the dog IgH locus, in which 80 germline VH segments formed three gene families. As expected, the dog VH1 family (human VH3 family analogue) was predominant at both the genome and expression levels. Sequence analysis indicated that CDR3 variability and somatic hypermutations may contribute greatly to dog antibody diversification. 2. Materials and methods 2.1. Acquirement of dog germline V, D and J segments Available cDNA sequence (GenBank accession number XM 844149) of the dog VH gene was used to search for corresponding genomic sequences by a conventional TBLASTN approach in the Ensembl database (www.ensembl.org). A region of approximately 1.28 Mb from dog chromosome 8 was found to contain potential VH, DH and JH gene segments. Combined with the specificity of the conserved recombination signal sequence (RSS) motif adjacent to the V, D or J gene segments, FUZZNUC, an on-line software package(http://anabench.bcm.umontreal.ca/anabench/ Anabench-Jsp/Applications/fuzznuc.jsp?APPLICATIONID= 81&APPLICATIONNA ME=fuzznuc), was used to identify these gene segments. 2.2. Dogs Three healthy Chinese rural dogs were selected for this experiment. The dogs were euthanized after the experiment was complete. The study was conducted with the approval of the State Ethical Commission, and met the
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International Guiding Principles for Biomedical Research Involving Animals. 2.3. Isolation of total RNA Spleen specimens were collected and frozen immediately in liquid nitrogen. Total RNA was extracted using the TRlzol Reagent (Invitrogen, USA). Equal amounts of RNA samples from the three dogs were pooled to amplify dog cDNA sequences. 2.4. Primer design Based on the characterized canine IGHC genes (GenBank accession numbers: IgM-AAEX02009527, IgG-AF354264, IgA-L36871, IgD-XM548647, and IgE-L36872), specific primers for the amplification of five classes of the immunoglobulin V region are displayed in Table 1. 2.5. 5 RACE of IGHV genes The 5 RACE system (Invitrogen, USA) was utilized to determine the immunoglobulin V region transcripts. First-stranded cDNA was synthesized using SuperscriptTM II reverse transcriptase with the Igs-GSP1 primer (Table 1). Double-stranded DNA (dsDNA) was obtained from cDNA by PCR with the primer sets, AAP (5 -ggccacgcgtcgactagtacgggggggggg-3 ) and Igs-GSP2 (Table 1). Nested PCR was performed with the primers, UAP (5 -ggccacgcgtcgactagtac-3 ) and Igs-GSP3 (Table 1). 2.6. Sequence analysis Editing and comparison of all sequences were performed using the DNAstar program. Multiple sequence alignments were made by the Clustal W method and optimized manually. 2.7. Diversity index Referring to the methods of Kabat et al. (1991), diversity index (DI) is the number of different amino acids at a particular position divided by the frequency of the most common amino acid at that position. 2.8. VH genes family definition Immunoglobulin VH genes were divided into families based on their nucleotide similarities. Criteria have been
Table 1 Primer sequences used in the 5 RACE system. GSP1 IgM IgG IgA IgD IgE
GSP2
5 -gaaccaagacacggaaat-3 5 -agggcactgtcaccat-3 5 -tgcttccagtcttcg-3 5 -tccggtatctttagcatc-3 5 -aggtcttgttgatggc-3
GSP3
5 -gggcacgttcacagatttgtcaccat-3 5 -gcggacggacgggaaggtg-3 5 -ctggatgggttccttccctttgg-3 5 -tgtcgtgacccgcacagaat-3 5 -tggctgacgatgctgtggag-3
5 -ggaaggtcttaatgtcctggttgtt-3 5 -gccagtgggaaaaccgaggg-3 5 -ggcagaggctcagcgggaacac-3 5 -gaccaagggagcagaagtgacgat-3 5 -gcgatgttgtctttacagcaggaggc-3
GSP1 was the gene specific primer for cDNA synthesis. GSP2 and GSP3 were antisense primers for PCR amplification. Sense primers (AAP and UAP) were supplied in this system.
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established that the same family members share more than 80% nucleotide identity, those sequences with less than 70% identity belong to different families, and those having between 70% and 80% similarity are examined on a case-by-case basis (Brodeur and Riblet, 1984). 3. Results 3.1. VH genes 3.1.1. Germline VH segments The publication of dog genome information in the Ensembl database allowed us to annotate its germline VH, DH and JH minigenes. A total of 80 VH segments were identified and located in a 1.2Mb region at the end of the dog chromosome 8 (Fig. 1). No significant matches were found outside of this region. In order to clarify the relations among dog germline VH segments, all the sequences were processed for pair comparison (results not shown). Considering sequence truncations (VH5, VH42, VH49 and VH76), we placed sequences having greater than 70% identity in the same family, the dog VH1 family composed of the upper 76 sequences shown in Fig. 2. VH51, VH64 and VH66, which showed greater than 80% nucleotide similarity, were classified into the dog VH2 family. For VH80, an identity values below 70% was found with all other sequences, indicating that it belongs to a single member family called dog VH3 (Fig. 2).
3.1.2. Prediction of the functional VH genes We have defined a VH gene as potentially functional if the gene retains an open reading frame (ORF), leader exons, a downstream RSS, and essential amino acid residues such as Cys residues, even though no corresponding transcripts were identified in later experiments. The gene was considered a pseudogene if it contained frame shifts or in-frame stop codons (Lefranc, 1998). Based on the above criteria, of 80 canine germline VH segments, 41 were regarded as potentially functional genes whose formal products are shown in Fig. 3. The remaining 39 segments showed various structural defects. 3.1.3. VH transcripts We investigated the usage of germline VH gene segments. A total of 111 positive clones from 26 , 22 ␥, 23 ␣, 16 ␦, and 24 chains containing almost full-length variable regions were obtained by 5 RACE-PCR. All the sequences have been deposited in the GenBank database and assigned accession numbers FJ197707–FJ197817. Comparing these expressed VH sequences with germline segments, major differences were found in the base composition. The differences were attributed to somatic hypermutation or individual differences. It is difficult to make an accurate assessment of the use of germline VH segments, but certain segments may be used more frequently than others. For example, there are 30 VH transcripts possessing high similarity with the germline VH62 (human VH3-21 homology). Another 26
Fig. 1. Dog IGHV locus. The organization of dog IGHV genes is showed to scale. Forty-one functional VH genes are shown as filled bars; 39 VH pseudogenes are represented with open bars and attached with the letter P; DH and JH are indicated with grey boxes and C genes with a black box. These VH genes are numbered based on the family to which they belong and their position in the locus. The number before VH refers to the family, while the number after VH denotes the genomic position.
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Fig. 2. Multiple sequences alignment of all the dog germline VH segments. 80 germline VH genes in dog genome were divided into three VH families, where family 1 comprises the upper 76 sequences, family 2 includes VH51, VH64 and VH66, and family 3 contains VH80. At the end of each sequence, the serial number of the gene is given. Identifies with respect to the majority of the nucleotides are represented by dots; gaps are represented by dashes. Designation of framework regions (FR) and complementarity-determining regions (CDR) are based on the IMGT numbering system (http://imgt.cines.fr/).
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Fig. 2. (Continued )
transcripts showed similarity with germline VH44 (human VH3-23 homology). The other segments were duplicated less than 13 times. Undoubtedly, the most expressed is the dog VH1 family, i.e. the human VH3 family analogue. From 111 transcripts, only clone G6 was derived from the dog VH2 family. In addition, no transcript of dog VH3 family was found.
3.1.4. High diversity index of the expressed VH genes The diversity index (DI) in the variable region (FR1–FR3) from expressed VH genes (excluding clone G6) is shown in Fig. 4. The DI of chain ␣ is the highest at 109.25 in the position 57 amino acid, followed by 91.2 ( in position 59), 89.25 (␥ in position 57), 70.57 ( in positions 50 and 54) and 35.2 (␦ in positions 50 and 54). As expected, variation was concentrated in the CDRs.
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Fig. 2. (Continued ).
3.2. DH genes 3.2.1. Germline DH segments Six germline DH segments of the dog were acquired and shown in Fig. 5. Except for DH5 with 3 -RSS including a 13-bp spacer, other five DH segments have a 5 - and 3 RSS with a 12-bp spacer. In addition, DH2, 3, 4, 5 and 6 have conserved 3 heptamers (CACAGTG). 5 heptamers of DH3, 4, 5, 6 and the 3 heptamer of DH1 differ from the consensus (5 : CACTGTG and 3 : CACAGTG) by A, G, G, A and CA, respectively. Consistently, the 5 and 3 nonamers of each DH gene segment are T-rich and A-rich, respectively. 3.2.2. DH transcripts The cDNA transcripts of the D region were compared with genomic sequences. Clones possessing <5 bp
coincidence with germline segments were not involved. Representing clones that utilized longer and relatively definite DH1–DH6 segments are presented in Fig. 6. Only clone G102 has the full-length coding region of the germline segment DH2. Various numbers of nucleotides removal and addition were found at the junction of VH, DH and JH segments. The addition of nucleotides was prominent. N-region additions were located between V–D junctions, as well as between D–J junctions, and ranged from 1 to 23 bp in length. A strong correlation was apparent between the length of the CDR3 and the number of N nucleotides. Most of the P-region additions, one or two nucleotides in length, were also identified. Nucleotide deletions from the D segments were found at either the 5 or the 3 ends, or from both the 5 and 3 ends. In addition, the D region of clone G118 seems to be a
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Fig. 3. Alignment of deduced amino acid sequences of 41 dog functional VH genes. The amino acid sequences were deduced from the obtained germline VH genes based on the codon usage. All positions where the amino acid residues are identical to those of majority are shown as small dots. Gaps and missing data are indicated by hyphens. The Designation of framework regions (FR) and complementarity determining regions (CDR) referred to the IMGT numbering system (http://imgt.cines.fr/).
fusion of DH3 and DH1. Interestingly, two groups of transcripts involved in germline DH4, clones AA5 and AA28 and clones E7, E21, E28 and E72 showed an identical CDR3.
of somatic mutation. In addition, this panel also illustrates the predominant usages of two JH segments, 1 and 2, in the rearrangements. 4. Discussion
3.2.3. Length and amino acid composition of CDR3 CDR3 lengths of the sequences obtained in this work ranged from 6 to 26 amino acid residues (solid column in Fig. 7), with a peak of 14 residues. Most of the loops ranged from 7 to 17 amino acids. The average length was 13.1 ± 3.59 residues. Three critical amino acids of canine CDR3 loops were analyzed: cysteine, glycine and serine residues. Dog CDR3 loops had 17 cysteine residues (1.2%), 149 glycine residues (10.41%), and 103 serine residues (7.2%) out of 1431 amino acids analyzed. In cattle, sheep, pigs and horses, the proportions of cysteine and glycine residues were 0.2%, 1.2%, 4.3%, and 6.4% and 17.0%, 13.2%, 12,0%, and 20.5%, respectively (Almagro et al., 2006). 3.3. JH segments There were three germline JH gene segments found in the dog IgH locus (Fig. 8). JH1, JH2 and JH3 segments were conserved in the 3 region, whereas in V–J junctions with 4, 3 and 5 different amino acid residues before tyrosine (Y) and tryptophan (W), respectively. In addition, JH1 and JH2 segments share higher amino acid homology than JH3. All JH encoded sequences were readily assigned to germline JH1-JH3 segments (Fig. 9). The small nucleotide variations observed in these JH sequences may be the effect
In the present study, we have characterized the dog immunoglobulin VH repertoire. Chromosome 8 of the dog displayed 80 VH genes, about half of which are pseudogenes (39/80). In the pool of human immunoglobulin VH gene segments, pseudogenes amount to roughly 30% of the total number of genes (Matsuda et al., 1998; VargasMadrazo et al., 1995). Among 141 VH gene segments from the mouse strain C57BL/6J, 92 are functional(de Bono et al., 2004). Pseudogenes in the Ig V region constitute a potential donor pool for gene conversion during evolution (Almagro et al., 1996; Haino et al., 1994; Vargas-Madrazo et al., 1995). Although no gene conversions were found in the 39 dog VH pseudogenes, pseudogenes reserves during evolution should be useful for dog immunoglobulin diversity. Alignment of VH gene sequences indicates that the vast majority have a high nucleotide similarity. The largest majority of VH genes are of the dog VH1 family, and the remainder are organized into two other groups, dog VH2 and VH3 family, with a few VH genes each. The three VH gene families were interspersed throughout the dog IgH locus. Earlier publications described the VH gene families as clans (Kirkham et al., 1992; Ota and Nei, 1994). The mammalian VH genes are classified into three clans, I, II, and
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Fig. 4. Comparison of the diversity index (DI) of VH transcripts for five classes of immunoglobulins. Horizontal axis represents the amino acid residue position. Vertical axis indicates DI. The calculation of DI is described in Kabat et al. (1991). DI is defined as the number of different amino acids at one position divided by the frequency of the most common amino acid at that position. The DI at position 57 of ␣ transcripts is 109.25, followed by 91.2 ( at position 59), 89.25 (␥ at position 57), 70.57 ( at positions 50 and 54) and 35.2 (␦ at positions 50 and 54).
III. Clan III is considered the most ancient (Johansson et al., 2002; Ota and Nei, 1994). When compared with the human counterpart, the dog VH1 family corresponds to human VH3 (a member of clan III), the dog VH2 family corresponds with human VH1 (a member of clan I), and the dog VH3 family corresponds with human VH4 (a member of clan II). The three dog VH gene families are classified into three different clans, but the most majority is in clan III. It has been reported that a single family belonging to clan III is expressed in some species such as camel, swine, rabbit, and chicken (Currier et al., 1988; Nguyen et al., 2000; Parvari et al., 1988; Sun et al., 1994). Bovine also uses a single VH family, which belongs to clan II (Berens et al.,
1997; Sinclair et al., 1997). In sheep, nine VH gene families belong to three clans, but the most are homologous to clan II (Charlton et al., 2000). The horse IgH locus includes at least three VH families, two of which belong to clan II, while the other was classified into clan I. Therefore, a close homology among bovine, sheep and horse IGHV genes exists. Similarly, the most primitive mammalian, platypus VH genes are also classified into three groups, the majority of which belong to a single family (clan III) (Gambon-Deza et al., 2009). The marsupial opossum IgH locus consists of only three V gene families, all belonging to clan III (Wang et al., 2009). As far as mammals are concerned, clan III seems to be the largest group.
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Fig. 5. Dog germline DH segments. The nonamer (9-mer) and heptamer (7-mer) sequences are underlined and heptamer components different from consensus (5 : CACTGTG and 3 : CACAGTG) are boxed. The 3 -RSS of the segment DH5 was intervened with 13 bp spacer (shadowed). The predicted amino acids of three reading frames of D segments are displayed.
As expected, the dog VH1 family (human VH3 family analogue) were used prevalently at the expression level (110/111). The other two proposed VH families, 2 and 3, were rarely observed, and only one cDNA sequence (clone G6) belonging to VH2 family was detected. To exclude the influence of breed on VH usage bias, all available IGHV genes sequences in the NCBI database from other studies were analyzed. Besides clone G6 in our study, only one mRNA clone (XM 843643) from the boxer breed belongs to the dog VH2 family. All other sequences originate from the dog VH1 family. Interestingly, it was demonstrated
that the bias is focused on some gene segments of the VH1 family, VH62 and VH44, human VH3-21 and VH3-23 homology, respectively, which have been demonstrated to be expressed at a high frequency in some cases (Bertoni et al., 2004; Brezinschek et al., 1997; Walsh et al., 2003). Unexpectedly, the dog diversity index (DI) value was very high. In contrast, the DI value of sheep expressed VH genes is much lower, 17 in CDR1 and 40 in CDR2 (Dufour et al., 1996). The diversity index from 51 human functional germline VH genes in position 50 (CDR2) is 70 (Cook and Tomlinson, 1995). The results from the adult cattle chain
Fig. 6. Comparison of CDR3 sequences between cDNA clones and germline segments. CDR3 sequences of 39 representing clones are presented and compared with germline DH segments. The extent of VH and JH sequences is based on genomic information and common sequences motifs of CDR3 codons. N and P (PD and PJ) addition are displayed. The clones G118, G102, AA5, AA28, E7, E21, E28 and E72 and corresponding sequences are shadowed. The D region of clone G118 seems to be a fusion of DH3 and DH1. Clone G102 used the full-length coding region of germline segment DH2. Clones AA5 and AA28 and clones E7, E21, E28 and E72 showed an identical CDR3.
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Fig. 7. Length distribution of dog CDR3. 111 cDNA sequences were analyzed for CDR3. The y-axis represents the relative frequency of each loop length in percentage and the x-axis indicates the length of the loop.
Fig. 8. Dog germline JH segments. Nucleotide and corresponding amino acid sequences of three JH segments along with RSSs are shown.
transcript showed a DI value of 73 in position 35 (CDR1) (Berens et al., 1997). Thus, a high variability in the variable region excluding CDR3 suggests that dog species may implement antibody diversification by somatic hypermutation.
The length and amino acid composition of CDR3 are considered to be important in antigen binding (Zemlin et al., 2003). The potential coding regions of dog germline DH segments are 11–31 bp in length. In humans, it is proposed that coding regions of DH segments may be characterized
Fig. 9. Alignment of JH transcripts. All JH sequences were transcribed from three germline JH segments. JH1 and JH2 are dominantly used. The dots represent those nucleotides that are identical to majority.
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by the character of their amino acids (Corbett et al., 1997). Inspection indicates that dog DH coding regions are composed of polar/hydrophilic amino acids or a stop codon(s) (details not shown). In humans and mice, the germline DH gene segments are divided into families (Kurosawa and Tonegawa, 1982; Siebenlist et al., 1981). The members of an identical family share nucleotide homology in the coding regions, RSS elements, and flanking regions (Corbett et al., 1997). However, our results indicate that six dog DH segments belong to single member gene families. In addition, compared with human and mouse, the dog DH segments are relatively few. Immunoglobulin diversification can be enhanced by inserting or deleting nucleotides at the breakpoints during site-specific recombination (Lieber, 1991). Nucleotide variations such as replacement, deletion or/and insertion are involved in CDR3 of the listed 39 transcripts. Undoubtedly, more complex events must have taken place at the V/D/J junction of the unlisted 72 clones having a less nucleotide match with known D segments. Thus, an increase in CDR3 diversity may be more important for generating variability to compensate for a limited germline VH repertoire. The immunoglobulin heavy chain V region is one of the most complex regions in the genome. Understanding gene organization and detailed structure of the region is an important aspect for understanding dog immunglobulin diversity. The study lays the foundations for further work on dog mmunglobulin. For further studies, VH gene segment usage at specific pathogen status may help in understanding the mechanism and the biological impact underlying dog antibody diversity. Additionally, knowing of gene segment organization of the light chain is essential to comprehensive understanding dog immunoglobulin feature. 5. Conclusions The study of the V gene repertoire is critical to understanding the nature of antibody molecules. In this study, we analyzed the gene organization of the dog IGHV locus. Eighty VH segments belonging to three gene families were characterized. Compared with the human and mouse VH sequences, dog should be considered a species with limited germline VH diversity. Subsequently, we isolated and analyzed a substantial number of dog expressed VH genes from five classes of immunoglobulin molecules. A preferential usage of a few germline VH segments was indicated. Complex and diverse CDR3 may contribute to dog immunoglobulin variability. The results generated from our study will provide new elements for analysis of other species immunglobulin and evolutionary relationships. Acknowledgement This research was supported by the National Science Fund (30972085). References Almagro, J.C., Dominguez-Martinez, V., Lara-Ochoa, F., Vargas-Madrazo, E., 1996. Structural repertoire in human VL pseudogenes of immunoglob-
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