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Sequence diversity of the MHC Ⅱ DRB gene in Chinese tree shrews (Tupaia belangeri chinensis)
Biochemical Systematics and Ecology 61 (2015) 417e423
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Sequence diversity of the MHC Ⅱ DRB gene in Chinese tree shrews (Tupaia belangeri chinensis) Zhang-qiong Huang a, b, Xiao-mei Sun a, Jie-jie Dai a, Ming-liang Gu c, You-song Ye a, Yu-feng Yao a, Rui-ju Jiang a, Kai-li Ma a, b, * a
Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infection Diseases, Kunming 650118, China b Institute of Basic Medical Sciences, Neuroscience Center, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China c Beijing Key Laboratory of Genome and Precision Medicine Technologies, CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2015 Received in revised form 8 July 2015 Accepted 11 July 2015 Available online xxx
The major histocompatibility complex (MHC) is a cluster of genes involved in vertebrate immune response regulation. MHC class I and II cell surface proteins are crucial for discrimination of self versus non-self by the adaptive immune system. Due to their special phylogenetic position within the Euarchontoglires and as a relative of primates, tree shrews have been proposed as an alternative experimental animal model for biomedical studies. However, information about the genetic structure of the tree shrew populations is largely unknown. In this study, we characterized diversity in exon 2 of the MHC II DRB gene isolated from Chinese tree shrews (Tupaia belangeri chinensis). We identified 12 different DRB exon 2 alleles from 15 Chinese tree shrews, 1 to 4 alleles were observed per individual with high levels of sequence divergence between alleles. There were more nonsynonymous than synonymous substitutions in the functionally important antigenbinding site (dN/dS ¼ 2.7952, P < 0.01), indicating that the DRB exon 2 in Chinese tree shrews has been influenced by positive selection. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Major histocompatibility complex Tupaia belangeri chinensis DRB exon 2 Diversity Antigen binding region Positive selection
1. Introduction Tree shrews (Tupaia belangeri) are small squirrel-like mammals that were formerly placed in the Primate order despite the lack of derived features that are diagnostic of primate species (Sargis, 2004). T. belangeri are currently classified as belonging to the family Tupaiidae in the order Scandentia, and together with Dermoptera and Primates, form the group Euarchonta (Murphy et al., 2001; Wilson and Reeder, 2005). Recent whole-genome data have shown that tree shrews are phylogenetically close to primates (Fan et al., 2013; Lindblad-Toh et al., 2011; Song et al., 2012). Given that tree shrews are easy to raise, cheap to maintain, have good reproduction ability and a small body size, and are a close relative of primates, this animal may be a useful alternative animal model for biomedical research. For example, tree shrews have been used in creating animal models for hepatitis B and hepatitis C infection (Amako et al., 2010; Cao et al., 2003; Xu et al., 2007). Tree shrews were also
* Corresponding author. E-mail address: [email protected] (K.-l. Ma). http://dx.doi.org/10.1016/j.bse.2015.07.007 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
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successfully used in studies of human myopia pathogenesis and psychosocial stress diseases (Cao et al., 2003; Fuchs, 2005; Norton et al., 2006). In addition, our previous work showed that the tree shrew might be a good model for investigating brain function and human neurodegenerative diseases (Ma et al., 2013). In vertebrate genomes, major histocompatibility complex (MHC) genes encode molecules responsible for the recognition and presentation of foreign antigens and thus play an important role in immune functions. Traditionally, the MHC gene family has been divided into three classes (I, II, and Ⅲ), which contain groups of genes with a variety of functions. Specifically, class I and II genes code for proteins that bind and present peptides on the cell surface (Klein et al., 1993). MHC I molecules are responsible for the recognition of endogenous and intracellular antigens, such as viruses and cancer cells (Hughes and Yeager, 1998; Sommer, 2005), whereas MHC II molecules are responsible for the recognition of extracellular pathogens such as bacteria and nematodes (Hughes and Yeager, 1998). Given their immunological function, MHC genes provide a genetic system for studying disease dynamics in vertebrates (Bernatchez and Landry, 2003). MHC II is one of the most polymorphic complexes of the vertebrate genome and the majority of the polymorphic sites are clustered in the antigen-binding region (Klein et al., 1993). MHC gene families, including the highly polymorphic MHC II DQ, DP, and DR genes (especially exon 2 of MHC II DRB) have been well defined. Such research has focused on the MHC in mammals and birds, including the gerbil (Gerbillurus paeba) (Harf and Sommer, 2005), the Eurasian beaver (Castor fiber) (Babik et al., 2005), the gray mouse lemur (Microcebus murinus) (Schad et al., 2004), the chacma baboon (Papio ursinus) (Huchard et al., 2006), and the red-winged blackbird (Agelaius phoeniceus) (Edwards et al., 2000). Additionally, limited information has been reported about sequences of DRB introns 4 and 5 of the German tree shrew and MHC II partial genes of the northern tree shrew native to Thailand (Kupfermann et al., 1999; Oppelt et al., 2010). However, MHC II DRB genes have not yet been well defined for the Chinese tree shrew (T. belangeri chinensis). The Chinese tree shrew (Tupaia balengeri chinensis, T. b. chinensis), which is from the nominal subspecies T. balengeri, is the most widely distributed in China and is thus becoming representative of the Chinese tree shrew. According to geographical environment and morphological characteristics, other researchers (Wang, 1987) have suggested that the Chinese tree shrew could be divided into six subspecies. T. b. chinensis is distributed across much of Yunnan, the Yun-Gui plateau, and southwest Sichuan; T. b. gaoligongensis is distributed in the middle and north Gaoligong Mountain; T. b. modesta is distributed on Hainan Island; T. b. tonquinia is distributed in southwest Guangxi; T. b. yaoshanensis is distributed in northwest Guangxi; T. b. yunalis is distributed in the middle and south Yunnan, northwest Guangxi and southwest Guizhou. Despite an increasing interest in using the tree shrew to establish animal models for medical and biological research, the complete genetic sequence of this animal has not yet been published. Specifically, the MHC II molecules have not been reported for T. b. chinensis. In this study, we provide the first description of the characteristics of exon 2 of MHC II DRB from a representative of the mammalian order Scandentia, the T. b. chinensis. We designed a pair of primers based on prosimian sequences that have successfully amplified fragments in rodents (Huchard et al., 2006; Oppelt et al., 2010). We amplified fragments of the MHC II DRB exon 2 gene from the T. b. chinensis, then cloned and sequenced the resulting fragments. This research aims to understand the sequence structure and diversity of the MHC II DRB exon 2 gene and to provide more useful information for studying tree shrew biology and modeling human diseases using this rising experimental animal.
2. Materials and methods 2.1. Study animals In this study, 15 Chinese tree shrews were randomly selected from a group of 3 populations originating from different suburbs of Kunming, including Fuming, Xundian, and Songming, Yunnan Province, China. Animals were raised at the center of the Tree Shrews Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College. All animal studies were approved by the Institutional Ethics Committee and were performed in accordance with the ethical standards detailed in the 1964 Declaration of Helsinki and its later amendments.
2.2. DNA extraction and PCR amplification Genomic DNA was extracted from blood samples using a standard phenol chloroform method. A 171-bp fragment of exon 2 of MHC II DRB was amplified using primers designed from published prosimian sequences. The primer sequences are as follows: forward 50 -GAGTGTCATTTCTACAACGGGACG-30 and reverse 50 -GATCCCGTAGTTGTGTCTGCA-30 . The primers were designed based on prosimian sequences that have successfully amplified fragments in rodents (Huchard et al., 2006; Oppelt et al., 2010). These primers amplify a region that contains most of the polymorphic antigen-binding region. PCR reactions were performed with 5 mL DNA, 2.5 mL buffer, 0.8 mM of each primer, 200 mM of each dNTP and 0.5 U polymerase, in a final volume of 25 mL. PCR amplification conditions were as follows: denaturation at 94 C for 2 min, followed by 35 cycles of 40 s at 94 C, 40 s at 55e65 C, and 40 s at 72 C, with a final elongation step of 2 min at 72 C. Amplified products were visualized on an agarose gel stained with ethidium bromide.
Z.-q. Huang et al. / Biochemical Systematics and Ecology 61 (2015) 417e423
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2.3. Cloning and sequencing The PCR products were cleaned using a QIAquick PCR Purification Kit (Takara Biotechnology, Dalian). All purified PCR products were cloned using the pMD-19 T-Easy vector system (Takara Biotechnology, Dalian) and transformed into competent DH5a cells (Tiangen, Beijing) according to the manufacturer's instructions. Recombinant clones were detected by blue/white screening and 15 to 20 colonies were selected for sequencing. Agarose gel electrophoresis was carried out with 5 mL of the PCR product. 2.4. Sequence analysis Each sequence was verified as a new allele if it was obtained from at least 3 clones, and only those from a single individual were confirmed as an allele through a second independent amplification and cloning procedure. This methodology should remove the potential cloning errors that occur from the use of Taq DNA polymerase, which can result in recombinant alleles, singleton mutations, or misincorporation (Saitoh and Chen, 2008). Sequences were aligned using Clustal X and manually checked to ensure that the sequences were different. We conducted a BLAST search to find the most homologous sequences that are publicly available. Study sequences were compared with sequences from the northern tree shrew (GU825757), Homo sapiens (EF495129), Gerbillurus paeba (AY693636), Pan troglodytes (M96124), M. murinus (EU137092), Macaca mulatta (AY487263.1), and Macaca fascicularis (FN433724.1). The pairwise amino acid differences, as well as the average rates of dN and dS per site, were calculated using Mega 5.0 (Tamura et al., 2011). The locations of the putative ABS and non-ABS were inferred from the molecular structure of human MHC II (Brown et al., 1993). A Z-test was performed to test whether positive selection had shaped the evolution of the assayed fragment of DRB exon 2. We constructed a neighbor-joining tree with K2P nucleotide distances in Mega 5.0 to establish phylogenetic relationships for the tree shrew MHC DRB alleles. In addition, as transpecific polymorphisms are common for MHC II genes (Murphy et al., 2001), close relatives of tree shrews were added to our analysis, including the northern tree shrew (T.belangeri), Gerbillurus paeba, P. troglodytes, M. murinus, M. mulatta, M. fascicularis, and H. sapiens sequences. Due to the high degree of sequence similarity between most Tube-DRB alleles, 1000 bootstraps were used to determine the strength of support for the phylogenetic trees. A Sus scrofa MHC II allele served as an outgroup (Accession number: D78150.1). 3. Results In total, 255 clones from 15 Chinese tree shrews were sequenced with an average of 17 clones (a range of 15e20) per individual. After removal of potential artificial polymorphisms, we confirmed the presence 12 unique alleles of MHC II DRB exon 2 in Chinese tree shrews. Ten new alleles were labeled Tube-DRB*30 to Tube-DRB*39, following the nomenclature of Klein et al. (1990). The other two sequences were identical to Tube-DRB2*15 and Tube-DRB3*20 from the northern tree shrew (T. belangeri), so we named them Tube-DRB2*15 and Tube-DRB3*20 for consistency with the northern tree shrew. All sequences were submitted to GenBank (accession numbers KF234452eKF234463). Comparison with other MHC sequences available in GenBank confirmed that all amplified sequences were part of exon 2 of MHC II DRB. Of the 12 unique alleles, Tube-DRB*15 and Tube-DRB*30 were observed in more than three individuals (prevalence of 40%), Tube-DRB*33, *34, *35, *36, and *37 were observed in two individuals, and the remaining Tube-DRB*20, *31, *38, and *39 were observed in only one individual. The allelic frequencies of the MHC II DRB exon 2 gene in the Chinese tree shrew are given in Table 1. Between one and four alleles were observed in each individual, and among these individuals, F5 had 4 alleles, which was the highest allelic frequency (0.1538). An analysis of the 12 alleles showed that 39.7% (68 out of 171) of the nucleotides were variable and 56.1% (32 out of 57) of the amino acid positions were variable (Fig. 1). In the ABS, 93.3% (14 out of 15) of the amino acids were variable. In contrast,
Table 1 Allele frequencies of the MHC II gene DRB exon 2 in T. b. chinensis. Code of sequences
Code of individual
Allelic number
Allele frequency
Tube-DRB2*15 Tube-DRB3*20 Tube-DRB*30 Tube-DRB*31 Tube-DRB*32 Tube-DRB*33 Tube-DRB*34 Tube-DRB*35 Tube-DRB*36 Tube-DRB*37 Tube-DRB*38 Tube-DRB*39
D6 A6 A4 F5 D8 D6 A4 A6 D2 C1 B6 A7 F5 C1 F1 A3 D6 D2 F5 F7 C7 F5 A6 T2 D7 D7
4 1 5 1 3 2 2 2 2 2 1 1
0.1538 0.0385 0.1923 0.0385 0.1154 0.0769 0.0769 0.0769 0.0769 0.0769 0.0385 0.0385
420
1
2
3
4
5
6
7
8
9
E
R
V
R
F
L
F
R
D
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 I
Y
N
R
E
E
V
V
R
F
D
S
D
V
G
E
F
R
A
V
T
E
L
G
R
F
R
A
E
Y
W
N
R
Q
W
D
I
L
E
Q
K
R
G
R
V
D
N
F
Tube-DRB3*20
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Q
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V
A
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Y
V
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Y
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Tube-DRB3*30
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Tube-DRB3*31
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Chinese
Tube-DRB3*32
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tree
Tube-DRB3*33
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Tube-DRB3*35
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Tube-DRB3*36
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Tube-DRB3*37
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Tube-DRB3*38
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Tube-DRB3*39
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Tube-DRB1*29
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HLA-DRB1*03
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Pan-DRB3*0207
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Other Mamu-DRB1*0414
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Fig. 1. The amino acid sequences of the 12 unique alleles of T. b. chinensis DRB exon 2. A dot indicates a residue identity with the first sequence. Putative antigen binding sites (ABS) (according to Brown et al., 1993) are marked with asterisks and are shaded gray. A BLAST search using all identified sequences confirmed the homology of DRB sequences to those of Homo sapiens (GenBank: EF495129), Gerbillurus paeba (GenBank: AY693636), Pan troglodytes (GenBank: M96124), Microcebus murinus (GenBank: EU137092), Macaca mulatta (GenBank: AY487263.1), Macaca fascicularis (GenBank: FN433724.1) and T. belangeri (northern tree shrew) (GU825757).
Z.-q. Huang et al. / Biochemical Systematics and Ecology 61 (2015) 417e423
Consensus Tube-DRB2*15
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421
Fig. 2. The neighbor-joining phylogenetic tree based on the twelve sequences of exon 2 of the MHC II DRB gene of T. b. chinensis is shown from a matrix of Kimura 2-parameter nucleotide distances (K2P). A Sus scrofa MHC II allele served as an outgroup (accession number D78150.1). The numbers shown indicate bootstrap significance values 50% (1000 replicates).
only 18 (42.9%) of the 42 amino acids were polymorphic in the noneABS. The alleles differed due to substitutions occurring between nucleotide positions 1 and 171. The number of pairwise nucleotide differences between pairs of alleles ranged from 1 (Tube-DRB*34 vs Tube-DRB*36 and Tube-DRB*35 vs Tube-DRB*36) to 48 (Tube-DRB*38 vs Tube-DRB*30) and the number of pairwise amino acid differences ranged from 0 (Tube-DRB*38 vs Tube-DRB*20) to 25 (5 pairs). No insertions, deletions or stop codons were detected in DRB exon 2 in the tree shrew, suggesting it was functional. When analyzed in a systematic BLAST search, sequences of Tube-DRB*15, *33, *35, and *36 were found to be the most similar to primate DRB sequences, with the highest homology rate of 88.30%. The sequences of Tube-DRB*32, *34 and *38 were similar to the DRB sequence from H. sapiens, with a homology rate of 87.13%. In contrast, the homology rates between Tube-DRB*30, *31 and other species were lower, with an average homology rate of 78.14%. The neighbor-joining phylogenetic tree based on the Chinese tree shrew DRB sequences is shown from a matrix of Kimura 2-parameter nucleotide distances (K2P) (Fig. 2). Bootstrap values 50% (1000 replicates) are shown. Tube-DRB2*15, TubeDRB*36, Tube-DRB*33, Tube-DRB*35 and Tube-DRB*32 belong to one branch. Tube-DRB*30, Tube-DRB*31, Tube-DRB*37, and Tube-DRB*39 are gathered in one branch. However, Tube-DRB3*20, Tube-DRB*34, and Tube-DRB*38 are arranged in another further branch. The phylogenetic tree constructed using the Poisson-corrected amino acid distances method in Mega 5.0 was very similar to our tree model. To examine the modes of selection acting on the MHC II DRB exon 2, the proportion of dN and dS was calculated for each of three regions: the whole exon 2 sequences, the ABS sequences and the non-ABS sequences. In the ABS, dN was higher than dS (dN/dS ¼ 2.7952,P < 0.01, Table 2). In contrast, there was an excess of dS for non-ABS (dN/dS ¼ 0.5459, P > 0.05, Table 2). When all nucleotide positions were considered, we found only a small excess in dS (dN/dS ¼ 0.9528, P > 0.05, Table 2).
Table 2 The rates of nonsynonymous substitutions (dN) and synonymous substitutions (dS) in the antigen binding site (ABS) and non-antigen binding site (non-ABS) and their ratio for DRB exon 2 sequences in T. b. chinensis. Note: ABS consists of 15 residues. Note: Non-ABS consists of 42 residues. Note: Results of the Z-test for positive selection (standard errors obtained through 1000 bootstrap replicates). Site
dN ± S.E.
dS ± S.E.
dN/dS
P
ABS N ¼ 15 Non-ABS N ¼ 42 All alleles N ¼ 57
0.587 ± 0.0045 0.119 ± 0.00096 0.202 ± 0.00148
0.210 ± 0.0021 0.218 ± 0.0018 0.212 ± 0.0015
2.7952 0.5459 0.9528
0.000042 0.0598 0.8589
422
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4. Discussion There are several well-documented examinations of exon 2 of the DRB gene in some populations (Edwards et al., 2000; Harf and Sommer, 2005; Huchard et al., 2006; Schad et al., 2004); however, little is known about this exon in the Chinese tree shrew (T. b. chinensis). This study represents the first investigation of MHC diversity in a Chinese tree shrew. We obtained 12 different sequences of exon 2 of MHC Ⅱ DRB from 15 Chinese tree shrews. DRB alleles from the Chinese tree shrew showed high levels of sequence divergence for an intraspecific comparison and all alleles had a unique nucleotide sequence. TubeDRB3*20 and Tube-DRB*38 displayed the same peptide sequences showed that there occurred synonymous mutation between the two alleles. More than 3 alleles were found in 3 Chinese tree shrews (such as F5,D6,and A6), indicating that some haplotypes in this population consist of at least two DRB loci and are highly polymorphic. Two alleles, Tube-DRB2*15 and Tube-DRB3*20, were found to be shared between the Chinese tree shrew and the northern tree shrew, demonstrating that there has been a relative evolutionary stability in the tree shrew (T. belangeri). That is, these alleles may have become common to all the tree shrews, because the tree shrews in different environments generate the same immune rejection response to facing the same infectious pathogens and parasites. Another cause could be that the alleles became universal in the ancestral population of northern tree shrew or Chinese tree shrew by drift alone and then spread to other regions of Southeast Asia by migration. Another 10 new alleles were detected in this study, which are likely to be Chinese tree shrew-specific alleles, and reflect the rich polymorphisms of the MHC Ⅱ DRB gene of the Chinese tree shrew. These are distinct from those of the northern tree shrew and this finding is consistent with the research results on MHC genes of other species (Qiu et al., 2008). DRB alleles from the northern tree shrew showed a rich genetic diversity, but only one of the alleles was transcribed. TubeDRB1*21 contained a triplet deletion, and Tube-DRB4*27 had a stop codon within the sequence. However, because no insertions/deletions or stop codons were detected in our samples, it is reasonable to suppose that they have biological functions in the body and are not pseudogene copies. The homology between most Chinese tree shrew MHC II DRB gene sequences and primates was higher than the homology between these gene sequences of other species. We inferred a relatively close phylogenetic relationship between Tube-DRB*15, *36, *33, *35 and *32 and primate MHC II DRB genes. Tree shrew MHC might be orthologous to the ancestral primate MHC II DRB gene, this finding is consistent with previous results (Fan et al., 2013; Lindblad-Toh et al., 2011; Song et al., 2012). We found evidence of positive selection acting on MHC with rates of non-synonymous substitutions greater than synonymous substitutions within the ABS (Table 2). Polymorphism was highest in the functionally important antigen recognition and binding sites. In these positions, more dN than dS were observed at the ABS. Any dS will change the amino acid sequence of the peptide, thus enabling the molecule to bind a diverse array of antigens (Brown et al., 1993; Musolf et al., 2004). This is considered to be a clear sign of positive selection (Hughes and Nei, 1988, 1989) and is in agreement with other MHC classes (Archie et al., 2010; Hauswaldt et al., 2007). In contrast, the other two regions show no signs of directed selection, which is also in accordance with those data observed for the northern tree shrew (Oppelt et al., 2010). Some variation hypotheses have been proposed to explain how the high MHC polymorphisms are maintained, including overdominance (i.e., heterozygote advantage), frequency-dependent selection (i.e., rare allele advantage) (Hughes and Nei, 1989), and sexual reproduction and mate choice (Bernatchez and Landry, 2003; Lampert et al., 2009); however, a combination of selection methods may be responsible for the extent of polymorphism observed and maintained within the MHC (Bernatchez and Landry, 2003). Further studies on allele-specific transcription will be our on-going goal in the near future. The topology of the Chinese tree shrew's and its close relatives' DRB sequences revealed four main branches (Fig. 2), and they seem close to each other. Alternatively, related species may show similarities at MHC alleles because of ancient transspecies polymorphisms caused by balancing selection. This result is consistent with the trans-species hypothesis presented by Klein (Klein, 1987). As interlocus allelic exchange is known to occur at MHC loci, it was impossible to assign alleles to individual loci without more detailed genomic information. Therefore, in our phylogenetic analysis, we treated all alleles detected in the Chinese tree shrew as representing the DRB locus and will obtain more samples and genomic information for future studies. In summary, our work is the first study of MHC variation in a Chinese tree shrew. The results presented here are based on the analysis of a functionally important region of the MHC. Our data on exon 2 of the MHC II DRB gene in the Chinese tree shrew show the genetic diversity of this animal resource and gives evidence of a positive selection processes at the ABS. These results provide a foundation for understanding the genetic basis for the use of this animal as a potential model for biomedical research.
Acknowledgments This work was supported by Yunnan Natural Science Foundation (No.2011FZ211; No.2013FZ132), National Natural Science Foundation of China Grants (No. 81301073; No.U1402221), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No.20121106120056), the PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (No.3332013082; No.3332013146; No.33320140191), the Fund for Institute of Medical Biology, Chinese Academy of Medical Sciences (No.IMB2013YB01, 2014IMB01ZD), the Fund for Institute of Pathogen Biology, Chinese Academy of Medical Sciences (No. 2015IPB201), Technology innovation talents in Yunnan Province (No.2014HB065), and the National Science and Technology Support Project (No.2009BAI83B02-21; No.2011BAI15B01-21).
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Sequence diversity of the MHC Ⅱ DRB gene in Chinese tree shrews (Tupaia belangeri chinensis) Zhang-qiong Huang a, b, Xiao-mei Sun a, Jie-jie Dai a, Ming-liang Gu c, You-song Ye a, Yu-feng Yao a, Rui-ju Jiang a, Kai-li Ma a, b, * a
Institute of Medical Biology, Chinese Academy of Medical Sciences, Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infection Diseases, Kunming 650118, China b Institute of Basic Medical Sciences, Neuroscience Center, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100005, China c Beijing Key Laboratory of Genome and Precision Medicine Technologies, CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 April 2015 Received in revised form 8 July 2015 Accepted 11 July 2015 Available online xxx
The major histocompatibility complex (MHC) is a cluster of genes involved in vertebrate immune response regulation. MHC class I and II cell surface proteins are crucial for discrimination of self versus non-self by the adaptive immune system. Due to their special phylogenetic position within the Euarchontoglires and as a relative of primates, tree shrews have been proposed as an alternative experimental animal model for biomedical studies. However, information about the genetic structure of the tree shrew populations is largely unknown. In this study, we characterized diversity in exon 2 of the MHC II DRB gene isolated from Chinese tree shrews (Tupaia belangeri chinensis). We identified 12 different DRB exon 2 alleles from 15 Chinese tree shrews, 1 to 4 alleles were observed per individual with high levels of sequence divergence between alleles. There were more nonsynonymous than synonymous substitutions in the functionally important antigenbinding site (dN/dS ¼ 2.7952, P < 0.01), indicating that the DRB exon 2 in Chinese tree shrews has been influenced by positive selection. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Major histocompatibility complex Tupaia belangeri chinensis DRB exon 2 Diversity Antigen binding region Positive selection
1. Introduction Tree shrews (Tupaia belangeri) are small squirrel-like mammals that were formerly placed in the Primate order despite the lack of derived features that are diagnostic of primate species (Sargis, 2004). T. belangeri are currently classified as belonging to the family Tupaiidae in the order Scandentia, and together with Dermoptera and Primates, form the group Euarchonta (Murphy et al., 2001; Wilson and Reeder, 2005). Recent whole-genome data have shown that tree shrews are phylogenetically close to primates (Fan et al., 2013; Lindblad-Toh et al., 2011; Song et al., 2012). Given that tree shrews are easy to raise, cheap to maintain, have good reproduction ability and a small body size, and are a close relative of primates, this animal may be a useful alternative animal model for biomedical research. For example, tree shrews have been used in creating animal models for hepatitis B and hepatitis C infection (Amako et al., 2010; Cao et al., 2003; Xu et al., 2007). Tree shrews were also
* Corresponding author. E-mail address: [email protected] (K.-l. Ma). http://dx.doi.org/10.1016/j.bse.2015.07.007 0305-1978/© 2015 Elsevier Ltd. All rights reserved.
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successfully used in studies of human myopia pathogenesis and psychosocial stress diseases (Cao et al., 2003; Fuchs, 2005; Norton et al., 2006). In addition, our previous work showed that the tree shrew might be a good model for investigating brain function and human neurodegenerative diseases (Ma et al., 2013). In vertebrate genomes, major histocompatibility complex (MHC) genes encode molecules responsible for the recognition and presentation of foreign antigens and thus play an important role in immune functions. Traditionally, the MHC gene family has been divided into three classes (I, II, and Ⅲ), which contain groups of genes with a variety of functions. Specifically, class I and II genes code for proteins that bind and present peptides on the cell surface (Klein et al., 1993). MHC I molecules are responsible for the recognition of endogenous and intracellular antigens, such as viruses and cancer cells (Hughes and Yeager, 1998; Sommer, 2005), whereas MHC II molecules are responsible for the recognition of extracellular pathogens such as bacteria and nematodes (Hughes and Yeager, 1998). Given their immunological function, MHC genes provide a genetic system for studying disease dynamics in vertebrates (Bernatchez and Landry, 2003). MHC II is one of the most polymorphic complexes of the vertebrate genome and the majority of the polymorphic sites are clustered in the antigen-binding region (Klein et al., 1993). MHC gene families, including the highly polymorphic MHC II DQ, DP, and DR genes (especially exon 2 of MHC II DRB) have been well defined. Such research has focused on the MHC in mammals and birds, including the gerbil (Gerbillurus paeba) (Harf and Sommer, 2005), the Eurasian beaver (Castor fiber) (Babik et al., 2005), the gray mouse lemur (Microcebus murinus) (Schad et al., 2004), the chacma baboon (Papio ursinus) (Huchard et al., 2006), and the red-winged blackbird (Agelaius phoeniceus) (Edwards et al., 2000). Additionally, limited information has been reported about sequences of DRB introns 4 and 5 of the German tree shrew and MHC II partial genes of the northern tree shrew native to Thailand (Kupfermann et al., 1999; Oppelt et al., 2010). However, MHC II DRB genes have not yet been well defined for the Chinese tree shrew (T. belangeri chinensis). The Chinese tree shrew (Tupaia balengeri chinensis, T. b. chinensis), which is from the nominal subspecies T. balengeri, is the most widely distributed in China and is thus becoming representative of the Chinese tree shrew. According to geographical environment and morphological characteristics, other researchers (Wang, 1987) have suggested that the Chinese tree shrew could be divided into six subspecies. T. b. chinensis is distributed across much of Yunnan, the Yun-Gui plateau, and southwest Sichuan; T. b. gaoligongensis is distributed in the middle and north Gaoligong Mountain; T. b. modesta is distributed on Hainan Island; T. b. tonquinia is distributed in southwest Guangxi; T. b. yaoshanensis is distributed in northwest Guangxi; T. b. yunalis is distributed in the middle and south Yunnan, northwest Guangxi and southwest Guizhou. Despite an increasing interest in using the tree shrew to establish animal models for medical and biological research, the complete genetic sequence of this animal has not yet been published. Specifically, the MHC II molecules have not been reported for T. b. chinensis. In this study, we provide the first description of the characteristics of exon 2 of MHC II DRB from a representative of the mammalian order Scandentia, the T. b. chinensis. We designed a pair of primers based on prosimian sequences that have successfully amplified fragments in rodents (Huchard et al., 2006; Oppelt et al., 2010). We amplified fragments of the MHC II DRB exon 2 gene from the T. b. chinensis, then cloned and sequenced the resulting fragments. This research aims to understand the sequence structure and diversity of the MHC II DRB exon 2 gene and to provide more useful information for studying tree shrew biology and modeling human diseases using this rising experimental animal.
2. Materials and methods 2.1. Study animals In this study, 15 Chinese tree shrews were randomly selected from a group of 3 populations originating from different suburbs of Kunming, including Fuming, Xundian, and Songming, Yunnan Province, China. Animals were raised at the center of the Tree Shrews Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College. All animal studies were approved by the Institutional Ethics Committee and were performed in accordance with the ethical standards detailed in the 1964 Declaration of Helsinki and its later amendments.
2.2. DNA extraction and PCR amplification Genomic DNA was extracted from blood samples using a standard phenol chloroform method. A 171-bp fragment of exon 2 of MHC II DRB was amplified using primers designed from published prosimian sequences. The primer sequences are as follows: forward 50 -GAGTGTCATTTCTACAACGGGACG-30 and reverse 50 -GATCCCGTAGTTGTGTCTGCA-30 . The primers were designed based on prosimian sequences that have successfully amplified fragments in rodents (Huchard et al., 2006; Oppelt et al., 2010). These primers amplify a region that contains most of the polymorphic antigen-binding region. PCR reactions were performed with 5 mL DNA, 2.5 mL buffer, 0.8 mM of each primer, 200 mM of each dNTP and 0.5 U polymerase, in a final volume of 25 mL. PCR amplification conditions were as follows: denaturation at 94 C for 2 min, followed by 35 cycles of 40 s at 94 C, 40 s at 55e65 C, and 40 s at 72 C, with a final elongation step of 2 min at 72 C. Amplified products were visualized on an agarose gel stained with ethidium bromide.
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2.3. Cloning and sequencing The PCR products were cleaned using a QIAquick PCR Purification Kit (Takara Biotechnology, Dalian). All purified PCR products were cloned using the pMD-19 T-Easy vector system (Takara Biotechnology, Dalian) and transformed into competent DH5a cells (Tiangen, Beijing) according to the manufacturer's instructions. Recombinant clones were detected by blue/white screening and 15 to 20 colonies were selected for sequencing. Agarose gel electrophoresis was carried out with 5 mL of the PCR product. 2.4. Sequence analysis Each sequence was verified as a new allele if it was obtained from at least 3 clones, and only those from a single individual were confirmed as an allele through a second independent amplification and cloning procedure. This methodology should remove the potential cloning errors that occur from the use of Taq DNA polymerase, which can result in recombinant alleles, singleton mutations, or misincorporation (Saitoh and Chen, 2008). Sequences were aligned using Clustal X and manually checked to ensure that the sequences were different. We conducted a BLAST search to find the most homologous sequences that are publicly available. Study sequences were compared with sequences from the northern tree shrew (GU825757), Homo sapiens (EF495129), Gerbillurus paeba (AY693636), Pan troglodytes (M96124), M. murinus (EU137092), Macaca mulatta (AY487263.1), and Macaca fascicularis (FN433724.1). The pairwise amino acid differences, as well as the average rates of dN and dS per site, were calculated using Mega 5.0 (Tamura et al., 2011). The locations of the putative ABS and non-ABS were inferred from the molecular structure of human MHC II (Brown et al., 1993). A Z-test was performed to test whether positive selection had shaped the evolution of the assayed fragment of DRB exon 2. We constructed a neighbor-joining tree with K2P nucleotide distances in Mega 5.0 to establish phylogenetic relationships for the tree shrew MHC DRB alleles. In addition, as transpecific polymorphisms are common for MHC II genes (Murphy et al., 2001), close relatives of tree shrews were added to our analysis, including the northern tree shrew (T.belangeri), Gerbillurus paeba, P. troglodytes, M. murinus, M. mulatta, M. fascicularis, and H. sapiens sequences. Due to the high degree of sequence similarity between most Tube-DRB alleles, 1000 bootstraps were used to determine the strength of support for the phylogenetic trees. A Sus scrofa MHC II allele served as an outgroup (Accession number: D78150.1). 3. Results In total, 255 clones from 15 Chinese tree shrews were sequenced with an average of 17 clones (a range of 15e20) per individual. After removal of potential artificial polymorphisms, we confirmed the presence 12 unique alleles of MHC II DRB exon 2 in Chinese tree shrews. Ten new alleles were labeled Tube-DRB*30 to Tube-DRB*39, following the nomenclature of Klein et al. (1990). The other two sequences were identical to Tube-DRB2*15 and Tube-DRB3*20 from the northern tree shrew (T. belangeri), so we named them Tube-DRB2*15 and Tube-DRB3*20 for consistency with the northern tree shrew. All sequences were submitted to GenBank (accession numbers KF234452eKF234463). Comparison with other MHC sequences available in GenBank confirmed that all amplified sequences were part of exon 2 of MHC II DRB. Of the 12 unique alleles, Tube-DRB*15 and Tube-DRB*30 were observed in more than three individuals (prevalence of 40%), Tube-DRB*33, *34, *35, *36, and *37 were observed in two individuals, and the remaining Tube-DRB*20, *31, *38, and *39 were observed in only one individual. The allelic frequencies of the MHC II DRB exon 2 gene in the Chinese tree shrew are given in Table 1. Between one and four alleles were observed in each individual, and among these individuals, F5 had 4 alleles, which was the highest allelic frequency (0.1538). An analysis of the 12 alleles showed that 39.7% (68 out of 171) of the nucleotides were variable and 56.1% (32 out of 57) of the amino acid positions were variable (Fig. 1). In the ABS, 93.3% (14 out of 15) of the amino acids were variable. In contrast,
Table 1 Allele frequencies of the MHC II gene DRB exon 2 in T. b. chinensis. Code of sequences
Code of individual
Allelic number
Allele frequency
Tube-DRB2*15 Tube-DRB3*20 Tube-DRB*30 Tube-DRB*31 Tube-DRB*32 Tube-DRB*33 Tube-DRB*34 Tube-DRB*35 Tube-DRB*36 Tube-DRB*37 Tube-DRB*38 Tube-DRB*39
D6 A6 A4 F5 D8 D6 A4 A6 D2 C1 B6 A7 F5 C1 F1 A3 D6 D2 F5 F7 C7 F5 A6 T2 D7 D7
4 1 5 1 3 2 2 2 2 2 1 1
0.1538 0.0385 0.1923 0.0385 0.1154 0.0769 0.0769 0.0769 0.0769 0.0769 0.0385 0.0385
420
1
2
3
4
5
6
7
8
9
E
R
V
R
F
L
F
R
D
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 I
Y
N
R
E
E
V
V
R
F
D
S
D
V
G
E
F
R
A
V
T
E
L
G
R
F
R
A
E
Y
W
N
R
Q
W
D
I
L
E
Q
K
R
G
R
V
D
N
F
Tube-DRB3*20
.
Q
.
.
Y
V
A
.
Y
V
.
.
.
.
.
Y
.
.
.
.
.
.
.
.
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Fig. 1. The amino acid sequences of the 12 unique alleles of T. b. chinensis DRB exon 2. A dot indicates a residue identity with the first sequence. Putative antigen binding sites (ABS) (according to Brown et al., 1993) are marked with asterisks and are shaded gray. A BLAST search using all identified sequences confirmed the homology of DRB sequences to those of Homo sapiens (GenBank: EF495129), Gerbillurus paeba (GenBank: AY693636), Pan troglodytes (GenBank: M96124), Microcebus murinus (GenBank: EU137092), Macaca mulatta (GenBank: AY487263.1), Macaca fascicularis (GenBank: FN433724.1) and T. belangeri (northern tree shrew) (GU825757).
Z.-q. Huang et al. / Biochemical Systematics and Ecology 61 (2015) 417e423
Consensus Tube-DRB2*15
Z.-q. Huang et al. / Biochemical Systematics and Ecology 61 (2015) 417e423
421
Fig. 2. The neighbor-joining phylogenetic tree based on the twelve sequences of exon 2 of the MHC II DRB gene of T. b. chinensis is shown from a matrix of Kimura 2-parameter nucleotide distances (K2P). A Sus scrofa MHC II allele served as an outgroup (accession number D78150.1). The numbers shown indicate bootstrap significance values 50% (1000 replicates).
only 18 (42.9%) of the 42 amino acids were polymorphic in the noneABS. The alleles differed due to substitutions occurring between nucleotide positions 1 and 171. The number of pairwise nucleotide differences between pairs of alleles ranged from 1 (Tube-DRB*34 vs Tube-DRB*36 and Tube-DRB*35 vs Tube-DRB*36) to 48 (Tube-DRB*38 vs Tube-DRB*30) and the number of pairwise amino acid differences ranged from 0 (Tube-DRB*38 vs Tube-DRB*20) to 25 (5 pairs). No insertions, deletions or stop codons were detected in DRB exon 2 in the tree shrew, suggesting it was functional. When analyzed in a systematic BLAST search, sequences of Tube-DRB*15, *33, *35, and *36 were found to be the most similar to primate DRB sequences, with the highest homology rate of 88.30%. The sequences of Tube-DRB*32, *34 and *38 were similar to the DRB sequence from H. sapiens, with a homology rate of 87.13%. In contrast, the homology rates between Tube-DRB*30, *31 and other species were lower, with an average homology rate of 78.14%. The neighbor-joining phylogenetic tree based on the Chinese tree shrew DRB sequences is shown from a matrix of Kimura 2-parameter nucleotide distances (K2P) (Fig. 2). Bootstrap values 50% (1000 replicates) are shown. Tube-DRB2*15, TubeDRB*36, Tube-DRB*33, Tube-DRB*35 and Tube-DRB*32 belong to one branch. Tube-DRB*30, Tube-DRB*31, Tube-DRB*37, and Tube-DRB*39 are gathered in one branch. However, Tube-DRB3*20, Tube-DRB*34, and Tube-DRB*38 are arranged in another further branch. The phylogenetic tree constructed using the Poisson-corrected amino acid distances method in Mega 5.0 was very similar to our tree model. To examine the modes of selection acting on the MHC II DRB exon 2, the proportion of dN and dS was calculated for each of three regions: the whole exon 2 sequences, the ABS sequences and the non-ABS sequences. In the ABS, dN was higher than dS (dN/dS ¼ 2.7952,P < 0.01, Table 2). In contrast, there was an excess of dS for non-ABS (dN/dS ¼ 0.5459, P > 0.05, Table 2). When all nucleotide positions were considered, we found only a small excess in dS (dN/dS ¼ 0.9528, P > 0.05, Table 2).
Table 2 The rates of nonsynonymous substitutions (dN) and synonymous substitutions (dS) in the antigen binding site (ABS) and non-antigen binding site (non-ABS) and their ratio for DRB exon 2 sequences in T. b. chinensis. Note: ABS consists of 15 residues. Note: Non-ABS consists of 42 residues. Note: Results of the Z-test for positive selection (standard errors obtained through 1000 bootstrap replicates). Site
dN ± S.E.
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ABS N ¼ 15 Non-ABS N ¼ 42 All alleles N ¼ 57
0.587 ± 0.0045 0.119 ± 0.00096 0.202 ± 0.00148
0.210 ± 0.0021 0.218 ± 0.0018 0.212 ± 0.0015
2.7952 0.5459 0.9528
0.000042 0.0598 0.8589
422
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4. Discussion There are several well-documented examinations of exon 2 of the DRB gene in some populations (Edwards et al., 2000; Harf and Sommer, 2005; Huchard et al., 2006; Schad et al., 2004); however, little is known about this exon in the Chinese tree shrew (T. b. chinensis). This study represents the first investigation of MHC diversity in a Chinese tree shrew. We obtained 12 different sequences of exon 2 of MHC Ⅱ DRB from 15 Chinese tree shrews. DRB alleles from the Chinese tree shrew showed high levels of sequence divergence for an intraspecific comparison and all alleles had a unique nucleotide sequence. TubeDRB3*20 and Tube-DRB*38 displayed the same peptide sequences showed that there occurred synonymous mutation between the two alleles. More than 3 alleles were found in 3 Chinese tree shrews (such as F5,D6,and A6), indicating that some haplotypes in this population consist of at least two DRB loci and are highly polymorphic. Two alleles, Tube-DRB2*15 and Tube-DRB3*20, were found to be shared between the Chinese tree shrew and the northern tree shrew, demonstrating that there has been a relative evolutionary stability in the tree shrew (T. belangeri). That is, these alleles may have become common to all the tree shrews, because the tree shrews in different environments generate the same immune rejection response to facing the same infectious pathogens and parasites. Another cause could be that the alleles became universal in the ancestral population of northern tree shrew or Chinese tree shrew by drift alone and then spread to other regions of Southeast Asia by migration. Another 10 new alleles were detected in this study, which are likely to be Chinese tree shrew-specific alleles, and reflect the rich polymorphisms of the MHC Ⅱ DRB gene of the Chinese tree shrew. These are distinct from those of the northern tree shrew and this finding is consistent with the research results on MHC genes of other species (Qiu et al., 2008). DRB alleles from the northern tree shrew showed a rich genetic diversity, but only one of the alleles was transcribed. TubeDRB1*21 contained a triplet deletion, and Tube-DRB4*27 had a stop codon within the sequence. However, because no insertions/deletions or stop codons were detected in our samples, it is reasonable to suppose that they have biological functions in the body and are not pseudogene copies. The homology between most Chinese tree shrew MHC II DRB gene sequences and primates was higher than the homology between these gene sequences of other species. We inferred a relatively close phylogenetic relationship between Tube-DRB*15, *36, *33, *35 and *32 and primate MHC II DRB genes. Tree shrew MHC might be orthologous to the ancestral primate MHC II DRB gene, this finding is consistent with previous results (Fan et al., 2013; Lindblad-Toh et al., 2011; Song et al., 2012). We found evidence of positive selection acting on MHC with rates of non-synonymous substitutions greater than synonymous substitutions within the ABS (Table 2). Polymorphism was highest in the functionally important antigen recognition and binding sites. In these positions, more dN than dS were observed at the ABS. Any dS will change the amino acid sequence of the peptide, thus enabling the molecule to bind a diverse array of antigens (Brown et al., 1993; Musolf et al., 2004). This is considered to be a clear sign of positive selection (Hughes and Nei, 1988, 1989) and is in agreement with other MHC classes (Archie et al., 2010; Hauswaldt et al., 2007). In contrast, the other two regions show no signs of directed selection, which is also in accordance with those data observed for the northern tree shrew (Oppelt et al., 2010). Some variation hypotheses have been proposed to explain how the high MHC polymorphisms are maintained, including overdominance (i.e., heterozygote advantage), frequency-dependent selection (i.e., rare allele advantage) (Hughes and Nei, 1989), and sexual reproduction and mate choice (Bernatchez and Landry, 2003; Lampert et al., 2009); however, a combination of selection methods may be responsible for the extent of polymorphism observed and maintained within the MHC (Bernatchez and Landry, 2003). Further studies on allele-specific transcription will be our on-going goal in the near future. The topology of the Chinese tree shrew's and its close relatives' DRB sequences revealed four main branches (Fig. 2), and they seem close to each other. Alternatively, related species may show similarities at MHC alleles because of ancient transspecies polymorphisms caused by balancing selection. This result is consistent with the trans-species hypothesis presented by Klein (Klein, 1987). As interlocus allelic exchange is known to occur at MHC loci, it was impossible to assign alleles to individual loci without more detailed genomic information. Therefore, in our phylogenetic analysis, we treated all alleles detected in the Chinese tree shrew as representing the DRB locus and will obtain more samples and genomic information for future studies. In summary, our work is the first study of MHC variation in a Chinese tree shrew. The results presented here are based on the analysis of a functionally important region of the MHC. Our data on exon 2 of the MHC II DRB gene in the Chinese tree shrew show the genetic diversity of this animal resource and gives evidence of a positive selection processes at the ABS. These results provide a foundation for understanding the genetic basis for the use of this animal as a potential model for biomedical research.
Acknowledgments This work was supported by Yunnan Natural Science Foundation (No.2011FZ211; No.2013FZ132), National Natural Science Foundation of China Grants (No. 81301073; No.U1402221), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No.20121106120056), the PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (No.3332013082; No.3332013146; No.33320140191), the Fund for Institute of Medical Biology, Chinese Academy of Medical Sciences (No.IMB2013YB01, 2014IMB01ZD), the Fund for Institute of Pathogen Biology, Chinese Academy of Medical Sciences (No. 2015IPB201), Technology innovation talents in Yunnan Province (No.2014HB065), and the National Science and Technology Support Project (No.2009BAI83B02-21; No.2011BAI15B01-21).
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