Sequence variation of the DQB allele in the cetacean MHC

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1 Mammal Study 28: (2003) the Mammalogical Society of Japan Sequence variation of the DQB allele in the cetacean MHC Kosuke Hayashi 1,*, Shin Nishida 1, Hideyoshi Yoshida 2, Mutsuo Goto 3, Luis A. Pastene 3 and Hiroko Koike 1 1 Graduate School of Social and Cultural Studies, Kyushu University, Ropponmatsu, Chuo-ku, Fukuoka , Japan 2 Cetacean Population Biology Section, National Research Institute of Far Seas Fisheries, Orido, Shimizu, Shizuoka , Japan 3 Institute of Cetacean Research, Toyoumi, Minato-ku, Tokyo, Japan Abstract. Putative nucleotide sequences for DQB exon 2 alleles of the major histocompatibility complex (MHC) were amplified and determined in the 16 cetacean species using the PCR technique. The 172 bp sequences amplified showed no multiple alleles more than two in each of the examined individuals. The sequences of the 31 cetacean DQB alleles detected were monophyletic with the HLA-DQB1, and were separated from the lineages of HLA- DQB2 and DQB3. These results suggest that the locus is the homolog to the human DQB1 gene. The observed frequency of nonsynonymous substitutions in the cetacean DQB sequences was significantly higher than that of synonymous substitutions. The amino acid variation at the putative peptide binding region (PBR) was considerably high. These results imply that positive selection has promoted its variability at the cetacean DQB gene as other mammalian MHC. The DQB gene tree showed that four Mysticeti alleles branched off from the clade consisted of Mysticeti alleles only and were included in the clade consisted of Odontoceti alleles. This suggests trans-species polymorphism in the cetacean MHC gene. Key words: Cetacea, DQB allele, major histocompatibility complex (MHC), positive selection, trans-species polymorphism. The major histocompatibility complex (MHC) is a large multigene coding glycoprotein, which plays a key role in the initiation of immune responses in vertebrates (Brown et al. 1988). Two types of MHC molecules are known, class I and class II MHC molecules. The class I molecules are found on all nucleated cells, and bind for endogenously derived peptides. The class II molecules are found only on antigen-presenting cells, and bind to exogenously derived peptides. These peptide-mhc complexes are recognised by T-lymphocytes. By binding to the complex, T-cells initiate adaptive immune responses (Edwards and Potts 1996). The human MHC, called HLA (human leucocytes antigen), spans about 4,000 kb of the genome (Germain and Margulies 1993; Harding 1996). Three class II loci (DP, DQ and DR) are known to produce functional heterodimer molecule which is made up of two glycopeptide chains ( and ). The MHC gene is known to be highly polymorphic in human, primates and other terrestrial species (Takahata and Nei 1990; Diaz et al. 2000). The crystal structures of HLA-DR 1 suggest that the general structure of peptides binding to class II proteins are very similar among different species (Brown et al. 1993), but the peptide-binding region (PBR) maintain higher polymorphism than the other regions (Nepom et al. 1996; Hoelzel et al. 1999; Diaz et al. 2000). In most mammals, genetic variations in the MHC class I and class II have been interpreted as an adaptation to a large number of pathogens (Klein and Takahata 1990; Hill et al. 1991). It is thought that high extent of polymorphism at MHC genes is maintained by natural selection such as through heterozygote advantage or * To whom correspondence should be addressed. Present address: Tottori-Higashi High School, Tottori, Japan. koikegsc@mbox.nc.kyushuu.ac.jp

2 90 Mammal Study 28 (2003) frequency-dependent selection (Hughes and Nei 1989, 1990, 1992; Takahata and Nei 1990; Edwards and Potts 1996). The deficiency of variation at the MHC loci may increase the risk of extinction of small, isolated populations (Yuhki and O Brien 1990). Therefore, it has been suggested that the allelic variety of MHC should be taken into account in conservation, such as management of captive breeding programs (Hughes 1991). It has been suggested that the pathogen environment for marine mammals may provide a week selective pressure on MHC variation due to a relatively low prevalence of infectious disease in the marine environment (Trowsdale et al. 1989; Hoelzel et al. 1993; Murray et al. 1995). For example, a study on the southern elephant seal (Mirounga leonina) also showed low restriction fragment length polymorphism (RFLP) (Slade 1992). However, Hoelzel et al. (1999) investigated sequence variation at exon 2 of the DQB gene in four Pinnipod species, and found that the southern elephant seal was comparable to that seen in human populations, while the northern elephant seal exhibited much less variation. These results shows that not every marine mammal species has a low MHC variation, and sequence analysis reveals genetic variation more precisely than RFLP analysis. MHC variations have been examined only in a few cetacean species. Trowsdale et al. (1989) analysed the MHC of the fin whale (Balaenoptera physalus) and the sei whale (B. borealis) by RFLP method, and indicated a relatively low degree of polymorphism in these two cetacean species. Sequence variation analysis at the class II DQB locus in beluga whales (Delphinapterus leucas) and narwhals (Monodon monoceros), also detected lower variation than those of the terrestrial mammals (Murray et al.1995; Murray and White 1998; Murray et al.1999). In this study, we conducted an intensive sequence analysis of the DQB exon in 16 cetacean species using the PCR technique. The DQB allelic variability, especially at the PBR, was investigated in order to understand positive selection in the MHC variation of the cetacean species. Materials and methods Tissue samples (muscle, liver or skin) were collected from 16 cetacean species including five species of Mysticeti and 11 species of Odontoceti (Table 1); one southern right whale, Eubalaena australis, one fin whale, Balaenoptera physalus, one blue whale, B. musculus, nine minke whales, B. acutorostrata, 11 Antarctic minke whales, B. bonaerensis, one sperm whale, Physeter macrocephalus, one Stejneger s beaked whale, Mesoplodon stejnegeri, one Hubbs beaked whale, M. carlhubbsi, one harbor porpoise, Phocoena phocoena, 50 finless porpoises, Neophocaena phocaenoides, one short-beaked common dolphin, Delphinus delphis, one bottlenose dolphin, Tursiops truncatus, one Risso s dolphin, Gramupus griseus, one irrawaddy dolphin, Orcaella brevirostris, two Pacific white-sided dolphins, Table 1. Samples used in this study *. Order Suborder Family Scientific name Common name N Source ** Cetacea Mysticeti Balaenidae Eubalaena australis Southern right whale 1 C Balaenopteridae Balaenoptera physalus Fin whale 1 C Balaenoptera musculus Blue whale 1 C Balaenoptera acutorostrata Minke whale 9 C Balaenoptera bonaerensis Antarctic minke whale 11 C Odontoceti Physeteridae Physeter macrocephalus Sperm whale 1 C Ziphiidae Mesoplodon stejnegeri Stejneger s beaked whale 1 C Mesoplodon carlhubbsi Hubbs beaked whale 1 S Phocoenidae Phocoena phocoena Harbor porpoise 1 S Neophocaena phocaenoides Finless porpoise 50 Y Delphinidae Delphinus delphis Short-beaked common dolphin 1 S Tursiops truncatus Bottlenose dolphin 1 M Gramupus griseus Risso s dolphin 1 M Orcaella brevirostris Irrawaddy dolphin 1 M Lagenorhynchus obliquidens Pacific white-sided dolphin 2 M Globicephala macrorhynchus Short-finned pilot whale 2 M * Rice (1998) and Kato et al. (2000) were referred for the scientific names and classification. ** C: The Institute of Cetacean Research, S : The National Science Museum, Tokyo, M: Marine World Uminonakamichi Aquarium, Y: Previous study by Yoshida et al. (2001)

3 Hayashi et al., DQB allele in the cetacean MHC 91 Lagenorhynchus obliquidens, and two short-finned pilot whales, Globicephala macrorhynchus. These samples were obtained from the Institute of Cetacean Research, National Museum of Natural Sciences and the Marine World Uminonakamichi Aquarium (Table 1). The finless porpoise samples were the same as used in the previous study on mitochondrial DNA sequences (Yoshida et al. 2001). About two to four mg of the tissue samples stored in 70% ethanol, and 100 l of blood samples kept in 50 mm EDTA-2Na were used for DNA extraction. For the protein digestion, 310 l of RSB buffer (10 mm Tris-HCl ph 7.4, 10 mm NaCl, 25 mm EDTA-2Na ph 7.4), 15 l of 10% SDS (sodium dodecyl sulfate), and 15 l of proteinase-k (20 mg/ml) were added, and incubated for two hours at 55 C on a rotator. The nucleic acids were extracted using the Iso-Quick DNA extraction kit (ORCA Research Inc.). DQB exon 2 region was amplified using the primer set, DQB-AMP-A (5'-CATGTGCTACTTCACCAACGG- 3') and DQB-AMP-B (5'-CTGGTAGTTGTGTCTGCA- CAC-3'), reported by Kimura and Sasazuki (1992). To improve the PCR specificity, we used the hot start technique (Chou et al.1992) using AmpliTaq Gold (PE Applied Biosystems). The employed PCR profile was as follows; preheating at 95 C for 12 minutes, and then 40 cycles of denature at 95 C for 30 seconds, annealing at 55 C for 45 seconds and extension at 72 C for 45 seconds. To sequence individual alleles correctly in heterozygous individuals, PCR products were cloned into pcr2.1 vector using the TOPO TA cloning kit (Invitrogen). Around ten to 15 colonies with a faint white colour were checked from each PCR product to examine whether the right products had been inserted. For the identification of an allele, at least two identical sequences from each PCR product were required, since a few clones would show a point mutation possibly by PCR artifacts (Gyllensten et al. 1990; Murray et al. 1995). Murray et al. (1995) suggests that if every allele was amplified at an equal frequency, by sequencing five clones, there is low probability (P = ) of not detecting both alleles in the case of heterozygote. After the insert check by PCR, the products with a right insert were purified by the PCR Product Pre-sequencing kit (USB), and cycle sequencing was performed using the DTCS Quick start kit containing Dye Terminators (Beckman Coulter). Sequences were determined up to ten clones for each individual with CEQ2000 DNA auto-sequencer Fig. 1. DQB allele sequences of 18 cetacean species. A dot indicates the same nucleotide as the Southern right whale. The shared codons show nonsynonymous substitutions.

4 92 Mammal Study 28 (2003) Fig. 2. Putative amino acid sequences translated from 172 bp sequences of the cetacean DQB allele. Amino acids are shown by the single-letter code. A dot indicates the same amino acid as the Southern right whale. The putative positions corresponding to the protein binding region are shaded. The boxes show the site of synonymous substitution for the cetacean species. The numbers across the sequence correspond to the amino acid position based on chain of the DR structure (Brown et al. 1993). (Beckman Coulter). Identified DQB allele sequences were aligned and translated into amino acid by using GENETYX-MAC, Ver (Software Developer Co. Ltd). Other genetic analyses were conducted using MEGA version 2.1 (Kumar et al. 2001). The number of synonymous substitutions per synonymous site (d s ) and number of nonsynonymous substitutions per nonsynonymous site (d n ) were calculated between detected DQB alleles by the Nei-Gojobori method with Jukes-Cantor correction and their standard errors were computed with 500 bootstrap replications (Jukes and Cantor 1969; Nei and Gojobori 1986). A neighbour-joining (NJ) tree (Saitou and Nei 1987) was constructed based on the P-distance, the proportion of number of amino acid differences between inferred amino acid sequences (Nei and Kumar 2000). HLA-DQB1, DQB2, DQB3 and bovine DQB1 were used as outgroup sequences. Results Detection of DQB alleles The samples from the16 cetacean species were successfully amplified, and 172 bp nucleotide sequences of the DQB alleles were determined (Fig. 1). The analysis of multiple clones from each individual did not reveal more than two different sequences, suggesting the success of amplification at a single DQB locus in the examined cetaceans. The observed numbers of alleles varied among the species. For example, eight alleles (Neph-a to h) were found in 50 finless porpoises, and six alleles (Babo-a to f ) were found in 11 Antarctic minke whales, while only one allele (Baac-a) was found in nine minke whales. In addition to these sequences, three white whale alleles and one narwhal allele had been deposited in the Genbank database. Consequently, a total of 35 allele sequences including the detected 31 alleles and the

5 Hayashi et al., DQB allele in the cetacean MHC 93 Table 2. The number of synonymous substitutions per synonymous site (d s), the number of nonsynonymous substitution per nonsubstitution site (d n) and their ratio at the peptide binding region (PBR), non-pbr and all the sequences of the analysed DQB exon 2 region. d s and d n are indicated as percentages. The numbers in parentheses are standard error calculated by bootstrap 500 replications. Suborder/Species No. of alleles PBR Non-PBR All d s d n d n/d s d s d n d n/d s d s d n d n/d s Mysticeti (5 species) (1.06) (6.13) (2.38) 5.48 (2.22) (1.88) 7.99 (2.14) 2.21 Odontoceti (13 species) (0.92) (6.64) (1.14) 4.34 (1.80) (0.88) 8.88 (2.08) 4.96 Total (18 species) (1.58) (7.23) (1.41) 5.35 (1.98) (1.12) 9.70 (2.30) 3.55 Fig. 3. Amino acid variability plot (Wu and Kabat 1970) for the 35 amino acid sequences of the cetacean DQB alleles. Filled bars indicate the peptide binding region. 2 and 3 sheets, and -helix are shaded. The numbers across the sequence correspond to the amino acid position based on chain of the DR structure (Brown et al. 1993). four alleles from the DNA database were obtained from the 18 cetacean species. These were used for the following analyses. Alignment of the 172 bp nucleotide sequences of the 35 DQB alleles indicated 50 variable sites (Fig. 1). All the mutations were base substitutions, and deletions, insertions or inversions were not found among the sequences. These 50 variable sites consisted of 13 synonymous substitution sites and 37 nonsynonymous substitution sites (Fig. 1). Amino acid alignment of the cetacean DQB genes The nucleotide sequence fragment of 172 bp corresponded to the 21st to 77th amino acids in the chain of the DR structure (Brown et al. 1993; Stern et al. 1994; Fig. 2). These 35 amino acid sequences showed higher similarity to the HLA-DQB1, than to the other human DQB genes (i.e., HLA-DQB2 and DQB3). The number of nonsynonymous substitutions per nonsynonymous site (d n ) in Mysticeti, Odontoceti and all the cetacean species was considerably higher than those for synonymous substitutions per synonymous site (d s ) (Table 2). Among the finless porpoise alleles, all the substitutions were nonsynonymous and no synonymous substitution was not found. The d n /d s ratio at the PBR were 16.4, 22.0 and 14.5 in Mysticeti, Odontoceti and all the cetacean species, respectively (Table 2). On the other hand, the ratio at non-pbr was 1.2, 2.0 and 1.7 respectively. Polymorphism at PBR The putative PBR of the cetacean DQB gene was estimated in comparison with the chain of the HLA-DR 1, of which molecular structure is well-known (Brown et al. 1993). For the 14 amino acids corresponding to the PBR, variation was detected at 13 sites, while for the

6 94 Mammal Study 28 (2003) Fig. 4. Neighbor-joining tree based on the P-distances among the 35 DQB allele sequences of the 18 cetacean species. Bootstrap replications were performed 1000 times. Only bootstrap values over 50% are shown. remaining 43 amino acids (here after referred to as non- PBR), it was detected only at 16 sites (Fig. 2). The amino acid variability plot (Wu and Kabat 1970) also demonstrated that amino acids at the PBR showed higher polymorphism than those at the non-pbr (Fig. 3). Most variability concentrated on the 2 and 3 sheets and -helix. Phylogenetic tree The NJ tree showed that all the 35 cetacean DQB

7 Hayashi et al., DQB allele in the cetacean MHC alleles formed a monophyletic group with the HLA- DQB1 allele, and not with HLA-DQB2 and DQB3 alleles, supporting that the amplified sequences are the homolog to the DQB1 gene in humans (Fig. 4). All the cetacean DQB alleles formed a monophylic group, and separated into the Mysticeti clade and the Odontoceti- Mysticeti mixed clade although most bootstrap values were low (Fig. 4). The Mysticeti clade consisted of six Mysticeti alleles (Fin whale, Baac-a, and Babo-a, b, c and d). In the Odontoceti-Mysticeti mixed clade, one Mysticeti allele (Southern right whale) was clustered with a Odontoceti allele (White whale), and three Mysticeti alleles (Blue whale, Babo-e and f ) were located within a clade mainly composed of the finless porpoise alleles. Each of the three alleles detected in the shortfinned pilot whale (Glma-a to c) shared the last node with a different species in Odontoceti with low bootstrap values for some nodes: Glma-a and the Risso s dolphin allele, Glma-b and the irrawaddy dolphin allele, Glma-c and the Pacific white-sided dolphin (Laob-b) formed a group, respectively. Discussion 95 The sequence analysis of 172 bp fragments amplified for the 16 cetacean species showed that multiple alleles more than two was not revealed in each of the examined individuals. The sequences of the 31 cetacean DQB alleles detected were monophyletic with the HLA-DQB1, and were separated from the lineages of HLA-DQB2 and DQB3. These results suggest that we succeeded in the amplification at a single DQB locus in the cetacean species, and indicate that the locus is the homolog to the human DQB1 locus as Hoelzel et al. (1999) reported. The DQB amino acid sequences inferred from the cetacean DQB alleles were highly variable at the PBR. This feature is a characteristic of genes encoding proteins with an antigen-presenting function. The ratio of d n to d s at the PBR of the DQB alleles were very high within the sub-orders of the new world monkey (Diaz et al. 2000). Hughes and Nei (1988, 1989) found that the nonsynonymous substitution rate was higher than the synonymous substitution rate in the codons encoding the PBR in human and mice, and suggested that positive selection has promoted their variability. The present results also suggest that the cetacean DQB detected herein is functionally important and selected positively. The phylogenetic relationship among the cetacean DQB alleles did not coincide with the taxonomic relationship. Alleles from different species and different genera are mixed together within the same allelic lineages (Klein et al. 1993). Some alleles from a species are more similar to the alleles of different species than each other. In the NJ tree, most DQB alleles shared a last node with other species alleles (Fig. 4). This pattern of evolution has been referred to as trans-species (Klein 1987). It is thought that the MHC diversity is maintained by balancing selection over long periods of evolutionary time (Klein and Takahata 1990; Takahata and Nei 1990; Gutierrez-Espeleta et al. 2001). Therefore, for example, for the three alleles of the short-finned pilot whale, they may have arisen before speciation and have been maintained since then. Successful amplification of the DQB1 exon 2 alleles in the 16 cetacean species indicated that the methodology of this study is widely applicable to the cetacean MHC analysis. The analysis of intraspecific polymorphism of this region will provide important information for the cetacean conservation genetics. Acknowledgments: This study was supported by the Institute of Cetacean Research. 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