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Characterisation of T cell antigen receptor α chain isotypes in the common carp
Fish & Shellfish Immunology 19 (2005) 205e216 www.elsevier.com/locate/fsi
Characterisation of T cell antigen receptor a chain isotypes in the common carp Etsuou Imaia, Jun Ishikawaa, Tadaaki Moritomob, Mitsuru Tomanaa,) a
Department of Applied Biological Science, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan b Department of Veterinary Science, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan Received 1 September 2004; revised 8 November 2004; accepted 17 November 2004 Available online 8 March 2005
Abstract T cell receptor a (TCRa) chain has been characterised in several teleost species to date. Here, a reverse transcriptionpolymerase chain reaction (RT-PCR) strategy was used to isolate cDNA clones encoding TCRa chain from an individual of the common carp (Cyprinus carpio.L.). The Va sequences identified were most similar to Va of other teleosts, and could be classified into as many as 14 Va families. For the Ja sequences, diversity comparable to that seen in other teleosts could be identified, and the J-region motif was well conserved. The Ca sequences demonstrated the highest similarity to zebrafish Ca and possessed a well-conserved transmembrane (TM) region. Two Ca isotypes with a complete C region were obtained, designated Ca1 and Ca2, with w70% similarity at the amino acid level (w85% identity at the nucleotide level), and, in addition, Ca2 contained two unique sequences, designated Ca2a and Ca2b, with 93% similarity (96% identity). Therefore, the results obtained using an individual clearly showed that carp possesses at least two Ca loci, possibly as a result of tetraploidisation. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: T cell receptor a; Isotype; Common carp; Evolution
1. Introduction T cell receptor (TCR) ab, comprised of a and b chains, can recognise an antigen (Ag)-derived peptide combined with a major histocompatibility complex (MHC) molecule. TCR molecule can be divided into constant (C) and variable (V) domains, the latter being responsible for the specific binding to Ag plus MHC molecule [1]. The Ag specificity of TCR in each T cell can be kept by an allelic exclusion, a mechanism by ) Corresponding author. Tel./fax: C81 466 84 3351. E-mail address: [email protected] (M. Tomana). 1050-4648/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2004.11.004
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which a T cell expresses only TCR molecules with a single specificity on the cell surface [2,3]. The C domain is encoded by a C segment gene, while the V domain by a V and joining (J) segment, or a V, diversity (D), and J segment, for TCRa or -b chains, respectively [1]. For mammals, the genomic organisation of TCRa is as follows: a cluster of Va segments is followed by a Ja cluster and a single Ca segment both in the same transcriptional orientation as the Va segments, and TCRd gene, a constituent of another type of TCR chain, exists between the Va and Ja cluster [4]. For teleosts, it has recently been reported that a Va cluster is followed by a Ca segment and a Ja cluster both in the inverted orientation as the Va segments, in addition, without TCRd gene between the Va cluster and Ca segment [5e7]. To date, TCRa sequences have been reported for various teleost species including rainbow trout (Oncorhynchus mykiss) [8], channel catfish (Ictalurus punctatus) [9], zebrafish (Danio rerio) [10e12], Atlantic cod (Gadus morhua) [13], Japanese pufferfish (Takifugu rubripes) [6], pufferfish (Spheroides nephelus) [14], puffer fish (Tetraodon nigroviridis) [7], Japanese flounder (Paralichthys olivaceus) [5], bicolor damselfish (Stegastes partitus) [15], and Atlantic salmon (Salmo salar) [16]. From those results, a considerable variety has been observed for both Va and Ja in teleosts, whereas a single Ca locus could be found in almost all teleosts examined to date, except for damselfish, in which the presence of two Ca loci was suggested by the result of Southern blots [15]. On the other hand, it has been shown that two Cb isotypes are present in most mammals examined, although the sequences of the two isotypes in each species seem to be highly similar to each other in a coding region [17]. Two Cb isotypes have also been observed in teleosts, bicolor damselfish [18], channel catfish [19] and Japanese flounder [5], and the existence of two isotypes was suggested for Atlantic cod [13]. For mammals, it would be expected that the presence of two Cb isotypes contributes to an increase in the number of Jb segments, due to the genomic organisation in which JbeCb is arranged in tandem following multiple Vb segments [20e22]. As for carp Ag receptor genes, an immunoglobulin (Ig) molecule, consisting of heavy (H) and light (L) chains, has been characterised thus far, and then three distinct CH sequences, probably representing two isotypes [23], and four IgL isotypes have been elucidated [24e26]. But carp TCR genes have not yet been reported until this study, where the cloning and characterisation of TCRa is described. 2. Materials and methods 2.1. Isolation of cDNA clones encoding Ja and partial Ca region Total RNA was extracted from thymus of a single common carp, according to the manufacturer’s instructions, using TRIzol reagent (Invitrogen, San Diego, USA). cDNA was synthesised with reverse transcriptase (SUPERSCRIPTÔ II; Invitrogen) using 2 mg of the total RNA as template and random primers (Invitrogen). Degenerated primers corresponding to conserved sequences in the 30 end of Va FR3 (forward) and in the C-terminal half of Ca (reverse) of TCRa genes found in other teleosts were used in reverse transcription PCR (RT-PCR). The sequences of primers were: Va(deg.) (A(I/L/V)Y(H/Y)CAL): GCI/ITI/TAY/YAY/TGY/GCI/YT (YZT or C; IZinosine); and Ca(deg.) ((F/L)LK(A/T)(I/V)(A/ V)FN(I/V)): Y/RTT/RAA/IRC/IAY/IGY/YTT/IAR/IA (RZA or G) (Fig. 1). Forty cycles of
Fig. 1. Position of primers for the isolation of carp TCRa gene sequences.
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amplification (95 (C for 0.5 min, 42.5 (C for 0.5 min, and 72 (C for 1 min) were performed in 25 ml of reaction mixture. The amplified DNA (about 400 bp in length) was gel-purified and cloned into pCR2.1 vector using a TA cloning kit (Invitrogen). 2.2. Isolation of cDNA clones encoding complete Va or Ca region using RACE method To amplify total RNA obtained above and gain entire Va or Ca region-containing cDNA clones using a SMARTÔ RACE cDNA Amplification kit (Clontech, Palo Alto, USA), the gene specific primers corresponding to the sequences within the Ca (for 50 RACE) or Ja (for 30 RACE) region, respectively, were employed. The sequences of the primers were: Ca (50 ) in clone Cpk1 (MSLGLYWLR): TCT/CAG/CCA/ ATA/GAG/GCC/CAG/TGA/CA; Jal in clone Cpl36 (FGTGTKLTVE): T/GGC/ACT/GGA/ACC/AAA/ CTG/ACT/GTT/G; Ja2 in clone Cpl42 (KLIFGSGTQ): G/CTG/ATT/TTT/GGG/TCT/GGC/ACA/CA; or Ja3 in clone Cpl70 (LQPDYGNNK): TG/CAG/CCG/GAT/TAT/GGG/AAC/AAC/AAG (Fig. 1). Clone Cpl70 was a chimeric Ca sequence between Ca2 and Ca1 possibly caused by PCR artifacts [27]. Special care was then taken when analysing sequences and possible chimeras were excluded from the data set. RACE was performed according to the manufacturer’s instructions. For both RACE, touchdown PCR was employed in 50 ml of reaction mixture: 5 s at 94 (C and 3 min at 72 (C for 5 cycles; 5 s at 94 (C, 10 s at 70 (C, and 3 min at 72 (C for 5 cycles; and 5 s at 94 (C, 10 s at 68 (C, and 3 min at 72 (C for 25 cycles. The PCR products (700e800 bp in length in 50 RACE, and 650e1050 bp in 30 RACE) were gel-purified and cloned into pCR2.1-TOPO vector (Invitrogen). 2.3. DNA sequencing and sequence analysis The PCR products were sequenced in both strands by the dideoxy method [28]. The deduced amino acid sequences were analysed using the tfasta program [29] against the DDBJ sequence database. Multiple alignments were made using the clustal W program [30]. The nucleotide sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers AB120568eAB120575, AB120577eAB120610, and AB120612eAB120623. 2.4. Southern blot analysis Carp erythrocyte DNA was extracted [31] and digested with EcoRI or HindIII (Takara, Shiga, Japan). The DNA (10 mg/lane) was separated on an 0.8% agarose gel and transferred to a nylon membrane (Hybond-NC, Amersham Biosciences, Piscataway, USA) by blotting in 0.4 M NaOH. The filter was fixed by UV cross-linking, prehybridised, and hybridised with 32P-labeled random-primed probes in a medium containing 0.5 M NaH2PO4/NaHPO4, (pH 7.2), 1 mM ethylenediaminetetraacetate (EDTA), and 7% (w/v) SDS at 65 (C overnight. Probes specific for sequences of Va1, Va2, and Va4 families, and 30 -UT of Ca1 and Ca2 isotypes (w700 bp in a full length), where only w60 bp at the 30 end of each Ca sequence was included, were used. The filter was washed in 40 mM NaH2PO4/NaHPO4 and 1% SDS at 65 (C for 10 min, and the radioactivity was detected with a phosphoimager (Molecular Dynamics, Sunnyvale, USA). 2.5. Northern blot analysis Total RNA was extracted from organs of brain, liver, spleen, mesonephros, pronephros, and thymus as described above. Poly(A)CRNA was purified from total RNA using the OligotexÔ-dT30CSuperD mRNA Purification kit (Takara) and then the RNA (2 mg/lane) was separated on 1.5% agarose gels containing formaldehyde and transferred to Hybond-XL membrane (Amersham Biosciences) by blotting in 10! SSC. The filter was fixed by UV cross-linking, prehybridised, and hybridised as described above for the Southern
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blot. A probe encompassing Va, Ja, and the Ca region of clone Cpl16 was used. The filter was washed according to the Hybond-XL manual, and the radioactivity was detected with a phosphoimager (Molecular Dynamics).
3. Results 3.1. Va and Ja region analysis To gain carp TCRa sequences, RT-PCR was employed using degenerated primers both in the 30 end of Va FR3 and in the C-terminal half of Ca (Fig. 1). Then several TCRa-coding clones with almost the same nucleotide sequences in the C region, represented by clone Cpk1, were obtained judging from a high similarity with corresponding Ca sequences in other teleosts (data not shown). Next, using a primer near the 30 ends of the Ca sequence in clone Cpk1, the 50 RACE method was performed to identify clones encoding an entire Va region (Fig. 1). As a result, a total of 34 unique Va sequences were identified and then could be classified into 14 Va families, defined as possessing less than 70% nucleotide identity in Va segments (Fig. 2). All the Va sequences tested showed the highest similarity to other teleost Va sequences each (data not shown). The number of Va segments within each family identified here was as follows: 8 (Va1 family), 5 (Va2), 4 (Va4 and Va5), 3 (Va3), 2(Va6), and 1 (Va7e14). The size of the CDR1 and CDR2 loops in the Va varied from 4 to 7 and from 5 to 13 amino acid residues, respectively. It is noted that the length of CDR2 in clone Cpl47 (Va5 family) is extremely long among the carp clones identified here, with 13 amino acid residues. In addition, the other three Va5 sequences cloned also possessed 13 amino acid residues for the CDR2 (data not shown). All Va sequences tested included the Cys23 and Cys104 residues, which likely form the intradomain disulphide bridge, typical features of TCR molecules, FR2 (WYRQ) and FR3 (YYC) motif, were well observed within each Va sequence. The data set of functional Ja examined is comprised of a total of 43 different clones obtained by the RT-PCR and 50 RACE (Fig. 3). Among them 40 clones show an in-frame VeJ and JeC junction, while the remaining three clones have an out-of-frame VeJ (clone Cpl48) and JeC junction (Cpl31 and Cpl42), and they might have been caused by an inappropriate VeJ recombination and JeC splicing, respectively. The 43 different Ja segments examined could be divided into 37 Jas with less than 70% amino acid similarity between them. The four groups of Ja showing a high similarity among them, particularly in the 30 end of the sequences, could be found, and members of each group used the same Ca isotype: either Ca1 or Ca2 (as mentioned below, two isotypes could be identified in this study). On the other hand, each Va family consisting of two or more members
Fig. 2. Multiple alignment of the amino acid sequences of the carp TCRa V region. The same residues as the sequence shown at the top are denoted by dots, and a gap introduced to maximise homology is indicated by a hyphen. Putative FR/CDR regions are indicated above sequences. Cysteine residues involved in intradomain interaction are numbered. The Va family of each clone is shown in parentheses.
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Fig. 3. Multiple alignment of the amino acid sequences of the carp TCRa J region. Amino acids in the J-region motif are indicated below the sequences. The Ca isotype linked with each Ja is shown in parentheses. Asterisks indicate an out-of-frame transcript. Four groups of Ja clones demonstrating a high similarity among them are underlined. 37 Jas with less than 70% amino acid similarity between them are numbered.
used both Ca isotypes, Ca1 and Ca2. The J-region motif, Lys-Leu/Ile-X-Phe-Gly-X-Gly-Thr-X-Leu (K(I/ L)XFGXGTXL), was well conserved. As shown in Fig. 4, Ja nucleotide sequences were aligned according to the IMGT numbering system [32] based on the Ja data set, except for the RT-PCR-derived clones, because Ala105 and Leu106 are included in the primer sequence used for this method. Variation could be seen well throughout positions 107e114. In clone Cpl48, the presence of an extra G nucleotide between positions 108 and 109 might result in an out-of-frame VeJ junction.
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Fig. 4. Multiple alignment of the nucleotide sequences of the carp TCRa J region. Asterisks indicate an out-of-frame transcript. Codons are numbered according to Lefranc et al. [32].
3.2. Ca region analysis As described above, RT-PCR was initially employed to obtain carp TCRa sequences encompassing the region from Va FR3 to the site near the 30 end of Ca. Then, the Ca sequences identified were all derived from a unique Ca isotype, designated Ca1. In 50 RACE, however, in addition to Ca1, another isotype of incomplete Ca sequences (about 80 amino acids in length) could be identified, designated Ca2, which could be classified into Ca2a and Ca2b subtypes due to the less degree of amino acid difference between them (data not shown). For each isotype, many clones were identified in this 50 RACE (17 clones for Ca1, 13 for Ca2a, 7 for Ca2b), and the amino acid similarity in the incomplete Ca sequences between them was about 60%. Next, using primers designed on the basis of the sequences of Ja joined to the respective Ca isotype, 30 RACE was employed to identify each complete Ca sequence: Ja1 primer for Ca1, Ja2 for Ca2, and Ja3 for the Ca2/Ca1 chimera as mentioned in Section 2 (Fig. 1). Then, Ca1, Ca2a, and Ca2b sequences could be identified, with four, five, and two clones, respectively. Ca1 sequences were obtained using Ja1 primer as expected, while Ca2 sequences were obtained both using Ja2 and Ja3. The representative clones of each isotype/subtype are shown in Fig. 5. When compared in the corresponding region of Ca, the amino acid sequence of clone Cpm73 (Ca1) was identical with those of Cpl16 (50 RACE products) and Cpk1 (RTPCR), while clones Cpm91 (Ca2a) and Cpm95 (Ca2b) sequences showed only one amino acid replacement with Cpl21 and Cpl63 (both from 50 RACE), respectively. The sequence similarity at the amino acid level was about 70% between Ca1 and Ca2 isotypes (about 85% identity at the nucleotide level), and 93% between Ca2a and Ca2b subtypes (96% nucleotide identity). It should be noted that sequences at the 30 end of Ca (35 amino acid residues in length) were identical among the representatives of the distinct types. When compared with other species, carp Ca sequences showed the highest similarity to zebrafish followed
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Fig. 5. Multiple alignment of the Ca amino acid sequences of the carp and other vertebrate species. Characteristic residues are numbered. The putative transmembrane (TM) region is underlined in clone Cpm95. Residues that match the CART motif are underlined in clone Cpm73. Percentages of amino acid similarity between clone Cpm91 and the others, and Cpm73 and the others are indicated. Accession numbers are: zebrafish (AF246174); catfish (IPU58505); Japanese pufferfish (AF269222); pufferfish (SNU22676); flounder (AB053369); cod (GMO133845); trout (OMU50991); mouse (MMTCRAI1); human (HSTCRAC); axolotl (AMU50992); chicken (GDU04611); skate (REU75768).
by trout and catfish Ca. The carp Ca C-terminal region is well conserved together with other species compared, and this likely represents the transmembrane (TM) region, according to the SOSUI program predicting TM segments [33], then followed by the cytoplasmic tail region. The carp TM includes the conserved antigen receptor TM motif (CART) which is thought to play a role in the assembly and/or signalling of the TCR/CD3 complex [34], and the two charged residues at positions 239 (Arg) and 244 (Lys), possibly involved in the association of the TCR with CD3 molecules [35,36]. Clone Cpm85 belonging to the Ca2 isotype might be a pseudo-product because one putative nucleotide insertion into the site around 30 amino acids upstream of the 30 end in normal Ca2 is recognised (data not shown). This insertion results in the loss of the TM region, which is well conserved among different taxa as described above, although the coding region apparently becomes 66 amino acids longer in length compared with that of normal Ca. Among 50 RACE products, this type of nucleotide insertion could also be found in a total of four clones. Regarding a 30 -untranslated region (30 -UT), the sequences of Ca1 and Ca2 isotypes could be clearly distinguished from one another (70e82% nucleotide identity) as well as in their C-region (data not shown).
3.3. Southern blot analysis The diversity and organisation of carp TCRa gene were examined by Southern blot analyses using probes specific for Va sequences, and 30 -UT of Ca1 and Ca2 isotypes (Fig. 6). Instead of the Ca region,
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Fig. 6. Southern blot analyses of carp genomic DNA hybridised with Va- and 30 -UT-specific probes. The restriction enzymes used are EcoRI (E) and HindIII (H), and the position of size markers (in kb) is shown on the left.
30 -UT was chosen as the probe, because teleost Cas have been reported to be coded by three coding exons, and then the use of Ca probe possibly provides ambiguous interpretation of the result [6,7,16]. The Va families with many clones isolated, represented by Va1, Va2, and Va4, were examined here, and each Va probe hybridised with multiple bands, as expected. The blot hybridised with 30 -UT probes of Ca1 and Ca2 isotypes showed only one band each. In EcoRI digestion, distinct bands in position could be clearly recognised in the two blots. 3.4. Northern blot analysis Northern blotting was performed with mRNA from various organs including thymus, using a VJC probe (Fig. 7). The intense signal was observed in thymus at approximately 1.4 kb, corresponding to the expected size of complete TCRa transcripts, calculated from the analyses of both 50 and 30 RACE clones (data not shown). The relatively weak signal could be seen in pronephros.
4. Discussion In this study, two Ca isotypes, designated Ca1 and Ca2, could be identified from a single carp. Recently, Criscitiello et al. have suggested as well that two TCR Ca loci exist in damselfish, but the sequences were similar and distinguished by a single amino acid [15]. Thus, the presence of two Ca loci might not be an
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Fig. 7. Northern blot analyses of mRNA from various carp organs hybridised with a VJC probe. The position of size markers (in kb) is shown on the left.
unusual situation in teleosts. Additionally, two different Ca genes have recently been described for Xenopus [37]. The existence of two Ca isotypes in a fish, as in mammalian Cb, might contribute to an increase in the number of Ja segments, since each Ca isotype in carp seemed to be joined with a unique set of Ja segments (Fig. 3). In addition to a Ca variety observed in Ca1 and Ca2, with w70% amino acid sequence similarity, another variety derived from Ca2a and Ca2b could be demonstrated, with 93% similarity (Fig. 5). As the data set of carp Ca sequences was derived from an individual, the presence of more than two unique Ca sequences identified in this study means that carp Ca region is encoded by at least two loci. It is consistent with the results of Southern analyses using probes for 30 -UT of Ca1 and Ca2 (Fig. 6). Since the genomic sequence data is not available for the Cas at present, it remains unknown whether the Cas are in tandem organisation or not. The presence of at least two Ca loci in carp might have been affected by a mechanism such as a tetraploidisation event, as well known for teleosts, including the Cyprinidae family [38e43]. For the relationship between Ca2a and Ca2b, although two unique sequences with a few substitutions were identified in Ca2a (data not shown), the possibility of Taq polymerase errors could not be excluded for the extent of difference observed; at present it is unknown whether the two subtypes are located on the same (alleles) or different loci (isotypes). As shown in Fig. 6, an apparent single band could be seen using a probe specific for the 30 -UT of Ca2, where the sequences employed were highly similar between Ca2a and Ca2b enough to cause cross-hybridisation (data not shown), suggesting that the two subtypes represent alleles, or are closely linked in a genomic organisation even if they are isotypes. The recent reports of teleost TCRas from several species have indicated the existence of high levels of polymorphism in the constant region [11,15]. Recently it has been reported that zebrafish, belonging to the same Cyprinidae family as carp, possessed several distinct Ca sequences, where the degree of amino acid sequence similarity observed corresponds to that found between the carp Ca2 subtypes [10e12]. But it is unclear at present if the difference found in zebrafish Cas represents an allelic variation or isotype, due to the data sets derived from pooled zebrafish but not from an individual.
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It is currently uncertain whether or not Ca1 and Ca2 are each linked with independent Va clusters. But if this is the case, it would be expected that a chance of producing two (or more) productive TCRa chains with different specificities within a single T cell increased in carp compared to mammals, with a single Ca locus, and the carp immune system might require a strict allelic exclusion machinery responsible for a single TCRa chain expression on the cell surface. The allelic exclusion seems to occur at a post-translational level in mammalian a-chain [2,3,44,45], mainly in thymus organ, where the mRNA expression of TCRa was active compared with other organs examined as shown in Fig. 7, and the sequences identified in this study were derived from this organ. By immunocytochemical studies in carp, it has been demonstrated that thymus may be the primary lymphoid organ for T cells [46]. Variability of Va in five teleost species studied in detail, is as follows: 10 Va families (zebrafish) [12], 6 (trout and puffer fish) [7,8], 5 (salmon) [16], and 4 subfamilies (Japanese pufferfish) [6]. On the other hand, carp possessed as many as 14 Va families, which consisted of one to eight members (Fig. 2). The existence of extraordinary numbers, 87 families of Va genes, has also been mentioned for zebrafish as unpublished data [12]. Thus, an apparent increase in the Va diversity of a Cyprinidae family compared with those of other teleosts, might have been caused by the duplication of Va loci in the ancestor for carp and zebrafish. Furthermore, a total of 43 different Jas were characterised in this study (Fig. 3); the degree of Ja complexity seems to be comparable to that reported for other teleosts [5e8]. Among the Ja sequences analysed, a putative failure of JeC splicing was observed, and by the following reason the splicing error may be also applicable to Ca sequences represented by clone Cpm85, likely a pseudo-product (data not shown). This type of clone was characterised by one putative nucleotide insertion into the site around 30 amino acids upstream of the 30 end in normal Ca, and this site could correspond to the boundary between the second and third exon of Ca reported in takifugu [6] and Japanese flounder (accession number AB081557). It is, however, possible that the insertion is encoded in a Ca gene itself. It seems that a single Ca locus is permitted in most species examined to date. In this report and recent studies [15,37], however, two Ca isotypes could be identified, therefore, it is important to examine if the situation is accompanied by a novel machinery for allelic exclusion at the TCRa loci.
Acknowledgements This work was supported in part by a grant from the Ministry of Education, Science, Sports and Culture to promote advanced scientific research.
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Proc Natl Acad Sci U S A 1988;85(8):2444e8. [30] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22(22):4673e80. [31] Sambrook J, Frisch E, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. [32] Lefranc MP, Pommie C, Ruiz M, Giudicelli V, Foulquier E, Truong L, et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 2003;27(1):55e77. [33] Hirokawa T, Boon-Chieng S, Mitaku S. SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 1998;14(4):378e9. [34] Campbell KS, Backstrom BT, Tiefenthaler G, Palmer E. CART: a conserved antigen receptor transmembrane motif. Semin Immunol 1994;6(6):393e410. [35] Blumberg RS, Alarcon B, Sancho J, McDermott FV, Lopez P, Breitmeyer J, et al. Assembly and function of the T cell antigen receptor. Requirement of either the lysine or arginine residues in the transmembrane region of the a chain. J Biol Chem 1990; 265(23):14036e43.
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[36] John S, Banting GS, Goodfellow PN, Owen MJ. Surface expression of the T cell receptor complex requires charged residues within the a chain transmembrane region. Eur J Immunol 1989;19(2):335e9. [37] Haire RN, Kitzan Haindfield MK, Turpen JB, Litman GW. Structure and diversity of T-lymphocyte antigen receptors alpha and gamma in Xenopus. Immunogenetics 2002;54(6):431e8. [38] Ohno S, Muramoto J, Christian L, Atkin Z. Diploid-tetraploid relationship among old-world members of the fish family Cyprinidae. Chromosoma 1967;23:1e19. [39] Ojima Y, Hitotsumachi S. Cytogenetic studies in lower vertebrates, IV. A note on the chromosomes of the carp (Cyprinus carpio) in comparison with those of the funa and the goldfish (Carassius auratus). Jpn J Genet 1967;42:163e7. [40] Raicu P, Taisescu E, Cristian A. Diploid chromosome complement of the carp. Cytologia 1972;37:355e8. [41] Wolf U, Ritter H, Atkin N, Ohno S. Polyploidization in the fishes family Cyprinidae, Order Cypriniformes. Humangenetik 1969; 7:240e4. [42] David L, Blum S, Feldman MW, Lavi U, Hillel J. Recent duplication of the common carp (Cyprinus carpio L.) genome as revealed by analyses of microsatellite loci. Mol Biol Evol 2003;20(9):1425e34. [43] Van de Peer Y, Taylor JS, Meyer A. Are all fishes ancient polyploids? J Struct Funct Genomics 2003;3(1e4):65e73. [44] Alam SM, Gascoigne NR. Posttranslational regulation of TCR Va allelic exclusion during T cell differentiation. J Immunol 1998; 160(8):3883e90. [45] Couez D, Malissen M, Buferne M, Schmitt-Verhulst AM, Malissen B. Each of the two productive T cell receptor a-gene rearrangements found in both the A10 and BM 3.3 T cell clones give rise to an a chain which can contribute to the constitution of a surface-expressed ab dimer. Int Immunol 1991;3(7):719e29. [46] Romano N, TaverneThieles JJ, VanMaanen JC, Rombout J. Leucocyte subpopulations in developing carp (Cyprinus carpio L.): immunocytochemical studies. Fish Shellfish Immunol 1997;7(7):439e53.
Characterisation of T cell antigen receptor a chain isotypes in the common carp Etsuou Imaia, Jun Ishikawaa, Tadaaki Moritomob, Mitsuru Tomanaa,) a
Department of Applied Biological Science, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan b Department of Veterinary Science, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan Received 1 September 2004; revised 8 November 2004; accepted 17 November 2004 Available online 8 March 2005
Abstract T cell receptor a (TCRa) chain has been characterised in several teleost species to date. Here, a reverse transcriptionpolymerase chain reaction (RT-PCR) strategy was used to isolate cDNA clones encoding TCRa chain from an individual of the common carp (Cyprinus carpio.L.). The Va sequences identified were most similar to Va of other teleosts, and could be classified into as many as 14 Va families. For the Ja sequences, diversity comparable to that seen in other teleosts could be identified, and the J-region motif was well conserved. The Ca sequences demonstrated the highest similarity to zebrafish Ca and possessed a well-conserved transmembrane (TM) region. Two Ca isotypes with a complete C region were obtained, designated Ca1 and Ca2, with w70% similarity at the amino acid level (w85% identity at the nucleotide level), and, in addition, Ca2 contained two unique sequences, designated Ca2a and Ca2b, with 93% similarity (96% identity). Therefore, the results obtained using an individual clearly showed that carp possesses at least two Ca loci, possibly as a result of tetraploidisation. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: T cell receptor a; Isotype; Common carp; Evolution
1. Introduction T cell receptor (TCR) ab, comprised of a and b chains, can recognise an antigen (Ag)-derived peptide combined with a major histocompatibility complex (MHC) molecule. TCR molecule can be divided into constant (C) and variable (V) domains, the latter being responsible for the specific binding to Ag plus MHC molecule [1]. The Ag specificity of TCR in each T cell can be kept by an allelic exclusion, a mechanism by ) Corresponding author. Tel./fax: C81 466 84 3351. E-mail address: [email protected] (M. Tomana). 1050-4648/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2004.11.004
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which a T cell expresses only TCR molecules with a single specificity on the cell surface [2,3]. The C domain is encoded by a C segment gene, while the V domain by a V and joining (J) segment, or a V, diversity (D), and J segment, for TCRa or -b chains, respectively [1]. For mammals, the genomic organisation of TCRa is as follows: a cluster of Va segments is followed by a Ja cluster and a single Ca segment both in the same transcriptional orientation as the Va segments, and TCRd gene, a constituent of another type of TCR chain, exists between the Va and Ja cluster [4]. For teleosts, it has recently been reported that a Va cluster is followed by a Ca segment and a Ja cluster both in the inverted orientation as the Va segments, in addition, without TCRd gene between the Va cluster and Ca segment [5e7]. To date, TCRa sequences have been reported for various teleost species including rainbow trout (Oncorhynchus mykiss) [8], channel catfish (Ictalurus punctatus) [9], zebrafish (Danio rerio) [10e12], Atlantic cod (Gadus morhua) [13], Japanese pufferfish (Takifugu rubripes) [6], pufferfish (Spheroides nephelus) [14], puffer fish (Tetraodon nigroviridis) [7], Japanese flounder (Paralichthys olivaceus) [5], bicolor damselfish (Stegastes partitus) [15], and Atlantic salmon (Salmo salar) [16]. From those results, a considerable variety has been observed for both Va and Ja in teleosts, whereas a single Ca locus could be found in almost all teleosts examined to date, except for damselfish, in which the presence of two Ca loci was suggested by the result of Southern blots [15]. On the other hand, it has been shown that two Cb isotypes are present in most mammals examined, although the sequences of the two isotypes in each species seem to be highly similar to each other in a coding region [17]. Two Cb isotypes have also been observed in teleosts, bicolor damselfish [18], channel catfish [19] and Japanese flounder [5], and the existence of two isotypes was suggested for Atlantic cod [13]. For mammals, it would be expected that the presence of two Cb isotypes contributes to an increase in the number of Jb segments, due to the genomic organisation in which JbeCb is arranged in tandem following multiple Vb segments [20e22]. As for carp Ag receptor genes, an immunoglobulin (Ig) molecule, consisting of heavy (H) and light (L) chains, has been characterised thus far, and then three distinct CH sequences, probably representing two isotypes [23], and four IgL isotypes have been elucidated [24e26]. But carp TCR genes have not yet been reported until this study, where the cloning and characterisation of TCRa is described. 2. Materials and methods 2.1. Isolation of cDNA clones encoding Ja and partial Ca region Total RNA was extracted from thymus of a single common carp, according to the manufacturer’s instructions, using TRIzol reagent (Invitrogen, San Diego, USA). cDNA was synthesised with reverse transcriptase (SUPERSCRIPTÔ II; Invitrogen) using 2 mg of the total RNA as template and random primers (Invitrogen). Degenerated primers corresponding to conserved sequences in the 30 end of Va FR3 (forward) and in the C-terminal half of Ca (reverse) of TCRa genes found in other teleosts were used in reverse transcription PCR (RT-PCR). The sequences of primers were: Va(deg.) (A(I/L/V)Y(H/Y)CAL): GCI/ITI/TAY/YAY/TGY/GCI/YT (YZT or C; IZinosine); and Ca(deg.) ((F/L)LK(A/T)(I/V)(A/ V)FN(I/V)): Y/RTT/RAA/IRC/IAY/IGY/YTT/IAR/IA (RZA or G) (Fig. 1). Forty cycles of
Fig. 1. Position of primers for the isolation of carp TCRa gene sequences.
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amplification (95 (C for 0.5 min, 42.5 (C for 0.5 min, and 72 (C for 1 min) were performed in 25 ml of reaction mixture. The amplified DNA (about 400 bp in length) was gel-purified and cloned into pCR2.1 vector using a TA cloning kit (Invitrogen). 2.2. Isolation of cDNA clones encoding complete Va or Ca region using RACE method To amplify total RNA obtained above and gain entire Va or Ca region-containing cDNA clones using a SMARTÔ RACE cDNA Amplification kit (Clontech, Palo Alto, USA), the gene specific primers corresponding to the sequences within the Ca (for 50 RACE) or Ja (for 30 RACE) region, respectively, were employed. The sequences of the primers were: Ca (50 ) in clone Cpk1 (MSLGLYWLR): TCT/CAG/CCA/ ATA/GAG/GCC/CAG/TGA/CA; Jal in clone Cpl36 (FGTGTKLTVE): T/GGC/ACT/GGA/ACC/AAA/ CTG/ACT/GTT/G; Ja2 in clone Cpl42 (KLIFGSGTQ): G/CTG/ATT/TTT/GGG/TCT/GGC/ACA/CA; or Ja3 in clone Cpl70 (LQPDYGNNK): TG/CAG/CCG/GAT/TAT/GGG/AAC/AAC/AAG (Fig. 1). Clone Cpl70 was a chimeric Ca sequence between Ca2 and Ca1 possibly caused by PCR artifacts [27]. Special care was then taken when analysing sequences and possible chimeras were excluded from the data set. RACE was performed according to the manufacturer’s instructions. For both RACE, touchdown PCR was employed in 50 ml of reaction mixture: 5 s at 94 (C and 3 min at 72 (C for 5 cycles; 5 s at 94 (C, 10 s at 70 (C, and 3 min at 72 (C for 5 cycles; and 5 s at 94 (C, 10 s at 68 (C, and 3 min at 72 (C for 25 cycles. The PCR products (700e800 bp in length in 50 RACE, and 650e1050 bp in 30 RACE) were gel-purified and cloned into pCR2.1-TOPO vector (Invitrogen). 2.3. DNA sequencing and sequence analysis The PCR products were sequenced in both strands by the dideoxy method [28]. The deduced amino acid sequences were analysed using the tfasta program [29] against the DDBJ sequence database. Multiple alignments were made using the clustal W program [30]. The nucleotide sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers AB120568eAB120575, AB120577eAB120610, and AB120612eAB120623. 2.4. Southern blot analysis Carp erythrocyte DNA was extracted [31] and digested with EcoRI or HindIII (Takara, Shiga, Japan). The DNA (10 mg/lane) was separated on an 0.8% agarose gel and transferred to a nylon membrane (Hybond-NC, Amersham Biosciences, Piscataway, USA) by blotting in 0.4 M NaOH. The filter was fixed by UV cross-linking, prehybridised, and hybridised with 32P-labeled random-primed probes in a medium containing 0.5 M NaH2PO4/NaHPO4, (pH 7.2), 1 mM ethylenediaminetetraacetate (EDTA), and 7% (w/v) SDS at 65 (C overnight. Probes specific for sequences of Va1, Va2, and Va4 families, and 30 -UT of Ca1 and Ca2 isotypes (w700 bp in a full length), where only w60 bp at the 30 end of each Ca sequence was included, were used. The filter was washed in 40 mM NaH2PO4/NaHPO4 and 1% SDS at 65 (C for 10 min, and the radioactivity was detected with a phosphoimager (Molecular Dynamics, Sunnyvale, USA). 2.5. Northern blot analysis Total RNA was extracted from organs of brain, liver, spleen, mesonephros, pronephros, and thymus as described above. Poly(A)CRNA was purified from total RNA using the OligotexÔ-dT30CSuperD mRNA Purification kit (Takara) and then the RNA (2 mg/lane) was separated on 1.5% agarose gels containing formaldehyde and transferred to Hybond-XL membrane (Amersham Biosciences) by blotting in 10! SSC. The filter was fixed by UV cross-linking, prehybridised, and hybridised as described above for the Southern
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blot. A probe encompassing Va, Ja, and the Ca region of clone Cpl16 was used. The filter was washed according to the Hybond-XL manual, and the radioactivity was detected with a phosphoimager (Molecular Dynamics).
3. Results 3.1. Va and Ja region analysis To gain carp TCRa sequences, RT-PCR was employed using degenerated primers both in the 30 end of Va FR3 and in the C-terminal half of Ca (Fig. 1). Then several TCRa-coding clones with almost the same nucleotide sequences in the C region, represented by clone Cpk1, were obtained judging from a high similarity with corresponding Ca sequences in other teleosts (data not shown). Next, using a primer near the 30 ends of the Ca sequence in clone Cpk1, the 50 RACE method was performed to identify clones encoding an entire Va region (Fig. 1). As a result, a total of 34 unique Va sequences were identified and then could be classified into 14 Va families, defined as possessing less than 70% nucleotide identity in Va segments (Fig. 2). All the Va sequences tested showed the highest similarity to other teleost Va sequences each (data not shown). The number of Va segments within each family identified here was as follows: 8 (Va1 family), 5 (Va2), 4 (Va4 and Va5), 3 (Va3), 2(Va6), and 1 (Va7e14). The size of the CDR1 and CDR2 loops in the Va varied from 4 to 7 and from 5 to 13 amino acid residues, respectively. It is noted that the length of CDR2 in clone Cpl47 (Va5 family) is extremely long among the carp clones identified here, with 13 amino acid residues. In addition, the other three Va5 sequences cloned also possessed 13 amino acid residues for the CDR2 (data not shown). All Va sequences tested included the Cys23 and Cys104 residues, which likely form the intradomain disulphide bridge, typical features of TCR molecules, FR2 (WYRQ) and FR3 (YYC) motif, were well observed within each Va sequence. The data set of functional Ja examined is comprised of a total of 43 different clones obtained by the RT-PCR and 50 RACE (Fig. 3). Among them 40 clones show an in-frame VeJ and JeC junction, while the remaining three clones have an out-of-frame VeJ (clone Cpl48) and JeC junction (Cpl31 and Cpl42), and they might have been caused by an inappropriate VeJ recombination and JeC splicing, respectively. The 43 different Ja segments examined could be divided into 37 Jas with less than 70% amino acid similarity between them. The four groups of Ja showing a high similarity among them, particularly in the 30 end of the sequences, could be found, and members of each group used the same Ca isotype: either Ca1 or Ca2 (as mentioned below, two isotypes could be identified in this study). On the other hand, each Va family consisting of two or more members
Fig. 2. Multiple alignment of the amino acid sequences of the carp TCRa V region. The same residues as the sequence shown at the top are denoted by dots, and a gap introduced to maximise homology is indicated by a hyphen. Putative FR/CDR regions are indicated above sequences. Cysteine residues involved in intradomain interaction are numbered. The Va family of each clone is shown in parentheses.
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Fig. 3. Multiple alignment of the amino acid sequences of the carp TCRa J region. Amino acids in the J-region motif are indicated below the sequences. The Ca isotype linked with each Ja is shown in parentheses. Asterisks indicate an out-of-frame transcript. Four groups of Ja clones demonstrating a high similarity among them are underlined. 37 Jas with less than 70% amino acid similarity between them are numbered.
used both Ca isotypes, Ca1 and Ca2. The J-region motif, Lys-Leu/Ile-X-Phe-Gly-X-Gly-Thr-X-Leu (K(I/ L)XFGXGTXL), was well conserved. As shown in Fig. 4, Ja nucleotide sequences were aligned according to the IMGT numbering system [32] based on the Ja data set, except for the RT-PCR-derived clones, because Ala105 and Leu106 are included in the primer sequence used for this method. Variation could be seen well throughout positions 107e114. In clone Cpl48, the presence of an extra G nucleotide between positions 108 and 109 might result in an out-of-frame VeJ junction.
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Fig. 4. Multiple alignment of the nucleotide sequences of the carp TCRa J region. Asterisks indicate an out-of-frame transcript. Codons are numbered according to Lefranc et al. [32].
3.2. Ca region analysis As described above, RT-PCR was initially employed to obtain carp TCRa sequences encompassing the region from Va FR3 to the site near the 30 end of Ca. Then, the Ca sequences identified were all derived from a unique Ca isotype, designated Ca1. In 50 RACE, however, in addition to Ca1, another isotype of incomplete Ca sequences (about 80 amino acids in length) could be identified, designated Ca2, which could be classified into Ca2a and Ca2b subtypes due to the less degree of amino acid difference between them (data not shown). For each isotype, many clones were identified in this 50 RACE (17 clones for Ca1, 13 for Ca2a, 7 for Ca2b), and the amino acid similarity in the incomplete Ca sequences between them was about 60%. Next, using primers designed on the basis of the sequences of Ja joined to the respective Ca isotype, 30 RACE was employed to identify each complete Ca sequence: Ja1 primer for Ca1, Ja2 for Ca2, and Ja3 for the Ca2/Ca1 chimera as mentioned in Section 2 (Fig. 1). Then, Ca1, Ca2a, and Ca2b sequences could be identified, with four, five, and two clones, respectively. Ca1 sequences were obtained using Ja1 primer as expected, while Ca2 sequences were obtained both using Ja2 and Ja3. The representative clones of each isotype/subtype are shown in Fig. 5. When compared in the corresponding region of Ca, the amino acid sequence of clone Cpm73 (Ca1) was identical with those of Cpl16 (50 RACE products) and Cpk1 (RTPCR), while clones Cpm91 (Ca2a) and Cpm95 (Ca2b) sequences showed only one amino acid replacement with Cpl21 and Cpl63 (both from 50 RACE), respectively. The sequence similarity at the amino acid level was about 70% between Ca1 and Ca2 isotypes (about 85% identity at the nucleotide level), and 93% between Ca2a and Ca2b subtypes (96% nucleotide identity). It should be noted that sequences at the 30 end of Ca (35 amino acid residues in length) were identical among the representatives of the distinct types. When compared with other species, carp Ca sequences showed the highest similarity to zebrafish followed
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Fig. 5. Multiple alignment of the Ca amino acid sequences of the carp and other vertebrate species. Characteristic residues are numbered. The putative transmembrane (TM) region is underlined in clone Cpm95. Residues that match the CART motif are underlined in clone Cpm73. Percentages of amino acid similarity between clone Cpm91 and the others, and Cpm73 and the others are indicated. Accession numbers are: zebrafish (AF246174); catfish (IPU58505); Japanese pufferfish (AF269222); pufferfish (SNU22676); flounder (AB053369); cod (GMO133845); trout (OMU50991); mouse (MMTCRAI1); human (HSTCRAC); axolotl (AMU50992); chicken (GDU04611); skate (REU75768).
by trout and catfish Ca. The carp Ca C-terminal region is well conserved together with other species compared, and this likely represents the transmembrane (TM) region, according to the SOSUI program predicting TM segments [33], then followed by the cytoplasmic tail region. The carp TM includes the conserved antigen receptor TM motif (CART) which is thought to play a role in the assembly and/or signalling of the TCR/CD3 complex [34], and the two charged residues at positions 239 (Arg) and 244 (Lys), possibly involved in the association of the TCR with CD3 molecules [35,36]. Clone Cpm85 belonging to the Ca2 isotype might be a pseudo-product because one putative nucleotide insertion into the site around 30 amino acids upstream of the 30 end in normal Ca2 is recognised (data not shown). This insertion results in the loss of the TM region, which is well conserved among different taxa as described above, although the coding region apparently becomes 66 amino acids longer in length compared with that of normal Ca. Among 50 RACE products, this type of nucleotide insertion could also be found in a total of four clones. Regarding a 30 -untranslated region (30 -UT), the sequences of Ca1 and Ca2 isotypes could be clearly distinguished from one another (70e82% nucleotide identity) as well as in their C-region (data not shown).
3.3. Southern blot analysis The diversity and organisation of carp TCRa gene were examined by Southern blot analyses using probes specific for Va sequences, and 30 -UT of Ca1 and Ca2 isotypes (Fig. 6). Instead of the Ca region,
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Fig. 6. Southern blot analyses of carp genomic DNA hybridised with Va- and 30 -UT-specific probes. The restriction enzymes used are EcoRI (E) and HindIII (H), and the position of size markers (in kb) is shown on the left.
30 -UT was chosen as the probe, because teleost Cas have been reported to be coded by three coding exons, and then the use of Ca probe possibly provides ambiguous interpretation of the result [6,7,16]. The Va families with many clones isolated, represented by Va1, Va2, and Va4, were examined here, and each Va probe hybridised with multiple bands, as expected. The blot hybridised with 30 -UT probes of Ca1 and Ca2 isotypes showed only one band each. In EcoRI digestion, distinct bands in position could be clearly recognised in the two blots. 3.4. Northern blot analysis Northern blotting was performed with mRNA from various organs including thymus, using a VJC probe (Fig. 7). The intense signal was observed in thymus at approximately 1.4 kb, corresponding to the expected size of complete TCRa transcripts, calculated from the analyses of both 50 and 30 RACE clones (data not shown). The relatively weak signal could be seen in pronephros.
4. Discussion In this study, two Ca isotypes, designated Ca1 and Ca2, could be identified from a single carp. Recently, Criscitiello et al. have suggested as well that two TCR Ca loci exist in damselfish, but the sequences were similar and distinguished by a single amino acid [15]. Thus, the presence of two Ca loci might not be an
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Fig. 7. Northern blot analyses of mRNA from various carp organs hybridised with a VJC probe. The position of size markers (in kb) is shown on the left.
unusual situation in teleosts. Additionally, two different Ca genes have recently been described for Xenopus [37]. The existence of two Ca isotypes in a fish, as in mammalian Cb, might contribute to an increase in the number of Ja segments, since each Ca isotype in carp seemed to be joined with a unique set of Ja segments (Fig. 3). In addition to a Ca variety observed in Ca1 and Ca2, with w70% amino acid sequence similarity, another variety derived from Ca2a and Ca2b could be demonstrated, with 93% similarity (Fig. 5). As the data set of carp Ca sequences was derived from an individual, the presence of more than two unique Ca sequences identified in this study means that carp Ca region is encoded by at least two loci. It is consistent with the results of Southern analyses using probes for 30 -UT of Ca1 and Ca2 (Fig. 6). Since the genomic sequence data is not available for the Cas at present, it remains unknown whether the Cas are in tandem organisation or not. The presence of at least two Ca loci in carp might have been affected by a mechanism such as a tetraploidisation event, as well known for teleosts, including the Cyprinidae family [38e43]. For the relationship between Ca2a and Ca2b, although two unique sequences with a few substitutions were identified in Ca2a (data not shown), the possibility of Taq polymerase errors could not be excluded for the extent of difference observed; at present it is unknown whether the two subtypes are located on the same (alleles) or different loci (isotypes). As shown in Fig. 6, an apparent single band could be seen using a probe specific for the 30 -UT of Ca2, where the sequences employed were highly similar between Ca2a and Ca2b enough to cause cross-hybridisation (data not shown), suggesting that the two subtypes represent alleles, or are closely linked in a genomic organisation even if they are isotypes. The recent reports of teleost TCRas from several species have indicated the existence of high levels of polymorphism in the constant region [11,15]. Recently it has been reported that zebrafish, belonging to the same Cyprinidae family as carp, possessed several distinct Ca sequences, where the degree of amino acid sequence similarity observed corresponds to that found between the carp Ca2 subtypes [10e12]. But it is unclear at present if the difference found in zebrafish Cas represents an allelic variation or isotype, due to the data sets derived from pooled zebrafish but not from an individual.
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It is currently uncertain whether or not Ca1 and Ca2 are each linked with independent Va clusters. But if this is the case, it would be expected that a chance of producing two (or more) productive TCRa chains with different specificities within a single T cell increased in carp compared to mammals, with a single Ca locus, and the carp immune system might require a strict allelic exclusion machinery responsible for a single TCRa chain expression on the cell surface. The allelic exclusion seems to occur at a post-translational level in mammalian a-chain [2,3,44,45], mainly in thymus organ, where the mRNA expression of TCRa was active compared with other organs examined as shown in Fig. 7, and the sequences identified in this study were derived from this organ. By immunocytochemical studies in carp, it has been demonstrated that thymus may be the primary lymphoid organ for T cells [46]. Variability of Va in five teleost species studied in detail, is as follows: 10 Va families (zebrafish) [12], 6 (trout and puffer fish) [7,8], 5 (salmon) [16], and 4 subfamilies (Japanese pufferfish) [6]. On the other hand, carp possessed as many as 14 Va families, which consisted of one to eight members (Fig. 2). The existence of extraordinary numbers, 87 families of Va genes, has also been mentioned for zebrafish as unpublished data [12]. Thus, an apparent increase in the Va diversity of a Cyprinidae family compared with those of other teleosts, might have been caused by the duplication of Va loci in the ancestor for carp and zebrafish. Furthermore, a total of 43 different Jas were characterised in this study (Fig. 3); the degree of Ja complexity seems to be comparable to that reported for other teleosts [5e8]. Among the Ja sequences analysed, a putative failure of JeC splicing was observed, and by the following reason the splicing error may be also applicable to Ca sequences represented by clone Cpm85, likely a pseudo-product (data not shown). This type of clone was characterised by one putative nucleotide insertion into the site around 30 amino acids upstream of the 30 end in normal Ca, and this site could correspond to the boundary between the second and third exon of Ca reported in takifugu [6] and Japanese flounder (accession number AB081557). It is, however, possible that the insertion is encoded in a Ca gene itself. It seems that a single Ca locus is permitted in most species examined to date. In this report and recent studies [15,37], however, two Ca isotypes could be identified, therefore, it is important to examine if the situation is accompanied by a novel machinery for allelic exclusion at the TCRa loci.
Acknowledgements This work was supported in part by a grant from the Ministry of Education, Science, Sports and Culture to promote advanced scientific research.
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