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Molecular cloning and expression analysis of the putative carp (Cyprinus carpio) pre-B cell enhancing factor
Fish & Shellfish Immunology (2000) 10, 383–385 doi:10.1006/fsim.2000.0263 Available online at http://www.idealibrary.com on
SHORT COMMUNICATION Molecular cloning and expression analysis of the putative carp (Cyprinus carpio) pre-B cell enhancing factor K. FUJIKI*, D.-H. SHIN, M. NAKAO
AND
T. YANO
Laboratory of Marine Biochemistry, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812–8581, Japan (Received 28 October 1999, accepted after revision 17 January 2000) Key words:
carp, pre-B cell enhancing factor, cytokine, suppression subtractive hybridisation, sodium alginate, scleroglucan.
Suppression subtractive hybridisation (SSH) is a powerful means to identify genes of cytokines and other genes expressing small amount of mRNA, as the SSH utilises PCR for the amplification of the subtracted and equalised gene fragments (Diatchenko et al., 1996). By using SSH, full-length cDNAs encoding carp cytokines homologous to mammalian CC chemokine (S-84), allograft inflammatory factor-1 (L-68), natural killer cell enhancing factor (L-128) and interleukin-1 (C-44) (Fujiki et al., 1999, 2000) have been cloned. In the SSH between carp leucocytes collected before and after dynamic migration elicited by sodium alginate (Fujiki et al., 1999), the generated cDNA fragments encoded some cytokines including one similar to human pre-B cell enhancing factor (PBEF), which remained to be fully sequenced and characterised. In the present work, using this fragment as a probe, a full-length clone named S-63 was isolated from the ZAP Express cDNA library (Stratagene) as previously described (Fujiki et al., 2000), and its expression in vivo was evaluated by RT-PCR. Clone S-63 is 2051 bp-long including 5 and 3 untranslated regions and the entire coding region for 493 amino acids. The coding region is 72·3 and 86·2% identical to that of human PBEF (GenBank accession No. P43490) at the nucleotide level (data not shown) and the amino acid level (Fig. 1), respectively. This strongly indicates that S-63 is a carp homologue of human PBEF. Like human PBEF, carp PBEF lacks a signal sequence as determined by the SignalP program (Nielsen et al., 1997), though human PBEF is indicated to be a secreted protein. Two N-glycosylation sites present in human PBEF are conserved in the carp homologue. Carp PBEF has five cysteines, one less than human PBEF. The isoelectric point calculated by the Compute pI/Mw program (Wilkins et al., 1998) is 6·27, similar to 6·69 of human PBEF. To examine if carp PBEF is upregulated in response to immunopotentiators in vivo, RT-PCR was performed on PBEF mRNA in head kidney cells (HKC) and peritoneal cells (PC) of carp which had been injected with sodium alginate (SA, 2 mg 100 g 1 body weight) and scleroglucan (SG, 1 mg 100 g 1 body weight) 48 h earlier, as previously described (Fujiki et al., 2000). Oligonucleotides used as PCR primers for carp PBEF (327 bp) were: sense primer 5 -CCATCGAGATCAAGGCTGTG (369–388), and antisense * Corresponding author. Present address: Department of Biological Sciences, University of Windsor, 401 Sunset Ave., Windsor, Ontario N9B 3P4, Canada. E-mail: [email protected] The nucleotide sequence data reported in this paper has been deposited to the DDBJ, EMBL and GenBank nucleotide sequence databases, with the following accession number AB027712. 1050–4648/00/040383+03 $35.00/0
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2000 Academic Press
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K. FUJIKI ET AL.
Fig. 1. Alignment of the deduced amino acid sequence of clone S-63 with that of human pre-B cell enhancing factor (PBEF). The right most numbers presented are amino acid numbers of clone S-63. Asterisks indicate identical amino acid residues. Dashes indicate gaps introduced for optimal alignment. The N-glycosylation sites are denoted by the upper line. The amino acid identity (%) is shown in parenthesis at the end of human PBEF.
Fig. 2. RT-PCR analysed mRNA expression of carp pre-B cell enhancing factor (PBEF) in the head kidney and peritoneal cells of fish injected with saline, sodium alginate (SA) or scleroglucan (SG) 48 h earlier. PCR amplification was performed using two numbers of cycles to show that at least under the lower numbers of cycles amplification did not reach the plateau. Fish ID numbers identify the individual fish. Injections were given intraperitoneally at 2 mg 100 g 1 (SA) and 1 mg 100 g 1 (SG). S11 indicates 40S ribosomal protein S11 used to confirm equal total RNA concentrations.
primer 5 -CCACTGTGTCTGTTCCTTTG (676–695). The numbers in parenthesis represent the nucleotide numbers of carp PBEF submitted to GenBank. As shown in Fig. 2, expression of ribosomal protein S11 mRNA was nearly constant in all experimental samples, indicating that the total RNA concentrations were almost the same among the samples. Carp PBEF expression in HKC and PC of SA-injected fish and SG-injected fish was 2·3–2·7-fold higher than in HKC of saline-injected fish, when the intensities of the bands were determined by the NIH Image program version 1·52. There was no
CLONING AND EXPRESSION ANALYSIS OF CARP PBEF
385
marked di#erence between the e#ectiveness of SA and SG on the expression of carp PBEF. No amplification was seen when the carp genome was used as template under the same PCR conditions (data not shown), indicating that the amplified cDNA was derived only from mRNA. The observation that expression of PBEF was higher in the SA-elicited PC than in the saline-injected control fish HKC is consistent with the fact that PBEF cDNA fragment was generated by SSH between these cells. Peritoneal cells of the saline control fish were not examined because they were considered to be identical to resident peritoneal cells which were not comparable to SA- and SG-elicited PC. PBEF has been cloned only from human. Human PBEF is expressed in a wide variety of tissues including liver, muscle, bone marrow, peripheral blood lymphocytes, heart, placenta, lung and kidney. Expression in lymphocytes is upregulated by mitogen stimulation in vitro. PBEF acts synergistically with IL-7 or stem cell factor to induce pre-B cell colony formation. However, additional biological activities are expected for PBEF because of its ubiquitous expression (Samal et al., 1994). Carp PBEF shows strikingly high amino acid identity to human PBEF. This high conservation through evolution suggests that PBEF probably plays one or more important biological roles besides its synergistic role in pre-B cell colony formation. Carp PBEF may be involved in innate immunity as its mRNA expression was increased by the injection of non-specific immunostimulants in the present study. In teleosts, little information on molecular mechanisms in haematopoiesis is available. Carp PBEF may be a useful probe to investigate the teleost haematopoiesis, especially di#erentiation during lymphopoiesis. We are grateful to C. J. Bayne for helpful comments. This work was supported in part by Grants-in Aid for Scientific Research (10556046 to T. Y., 9113 to K. F.) from The Ministry of Education, Science, Sports and Culture of Japan.
References Diatchenko, L., Lau, Y.-F. C., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D. & Siebert, P. D. (1996). Suppression subtractive hybridization: a method for generating di#erentially regulated or tissuespecific cDNA probes and libraries. Proceedings of the National Academy of Sciences USA 93, 6025–6030. Fujiki, K., Shin, D.-H., Nakao, M. & Yano, T. (1999). Molecular cloning of carp (Cyprinus carpio) CC chemokine, CXC chemokine receptors, allograft inflammatory factor-1 and natural killer cell enhancing factor by use of suppression subtractive hybridization. Immunogenetics 49, 909–914. Fujiki, K., Shin, D.-H., Nakao, M. & Yano, T. (2000). Molecular cloning and expression analysis of carp (Cyprinus carpio) interleukin-1, high a$nity immunoglobulin E Fc receptor subunit and serum amyloid A. Fish & Shellfish Immunology 10, 229–242. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10, 1–6. Samal, B., Sun, Y., Stearns, G., Xie, C., Suggs, S. & McNiece, I. (1994). Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony enhancing factor. Molecular and Cellular Biology 14, 1431–1437. Wilkins, M. R., Gasteiger, E., Bairoch, A., Sanchez, J.-C., Williams, K. L., Appel, R. D. & Hochstrasser, D. F. (1998). Protein identification and analysis tools in the ExPASy server. In 2-D Proteome Analysis Protocols (A. J. Link, ed) New Jersey: Humana Press.
SHORT COMMUNICATION Molecular cloning and expression analysis of the putative carp (Cyprinus carpio) pre-B cell enhancing factor K. FUJIKI*, D.-H. SHIN, M. NAKAO
AND
T. YANO
Laboratory of Marine Biochemistry, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812–8581, Japan (Received 28 October 1999, accepted after revision 17 January 2000) Key words:
carp, pre-B cell enhancing factor, cytokine, suppression subtractive hybridisation, sodium alginate, scleroglucan.
Suppression subtractive hybridisation (SSH) is a powerful means to identify genes of cytokines and other genes expressing small amount of mRNA, as the SSH utilises PCR for the amplification of the subtracted and equalised gene fragments (Diatchenko et al., 1996). By using SSH, full-length cDNAs encoding carp cytokines homologous to mammalian CC chemokine (S-84), allograft inflammatory factor-1 (L-68), natural killer cell enhancing factor (L-128) and interleukin-1 (C-44) (Fujiki et al., 1999, 2000) have been cloned. In the SSH between carp leucocytes collected before and after dynamic migration elicited by sodium alginate (Fujiki et al., 1999), the generated cDNA fragments encoded some cytokines including one similar to human pre-B cell enhancing factor (PBEF), which remained to be fully sequenced and characterised. In the present work, using this fragment as a probe, a full-length clone named S-63 was isolated from the ZAP Express cDNA library (Stratagene) as previously described (Fujiki et al., 2000), and its expression in vivo was evaluated by RT-PCR. Clone S-63 is 2051 bp-long including 5 and 3 untranslated regions and the entire coding region for 493 amino acids. The coding region is 72·3 and 86·2% identical to that of human PBEF (GenBank accession No. P43490) at the nucleotide level (data not shown) and the amino acid level (Fig. 1), respectively. This strongly indicates that S-63 is a carp homologue of human PBEF. Like human PBEF, carp PBEF lacks a signal sequence as determined by the SignalP program (Nielsen et al., 1997), though human PBEF is indicated to be a secreted protein. Two N-glycosylation sites present in human PBEF are conserved in the carp homologue. Carp PBEF has five cysteines, one less than human PBEF. The isoelectric point calculated by the Compute pI/Mw program (Wilkins et al., 1998) is 6·27, similar to 6·69 of human PBEF. To examine if carp PBEF is upregulated in response to immunopotentiators in vivo, RT-PCR was performed on PBEF mRNA in head kidney cells (HKC) and peritoneal cells (PC) of carp which had been injected with sodium alginate (SA, 2 mg 100 g 1 body weight) and scleroglucan (SG, 1 mg 100 g 1 body weight) 48 h earlier, as previously described (Fujiki et al., 2000). Oligonucleotides used as PCR primers for carp PBEF (327 bp) were: sense primer 5 -CCATCGAGATCAAGGCTGTG (369–388), and antisense * Corresponding author. Present address: Department of Biological Sciences, University of Windsor, 401 Sunset Ave., Windsor, Ontario N9B 3P4, Canada. E-mail: [email protected] The nucleotide sequence data reported in this paper has been deposited to the DDBJ, EMBL and GenBank nucleotide sequence databases, with the following accession number AB027712. 1050–4648/00/040383+03 $35.00/0
383
2000 Academic Press
384
K. FUJIKI ET AL.
Fig. 1. Alignment of the deduced amino acid sequence of clone S-63 with that of human pre-B cell enhancing factor (PBEF). The right most numbers presented are amino acid numbers of clone S-63. Asterisks indicate identical amino acid residues. Dashes indicate gaps introduced for optimal alignment. The N-glycosylation sites are denoted by the upper line. The amino acid identity (%) is shown in parenthesis at the end of human PBEF.
Fig. 2. RT-PCR analysed mRNA expression of carp pre-B cell enhancing factor (PBEF) in the head kidney and peritoneal cells of fish injected with saline, sodium alginate (SA) or scleroglucan (SG) 48 h earlier. PCR amplification was performed using two numbers of cycles to show that at least under the lower numbers of cycles amplification did not reach the plateau. Fish ID numbers identify the individual fish. Injections were given intraperitoneally at 2 mg 100 g 1 (SA) and 1 mg 100 g 1 (SG). S11 indicates 40S ribosomal protein S11 used to confirm equal total RNA concentrations.
primer 5 -CCACTGTGTCTGTTCCTTTG (676–695). The numbers in parenthesis represent the nucleotide numbers of carp PBEF submitted to GenBank. As shown in Fig. 2, expression of ribosomal protein S11 mRNA was nearly constant in all experimental samples, indicating that the total RNA concentrations were almost the same among the samples. Carp PBEF expression in HKC and PC of SA-injected fish and SG-injected fish was 2·3–2·7-fold higher than in HKC of saline-injected fish, when the intensities of the bands were determined by the NIH Image program version 1·52. There was no
CLONING AND EXPRESSION ANALYSIS OF CARP PBEF
385
marked di#erence between the e#ectiveness of SA and SG on the expression of carp PBEF. No amplification was seen when the carp genome was used as template under the same PCR conditions (data not shown), indicating that the amplified cDNA was derived only from mRNA. The observation that expression of PBEF was higher in the SA-elicited PC than in the saline-injected control fish HKC is consistent with the fact that PBEF cDNA fragment was generated by SSH between these cells. Peritoneal cells of the saline control fish were not examined because they were considered to be identical to resident peritoneal cells which were not comparable to SA- and SG-elicited PC. PBEF has been cloned only from human. Human PBEF is expressed in a wide variety of tissues including liver, muscle, bone marrow, peripheral blood lymphocytes, heart, placenta, lung and kidney. Expression in lymphocytes is upregulated by mitogen stimulation in vitro. PBEF acts synergistically with IL-7 or stem cell factor to induce pre-B cell colony formation. However, additional biological activities are expected for PBEF because of its ubiquitous expression (Samal et al., 1994). Carp PBEF shows strikingly high amino acid identity to human PBEF. This high conservation through evolution suggests that PBEF probably plays one or more important biological roles besides its synergistic role in pre-B cell colony formation. Carp PBEF may be involved in innate immunity as its mRNA expression was increased by the injection of non-specific immunostimulants in the present study. In teleosts, little information on molecular mechanisms in haematopoiesis is available. Carp PBEF may be a useful probe to investigate the teleost haematopoiesis, especially di#erentiation during lymphopoiesis. We are grateful to C. J. Bayne for helpful comments. This work was supported in part by Grants-in Aid for Scientific Research (10556046 to T. Y., 9113 to K. F.) from The Ministry of Education, Science, Sports and Culture of Japan.
References Diatchenko, L., Lau, Y.-F. C., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D. & Siebert, P. D. (1996). Suppression subtractive hybridization: a method for generating di#erentially regulated or tissuespecific cDNA probes and libraries. Proceedings of the National Academy of Sciences USA 93, 6025–6030. Fujiki, K., Shin, D.-H., Nakao, M. & Yano, T. (1999). Molecular cloning of carp (Cyprinus carpio) CC chemokine, CXC chemokine receptors, allograft inflammatory factor-1 and natural killer cell enhancing factor by use of suppression subtractive hybridization. Immunogenetics 49, 909–914. Fujiki, K., Shin, D.-H., Nakao, M. & Yano, T. (2000). Molecular cloning and expression analysis of carp (Cyprinus carpio) interleukin-1, high a$nity immunoglobulin E Fc receptor subunit and serum amyloid A. Fish & Shellfish Immunology 10, 229–242. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10, 1–6. Samal, B., Sun, Y., Stearns, G., Xie, C., Suggs, S. & McNiece, I. (1994). Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony enhancing factor. Molecular and Cellular Biology 14, 1431–1437. Wilkins, M. R., Gasteiger, E., Bairoch, A., Sanchez, J.-C., Williams, K. L., Appel, R. D. & Hochstrasser, D. F. (1998). Protein identification and analysis tools in the ExPASy server. In 2-D Proteome Analysis Protocols (A. J. Link, ed) New Jersey: Humana Press.