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Genomic structure of the sponge, Halichondria okadai calcyphosine gene
Gene 298 (2002) 21–27 www.elsevier.com/locate/gene
Genomic structure of the sponge, Halichondria okadai calcyphosine gene Hajime Julie Yuasa a,*, Akiko Nakatomi b, Tomohiko Suzuki a, Michio Yazawa b a
b
Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan Received 12 April 2002; received in revised form 7 August 2002; accepted 20 August 2002 Received by T. Gojobori
Abstract Calcyphosine is an EF-hand Ca 21-binding protein, which was first isolated from the canine thyroid. It is phosphorylated in a cyclic AMP (cAMP)-dependent manner; then it is thought to be implicated in the cross-signaling between the cAMP and calcium-phosphatidylinositol cascades. Here, we isolated the DNA complementary to RNA (cDNA) of an EF-hand Ca 21-binding protein from the sponge, Halichondria okadai and determined its genomic structure. The deduced sequence of the sponge Ca 21-binding protein showed significant similarity (about 40% identity) with those of mammal calcyphosines, and the intron positions were well conserved between the sponge and human calcyphosine genes. We considered that the isolated cDNA was that of sponge calcyphosine, and that sponge and mammalian calcyphosines evolved from a common ancestor gene. Recent cDNA projects have revealed that a calcyphosine cDNA is also expressed by human, mouse, and the ascidia. These cDNAs have more than 60% identity with sponge calcyphosine and each other, and all are composed of 208 amino acid residues. On the constructed phylogenetic trees, calcyphosines are essentially divided into two groups, types-I and -II calcyphosines. Type-I calcyphosine may be specific to mammals, and type-II is widely distributed among metazoan species. This suggests that type-II calcyphosine is a rather ancient gene with some essential function. q 2002 Elsevier Science B.V. All rights reserved. Keywords: EF-hand protein; Molecular evolution; Sponge (Porifera)
1. Introduction There are variously evolved metazoans, and they have independently acquired their original genes that had specific functions during evolution of each lineage. On the other hand, the common ancestor of metazoans must have already possessed at least a minimal set of genes essential for life. It can be easily expected that most of such essential elements have been inherited by today’s descendants. Calcyphosine is a Ca 21-binding protein that was originally detected in the canine thyroid (Lecocq et al., 1979; Lamy et al., 1986). It has four EF-hand calcium binding domains and is phosphorylated in a cyclic AMP (cAMP)-dependent manner. Although the exact function of calcyphosine is unclear, it is implicated in cross signaling between cAMP and calciumphosphatidylinositol cascades. Calcyphosine is also expressed in brain, salivary glands, and lung of dog (Lefort et al., 1989). Homologues of the canine calcyphosine have been isolated from the human thyroid (El Housni et al., 1997) and rabbit olfactory receptor neurons (R2D5 antigen; Nemoto et al., Abbreviations: CaM, calmodulin; cAMP, cyclic AMP; EST, expressed sequence tag; TnC, troponin C * Corresponding author. Tel.: 181-88-844-8464; fax: 181-88-844-8356. E-mail address: [email protected] (H.J. Yuasa).
1993), but it may be absent from the mouse and five other rodents (Cle´ment et al., 1997). Mammalian calcyphosines are highly homologous (more than 86% identity). CCBP-23 is a protein that have been isolated from crustacean abdominal muscle, which also has significant identity (over 40%) with mammal calcyphosines, although CCBP-23 is not apparently phosphorylated (Sauter et al., 1995). All metazoan phyla are thought to have evolved from the common ancestor, Urmetazoa, and the phylum Porifera is thought to be the earliest among them to have diverged (Mu¨ller, 2001; Mu¨ller et al., 2001). The sponges that belong to the Porifera are the simplest animals and they lack muscles, nerves, and other specialized organs such as the thyroid, brain, salivary glands, or lungs. Here, we isolated calcyphosine DNA complementary to RNA (cDNA) from the sponge, Halichondria okadai and determined its genomic structure. We also propose a scheme to explain calcyphosine evolution.
2. Materials and methods 2.1. Cloning of the sponge Ca 21-binding protein (calcyphosine) cDNA Single-stranded cDNA of the sponge, H. okadai was
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00920-4
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H.J. Yuasa et al. / Gene 298 (2002) 21–27
Fig. 1. cDNA and deduced amino acid sequences of the sponge, H. okadai calcyphosine. Four canonical EF-hand Ca 21 binding domains (site I–IV) are boxed and three insertions within the fourth EF-hand domain are shaded. Downward arrows indicate positions of introns in genome. The determined nucleotide sequence has been submitted to DDBJ under the accession number, AB081565 (H. okadai calcyphosine cDNA).
prepared as previously described (Yuasa et al., 2001). The 3 0 half of sponge Ca 21-binding protein (calcyphosine) cDNA was amplified by polymerase chain reaction (PCR) using Ex Taq DNA polymerase (Takara). The redundant oligomer used for amplification was 5 0 -TTYGAYAARGAYGGNGAYGG3 0 , which was originally designed based on consensus amino acid sequences from the metazoan calmodulins (CaMs), FDKDGDG. The oligo-dT adaptor, 5 0 -GGGATCCGAATTCT17-3 0 was used as another primer. The 5 0 -untranslated region of the cDNA was amplified by 5 0 -rapid amplification of cDNA ends (RACE). The poly-C tail was added to the 3 0 -end of cDNAs using terminal deoxynucleotidyl transferase, and PCR proceeded with primer sets, oligo-dG adapter, 5 0 -GAATTCG15-3 0 and the reverse primer R1, 5 0 TACATGTGCATACACCTG-3 0 (complementary to the sequence from nt 751 to 768, see Fig. 1). 21
2.2. Amplification of sponge Ca -binding protein (calcyphosine) gene Genomic DNA from the sponge was prepared with the conventional phenol-chloroform method. The genomic fragment of the sponge Ca 21-binding protein (calcyphosine) gene was amplified by PCR using Ex Taq DNA polymerase (Takara). The primers used were the forward primer F1, 5 0 -
TGAACACGGAGCTGACAA-3 0 (corresponding to the sequence from nt 249 to 232, see Fig. 1) and the reverse primer R1. 2.3. DNA sequencing The nucleotide sequences were determined by the dideoxy chain termination method with a Thermo Sequenase dye terminator cycle sequencing premix kit, v2.0 (Amersham Pharmacia Biotech) using an automated DNA sequencer (ABI PRISMTM377). 3. Results and discussion 3.1. Sequence of the sponge calcyphosine cDNA The partial cDNA of a sponge calcium binding protein was amplified by PCR as a by-product of sponge CaM cDNA amplification (Yuasa et al., 2001). The redundant oligomer, which was designed based on consensus amino acid sequence among metazoan CaMs, FDKDGDG (residue 20–26, within the first EF-hand domain of CaMs), was annealed to the sequence encoding FDKDGNG (residue 86–93, within the second EF-hand domain of sponge calcyphosine, see Fig. 1). The partial cDNA was encoding a
H.J. Yuasa et al. / Gene 298 (2002) 21–27
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Fig. 2. (a) Amplification of sponge calcyphosine gene. PCR was performed with primers F1 and R1. A single product (about 3 kbp) was amplified. Left, size markers in kbp based on molecular markers. (b) Exon/intron organization of sponge calcyphosine gene. Exons are shown in boxes and introns are bars. Position and length of each intron (In I–V) are indicated. The determined nucleotide sequences have been submitted to DDBJ under the accession number, AB081566 (H. okadai calcyphosine gene).
typical EF-hand protein, then we determined entire cDNA sequence using 5 0 -RACE. The cDNA was composed of 850 nucleotides, encoding 208 amino acid residues including the initial Met. A homology search within SwissProt (protein data bank) showed that the deduced sequence had significant similarity (about 40% identity) with mammalian calcyphosines and with crayfish CCBP-23, both of which showed 44% identity to each other. Then we refer to the isolated cDNA as sponge calcyphosine below. Two of four EF-hand domains of sponge calcyphosine obviously deviated from the canonical EF-hand sequence (Kawasaki and Kretsinger, 1995). Within the first domain, the central Gly, which is thought to be essential for the sharp bend in the center of the EF-hand, was replaced by Lys. Substitution at this position is frequent among calcyphosines. The fourth domain contains three insertions (Fig. 1, shaded residues). The same domain of mammal calcyphosines and CCBP-23 is also separated by two or by three insertions respectively (El Housni et al., 1997; Sauter et al., 1995). Thus, the first and fourth EF-hand domains of calcyphosines have probably lost Ca 21-binding ability. However, among the four
EF-hand domains, the fourth is most conserved among species (Fig. 3). The fourth EF-hand domain of calcyphosine may be functionally involved in a process other than Ca 21-binding. Mammalian calcyphosines are phosphorylated in a cAMP dependant manner, and a putative phosphorylation site for protein kinase A is located within the first EF-hand domain (Arg/Gly37-Ser-Arg-Ser, in which the last Ser is thought to be phosphorylated). This supposed phosphorylation site is not conserved in either sponge calcyphosine or in CCBP-23. Indeed, the results of ESI-MS analysis suggest that CCBP-23 is not phosphorylated (Sauter et al., 1995). Thus, sponge calcyphosine might not be phosphorylated either. 3.2. Genomic structure of the sponge calcyphosine gene We amplified a single 3 kbp fragment of the sponge calcyphosine gene by PCR using the primers, F1 and R1 (Fig. 2a). Sequencing revealed that this product is composed of 3186 nucleotides and that it contains six exons and five introns. The exon/intron structure of the sponge calcypho-
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H.J. Yuasa et al. / Gene 298 (2002) 21–27
Table 1 Intron positions of calcyphosine family and TnC superfamily genes a Positions of introns Calcyphosine family Type-I calcyphosine Human Type-II calcyphosine Fruit fly Sponge Calcyphosine 2 Human Troponin C superfamily Calmodulin Human (III) Chicken (I,II) Amphioxus Sea hare Fruit fly Sponge Troponin C Human (f) Human (s/c) Amphioxus Ascidian Scallop Fruit fly Sandworm Spec Sea urchin Myosin essential light chain Human Fruit fly Parvalbumin Human
1.04/2
–
2.28/0
–
4.17/0
1.04/2 1.04/2
2.08/2 2.01/0
2.28/0 2.28/0
– 3.24/0
4.17/0 4.17/0
–
–
–
–
210/0 (M) 210/0 (M) 210/0 (M) 210/0 (M) 210/0 (M) 210/0 (M)
1.01/1 1.01/1 1.01/1 1.01/1 – –
2.13/1 2.13/1 2.13/1 2.13/1 2.13/1 –
3.12/0 3.12/0 – – – –
4.21/1 4.21/1 4.21/1 4.21/1 4.21/1 –
217/0 (M) 210/0 217/0 (M) 210/0(M) 212/0 (M) 210/0 213/0
1.01/1 1.01/1 1.01/1 1.01/1 1.01/1 – 1.01/1
2.13/1 2.13/1 2.13/1 2.13/1 2.13/1 2.13/1 2.13/1
3.11/2 3.11/2 3.24/0 3.24/0 – – 3.23/2
4.21/1 4.21/1 4.21/1 4.21/1 4.21/1 4.21/1 4.21/1
212/0 (M)
1.01/1
2.13/1
3.18/2
4.21/1
209/0 (M) 208/0 (M)
1.01/1 1.02/1
2.12/1 –
3 1 01/1 3 1 01/1
4.21/1 4.21/1
–
2.11/1
3.23/2
4.21/1
–
–
(intron-less)
a (M) refers to intron is inserted just after the initiation codon, ATG. (–) refers to absence of intron. Human (f), human fast skeletal TnC; and Human (s/c), human slow-cardiac TnC.
sine gene is shown in Fig. 2b. A comparison with the cDNA sequence (Fig. 1) determined that an AAA codon encoding Lys-14 was changed to AAG, an AAC codon encoding Asn92 changed to AAT, and that T-739 was changed to C, which may be due to individual differences or allelic polymorphisms. All introns conformed to the GT-AG rule and the positions of the introns are 1.04/2, 2.01/0, 2.28/0, 3.24/2, and 4.17/0 1 (residues inserted within the fourth EF-hand domain were not counted). The human calcyphosine gene has only three introns, but their positions are identical to three of five introns of the sponge calcyphosine gene (1.04/ 2, 2.28/0, and 4.17/0; Lamerdin, 1998). Furthermore, a fruit fly gene encoding Drosophila melanogaster CG10126 1 The positions of introns are indicated according to the nomenclature of Kretsinger and Nakayama (1993). The first number corresponds to the EFhand domain numbered sequentially from N to C. The second number (following the period) represents the residue number of intron insertion in the EF-hand domain, which as a rule consists of 29 residues. The last number (following the slash) is phase: 0 means that the intron lies between triplet codons, 1 means between the first and second nucleotides of the codon, and 2 means between the second and third. For instance, 1.04/2 means insertion in domain I, fourth residue and phase 2.
protein (Adams et al., 2000), which shows significant similarity with calcyphosines, has four introns (1.04/2, 2.08/2, 2.28/0, and 4.17/0) and three of them are inserted at positions identical to those of the insertions in the sponge/human calcyphosine genes. These suggest that the mammal and sponge calcyphosines, as well as the Drosophila gene encoding CG10126 have evolved from a common ancestor. As far as we know, these intron positions are unique among EF-hand genes, so they should be classified as members of the calcyphosine family. In addition, calcyphosines show some similarity with CaM, which also has four EF-hand domains per molecule, and Lefort et al. (1989) and Cle´ ment et al. (1997) have suggested a rather close relationship between them. Indeed the chordate CaM genes essentially possess five introns (Yuasa et al., 2001), as do sponge calcyphosine gene. However, CaM belongs to the troponin C (TnC) superfamily in which the intron positions are highly conserved (Yuasa et al., 2001). The intron positions of calcyphosines are completely different from those of the TnC superfamily, so we infer that the calcyphosine and CaM (or TnC superfamily) genes are not that closely related. Recently, the cDNA sequence of the novel human
H.J. Yuasa et al. / Gene 298 (2002) 21–27
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Fig. 3. Amino acid sequences of metazoan calcyphosines aligned by the Clustal W 1.7 program (Thompson et al., 1994). Four canonical EF-hand Ca 21 binding domains (site I–IV) are shown by # and three insertions within the fourth EF-hand domain are shown by dots (.). The residues conserved in all chains are indicated by asterisks (*) and gaps are inserted for maximal similarity and shown by bars (2). The supposed phosphorylation sites of mammal calcyphosines (type-I calcyphosines) are underlined. Sponge, sponge calcyphosine (this work); Human II, human clone: MGC 26610 (DDBJ/EMBL/GenBank accession no. BC017586); Mouse, adult mouse testis cDNA, clone:1700028N11 (accession no. AK006467); Ascidia, C. intestinalis expressed sequence tag clone: cluster ID:03389 (Satou et al., 2002); Fruit fly, Drosophila melanogaster CG10126 protein (accession no. AE003697); Hornworm, the tobacco hornworm Manduca sexta calcyphosine-like protein (accession no. AF117582) to which Drosophila CG10126 shows the highest similarity; Dog, dog calcyphosine (Lefort et al., 1989); Human, human calcyphosine (El Housni et al., 1997); Rabbit, rabbit R2D5 antigen (Nemoto et al., 1993); Crayfish, crayfish CCBP-23 (Sauter et al., 1995); Calcy 2, human calcyphosine 2 (Wang et al., 2002). (1) means that some N-terminal residues are truncated.
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H.J. Yuasa et al. / Gene 298 (2002) 21–27
Fig. 4. Phylogenetic trees based on the alignment of data in Fig. 3 (only four EF-hand domains and three interdomain regions were used). Above, the tree constructed using the PHYLIP package (Felsenstein, 1993) and Neighbor-Joining method was used. Numbers at forks indicate percentage of 1000 bootstrap resamplings that support these topological elements. Below, the maximum likelihood tree constructed with TREE-PUZZLE 5.0 (Schmidt et al., 2000). Numbers at forks indicate the support values for internal branches. Human calcyphosine 2 (Wang et al., 2002) was used as an outgroup.
protein, calcyphosine 2 has been reported (Wang et al., 2002). Human calcyphosine 2 consists of 382 amino acids residues (about twice the size of other calcyphosines) and its C-terminal half shows about 30% identity with other calcy-
phosines. Since the human calcyphosine 2 gene is mapped to the human genome 12q15, we aligned the sequences of the cDNA and a human chromosome 12q BAC (bacterial artificial chromosome) clone (Worley, 2001). Our results
H.J. Yuasa et al. / Gene 298 (2002) 21–27
showed that the human calcyphosine 2 gene is intron-less (data not shown). The intron positions of the calcyphosine family and several troponin C super family genes are summarized in Table 1. 3.3. Evolution of the calcyphosine family A homology search that included deduced amino acid sequences identified the adult mouse testis cDNA clone, 1700028N11 (Adachi et al., 2000) and the human testis cDNA clone MGC:26610 (Strausberg, 2001). The databases defined the initiation Met of these cDNAs as Met-51 (Fig. 3). However, both cDNAs have an in-frame Met (Met-1) at 50 residues upstream from Met-51, and if Met-1 is the real initiation point, the product from these genes must be composed of 208 residues. Furthermore, the Ciona intestinalis cDNA projects (Satou et al., 2002) have revealed that this ascidian also has the calcyphosine gene (cluster ID:03389). These deduced sequences show more than 60% identity with sponge calcyphosine and between each other, and they are all composed of 208 amino acid residues like sponge calcyphosine. All known calcyphosine sequences are aligned in Fig. 3, and phylogenetic trees calculated based on these data are shown in Fig. 4. The human calcyphosine 2 was also localized farther out than the calcyphosine/CCBP23 cluster on the tree constructed by Wang et al. (2002), so we considered it was suitable as an outgroup. The phylogenetic trees were constructed using two methods (neighbor-joining or maximum likelihood), and the topologies of both trees are similar. There are two major clusters; one is composed of three known mammal calcyphosines (named type-I) and the other includes 208residual and insect calcyphosines (named type-II). Type-II calcyphosine is widely distributed among metazoan species, suggesting that it is a rather ancient gene which has some essential function. On the other hand, type-I calcyphosines may be a mammalian specific isoform. Types-I and -II calcyphosines are paralogous, and the former may be rather divergent genes that have acquired new functions (such as cross-signaling between cAMP and calcium-phosphatidylinositol cascades) for higher organs (such as the thyroid, brain, salivary glands and lungs) that the sponge lacks. If this notion is correct, more species of metazoan should have inherited the type-II calcyphosines. A search for type-II calcyphosines among various species will help clarify the evolution of calcyphosine. Acknowledgements This study was supported in part by a Grant-in-Aid for general scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Yuasa, H.J. (13740476).
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References Adachi et al., 2000. DDBJ/EMBL/GenBank Accession No. AK006467. Adams, M.D., Celniker, S.E., Gibbs, R.A., Rubin, G.M., Venter, C.J., 2000. DDBJ/EMBL/GenBank Accession No. AE003697. Cle´ ment, S., Dumont, J.E., Schurmans, S., 1997. Loss of calcyphosine gene expression in mouse and other rodents. Biochem. Biophys. Res. Commun. 232, 407–413. El Housni, H., Radulescu, A., Lecocq, R., Dumont, J.E., Christophe, D., 1997. Cloning and sequence analysis of human calcyphosine complementary DNA. Biochim. Biophys. Acta 1352, 249–252. Felsenstein, J., 1993. PHYLIP (Phylogeny Inference Package) Version 3.5c. Distributed by the Author, Department of Genetics, University of Washington, Seattle. Kawasaki, H., Kretsinger, R.H., 1995. Calcium binding proteins I: EFhands. Prot. Profiles 2, 297–490. Kretsinger, R.H., Nakayama, S., 1993. Evolution of EF-hand calciummodulated proteins. IV. Exon shuffling did not determine the domain compositions of EF-hand proteins. J. Mol. Evol. 36, 477–488. Lamerdin, J.E., 1998. DDBJ/EMBL/GenBank Accession No. AC004602. Lamy, F., Roge, P.P., Lecocq, R., Dumont, J.E., 1986. Differential protein synthesis in the induction of thyroid cell proliferation by thyrotropin, epidermal growth factor or serum. Eur. J. Biochem. 155, 265–272. Lecocq, R., Lamy, F., Dumont, J.E., 1979. Pattern of protein phosphorylation in intact stimulated cells: thyrotropin and dog thyroid. Eur. J. Biochem. 102, 147–152. Lefort, A., Lecocq, R., Libert, F., Lamy, F., Swillens, S., Vassart, G., Dumont, J.E., 1989. Cloning and sequencing of a calcium-binding protein regulated by cyclic AMP in the thyroid. EMBO J. 8, 111–116. Mu¨ ller, W.E.G., 2001. How was metazoan threshold crossed? The hypothetical Urmetazoa. Comp. Biochem. Physiol. (Part A Mol. Integr. Physiol.) 129, 433–460. Mu¨ ller, W.E.G., Schro¨ der, H.C., Skorokhod, A., Bu¨ nz, C., Mu¨ ller, I.M., Grebenjuk, V.A., 2001. Contribution of sponge genes to unravel the genome of the hypothetical ancestor of Metazoa (Urmetazoa). Gene 276, 161–173. Nemoto, Y., Ikeda, J., Katoh, K., Koshimoto, H., Yoshihara, Y., Mori, K., 1993. R2D5 antigen: a calcium-binding phosphoprotein predominantly expressed in olfactory receptor neurons. J. Cell Biol. 123, 963–976. Satou, Y., Takatori, N., Fujiwara, S., Nishikata, T., Saiga, H., Kusakabe, T., Shin-i, T., Kohara, Y., Satoh, N., 2002. Ciona intestinalis cDNA projects: expressed sequence tag analyses and gene expression profiles during embryogenesis. Gene 287 (1–2), 83–96. Sauter, A., Staudenmann, W., Hughes, G.J., Heizmann, C.W., 1995. A novel EF-hand Ca 21-binding protein from abdominal muscle of crustaceans with similarity to calcyphosine from dog thyroidea. Eur. J. Biochem. 227, 97–101. Schmidt, H.A., Strimmer, K., Vingron, M., von Haeseler, A., 2000. TREEPUZZLE-5.0. Distributed by the Authors, Zoologisches Institut, Universita¨ t Muenchen, Muenchen, Germany. Strausberg, R., 2001. DDBJ/EMBL/GenBank Accession No. BC017586. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Worley, K.C., 2001. DDBJ/EMBL/GenBank Accession No. AC091534. Wang, S., Chen, J.Z., Zhang, Z., Huang, Q., Gu, S., Ying, K., Xie, Y., Mao, Y., 2002. Cloning, characterization, and expression of calcyphosine 2, a novel human gene encoding an EF-hand Ca( 21)-binding protein. Biochem. Biophys. Res. Commun. 291 (2), 414–420. Yuasa, H.J., Suzuki, T., Yazawa, M., 2001. Structural organization of lower marine non-vertebrate calmodulin genes. Gene 279, 205–212.
Genomic structure of the sponge, Halichondria okadai calcyphosine gene Hajime Julie Yuasa a,*, Akiko Nakatomi b, Tomohiko Suzuki a, Michio Yazawa b a
b
Laboratory of Biochemistry, Faculty of Science, Kochi University, Kochi 780-8520, Japan Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan Received 12 April 2002; received in revised form 7 August 2002; accepted 20 August 2002 Received by T. Gojobori
Abstract Calcyphosine is an EF-hand Ca 21-binding protein, which was first isolated from the canine thyroid. It is phosphorylated in a cyclic AMP (cAMP)-dependent manner; then it is thought to be implicated in the cross-signaling between the cAMP and calcium-phosphatidylinositol cascades. Here, we isolated the DNA complementary to RNA (cDNA) of an EF-hand Ca 21-binding protein from the sponge, Halichondria okadai and determined its genomic structure. The deduced sequence of the sponge Ca 21-binding protein showed significant similarity (about 40% identity) with those of mammal calcyphosines, and the intron positions were well conserved between the sponge and human calcyphosine genes. We considered that the isolated cDNA was that of sponge calcyphosine, and that sponge and mammalian calcyphosines evolved from a common ancestor gene. Recent cDNA projects have revealed that a calcyphosine cDNA is also expressed by human, mouse, and the ascidia. These cDNAs have more than 60% identity with sponge calcyphosine and each other, and all are composed of 208 amino acid residues. On the constructed phylogenetic trees, calcyphosines are essentially divided into two groups, types-I and -II calcyphosines. Type-I calcyphosine may be specific to mammals, and type-II is widely distributed among metazoan species. This suggests that type-II calcyphosine is a rather ancient gene with some essential function. q 2002 Elsevier Science B.V. All rights reserved. Keywords: EF-hand protein; Molecular evolution; Sponge (Porifera)
1. Introduction There are variously evolved metazoans, and they have independently acquired their original genes that had specific functions during evolution of each lineage. On the other hand, the common ancestor of metazoans must have already possessed at least a minimal set of genes essential for life. It can be easily expected that most of such essential elements have been inherited by today’s descendants. Calcyphosine is a Ca 21-binding protein that was originally detected in the canine thyroid (Lecocq et al., 1979; Lamy et al., 1986). It has four EF-hand calcium binding domains and is phosphorylated in a cyclic AMP (cAMP)-dependent manner. Although the exact function of calcyphosine is unclear, it is implicated in cross signaling between cAMP and calciumphosphatidylinositol cascades. Calcyphosine is also expressed in brain, salivary glands, and lung of dog (Lefort et al., 1989). Homologues of the canine calcyphosine have been isolated from the human thyroid (El Housni et al., 1997) and rabbit olfactory receptor neurons (R2D5 antigen; Nemoto et al., Abbreviations: CaM, calmodulin; cAMP, cyclic AMP; EST, expressed sequence tag; TnC, troponin C * Corresponding author. Tel.: 181-88-844-8464; fax: 181-88-844-8356. E-mail address: [email protected] (H.J. Yuasa).
1993), but it may be absent from the mouse and five other rodents (Cle´ment et al., 1997). Mammalian calcyphosines are highly homologous (more than 86% identity). CCBP-23 is a protein that have been isolated from crustacean abdominal muscle, which also has significant identity (over 40%) with mammal calcyphosines, although CCBP-23 is not apparently phosphorylated (Sauter et al., 1995). All metazoan phyla are thought to have evolved from the common ancestor, Urmetazoa, and the phylum Porifera is thought to be the earliest among them to have diverged (Mu¨ller, 2001; Mu¨ller et al., 2001). The sponges that belong to the Porifera are the simplest animals and they lack muscles, nerves, and other specialized organs such as the thyroid, brain, salivary glands, or lungs. Here, we isolated calcyphosine DNA complementary to RNA (cDNA) from the sponge, Halichondria okadai and determined its genomic structure. We also propose a scheme to explain calcyphosine evolution.
2. Materials and methods 2.1. Cloning of the sponge Ca 21-binding protein (calcyphosine) cDNA Single-stranded cDNA of the sponge, H. okadai was
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(02)00920-4
22
H.J. Yuasa et al. / Gene 298 (2002) 21–27
Fig. 1. cDNA and deduced amino acid sequences of the sponge, H. okadai calcyphosine. Four canonical EF-hand Ca 21 binding domains (site I–IV) are boxed and three insertions within the fourth EF-hand domain are shaded. Downward arrows indicate positions of introns in genome. The determined nucleotide sequence has been submitted to DDBJ under the accession number, AB081565 (H. okadai calcyphosine cDNA).
prepared as previously described (Yuasa et al., 2001). The 3 0 half of sponge Ca 21-binding protein (calcyphosine) cDNA was amplified by polymerase chain reaction (PCR) using Ex Taq DNA polymerase (Takara). The redundant oligomer used for amplification was 5 0 -TTYGAYAARGAYGGNGAYGG3 0 , which was originally designed based on consensus amino acid sequences from the metazoan calmodulins (CaMs), FDKDGDG. The oligo-dT adaptor, 5 0 -GGGATCCGAATTCT17-3 0 was used as another primer. The 5 0 -untranslated region of the cDNA was amplified by 5 0 -rapid amplification of cDNA ends (RACE). The poly-C tail was added to the 3 0 -end of cDNAs using terminal deoxynucleotidyl transferase, and PCR proceeded with primer sets, oligo-dG adapter, 5 0 -GAATTCG15-3 0 and the reverse primer R1, 5 0 TACATGTGCATACACCTG-3 0 (complementary to the sequence from nt 751 to 768, see Fig. 1). 21
2.2. Amplification of sponge Ca -binding protein (calcyphosine) gene Genomic DNA from the sponge was prepared with the conventional phenol-chloroform method. The genomic fragment of the sponge Ca 21-binding protein (calcyphosine) gene was amplified by PCR using Ex Taq DNA polymerase (Takara). The primers used were the forward primer F1, 5 0 -
TGAACACGGAGCTGACAA-3 0 (corresponding to the sequence from nt 249 to 232, see Fig. 1) and the reverse primer R1. 2.3. DNA sequencing The nucleotide sequences were determined by the dideoxy chain termination method with a Thermo Sequenase dye terminator cycle sequencing premix kit, v2.0 (Amersham Pharmacia Biotech) using an automated DNA sequencer (ABI PRISMTM377). 3. Results and discussion 3.1. Sequence of the sponge calcyphosine cDNA The partial cDNA of a sponge calcium binding protein was amplified by PCR as a by-product of sponge CaM cDNA amplification (Yuasa et al., 2001). The redundant oligomer, which was designed based on consensus amino acid sequence among metazoan CaMs, FDKDGDG (residue 20–26, within the first EF-hand domain of CaMs), was annealed to the sequence encoding FDKDGNG (residue 86–93, within the second EF-hand domain of sponge calcyphosine, see Fig. 1). The partial cDNA was encoding a
H.J. Yuasa et al. / Gene 298 (2002) 21–27
23
Fig. 2. (a) Amplification of sponge calcyphosine gene. PCR was performed with primers F1 and R1. A single product (about 3 kbp) was amplified. Left, size markers in kbp based on molecular markers. (b) Exon/intron organization of sponge calcyphosine gene. Exons are shown in boxes and introns are bars. Position and length of each intron (In I–V) are indicated. The determined nucleotide sequences have been submitted to DDBJ under the accession number, AB081566 (H. okadai calcyphosine gene).
typical EF-hand protein, then we determined entire cDNA sequence using 5 0 -RACE. The cDNA was composed of 850 nucleotides, encoding 208 amino acid residues including the initial Met. A homology search within SwissProt (protein data bank) showed that the deduced sequence had significant similarity (about 40% identity) with mammalian calcyphosines and with crayfish CCBP-23, both of which showed 44% identity to each other. Then we refer to the isolated cDNA as sponge calcyphosine below. Two of four EF-hand domains of sponge calcyphosine obviously deviated from the canonical EF-hand sequence (Kawasaki and Kretsinger, 1995). Within the first domain, the central Gly, which is thought to be essential for the sharp bend in the center of the EF-hand, was replaced by Lys. Substitution at this position is frequent among calcyphosines. The fourth domain contains three insertions (Fig. 1, shaded residues). The same domain of mammal calcyphosines and CCBP-23 is also separated by two or by three insertions respectively (El Housni et al., 1997; Sauter et al., 1995). Thus, the first and fourth EF-hand domains of calcyphosines have probably lost Ca 21-binding ability. However, among the four
EF-hand domains, the fourth is most conserved among species (Fig. 3). The fourth EF-hand domain of calcyphosine may be functionally involved in a process other than Ca 21-binding. Mammalian calcyphosines are phosphorylated in a cAMP dependant manner, and a putative phosphorylation site for protein kinase A is located within the first EF-hand domain (Arg/Gly37-Ser-Arg-Ser, in which the last Ser is thought to be phosphorylated). This supposed phosphorylation site is not conserved in either sponge calcyphosine or in CCBP-23. Indeed, the results of ESI-MS analysis suggest that CCBP-23 is not phosphorylated (Sauter et al., 1995). Thus, sponge calcyphosine might not be phosphorylated either. 3.2. Genomic structure of the sponge calcyphosine gene We amplified a single 3 kbp fragment of the sponge calcyphosine gene by PCR using the primers, F1 and R1 (Fig. 2a). Sequencing revealed that this product is composed of 3186 nucleotides and that it contains six exons and five introns. The exon/intron structure of the sponge calcypho-
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H.J. Yuasa et al. / Gene 298 (2002) 21–27
Table 1 Intron positions of calcyphosine family and TnC superfamily genes a Positions of introns Calcyphosine family Type-I calcyphosine Human Type-II calcyphosine Fruit fly Sponge Calcyphosine 2 Human Troponin C superfamily Calmodulin Human (III) Chicken (I,II) Amphioxus Sea hare Fruit fly Sponge Troponin C Human (f) Human (s/c) Amphioxus Ascidian Scallop Fruit fly Sandworm Spec Sea urchin Myosin essential light chain Human Fruit fly Parvalbumin Human
1.04/2
–
2.28/0
–
4.17/0
1.04/2 1.04/2
2.08/2 2.01/0
2.28/0 2.28/0
– 3.24/0
4.17/0 4.17/0
–
–
–
–
210/0 (M) 210/0 (M) 210/0 (M) 210/0 (M) 210/0 (M) 210/0 (M)
1.01/1 1.01/1 1.01/1 1.01/1 – –
2.13/1 2.13/1 2.13/1 2.13/1 2.13/1 –
3.12/0 3.12/0 – – – –
4.21/1 4.21/1 4.21/1 4.21/1 4.21/1 –
217/0 (M) 210/0 217/0 (M) 210/0(M) 212/0 (M) 210/0 213/0
1.01/1 1.01/1 1.01/1 1.01/1 1.01/1 – 1.01/1
2.13/1 2.13/1 2.13/1 2.13/1 2.13/1 2.13/1 2.13/1
3.11/2 3.11/2 3.24/0 3.24/0 – – 3.23/2
4.21/1 4.21/1 4.21/1 4.21/1 4.21/1 4.21/1 4.21/1
212/0 (M)
1.01/1
2.13/1
3.18/2
4.21/1
209/0 (M) 208/0 (M)
1.01/1 1.02/1
2.12/1 –
3 1 01/1 3 1 01/1
4.21/1 4.21/1
–
2.11/1
3.23/2
4.21/1
–
–
(intron-less)
a (M) refers to intron is inserted just after the initiation codon, ATG. (–) refers to absence of intron. Human (f), human fast skeletal TnC; and Human (s/c), human slow-cardiac TnC.
sine gene is shown in Fig. 2b. A comparison with the cDNA sequence (Fig. 1) determined that an AAA codon encoding Lys-14 was changed to AAG, an AAC codon encoding Asn92 changed to AAT, and that T-739 was changed to C, which may be due to individual differences or allelic polymorphisms. All introns conformed to the GT-AG rule and the positions of the introns are 1.04/2, 2.01/0, 2.28/0, 3.24/2, and 4.17/0 1 (residues inserted within the fourth EF-hand domain were not counted). The human calcyphosine gene has only three introns, but their positions are identical to three of five introns of the sponge calcyphosine gene (1.04/ 2, 2.28/0, and 4.17/0; Lamerdin, 1998). Furthermore, a fruit fly gene encoding Drosophila melanogaster CG10126 1 The positions of introns are indicated according to the nomenclature of Kretsinger and Nakayama (1993). The first number corresponds to the EFhand domain numbered sequentially from N to C. The second number (following the period) represents the residue number of intron insertion in the EF-hand domain, which as a rule consists of 29 residues. The last number (following the slash) is phase: 0 means that the intron lies between triplet codons, 1 means between the first and second nucleotides of the codon, and 2 means between the second and third. For instance, 1.04/2 means insertion in domain I, fourth residue and phase 2.
protein (Adams et al., 2000), which shows significant similarity with calcyphosines, has four introns (1.04/2, 2.08/2, 2.28/0, and 4.17/0) and three of them are inserted at positions identical to those of the insertions in the sponge/human calcyphosine genes. These suggest that the mammal and sponge calcyphosines, as well as the Drosophila gene encoding CG10126 have evolved from a common ancestor. As far as we know, these intron positions are unique among EF-hand genes, so they should be classified as members of the calcyphosine family. In addition, calcyphosines show some similarity with CaM, which also has four EF-hand domains per molecule, and Lefort et al. (1989) and Cle´ ment et al. (1997) have suggested a rather close relationship between them. Indeed the chordate CaM genes essentially possess five introns (Yuasa et al., 2001), as do sponge calcyphosine gene. However, CaM belongs to the troponin C (TnC) superfamily in which the intron positions are highly conserved (Yuasa et al., 2001). The intron positions of calcyphosines are completely different from those of the TnC superfamily, so we infer that the calcyphosine and CaM (or TnC superfamily) genes are not that closely related. Recently, the cDNA sequence of the novel human
H.J. Yuasa et al. / Gene 298 (2002) 21–27
25
Fig. 3. Amino acid sequences of metazoan calcyphosines aligned by the Clustal W 1.7 program (Thompson et al., 1994). Four canonical EF-hand Ca 21 binding domains (site I–IV) are shown by # and three insertions within the fourth EF-hand domain are shown by dots (.). The residues conserved in all chains are indicated by asterisks (*) and gaps are inserted for maximal similarity and shown by bars (2). The supposed phosphorylation sites of mammal calcyphosines (type-I calcyphosines) are underlined. Sponge, sponge calcyphosine (this work); Human II, human clone: MGC 26610 (DDBJ/EMBL/GenBank accession no. BC017586); Mouse, adult mouse testis cDNA, clone:1700028N11 (accession no. AK006467); Ascidia, C. intestinalis expressed sequence tag clone: cluster ID:03389 (Satou et al., 2002); Fruit fly, Drosophila melanogaster CG10126 protein (accession no. AE003697); Hornworm, the tobacco hornworm Manduca sexta calcyphosine-like protein (accession no. AF117582) to which Drosophila CG10126 shows the highest similarity; Dog, dog calcyphosine (Lefort et al., 1989); Human, human calcyphosine (El Housni et al., 1997); Rabbit, rabbit R2D5 antigen (Nemoto et al., 1993); Crayfish, crayfish CCBP-23 (Sauter et al., 1995); Calcy 2, human calcyphosine 2 (Wang et al., 2002). (1) means that some N-terminal residues are truncated.
26
H.J. Yuasa et al. / Gene 298 (2002) 21–27
Fig. 4. Phylogenetic trees based on the alignment of data in Fig. 3 (only four EF-hand domains and three interdomain regions were used). Above, the tree constructed using the PHYLIP package (Felsenstein, 1993) and Neighbor-Joining method was used. Numbers at forks indicate percentage of 1000 bootstrap resamplings that support these topological elements. Below, the maximum likelihood tree constructed with TREE-PUZZLE 5.0 (Schmidt et al., 2000). Numbers at forks indicate the support values for internal branches. Human calcyphosine 2 (Wang et al., 2002) was used as an outgroup.
protein, calcyphosine 2 has been reported (Wang et al., 2002). Human calcyphosine 2 consists of 382 amino acids residues (about twice the size of other calcyphosines) and its C-terminal half shows about 30% identity with other calcy-
phosines. Since the human calcyphosine 2 gene is mapped to the human genome 12q15, we aligned the sequences of the cDNA and a human chromosome 12q BAC (bacterial artificial chromosome) clone (Worley, 2001). Our results
H.J. Yuasa et al. / Gene 298 (2002) 21–27
showed that the human calcyphosine 2 gene is intron-less (data not shown). The intron positions of the calcyphosine family and several troponin C super family genes are summarized in Table 1. 3.3. Evolution of the calcyphosine family A homology search that included deduced amino acid sequences identified the adult mouse testis cDNA clone, 1700028N11 (Adachi et al., 2000) and the human testis cDNA clone MGC:26610 (Strausberg, 2001). The databases defined the initiation Met of these cDNAs as Met-51 (Fig. 3). However, both cDNAs have an in-frame Met (Met-1) at 50 residues upstream from Met-51, and if Met-1 is the real initiation point, the product from these genes must be composed of 208 residues. Furthermore, the Ciona intestinalis cDNA projects (Satou et al., 2002) have revealed that this ascidian also has the calcyphosine gene (cluster ID:03389). These deduced sequences show more than 60% identity with sponge calcyphosine and between each other, and they are all composed of 208 amino acid residues like sponge calcyphosine. All known calcyphosine sequences are aligned in Fig. 3, and phylogenetic trees calculated based on these data are shown in Fig. 4. The human calcyphosine 2 was also localized farther out than the calcyphosine/CCBP23 cluster on the tree constructed by Wang et al. (2002), so we considered it was suitable as an outgroup. The phylogenetic trees were constructed using two methods (neighbor-joining or maximum likelihood), and the topologies of both trees are similar. There are two major clusters; one is composed of three known mammal calcyphosines (named type-I) and the other includes 208residual and insect calcyphosines (named type-II). Type-II calcyphosine is widely distributed among metazoan species, suggesting that it is a rather ancient gene which has some essential function. On the other hand, type-I calcyphosines may be a mammalian specific isoform. Types-I and -II calcyphosines are paralogous, and the former may be rather divergent genes that have acquired new functions (such as cross-signaling between cAMP and calcium-phosphatidylinositol cascades) for higher organs (such as the thyroid, brain, salivary glands and lungs) that the sponge lacks. If this notion is correct, more species of metazoan should have inherited the type-II calcyphosines. A search for type-II calcyphosines among various species will help clarify the evolution of calcyphosine. Acknowledgements This study was supported in part by a Grant-in-Aid for general scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Yuasa, H.J. (13740476).
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