- Email: [email protected]
Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch
peptides 27 (2006) 1235–1240
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch Cao Zhijian 1, Xie Yun 1, Dai Chao, Zhu Shunyi, Yin Shijin, Wu Yingliang, Li Wenxin * State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China
article info
abstract
Article history:
Many studies have been carried on peptides and genes encoding scorpion toxins from the
Received 24 September 2005
venom of Mesobuthus martensii Karsch (synonym: Buthus martensii Karsch, BmK), such as
Received in revised form
Na+, K+ and Cl channel modulators. In this study, a novel calcium channel toxin-like gene
8 October 2005
BmCa1 was isolated and characterized from the venom of Mesobuthus martensii Karsch.
Accepted 10 October 2005
First, a partial cDNA sequence of the Ca2+ channel toxin-like gene was identified by random
Published on line 18 November 2005
sequencing method from a venomous gland cDNA library of Mesobuthus martensii Karsch. The full-length sequence of BmCa1 was then obtained by 5’RACE technique. The peptide
Keywords:
deduced from BmCa1 precursor nucleotide sequence contains a 27-residue signal peptide
Mesobuthus martensii Karsch
and a 37-residue mature peptide. Although BmCa1 and other scorpion toxins are different at
Calcium channel scorpion toxins
the gene and protein primary structure levels, BmCa1 has the same precursor nucleotide
Genomic organization and structure
organization and cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins. Genomic DNA sequence of BmCa1 was also cloned by PCR. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns of 72 bp and 1076 bp in length, respectively. BmCa1 is the first calcium channel toxin-like gene cloned from the venom of Mesobuthus martensii Karsch and potentially represents a novel class of calcium channel toxins in scorpion venoms. # 2005 Elsevier Inc. All rights reserved.
1.
Introduction
Scorpion venom is an important and rich source of polypeptides, most of which are modulators of ion channels (e.g., Na+, K+, Cl and Ca2+ channels) on the cell membrane [17]. Na+ channel scorpion toxins consist of 60–70 amino acids with four disulfide bridges [16]. Most of K+ channel scorpion toxins are short chain polypeptides (30–40 amino acids) with three or four disulfide bonds [18,21]. Almost all the Cl channel scorpion toxins comprise of about 35 amino acid residues with four
disulfide bridges [19]. Compared to these scorpion toxin families, calcium channel scorpion toxins have more diverse amino acid length and unique disulfide bridge organization [28]. In addition, the reported scorpion toxins mostly belong to the ligand families of Na+, K+ and Cl channels. Out of about 400 wild type toxins from natural scorpion venom, there are about 200 Na+ channel scorpion toxins, 150 K+ channel scorpion toxins, and 19 Cl channel scorpion toxins, but only 10 typical calcium channel toxins from several scorpion species classified into three subfamilies [3,13,20].
* Corresponding author. Tel.: +86 27 68752831; fax: +86 27 68752146. E-mail addresses: [email protected] (C. Zhijian), [email protected] (L. Wenxin). 1 These authors contribute equally to this article. 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.10.010
1236
peptides 27 (2006) 1235–1240
Chinese scorpion Mesobuthus martensii Karsch is widely distributed in China and is an important raw material in traditional Chinese medicines [8,27]. The venom of Mesobuthus martensii Karsch is one of the most studied scorpion venoms. To date, over 80 scorpion toxins and 10 scorpion toxin-like peptides have been isolated and identified from Mesobuthus martensii Karsch venom, which accounts for 25% of the total known scorpion toxins (90/400) [8,20]. These reported scorpion toxins from Mesobuthus martensii Karsch target Na+, K+ and Cl ion channels [8–10,23,24,26,30]. No calcium channel toxin peptides or genes have been isolated or cloned from the venom of Mesobuthus martensii Karsch, even though they are an important class of ion channel modulators. Here we report the isolation and characterization of a novel calcium channel scorpion toxin-like gene BmCa1 from the venom of Mesobuthus martensii Karsch. The peptide deduced from BmCa1 cDNA sequence contains a 27-aa signal peptide and a 37-aa mature peptide. Sequence analysis of BmCa1 genomic DNA showed that the BmCa1 gene consists of three exons separated by two introns, a genomic structure similar to that of the calcium channel scorpion toxin Opicalcine1 from Opistophthalmus carinatus. Our data suggest that BmCa1 is possibly a first member of calcium channel toxin-like gene from Mesobuthus martensii Karsch and that BmCa1 probably represents a novel class of calcium channel toxins.
2.
Materials and methods
2.1.
Screening of a cDNA library of venomous gland
The cDNA library prepared from the venomous gland of the Mesobuthus martensii Karsch was reported previously [29]. Random sequencing strategy was used to screen the cDNA library clones. Novel cDNA sequences encoding scorpion venom peptides were thus obtained and submitted to GenBank.
2.2.
50 RACE for scorpion toxin-like gene BmCa1
Double stranded cDNAs reported eariler [1] were used as 50 RACE template for amplifying the 50 end of the BmCa1 gene. Specific and universal primers for 5’RACE were 50 CCGTATCTGCAACAGTC-30 (corresponding to DCCRYG mature peptide coding region of BmCa1) and 50 -AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGGG-30 (50 anchor primer SMART IVTM Oligonucleotide), respectively, The PCR conditions for 50 RACE were the following: 10 cycles of denaturing at 94 8C for 1 min, annealing from 60 to 50 8C for 1 min and elongation at 72 8C for 1.5 min, and then 25 cycles of denaturing at 94 8C for 1 min, annealing at 53 8C for 1 min and elongation at 72 8C for 1.5 min, and finally terminated with a 5 min elongation at 72 8C. The PCR products of 50 RACE were purified and cloned into pGEM-T easy vector (Promega, USA). The clones containing inserts were analyzed by sequencing.
2.3.
Genomic DNA cloning and analysis
The total genomic DNA was isolated from whole scorpion Mesobuthus martensii Karsch as described before [2]. The
BmCa1 genomic DNA was amplified by PCR with the following primers: the forward primer (50 -ATTTGAGAAGCAGCGA-30 , corresponding to 50 UTR of BmCa1) and the reverse primer (50 -CCGTATCTGCAACAGTC-30 , 50 RACE specific primer). The PCR was carried out in a programmable heating chamber (Biometra, Germany) for 35 PCR cycles (94 8C for 1 min, 53 8C for 1 min and 72 8C for 1.5 min) followed by 5 min at 72 8C in 25 ml. The PCR products were purified after gel electrophoresis with the Gel Extraction Kit (Omega, USA) and the purified products were cloned into pGEM-T easy vector. A positive clone was sequenced with the universal T7 and SP6 promoter primers.
2.4.
Sequence analysis
Sequence analysis was performed with Generunr software. Multiple sequence alignment (MSA) was carried out with the DNAMAN program.
3.
Results
3.1. Isolation of a partial cDNA clone for the calcium channel scorpion toxin-like gene BmCa1 from Mesobuthus martensii Karsch Random sequencing strategy was used to screen a venomous cDNA library of Mesobuthus martensii Karsch. The positive clones with the inserts of about 300 bp were isolated and sequenced. A clone corresponding to BmCa1 was thus obtained (GenBank Accession Number: AF419253). Sequence search against the GenBank database (http://www.ncbi.nlm.nih.gov/) revealed that this cDNA shares low degrees of similarity with the other scorpion toxin or venom peptide genes. Sequence analysis suggested that this cDNA likely encodes a scorpion venom peptide and contains 174 bp of the open reading frame (ORF) and 68 bp 30 UTR. The intiation codon and 50 UTR are not present. A polyA signal ATTAAA was found 44 bp downstream to the terminal codon (Fig. 1). The deduced amino acid sequences has the same cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins (Maurocalcine from Scorpio maurus, Imperatoxin A from Pandinus imperator, Opicalcine 1 and Opicalcine 2 from Opistophthalmus carinatus) [6,7,28] and thus the gene was named calcium channel scorpion toxin-like gene BmCa1 from Mesobuthus martensii Karsch.
3.2.
Cloning of the full-length cDNA for BmCa1 by 50 RACE
The positive DNA band of BmCa1 50 RACE was amplified again by PCR (Fig. 2A) and was cloned to pGEM-T easy vector for sequencing. Sequence analysis showed that the cloned DNA contains the 50 UTR and initiation codon of BmCa1. A composite full length cDNA sequence for BmCa1 was obtained from the partial cDNA sequence and 50 RACE sequence (GenBank Accession Number: DQ201205). Sequence analysis showed that the full-length BmCa1 cDNA is 288 bp in length, including 25 bp 5’UTR, 68 bp 30 UTR and 195 bp ORF (Fig. 1). The BmCa1 cDNA shares low degrees of similarity with the other scorpion toxin genes. The A+T content of 30 UTR (78%) is higher
peptides 27 (2006) 1235–1240
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Fig. 1 – Genomic DNA, cDNA and the deduced amino acid sequences of BmCa1 from Chinese scorpion Mesobuthus martensii Karsch. The sequences of UTRs and introns are lowercase; a potential polyadenylation signal ATTAAA is shaded; the primers for 50 RACE and genomic amplifying are underlined twice; the flanking sequences of both introns are shown with red color; the deduced amino acids are in capital and shown below their corresponding nucleotide sequences; the putative signal peptide is underlined.
than those of 5’UTR and ORF (60% and 64%, respectively). Moreover, the sequence around its initiation codon (AAGATGAAT) is different from the highly conserved AAAATGAAT found in most scorpion venom peptides from Mesobuthus martensii Karsch [30].
The peptide deduced from the BmCa1 cDNA is 64 amino acids long, including a 27-aa signal peptide and a 37-aa residue mature peptide with three disulfide bridges (Fig. 1). BmCa1 shares the cysteine arrangement and the precursor organization with other scorpion venom-derived peptides that contain
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peptides 27 (2006) 1235–1240
3.3. Genomic organization of calcium channel scorpion toxin-like gene BmCa1
Fig. 2 – Electrophoretic analysis of 50 RACE and genomic DNA of BmCa1 from Mesobuthus martensii Karsch. (A) the product of 50 RACE. M is DNA marker (2-log DNA ladder from NEB); lane 1 is the amplifying products of 50 RACE; lanes 2 and 3 are the amplifying control of 50 RACE specific and universal primers, respectively. (B) the product of genomic DNA amplification. M is DNA marker (1 kb ladder from MBI fermentas); lanes 1 and 2 are the amplifying products of genomic DNA and corresponding cDNA of BmCa1, respectively.
A DNA band of approximately 1.4 kb corresponding to the BmCa1 gene was amplified from scorpion genomic DNA by using PCR (Fig. 2B). The PCR product was inserted into pGEM-T easy vector. A number of positive clones were obtained and one clone was selected at random for sequencing. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns (Fig. 1). A 72 bp short intron is located at the end of the signal peptide coding region, following the first base of Asp codon at position +67 nucleotide. The second intron is 1076 bp long and located within the mature peptide coding region. These two introns begin with GT and end with AG, consistent with previously reported scorpion toxin gene introns. The 50 splice donor sequences of these two introns are 50 -Gjgtaag-30 , while the 30 splice receptor of these are 50 ttcagjA-30 and 50 -tatagjT-30 , respectively. It is worth mentioning that the A+T content of these two introns (83% and 71%) is much higher than that of corresponding exons, and a number of A-runs and T-runs existed in both introns, which suggested that they may be playing a role in the splicing process [5]. The genomic sequence of BmCa1 has been deposited into GenBank (GenBank Accession Number: DQ206446).
4.
Discussion
4.1. Scorpion toxins or peptides from the venom of Mesobuthus martensii Karsch the inhibitory cysteine knot (ICK) motif [6,7,28]. The 27-aa long signal peptide is rich in hydrophobic amino acid residues (e.g. Phe, Leu, Ile), which is a characteristic of all signal peptides. The 37-aa mature peptide of BmCa1 has the common disulfide bridge motif: CX(3–7)CX(4–6)CX(0–5) CX(1–5)CX(4–13)C, which suggests that BmCa1 and the known calcium channel scorpion toxins share the conserved 3D structure with two or three bsheets [12]. BmCa1 shares only low degrees of similarity with other scorpion toxins at amino acid level. These results suggest that BmCa1 is likely a novel member of calcium channel scorpion toxin-like peptides. It is interesting to note that BmCa1 and another deduced peptide in the data bank, BmKCa, (GenBank Accession Number: DQ120790) from Mesobuthus martensii Karsch, contain the same 57-aa at Cterminus but have completely different amino acid residues at the very N-terminus (Fig. 3). It would be of interest to further explore the origin of the difference in the future.
BmCa1 is likely the first member of calcium channel toxin-like genes isolated from Chinese scorpion Mesobuthus martensii Karsch. BmCa1 shares low degrees of similarity with the other scorpion toxins, including calcium channel toxins. This suggests that BmCa1 may be a novel member of the calcium channel scorpion toxin superfamily and represents a new class of calcium channel scorpion toxin subfamily. This is the first example from a scorpion of the family Buthidae that suggests the possible presence of peptides with the ICK motif, thus far known to exist only in scorpions of the family Scorpionidae. To date, over 80 toxins or peptides have been isolated and characterized from the venom of Chinese scorpion Mesobuthus martensii Karsch [8,25]. Mesobuthus martensii Karsch is the most studied scorpion species in the world. Possani et al. proposed that there are about one hundred toxins or peptides in the venom of each scorpion species. At least 100000
Fig. 3 – Mutiple sequence alignments of the first subfamily members of calcium channel scorpion toxins. BmCa1 and BmKCa (GenBank Number: DQ120790) from Mesobuthus martensii Karsch; Maurocalcine from Scorpio maurus; Imperatoxin A from Pandinus imperator; Opicalcine 1 and Opicalcine 2 from Opistophthalmus carinatus. The first amino acid glycine (G) of mature peptides is indicated with the arrow.
peptides 27 (2006) 1235–1240
bioactive peptides exist in scorpion venoms out of 1500 scorpion species in the world [17]. This makes scorpion venom a source rich in polypeptides and drugs for treating a number of diseases. Recently, Pimenta et al. speculated that there are 380 different bioactive peptides in the venom of another scorpion of the Buthidae family, Tityus serrulatus [15]. An important question is how many toxin peptides and genes a scorpion venom actually contains. Toxin peptides and genes identified from the venom of scorpion Mesobuthus martensii Karsch so far represent only a small fraction of the scorpion venom. Advances in mass spectrometry and peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous novel peptide toxins from scorpion venoms [4]. This should help to discover the large number of unidentified peptides in the scorpion venom, which in turn would accelerate the understanding of the structure-function relationship of ion channels and help to develop novel therapeutic agents and strategies for ion channel-related diseases.
4.2.
Genomic organization and structure of BmCa1
To date, over 30 genomic sequences of scorpion toxins or venom peptides have been reported [11,22]. The genomic organization of most scorpion toxin genes is completely identical, namely, containing two exons and one intron. The first exon spans the 50 untranslated region and the stretch that codes for the first two-thirds of the signal peptide, followed by an intron and the second exon encoding the last third of the signal peptide, the mature toxin, and the 30 untranslated region [22]. Recently, some new genomic structures of scorpion toxins or venom peptides were reported [2,19]. However, only two scorpion toxin genes BmCa1 and Opicalcine1 [28] have the same genomic organization and structure. Both contain two introns (one intron located in the signal peptide coding region and the other in the mature peptide coding region). Moreover, four introns of both scorpion toxin genes belonged to the phase-I and phase-II, respectively. The locations of their introns are consistent with each other. It is worth noting that four introns of BmCa1 and Opicalcine have different length. The first intron of BmCa1 is similar in length with that of the short-chain scorpion toxin genes (K+ channel and Cl channel toxins) [22], while the second intron of BmCa1 is similar in length with that of long-chain scorpion toxin genes (mainly Na+ channel toxins) [14]. On the other hand, the disulfide-bridge number of calcium channel scorpion toxins is similar to that of most K+ channel scorpion toxins (3 disulfide bridges), while the cysteine residue arrangement of the first subfamily members of calcium channel scorpion toxins is similar to that of excitatory Na+ channel scorpion toxins and Cl channel scorpion toxins (cysteine-cysteine arrangement). All these common features suggest the strong conservation among different classes of scorpion toxins.
Acknowledgements This work is supported by the grants (Number: 30530140; 30500089; 30570045) from the National Natural Sciences Foundation of China to Li W.X., Wu Y.L. and Cao Z.J., and
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the Foundation of HuBei Provincial Key Laboratory of Biotechnology of Traditional Chinese Medicine, HuBei University. We also thank Dr. Shi Yunbo and Dr. Tasleem for his help in editing the paper.
references
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Cloning and characterization of a novel calcium channel toxin-like gene BmCa1 from Chinese scorpion Mesobuthus martensii Karsch Cao Zhijian 1, Xie Yun 1, Dai Chao, Zhu Shunyi, Yin Shijin, Wu Yingliang, Li Wenxin * State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China
article info
abstract
Article history:
Many studies have been carried on peptides and genes encoding scorpion toxins from the
Received 24 September 2005
venom of Mesobuthus martensii Karsch (synonym: Buthus martensii Karsch, BmK), such as
Received in revised form
Na+, K+ and Cl channel modulators. In this study, a novel calcium channel toxin-like gene
8 October 2005
BmCa1 was isolated and characterized from the venom of Mesobuthus martensii Karsch.
Accepted 10 October 2005
First, a partial cDNA sequence of the Ca2+ channel toxin-like gene was identified by random
Published on line 18 November 2005
sequencing method from a venomous gland cDNA library of Mesobuthus martensii Karsch. The full-length sequence of BmCa1 was then obtained by 5’RACE technique. The peptide
Keywords:
deduced from BmCa1 precursor nucleotide sequence contains a 27-residue signal peptide
Mesobuthus martensii Karsch
and a 37-residue mature peptide. Although BmCa1 and other scorpion toxins are different at
Calcium channel scorpion toxins
the gene and protein primary structure levels, BmCa1 has the same precursor nucleotide
Genomic organization and structure
organization and cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins. Genomic DNA sequence of BmCa1 was also cloned by PCR. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns of 72 bp and 1076 bp in length, respectively. BmCa1 is the first calcium channel toxin-like gene cloned from the venom of Mesobuthus martensii Karsch and potentially represents a novel class of calcium channel toxins in scorpion venoms. # 2005 Elsevier Inc. All rights reserved.
1.
Introduction
Scorpion venom is an important and rich source of polypeptides, most of which are modulators of ion channels (e.g., Na+, K+, Cl and Ca2+ channels) on the cell membrane [17]. Na+ channel scorpion toxins consist of 60–70 amino acids with four disulfide bridges [16]. Most of K+ channel scorpion toxins are short chain polypeptides (30–40 amino acids) with three or four disulfide bonds [18,21]. Almost all the Cl channel scorpion toxins comprise of about 35 amino acid residues with four
disulfide bridges [19]. Compared to these scorpion toxin families, calcium channel scorpion toxins have more diverse amino acid length and unique disulfide bridge organization [28]. In addition, the reported scorpion toxins mostly belong to the ligand families of Na+, K+ and Cl channels. Out of about 400 wild type toxins from natural scorpion venom, there are about 200 Na+ channel scorpion toxins, 150 K+ channel scorpion toxins, and 19 Cl channel scorpion toxins, but only 10 typical calcium channel toxins from several scorpion species classified into three subfamilies [3,13,20].
* Corresponding author. Tel.: +86 27 68752831; fax: +86 27 68752146. E-mail addresses: [email protected] (C. Zhijian), [email protected] (L. Wenxin). 1 These authors contribute equally to this article. 0196-9781/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.10.010
1236
peptides 27 (2006) 1235–1240
Chinese scorpion Mesobuthus martensii Karsch is widely distributed in China and is an important raw material in traditional Chinese medicines [8,27]. The venom of Mesobuthus martensii Karsch is one of the most studied scorpion venoms. To date, over 80 scorpion toxins and 10 scorpion toxin-like peptides have been isolated and identified from Mesobuthus martensii Karsch venom, which accounts for 25% of the total known scorpion toxins (90/400) [8,20]. These reported scorpion toxins from Mesobuthus martensii Karsch target Na+, K+ and Cl ion channels [8–10,23,24,26,30]. No calcium channel toxin peptides or genes have been isolated or cloned from the venom of Mesobuthus martensii Karsch, even though they are an important class of ion channel modulators. Here we report the isolation and characterization of a novel calcium channel scorpion toxin-like gene BmCa1 from the venom of Mesobuthus martensii Karsch. The peptide deduced from BmCa1 cDNA sequence contains a 27-aa signal peptide and a 37-aa mature peptide. Sequence analysis of BmCa1 genomic DNA showed that the BmCa1 gene consists of three exons separated by two introns, a genomic structure similar to that of the calcium channel scorpion toxin Opicalcine1 from Opistophthalmus carinatus. Our data suggest that BmCa1 is possibly a first member of calcium channel toxin-like gene from Mesobuthus martensii Karsch and that BmCa1 probably represents a novel class of calcium channel toxins.
2.
Materials and methods
2.1.
Screening of a cDNA library of venomous gland
The cDNA library prepared from the venomous gland of the Mesobuthus martensii Karsch was reported previously [29]. Random sequencing strategy was used to screen the cDNA library clones. Novel cDNA sequences encoding scorpion venom peptides were thus obtained and submitted to GenBank.
2.2.
50 RACE for scorpion toxin-like gene BmCa1
Double stranded cDNAs reported eariler [1] were used as 50 RACE template for amplifying the 50 end of the BmCa1 gene. Specific and universal primers for 5’RACE were 50 CCGTATCTGCAACAGTC-30 (corresponding to DCCRYG mature peptide coding region of BmCa1) and 50 -AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGGG-30 (50 anchor primer SMART IVTM Oligonucleotide), respectively, The PCR conditions for 50 RACE were the following: 10 cycles of denaturing at 94 8C for 1 min, annealing from 60 to 50 8C for 1 min and elongation at 72 8C for 1.5 min, and then 25 cycles of denaturing at 94 8C for 1 min, annealing at 53 8C for 1 min and elongation at 72 8C for 1.5 min, and finally terminated with a 5 min elongation at 72 8C. The PCR products of 50 RACE were purified and cloned into pGEM-T easy vector (Promega, USA). The clones containing inserts were analyzed by sequencing.
2.3.
Genomic DNA cloning and analysis
The total genomic DNA was isolated from whole scorpion Mesobuthus martensii Karsch as described before [2]. The
BmCa1 genomic DNA was amplified by PCR with the following primers: the forward primer (50 -ATTTGAGAAGCAGCGA-30 , corresponding to 50 UTR of BmCa1) and the reverse primer (50 -CCGTATCTGCAACAGTC-30 , 50 RACE specific primer). The PCR was carried out in a programmable heating chamber (Biometra, Germany) for 35 PCR cycles (94 8C for 1 min, 53 8C for 1 min and 72 8C for 1.5 min) followed by 5 min at 72 8C in 25 ml. The PCR products were purified after gel electrophoresis with the Gel Extraction Kit (Omega, USA) and the purified products were cloned into pGEM-T easy vector. A positive clone was sequenced with the universal T7 and SP6 promoter primers.
2.4.
Sequence analysis
Sequence analysis was performed with Generunr software. Multiple sequence alignment (MSA) was carried out with the DNAMAN program.
3.
Results
3.1. Isolation of a partial cDNA clone for the calcium channel scorpion toxin-like gene BmCa1 from Mesobuthus martensii Karsch Random sequencing strategy was used to screen a venomous cDNA library of Mesobuthus martensii Karsch. The positive clones with the inserts of about 300 bp were isolated and sequenced. A clone corresponding to BmCa1 was thus obtained (GenBank Accession Number: AF419253). Sequence search against the GenBank database (http://www.ncbi.nlm.nih.gov/) revealed that this cDNA shares low degrees of similarity with the other scorpion toxin or venom peptide genes. Sequence analysis suggested that this cDNA likely encodes a scorpion venom peptide and contains 174 bp of the open reading frame (ORF) and 68 bp 30 UTR. The intiation codon and 50 UTR are not present. A polyA signal ATTAAA was found 44 bp downstream to the terminal codon (Fig. 1). The deduced amino acid sequences has the same cysteine arrangement as that of the first subfamily members of calcium channel scorpion toxins (Maurocalcine from Scorpio maurus, Imperatoxin A from Pandinus imperator, Opicalcine 1 and Opicalcine 2 from Opistophthalmus carinatus) [6,7,28] and thus the gene was named calcium channel scorpion toxin-like gene BmCa1 from Mesobuthus martensii Karsch.
3.2.
Cloning of the full-length cDNA for BmCa1 by 50 RACE
The positive DNA band of BmCa1 50 RACE was amplified again by PCR (Fig. 2A) and was cloned to pGEM-T easy vector for sequencing. Sequence analysis showed that the cloned DNA contains the 50 UTR and initiation codon of BmCa1. A composite full length cDNA sequence for BmCa1 was obtained from the partial cDNA sequence and 50 RACE sequence (GenBank Accession Number: DQ201205). Sequence analysis showed that the full-length BmCa1 cDNA is 288 bp in length, including 25 bp 5’UTR, 68 bp 30 UTR and 195 bp ORF (Fig. 1). The BmCa1 cDNA shares low degrees of similarity with the other scorpion toxin genes. The A+T content of 30 UTR (78%) is higher
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Fig. 1 – Genomic DNA, cDNA and the deduced amino acid sequences of BmCa1 from Chinese scorpion Mesobuthus martensii Karsch. The sequences of UTRs and introns are lowercase; a potential polyadenylation signal ATTAAA is shaded; the primers for 50 RACE and genomic amplifying are underlined twice; the flanking sequences of both introns are shown with red color; the deduced amino acids are in capital and shown below their corresponding nucleotide sequences; the putative signal peptide is underlined.
than those of 5’UTR and ORF (60% and 64%, respectively). Moreover, the sequence around its initiation codon (AAGATGAAT) is different from the highly conserved AAAATGAAT found in most scorpion venom peptides from Mesobuthus martensii Karsch [30].
The peptide deduced from the BmCa1 cDNA is 64 amino acids long, including a 27-aa signal peptide and a 37-aa residue mature peptide with three disulfide bridges (Fig. 1). BmCa1 shares the cysteine arrangement and the precursor organization with other scorpion venom-derived peptides that contain
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3.3. Genomic organization of calcium channel scorpion toxin-like gene BmCa1
Fig. 2 – Electrophoretic analysis of 50 RACE and genomic DNA of BmCa1 from Mesobuthus martensii Karsch. (A) the product of 50 RACE. M is DNA marker (2-log DNA ladder from NEB); lane 1 is the amplifying products of 50 RACE; lanes 2 and 3 are the amplifying control of 50 RACE specific and universal primers, respectively. (B) the product of genomic DNA amplification. M is DNA marker (1 kb ladder from MBI fermentas); lanes 1 and 2 are the amplifying products of genomic DNA and corresponding cDNA of BmCa1, respectively.
A DNA band of approximately 1.4 kb corresponding to the BmCa1 gene was amplified from scorpion genomic DNA by using PCR (Fig. 2B). The PCR product was inserted into pGEM-T easy vector. A number of positive clones were obtained and one clone was selected at random for sequencing. Sequence analysis showed that BmCa1 gene consists of three exons separated by two introns (Fig. 1). A 72 bp short intron is located at the end of the signal peptide coding region, following the first base of Asp codon at position +67 nucleotide. The second intron is 1076 bp long and located within the mature peptide coding region. These two introns begin with GT and end with AG, consistent with previously reported scorpion toxin gene introns. The 50 splice donor sequences of these two introns are 50 -Gjgtaag-30 , while the 30 splice receptor of these are 50 ttcagjA-30 and 50 -tatagjT-30 , respectively. It is worth mentioning that the A+T content of these two introns (83% and 71%) is much higher than that of corresponding exons, and a number of A-runs and T-runs existed in both introns, which suggested that they may be playing a role in the splicing process [5]. The genomic sequence of BmCa1 has been deposited into GenBank (GenBank Accession Number: DQ206446).
4.
Discussion
4.1. Scorpion toxins or peptides from the venom of Mesobuthus martensii Karsch the inhibitory cysteine knot (ICK) motif [6,7,28]. The 27-aa long signal peptide is rich in hydrophobic amino acid residues (e.g. Phe, Leu, Ile), which is a characteristic of all signal peptides. The 37-aa mature peptide of BmCa1 has the common disulfide bridge motif: CX(3–7)CX(4–6)CX(0–5) CX(1–5)CX(4–13)C, which suggests that BmCa1 and the known calcium channel scorpion toxins share the conserved 3D structure with two or three bsheets [12]. BmCa1 shares only low degrees of similarity with other scorpion toxins at amino acid level. These results suggest that BmCa1 is likely a novel member of calcium channel scorpion toxin-like peptides. It is interesting to note that BmCa1 and another deduced peptide in the data bank, BmKCa, (GenBank Accession Number: DQ120790) from Mesobuthus martensii Karsch, contain the same 57-aa at Cterminus but have completely different amino acid residues at the very N-terminus (Fig. 3). It would be of interest to further explore the origin of the difference in the future.
BmCa1 is likely the first member of calcium channel toxin-like genes isolated from Chinese scorpion Mesobuthus martensii Karsch. BmCa1 shares low degrees of similarity with the other scorpion toxins, including calcium channel toxins. This suggests that BmCa1 may be a novel member of the calcium channel scorpion toxin superfamily and represents a new class of calcium channel scorpion toxin subfamily. This is the first example from a scorpion of the family Buthidae that suggests the possible presence of peptides with the ICK motif, thus far known to exist only in scorpions of the family Scorpionidae. To date, over 80 toxins or peptides have been isolated and characterized from the venom of Chinese scorpion Mesobuthus martensii Karsch [8,25]. Mesobuthus martensii Karsch is the most studied scorpion species in the world. Possani et al. proposed that there are about one hundred toxins or peptides in the venom of each scorpion species. At least 100000
Fig. 3 – Mutiple sequence alignments of the first subfamily members of calcium channel scorpion toxins. BmCa1 and BmKCa (GenBank Number: DQ120790) from Mesobuthus martensii Karsch; Maurocalcine from Scorpio maurus; Imperatoxin A from Pandinus imperator; Opicalcine 1 and Opicalcine 2 from Opistophthalmus carinatus. The first amino acid glycine (G) of mature peptides is indicated with the arrow.
peptides 27 (2006) 1235–1240
bioactive peptides exist in scorpion venoms out of 1500 scorpion species in the world [17]. This makes scorpion venom a source rich in polypeptides and drugs for treating a number of diseases. Recently, Pimenta et al. speculated that there are 380 different bioactive peptides in the venom of another scorpion of the Buthidae family, Tityus serrulatus [15]. An important question is how many toxin peptides and genes a scorpion venom actually contains. Toxin peptides and genes identified from the venom of scorpion Mesobuthus martensii Karsch so far represent only a small fraction of the scorpion venom. Advances in mass spectrometry and peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous novel peptide toxins from scorpion venoms [4]. This should help to discover the large number of unidentified peptides in the scorpion venom, which in turn would accelerate the understanding of the structure-function relationship of ion channels and help to develop novel therapeutic agents and strategies for ion channel-related diseases.
4.2.
Genomic organization and structure of BmCa1
To date, over 30 genomic sequences of scorpion toxins or venom peptides have been reported [11,22]. The genomic organization of most scorpion toxin genes is completely identical, namely, containing two exons and one intron. The first exon spans the 50 untranslated region and the stretch that codes for the first two-thirds of the signal peptide, followed by an intron and the second exon encoding the last third of the signal peptide, the mature toxin, and the 30 untranslated region [22]. Recently, some new genomic structures of scorpion toxins or venom peptides were reported [2,19]. However, only two scorpion toxin genes BmCa1 and Opicalcine1 [28] have the same genomic organization and structure. Both contain two introns (one intron located in the signal peptide coding region and the other in the mature peptide coding region). Moreover, four introns of both scorpion toxin genes belonged to the phase-I and phase-II, respectively. The locations of their introns are consistent with each other. It is worth noting that four introns of BmCa1 and Opicalcine have different length. The first intron of BmCa1 is similar in length with that of the short-chain scorpion toxin genes (K+ channel and Cl channel toxins) [22], while the second intron of BmCa1 is similar in length with that of long-chain scorpion toxin genes (mainly Na+ channel toxins) [14]. On the other hand, the disulfide-bridge number of calcium channel scorpion toxins is similar to that of most K+ channel scorpion toxins (3 disulfide bridges), while the cysteine residue arrangement of the first subfamily members of calcium channel scorpion toxins is similar to that of excitatory Na+ channel scorpion toxins and Cl channel scorpion toxins (cysteine-cysteine arrangement). All these common features suggest the strong conservation among different classes of scorpion toxins.
Acknowledgements This work is supported by the grants (Number: 30530140; 30500089; 30570045) from the National Natural Sciences Foundation of China to Li W.X., Wu Y.L. and Cao Z.J., and
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the Foundation of HuBei Provincial Key Laboratory of Biotechnology of Traditional Chinese Medicine, HuBei University. We also thank Dr. Shi Yunbo and Dr. Tasleem for his help in editing the paper.
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