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Genomic organization of four novel nondisulfide-bridged peptides from scorpion Mesobuthus martensii Karsch: Gaining insight into evolutionary mechanism
Peptides 26 (2005) 2427–2433
Genomic organization of four novel nondisulfide-bridged peptides from scorpion Mesobuthus martensii Karsch: Gaining insight into evolutionary mechanism夽 Feng Luo a , Xian-Chun Zeng a , Richard Hahin b , Zhi-Jian Cao a , Hui Liu a , Wen-Xin Li a,∗ a
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China b Department of Biological Sciences, Northern Illinois University, Dekalb, IL 60115-2861, USA Received 7 April 2005; received in revised form 2 June 2005; accepted 2 June 2005 Available online 22 July 2005
Abstract At least 25 nondisulfide-bridged peptides (NDBPs) have been identified and characterized from scorpions. However, the genomic organization of the genes that encode these peptides have not been reported yet. BmKa1, BmKa2 and BmKb1 are three novel genes that code for NDBPs identified by our group from Mesobuthus martensii Karsch. Based on their cDNA sequences, the genomic DNA sequences encoding these peptides were obtained using the PCR method. Sequence analysis showed that three distinct genomic structural patterns are used to encode these three peptides. The BmKa1 gene is not interrupted by any introns. However, the BmKa2 gene is composed of two exons, interrupted by a 67 bp intron that is located in the DNA region encoding the mature peptide. Two genomic homologues of the BmKb1 cDNA sequence, named BmKb1 and BmKb2, respectively, were obtained. The BmKb1 gene contains one intron of 593 bp, inserted into the DNA region that encodes the signal peptide. Similarly, the BmKb2 gene also contains an intron that interrupts the exon that encodes the NDBP signal peptide. The amino acid sequences deduced for BmKb2 and BmKb1 differ only at one position. The data suggest that the genomic organizational pattern of NDBPs displays more divergence than that exhibited by the genes that encode disulfide-bridged peptides from scorpions. © 2005 Elsevier Inc. All rights reserved. Keywords: Mesobuthus martensii Karsch; Nondisulfide-bridged peptide (NDBP); Genomic organization; BmKa1; BmKa2; BmKb1; BmKb2
1. Introduction During more than 400 millions years, scorpions have successfully developed a large variety of bioactive peptides to capture their preys or to protect them against their biological enemies. These peptides contain 13–76 amino acid residues, and could be simply classified into two types: disulfide-bridged or nondisulfide-bridged peptides (NDBPs). Scorpion toxins with disulfide bridges specifically interact with membrane-bound ionic channels including Na+ , K+ , Cl− and Ca2+ channels [7,10,16,23,27,30]. The toxins that 夽 Gene accession numbers: BmKa2 gene, AY648240; BmKb1 gene,
AY700566 (BmKb1 gene renamed as BmKb1 gene); BmKb2 gene, AY729077. ∗ Corresponding author. Tel.: +86 27 68752831; fax: +86 27 68752146. E-mail address: [email protected] (W.-X. Li). 0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.06.008
act to bind to and alter a Na+ -channel are composed of 60–76 amino acid residues linked by four disulfide bridges, whereas toxins that specifically bind to K+ - and Cl− -channels are short-chain peptides composed of 20–41 amino acid residues and stabilized by three or four disulfide bridges. The toxins that specifically alter Ca2+ -channels possess a much more variable amino acid length [23]. Recently, Zeng et al. isolated a compact anticoagulant-like, 64 residue, scorpion peptide toxin that possessed five disulfide bridges [34]. Currently, at least 180 Na+ -channel toxins, 120 K+ -channel toxins, 17 Cl− -channel toxins and 3 Ca2+ -channel toxins have been described [3,10]. These ion channel modulators have been widely used as tools for the identification, isolation and characterization of ion channel proteins, and for the investigation of the peptides’ biochemical, pharmacological and physiological functions [13].
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The scorpion venom peptides without disulfide bridges have been poorly characterized so far. Only 25 such peptides have been isolated or functionally characterized, including two bradykinin-potentiating peptides (peptide T and K12) from Buthus occitanus and Tityus serrulatus, respectively [11,20], 10 antimicrobial peptides: Parabutoporin from Parabuthus schlechteri [28], Pandinin 1 and Pandinin 2 from Pandinus imperator [5], Hadrurin from Hadrurus aztecus [26], IsCT and IsCT2 from Opisthacanthus madagascariensis [6], Opistoporin 1 and Opistoporin 2 from Opistophtalmus carinatus [21], BmKb1 and BmKn2 from Mesobuthus martensii [35], and 13 peptides with unknown functions: Bs10 from Buthus sindicus [1], BmKbpp, BmKn1, BmKa1 and BmKa2 from M. martensii [31,32,35], Tco36.14-2 and Clones 4–10 from Tityus costatus [9]. No information about the organization of genomic DNA encoding these peptides has been previously reported. In this paper, we have described the genomic organization of the genes for four NDBPs (BmKa1, BmKa2, BmKb1 and a newly identified peptide BmKb2) from M. martensii.
2. Materials and methods
2.3. PCR amplification To produce a reaction volume of 25 l, 2 g of scorpion genomic DNA was used as a template, and 1.0 unit of T4 Taq polymerase (Promega, USA) and 10 pmol of each primer were added to the reaction mixture. PCR amplification was performed using the following set of steps and conditions: pre-denaturing at 94 ◦ C for 5 min, 35 cycles under denaturing conditions at 94 ◦ C for 1 min, annealing at 55 ◦ C for 45 s, a 72 ◦ C extension for 1 min, followed by a final extension phase of 5 min at 72 ◦ C. 2.4. Cloning and DNA sequencing PCR products were separated using electrophoresis with a 1.8% agarose gel, and purified using a Gel extraction kit (Omega, USA). The purified products were ligated into a pGEM-T Easy vector (Promega, USA) according to the manufacturer’s protocol. After transformation into E. coli DH5␣ competent cells, the positive clones were screened, and sequenced using an ABI PRISMTM 3700 DNA Automatic Sequencer. The BmKa1 and BmKa2 genes were sequenced with a T7 promoter primer. BmKb1 and BmKb2 genes were sequenced with M13+ and M13− primers.
2.1. Preparation of genomic DNA from the scorpion 3. Results High molecular mass scorpion genomic DNA was purified from a single whole scorpion of M. martensii as previously described [33]. 2.2. Oligonucleotides According to the cDNA sequence determined for peptides BmKa1, BmKa2 and BmKb1 [35], three pairs of primers were designed and synthesized to amplify their genomic sequences, respectively. For BmKa1, the forward primer used was 5 -GTAGAAATGAAACCTCGAGTA-3 , which corresponds to a part of the 5 -UTR of BmKa1 cDNA and the DNA region encoding the first five amino acid residues of the BmKa1 precursor; the reverse primer used was 5 -TAGCAATGAAACATCAATCAG-3 , which corresponds to a partial 3 -UTR and the DNA region encoding the last two residues of the BmKa1 precursor. For BmKa2, the forward primer used was 5 TCAAGTAGTCAAAATGTCTTC-3 , which corresponds to a partial 5 -UTR and the DNA region encoding the first two residues of the BmKa2 precursor; the reverse primer used was 5 -ATTCCATATCATCAAAGTCCG-3 , which corresponds to the DNA sequence encoding the last seven residues of the BmKa2 precursor. For BmKb1, the forward primer used was 5 -CGAAACTCGGCCAAGATG3 , which corresponds to the 5 -UTR and initial codon of the BmKb1 cDNA; the reverse primer used was 5 AAACGACCGGAAGAGAGAC-3 , which corresponds to a partial 3 -UTR sequence of the BmKb1 cDNA.
3.1. Genomic organization of BmKa1 and BmKa2 genes Since the genomic and cDNA sequences of BmKa1 were fully consistent, this suggests that the BmKa1 gene does not possess an intron. The BmKa2 gene was found to contain two exons interrupted by an intron of 67 bp (Fig. 1A). The intron is located in and interrupts the DNA region that encodes the mature peptide; it is inserted between the first and the second base of a codon coding for Ala at position +11 in the mature peptide. This genomic organization differs from the organization of genes that encode for most of the disulfide-bridged scorpion peptides: the genes contain an intron located in the DNA region encoding the C-terminal part of signal peptide. The intron of BmKa2 is rich in A + T (up to 77.61%). The 5 splice donor and 3 splice acceptor of the intron are 5 -G/gtttgt-3 and 5 -tttcag/C-3 , respectively. These GT/AG splice junctions are highly conserved among the previously described genes that code for scorpion venom peptides [8,14,17]. A putative branch point, 5 -ttaat-3 was found 21 bp upstream of the 3 splice site. The nucleotide sequence of the two exons of BmKa2 gene is consistent with the corresponding cDNA sequence for BmKa2 except for a nonsense point mutation in a single codon (Fig. 1A). 3.2. Genomic organization of BmKb1 and BmKb2 genes The PCR amplification product for the BmKb1 primers, when viewed on electrophoretic gels, showed two distinct
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Fig. 1. Nucleotide sequence of cDNAs and the genomic genes encoding the precursor of NDBPs: BmKa2 (A), BmKb1 (B) and BmKb2 (C). The deduced amino acid sequence is shown below the nucleotide sequence. The signal peptide of NDBP precursor is underlined once. The posttranslational processing signal: Gly–Arg–Arg is boxed and printed in boldface. The propeptide of the precursor is underlined boldly once. Primers for the genomic DNA amplification are double-underlined. Exons are written in capital letters and intron sequence is written in lower-case letters. Compared with their corresponding cDNA sequences, the nucleotides mutated are boxed. A putative branch point, 5 -taat-3 or 5 -tcgat-3 , is in italic and bold. Numbers at the right margin indicate position of nucleotides or amino acids in the sequence.
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Fig. 2. (A) Nucleotide sequence alignment of BmKb1 cDNA, BmKb1 gene and BmKb2 gene. The nucleotide sequences of introns and exons are shown in the lower-case and upper-case letters, respectively. (B) Alignment of the precursors of BmKb1, BmKb1 and BmKb2.
similarly sized bands. The DNA in both bands was cloned and sequenced. The resultant DNA obtained represent two apparently different homologues of the BmKb1 gene, named BmKb1 and BmKb2, respectively. The BmKb1 genomic sequence contains an intron of 593 bp (Fig. 1B), located in the DNA region encoding N-terminal part of signal peptide; the intron interrupts the first and the second bases of the codon coding for Val at position −7. The BmKb2 gene also contains an intron located in the same position as the BmKb1
gene (Fig. 1C), but its size, 630 bp, is a little longer than that of BmKb1 gene. As shown in Fig. 2A, the sequences of BmKb1 and BmKb2 genes are consistently similar with the exception that the BmKb2 intron contains two additional short fragments inserted into the sequence at two separate positions. The amino acid sequences encoded by BmKb1 and BmKb2 only differ at one position. The introns of both BmKb1 and BmKb2 begin with GT and end with AG. The A + T content in BmKb1 and BmKb2 are 65.26 and 65.87%,
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respectively. Both genes 5 splice donors are 5 -G/gtgagt-3 , whereas their 3 splice acceptors are 5 -tttcag/T-3 . A putative branch point, 5 -tcgat-3 , is found 89 bp upstream from the 3 splice site for both of the genes (Fig. 1B and C). These structural features are similar to those previously described for other scorpion toxin genes [14,17,22,29]. A sequence comparison of the cDNA of BmKb1 with the exons of BmKb1 gene revealed three nucleotide substitutions at positions 764 (T → C), 766 (T → A) and 854 (C → G). The first two of these three substitutions led to the mutation of Tyr → Gln in the propeptide; the third substitution (854: C → G) appeared in the 3 -UTR (Figs. 1B and 2B). The corresponding sequence of the exons of the BmKb2 gene and BmKb1 cDNA differ at only three positions: 727, 801 and 803. The substitution at position 727 (BmKb1:T → BmKb2:C) led to the mutation of BmKb1:Phe → BmKb2:Ser in the mature peptide region (Figs. 1C and 2B).
4. Discussion Unexpected variability in the genomic architecture of genes encoding BmKb1 , BmKb2, BmKa1 and BmKa2 peptides was observed. Three architectural organizational types were found (Fig. 3): (a) a peptide gene without any intervening intron in its genomic sequence, e.g. BmKa1; (b) a gene with an intron that interrupts the DNA that encodes the mature peptide, e.g. BmKa2; (c) a gene with an intron that interrupts the DNA that encodes the signal peptide, e.g. BmKb1 and BmKb2. Thus, it appears that the genomic organization of the genes that produce NDBPs shows much greater diversity than observed for the corresponding genes that code for the disulfide-bridged toxins from scorpions. Our data gained insight into the evolutionary mechanism of NDBPs from scorpions. Three evolutionary models might explain the high divergence and intron polymorphism of the NDBPs. First, the “gene duplication and subsequent divergence” model explains much of the documented data observed for genes that code for disulfide-bridged peptides from scorpions [2,19,22,24,29,37,38], and also appears to
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explain the evolution of NDBP genes. The presence of two homologues of the BmKb1 gene (BmKb1 and BmKb2) in the scorpion genome suggests that the BmKb1 gene diverged after a duplication event by subsequent mutations. This inference was also confirmed by the work conducted by Passani’s group [9]. As shown in Fig. 4, this group identified four homologues of BmKa2, and three homologues of BmKb1 and BmKb2 from Brazilian scorpion T. costatus Karsch. The alignments of these peptides suggest that, just like the disulfide-bridged scorpion peptides, NDBPs also demonstrate evolutionary conserved region, and variable region which could accommodate replacements, deletions and mutations that confer on them diverse functional specificities or new functions [15,16,25]. Another possible, but less likely, explanation, exonshuffling, may explain the diversity of NDBP genes. The primary and secondary structure of BmKa1 and BmKa2 are quite similar and may well represent a conserved protein domain, and may have evolved from a common ancestor through intronic recombination events. However, the signature left behind by these domain shuffling events is the presence of domains flanked by in-phase introns [18], and they have not been observed in our data. These variations suggests the exon encoding the mature peptide might, but less likely, undergo exon-shuffling during evolution. Unlike other exon-shuffling events, by which two or more exons from different genes can be brought together ectopically [12], the exon-shuffling of NDBPs might occurr among autonomous modules. More likely, the variability of the NDBPs’ genomic organization could be explained by the “intron gain or loss” model [36]. This model suggests the naturally occurring exon-intron (donor) and intron-exon (acceptor) junction motif AG/GT is a preferred context for intron gain or loss, and the intron evolution is largely dominated by intron gain. Coghlan and Wolfe [4] demonstrated that 122 introns have been gained in Caenorhabditis genes, They also provide solid evidence that 28 of them are actually derived from “donor” introns present in the same genome, and a few of these new introns apparently derive from other introns in the same gene. Although its mechanism remains to be found out, the intron gain seems
Fig. 3. Genomic organizations of NDBP genes from scorpions. BmKa1, no intron; BmKa2, an intron in the DNA region encoding MP; BmKb1 /BmKb2, an intron in the DNA region encoding SP.
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Fig. 4. (A) Primary sequence alignment of the NDBPs of BmKb1, BmKb2 and Clones 4–6; (B) Alignment of the NDBPs of BmKa2 and Clones 7–10. The black and gray boxes represent conserved or homologous residues, respectively.
to be a common event in the eukaryotic evolution. We first found this event also frequently occurred in the evolution of NDBPs. Intron variability combined with alternative splicing of the introns might contribute significantly to the diversity of NDBPs from scorpions.
[10] [11]
[12]
Acknowledgements This work was supported by the Key Project of China Ministry of Science and Technology (2002BA49C), and the National Natural Science Foundation of China (30370349).
[13]
[14]
[15]
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Genomic organization of four novel nondisulfide-bridged peptides from scorpion Mesobuthus martensii Karsch: Gaining insight into evolutionary mechanism夽 Feng Luo a , Xian-Chun Zeng a , Richard Hahin b , Zhi-Jian Cao a , Hui Liu a , Wen-Xin Li a,∗ a
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China b Department of Biological Sciences, Northern Illinois University, Dekalb, IL 60115-2861, USA Received 7 April 2005; received in revised form 2 June 2005; accepted 2 June 2005 Available online 22 July 2005
Abstract At least 25 nondisulfide-bridged peptides (NDBPs) have been identified and characterized from scorpions. However, the genomic organization of the genes that encode these peptides have not been reported yet. BmKa1, BmKa2 and BmKb1 are three novel genes that code for NDBPs identified by our group from Mesobuthus martensii Karsch. Based on their cDNA sequences, the genomic DNA sequences encoding these peptides were obtained using the PCR method. Sequence analysis showed that three distinct genomic structural patterns are used to encode these three peptides. The BmKa1 gene is not interrupted by any introns. However, the BmKa2 gene is composed of two exons, interrupted by a 67 bp intron that is located in the DNA region encoding the mature peptide. Two genomic homologues of the BmKb1 cDNA sequence, named BmKb1 and BmKb2, respectively, were obtained. The BmKb1 gene contains one intron of 593 bp, inserted into the DNA region that encodes the signal peptide. Similarly, the BmKb2 gene also contains an intron that interrupts the exon that encodes the NDBP signal peptide. The amino acid sequences deduced for BmKb2 and BmKb1 differ only at one position. The data suggest that the genomic organizational pattern of NDBPs displays more divergence than that exhibited by the genes that encode disulfide-bridged peptides from scorpions. © 2005 Elsevier Inc. All rights reserved. Keywords: Mesobuthus martensii Karsch; Nondisulfide-bridged peptide (NDBP); Genomic organization; BmKa1; BmKa2; BmKb1; BmKb2
1. Introduction During more than 400 millions years, scorpions have successfully developed a large variety of bioactive peptides to capture their preys or to protect them against their biological enemies. These peptides contain 13–76 amino acid residues, and could be simply classified into two types: disulfide-bridged or nondisulfide-bridged peptides (NDBPs). Scorpion toxins with disulfide bridges specifically interact with membrane-bound ionic channels including Na+ , K+ , Cl− and Ca2+ channels [7,10,16,23,27,30]. The toxins that 夽 Gene accession numbers: BmKa2 gene, AY648240; BmKb1 gene,
AY700566 (BmKb1 gene renamed as BmKb1 gene); BmKb2 gene, AY729077. ∗ Corresponding author. Tel.: +86 27 68752831; fax: +86 27 68752146. E-mail address: [email protected] (W.-X. Li). 0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.06.008
act to bind to and alter a Na+ -channel are composed of 60–76 amino acid residues linked by four disulfide bridges, whereas toxins that specifically bind to K+ - and Cl− -channels are short-chain peptides composed of 20–41 amino acid residues and stabilized by three or four disulfide bridges. The toxins that specifically alter Ca2+ -channels possess a much more variable amino acid length [23]. Recently, Zeng et al. isolated a compact anticoagulant-like, 64 residue, scorpion peptide toxin that possessed five disulfide bridges [34]. Currently, at least 180 Na+ -channel toxins, 120 K+ -channel toxins, 17 Cl− -channel toxins and 3 Ca2+ -channel toxins have been described [3,10]. These ion channel modulators have been widely used as tools for the identification, isolation and characterization of ion channel proteins, and for the investigation of the peptides’ biochemical, pharmacological and physiological functions [13].
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The scorpion venom peptides without disulfide bridges have been poorly characterized so far. Only 25 such peptides have been isolated or functionally characterized, including two bradykinin-potentiating peptides (peptide T and K12) from Buthus occitanus and Tityus serrulatus, respectively [11,20], 10 antimicrobial peptides: Parabutoporin from Parabuthus schlechteri [28], Pandinin 1 and Pandinin 2 from Pandinus imperator [5], Hadrurin from Hadrurus aztecus [26], IsCT and IsCT2 from Opisthacanthus madagascariensis [6], Opistoporin 1 and Opistoporin 2 from Opistophtalmus carinatus [21], BmKb1 and BmKn2 from Mesobuthus martensii [35], and 13 peptides with unknown functions: Bs10 from Buthus sindicus [1], BmKbpp, BmKn1, BmKa1 and BmKa2 from M. martensii [31,32,35], Tco36.14-2 and Clones 4–10 from Tityus costatus [9]. No information about the organization of genomic DNA encoding these peptides has been previously reported. In this paper, we have described the genomic organization of the genes for four NDBPs (BmKa1, BmKa2, BmKb1 and a newly identified peptide BmKb2) from M. martensii.
2. Materials and methods
2.3. PCR amplification To produce a reaction volume of 25 l, 2 g of scorpion genomic DNA was used as a template, and 1.0 unit of T4 Taq polymerase (Promega, USA) and 10 pmol of each primer were added to the reaction mixture. PCR amplification was performed using the following set of steps and conditions: pre-denaturing at 94 ◦ C for 5 min, 35 cycles under denaturing conditions at 94 ◦ C for 1 min, annealing at 55 ◦ C for 45 s, a 72 ◦ C extension for 1 min, followed by a final extension phase of 5 min at 72 ◦ C. 2.4. Cloning and DNA sequencing PCR products were separated using electrophoresis with a 1.8% agarose gel, and purified using a Gel extraction kit (Omega, USA). The purified products were ligated into a pGEM-T Easy vector (Promega, USA) according to the manufacturer’s protocol. After transformation into E. coli DH5␣ competent cells, the positive clones were screened, and sequenced using an ABI PRISMTM 3700 DNA Automatic Sequencer. The BmKa1 and BmKa2 genes were sequenced with a T7 promoter primer. BmKb1 and BmKb2 genes were sequenced with M13+ and M13− primers.
2.1. Preparation of genomic DNA from the scorpion 3. Results High molecular mass scorpion genomic DNA was purified from a single whole scorpion of M. martensii as previously described [33]. 2.2. Oligonucleotides According to the cDNA sequence determined for peptides BmKa1, BmKa2 and BmKb1 [35], three pairs of primers were designed and synthesized to amplify their genomic sequences, respectively. For BmKa1, the forward primer used was 5 -GTAGAAATGAAACCTCGAGTA-3 , which corresponds to a part of the 5 -UTR of BmKa1 cDNA and the DNA region encoding the first five amino acid residues of the BmKa1 precursor; the reverse primer used was 5 -TAGCAATGAAACATCAATCAG-3 , which corresponds to a partial 3 -UTR and the DNA region encoding the last two residues of the BmKa1 precursor. For BmKa2, the forward primer used was 5 TCAAGTAGTCAAAATGTCTTC-3 , which corresponds to a partial 5 -UTR and the DNA region encoding the first two residues of the BmKa2 precursor; the reverse primer used was 5 -ATTCCATATCATCAAAGTCCG-3 , which corresponds to the DNA sequence encoding the last seven residues of the BmKa2 precursor. For BmKb1, the forward primer used was 5 -CGAAACTCGGCCAAGATG3 , which corresponds to the 5 -UTR and initial codon of the BmKb1 cDNA; the reverse primer used was 5 AAACGACCGGAAGAGAGAC-3 , which corresponds to a partial 3 -UTR sequence of the BmKb1 cDNA.
3.1. Genomic organization of BmKa1 and BmKa2 genes Since the genomic and cDNA sequences of BmKa1 were fully consistent, this suggests that the BmKa1 gene does not possess an intron. The BmKa2 gene was found to contain two exons interrupted by an intron of 67 bp (Fig. 1A). The intron is located in and interrupts the DNA region that encodes the mature peptide; it is inserted between the first and the second base of a codon coding for Ala at position +11 in the mature peptide. This genomic organization differs from the organization of genes that encode for most of the disulfide-bridged scorpion peptides: the genes contain an intron located in the DNA region encoding the C-terminal part of signal peptide. The intron of BmKa2 is rich in A + T (up to 77.61%). The 5 splice donor and 3 splice acceptor of the intron are 5 -G/gtttgt-3 and 5 -tttcag/C-3 , respectively. These GT/AG splice junctions are highly conserved among the previously described genes that code for scorpion venom peptides [8,14,17]. A putative branch point, 5 -ttaat-3 was found 21 bp upstream of the 3 splice site. The nucleotide sequence of the two exons of BmKa2 gene is consistent with the corresponding cDNA sequence for BmKa2 except for a nonsense point mutation in a single codon (Fig. 1A). 3.2. Genomic organization of BmKb1 and BmKb2 genes The PCR amplification product for the BmKb1 primers, when viewed on electrophoretic gels, showed two distinct
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Fig. 1. Nucleotide sequence of cDNAs and the genomic genes encoding the precursor of NDBPs: BmKa2 (A), BmKb1 (B) and BmKb2 (C). The deduced amino acid sequence is shown below the nucleotide sequence. The signal peptide of NDBP precursor is underlined once. The posttranslational processing signal: Gly–Arg–Arg is boxed and printed in boldface. The propeptide of the precursor is underlined boldly once. Primers for the genomic DNA amplification are double-underlined. Exons are written in capital letters and intron sequence is written in lower-case letters. Compared with their corresponding cDNA sequences, the nucleotides mutated are boxed. A putative branch point, 5 -taat-3 or 5 -tcgat-3 , is in italic and bold. Numbers at the right margin indicate position of nucleotides or amino acids in the sequence.
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Fig. 2. (A) Nucleotide sequence alignment of BmKb1 cDNA, BmKb1 gene and BmKb2 gene. The nucleotide sequences of introns and exons are shown in the lower-case and upper-case letters, respectively. (B) Alignment of the precursors of BmKb1, BmKb1 and BmKb2.
similarly sized bands. The DNA in both bands was cloned and sequenced. The resultant DNA obtained represent two apparently different homologues of the BmKb1 gene, named BmKb1 and BmKb2, respectively. The BmKb1 genomic sequence contains an intron of 593 bp (Fig. 1B), located in the DNA region encoding N-terminal part of signal peptide; the intron interrupts the first and the second bases of the codon coding for Val at position −7. The BmKb2 gene also contains an intron located in the same position as the BmKb1
gene (Fig. 1C), but its size, 630 bp, is a little longer than that of BmKb1 gene. As shown in Fig. 2A, the sequences of BmKb1 and BmKb2 genes are consistently similar with the exception that the BmKb2 intron contains two additional short fragments inserted into the sequence at two separate positions. The amino acid sequences encoded by BmKb1 and BmKb2 only differ at one position. The introns of both BmKb1 and BmKb2 begin with GT and end with AG. The A + T content in BmKb1 and BmKb2 are 65.26 and 65.87%,
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respectively. Both genes 5 splice donors are 5 -G/gtgagt-3 , whereas their 3 splice acceptors are 5 -tttcag/T-3 . A putative branch point, 5 -tcgat-3 , is found 89 bp upstream from the 3 splice site for both of the genes (Fig. 1B and C). These structural features are similar to those previously described for other scorpion toxin genes [14,17,22,29]. A sequence comparison of the cDNA of BmKb1 with the exons of BmKb1 gene revealed three nucleotide substitutions at positions 764 (T → C), 766 (T → A) and 854 (C → G). The first two of these three substitutions led to the mutation of Tyr → Gln in the propeptide; the third substitution (854: C → G) appeared in the 3 -UTR (Figs. 1B and 2B). The corresponding sequence of the exons of the BmKb2 gene and BmKb1 cDNA differ at only three positions: 727, 801 and 803. The substitution at position 727 (BmKb1:T → BmKb2:C) led to the mutation of BmKb1:Phe → BmKb2:Ser in the mature peptide region (Figs. 1C and 2B).
4. Discussion Unexpected variability in the genomic architecture of genes encoding BmKb1 , BmKb2, BmKa1 and BmKa2 peptides was observed. Three architectural organizational types were found (Fig. 3): (a) a peptide gene without any intervening intron in its genomic sequence, e.g. BmKa1; (b) a gene with an intron that interrupts the DNA that encodes the mature peptide, e.g. BmKa2; (c) a gene with an intron that interrupts the DNA that encodes the signal peptide, e.g. BmKb1 and BmKb2. Thus, it appears that the genomic organization of the genes that produce NDBPs shows much greater diversity than observed for the corresponding genes that code for the disulfide-bridged toxins from scorpions. Our data gained insight into the evolutionary mechanism of NDBPs from scorpions. Three evolutionary models might explain the high divergence and intron polymorphism of the NDBPs. First, the “gene duplication and subsequent divergence” model explains much of the documented data observed for genes that code for disulfide-bridged peptides from scorpions [2,19,22,24,29,37,38], and also appears to
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explain the evolution of NDBP genes. The presence of two homologues of the BmKb1 gene (BmKb1 and BmKb2) in the scorpion genome suggests that the BmKb1 gene diverged after a duplication event by subsequent mutations. This inference was also confirmed by the work conducted by Passani’s group [9]. As shown in Fig. 4, this group identified four homologues of BmKa2, and three homologues of BmKb1 and BmKb2 from Brazilian scorpion T. costatus Karsch. The alignments of these peptides suggest that, just like the disulfide-bridged scorpion peptides, NDBPs also demonstrate evolutionary conserved region, and variable region which could accommodate replacements, deletions and mutations that confer on them diverse functional specificities or new functions [15,16,25]. Another possible, but less likely, explanation, exonshuffling, may explain the diversity of NDBP genes. The primary and secondary structure of BmKa1 and BmKa2 are quite similar and may well represent a conserved protein domain, and may have evolved from a common ancestor through intronic recombination events. However, the signature left behind by these domain shuffling events is the presence of domains flanked by in-phase introns [18], and they have not been observed in our data. These variations suggests the exon encoding the mature peptide might, but less likely, undergo exon-shuffling during evolution. Unlike other exon-shuffling events, by which two or more exons from different genes can be brought together ectopically [12], the exon-shuffling of NDBPs might occurr among autonomous modules. More likely, the variability of the NDBPs’ genomic organization could be explained by the “intron gain or loss” model [36]. This model suggests the naturally occurring exon-intron (donor) and intron-exon (acceptor) junction motif AG/GT is a preferred context for intron gain or loss, and the intron evolution is largely dominated by intron gain. Coghlan and Wolfe [4] demonstrated that 122 introns have been gained in Caenorhabditis genes, They also provide solid evidence that 28 of them are actually derived from “donor” introns present in the same genome, and a few of these new introns apparently derive from other introns in the same gene. Although its mechanism remains to be found out, the intron gain seems
Fig. 3. Genomic organizations of NDBP genes from scorpions. BmKa1, no intron; BmKa2, an intron in the DNA region encoding MP; BmKb1 /BmKb2, an intron in the DNA region encoding SP.
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Fig. 4. (A) Primary sequence alignment of the NDBPs of BmKb1, BmKb2 and Clones 4–6; (B) Alignment of the NDBPs of BmKa2 and Clones 7–10. The black and gray boxes represent conserved or homologous residues, respectively.
to be a common event in the eukaryotic evolution. We first found this event also frequently occurred in the evolution of NDBPs. Intron variability combined with alternative splicing of the introns might contribute significantly to the diversity of NDBPs from scorpions.
[10] [11]
[12]
Acknowledgements This work was supported by the Key Project of China Ministry of Science and Technology (2002BA49C), and the National Natural Science Foundation of China (30370349).
[13]
[14]
[15]
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