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Molecular cloning of cDNAs encoding insecticidal neurotoxic peptides from the spider Phoneutria nigriventer
Toxicon 38 (2000) 1443±1449 www.elsevier.com/locate/toxicon
Molecular cloning of cDNAs encoding insecticidal neurotoxic peptides from the spider Phoneutria nigriventer C.L. Penaforte a,b, V.F. Prado b, M.A.M. Prado a, M.A. Romano-Silva a, P.E.M. GuimaraÄes a, L. De Marco a, M.V. Gomez a, E. Kalapothakis a,* a
DivisaÄo de Biologia Celular, Departamento de Farmacologia, Instituto de CieÃncias BioloÂgicas, Universidade Federal de Minas Gerais, Av. AntoÃnio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil b Departamento de BioquõÂmica-Imunologia, Instituto de CieÃncias BioloÂgicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil Received 5 August 1999; accepted 21 October 1999
Abstract From a Phoneutria nigriventer venom gland cDNA library several clones coding for the insect speci®c neurotoxin Tx4(6±1) were isolated. cDNA analysis showed that the encoded protein contained three distinct segments, comprising a signal sequence of 16 amino acids, followed by a glutamate-rich sequence of 18 amino acids and, ®nally, the coding region for the mature toxin. The deduced amino acid sequence for the mature polypeptide was identical to the protein sequence determined chemically. In addition, two new putative toxins called Pn4A and Pn4B were characterized and their predicted complete amino acid sequence revealed approximately 78% similarity to Tx4(6±1). # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Phoneutria nigriventer; Insecticidal neurotoxin; Tx4(6±1); Pn4A; Pn4B; cDNA
* Corresponding author. Tel.: +55-31-4992695; fax: +55-31-4992713. E-mail address: [email protected] (E. Kalapothakis). 0041-0101/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 2 3 7 - 8
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1. Introduction The South American solitary `armed' spider Phoneutria nigriventer is probably the most aggressive species found in Brazil (Lucas, 1988). Its venom contains numerous neurotoxic polypeptides which have been used as probes in the ®eld of pharmacology and biochemistry (Prado et al., 1996; Guatimosim et al., 1997). These neurotoxins are basic polypeptides of low molecular weight which bind with nanomolar anity and high speci®city to ion channels, subsequently altering their behavior (Cordeiro et al., 1995). The speci®city of spider toxins are also very valuable to the agriculture ®eld and considerable eort has been carried out in the use of natural toxins against pests (Stewart et al., 1991). Figueiredo et al. (1995) reported the isolation of Tx4(6±1), a neurotoxic component from the fraction PhTx4, which appears to be insect-speci®c. The complete amino acid sequence of Tx4(6±1) indicates that it is a single-chain polypeptide of 48 amino acids with a molecular weight of 5251. Injection of Tx4(6±1) induced variable symptoms depending on the species and dose injected. It is highly active against house ¯ies, producing neurotoxic eects at a dose as low as 0.02 ng/mg. Additionally, Tx4(6±1) was also active against cockroaches, at doses in the range of 0.5±2.5 ng/mg. The insects are knocked down immediately, are unable to reinvert and exhibit tremors with uncoordinated movements. To further characterize insecticidal neurotoxic peptides from the spider P. nigriventer, we decided to clone the cDNA coding for Tx4(6±1) and search for new isoforms. Standard recombinant DNA techniques (DNA puri®cation, phenol extraction, ethanol precipitation, electrophoresis, etc.) were carried out as described by Sambrook et al. (1989). A P. nigriventer venom gland cDNA library was constructed using 5 mg of mRNA using the cDNA synthesis Kit (Zap-cDNA synthesis Kit) and Gigapack II Packaging Extract (Stratagene). Using the cDNA library and the methods described by Kalapothakis et al. (1998a) the cDNA encoding for Tx4(6±1) was obtained. The deduced amino acid sequence obtained by cDNA sequencing was identical to the protein sequence described for Tx4(6±1) (Figueiredo et al., 1995) (Fig. 2). The nucleotide sequence displayed an open reading frame of 249 bp encoding a precursor polypeptide of 82 amino acids and a large 3 ' untranslated region composed of 127 bp. Clones were sequenced on both strands using the chain termination method of Sanger et al. (1977) with an Automated ALF DNA Sequencer (Pharmacia). Nucleic acid sequences were analyzed using the basic local alignment search tool program for amino acids (BLASTX) comparisons (Altschul et al., 1990) and PC/GENE (nucleic acid and protein sequence analysis software system). As depicted in Fig. 1, the sequence of nucleotides 1±48 code for a signal peptide composed of 16 amino acids residues. It has all the characteristics of a typical signal peptide (Perlman and Halvorson, 1983) including a consensus cleavage point for a signal peptidase. The signal peptide is followed by an intervening propeptide with 18 amino acids (nucleotides 49±102) rich in Glu residues and ending with an Arg residue. The region corresponding to nucleotides 103 to 246 encode the mature polypeptide with 48 amino acids residues. These sequence,
Fig. 1. Comparison of the nucleotide sequence of cDNAs encoding the insecticidal toxin Tx4(6±1) and its isoforms Pn4A and Pn4B. A point (.) indicates that the nucleotide is identical to the one above, where no identity occurs, the corresponding change is indicated. Gaps (-) were introduced in the nucleotide sequence to maximize homology. The coding region is shown in upper case, with the 5' and 3' untranslated region in lower case. The predicted amino acid sequence for the Tx4(6±1), Pn4A and Pn4B toxins are shown in boldface, with the signal sequence underlined. The Glu-rich region (propeptide) is shown in square brackets. Initiation (ATG) codons, stop codon (TAA) and polyadenylation signal (AATAAA) are designated by asterisks.
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which ends with a TAA termination codon, is followed by a 3 ' untranslated region including a putative AATAAA polyadenylation sequence between the termination codon and the ®nal poly(A) tail. The search for isoforms of P. nigriventer insecticidal peptides was carried out using the isolated Tx4(6±1) cDNA to screen the original library by plaque hybridization (20,000 plaques) applying low stringency conditions (Sambrook et al., 1989). DNA was labeled by the random primer method (Feinberg and Vogelstein, 1984) using 25 ng of DNA. The sequence of the probe used for the screening (Tx4(6±1) cDNA) is shown in Fig. 1. Thirty clones were isolated, sequenced and the results showed 15 clones coding for Tx4(6±1), 14 coding for an isoform termed Pn4A and only one coding for an isoform termed Pn4B. These cDNAs with 370 bp (Pn4A) and 372 bp (Pn4B) encode precursors of 47 and 51 amino acids, respectively. The nucleotide sequence coding for the signal peptide of the three clones described showed 100% similarity. Dierences and similarities at the nucleotide and amino acid sequences are shown in Figs. 1 and 2, respectively. Total RNA from P. nigriventer venom gland was examined by Northern blot analysis using cDNAs isolated from clones coding for Tx4(6±1), Pn4A and Pn4B. A band of approximately 400 bp was observed in each case, suggesting that we isolated full length cDNAs and high levels of mRNA for each isolated toxin (data not shown). Preparation of total RNA was performed as described by Chomczynski and Sacchi (1987). Electrophoresis of RNA (10 mg) was carried out after denaturation with formaldehyde as described by Sambrook et al. (1989).
Fig. 2. Structural analysis. Gaps (dashes) were introduced to maximize sequence similarities. (A) The hydrophobic region (signal peptide), the hydrophilic region (propeptide) of Tx4(6±1), Pn4A and Pn4B are compared with Pn2-5A, Tx2-1 and Pn2-1A (Kalapothakis et al., 1998a). The residues of Pn2-1A located in the propeptide between Glu-25 and Asp-53 (SRPNAMERSANDWIPTAPSAVERSADF) are indicated by an asterisk and were omitted to improve similarities. (B) Comparison of amino acid sequences of Tx4(6±1), Pn4A, Pn4B with Pn2-5A, Tx2-1, Pn2-1A and Tx2-6, Tx2-5 (Cordeiro et al., 1995). The previously determined amino acid sequence of Tx4(6±1) is underlined.
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In this work we report the nucleotide sequences of three cDNAs encoding neurotoxins and the processing they require to generate mature toxins. We identi®ed and analyzed by direct sequencing the insecticidal toxin Tx4(6±1) and two putative neurotoxins termed Pn4A and Pn4B. The reported structure of the cDNAs have a similar overall organization (Fig. 1) showing the presence of an open reading frames encoding signal peptides with several hydrophobic residues (Von Heijne, 1985) followed by a hydrophilic region (Fig. 2), highly enriched in Glu residues with an Arg residue at the C-terminus preceding the mature toxin. The presence of such Glu-rich sequence with Arg at the C-terminus has been described in other small spiders peptide precursors, including the o-Agatoxin IA from Agelenopsis aperta (Santos et al., 1992) and several toxins from P. nigriventer venom gland (Diniz et al., 1993; Kalapothakis et al., 1998a,b). In comparison to Tx4(6±1) and Pn4A, the clone Pn4B showed four extra amino acids at the Cterminal before the stop codon (Figs. 1 and 2). Our cDNA sequencing data, together with biochemical evidence, indicate that the processing of precursors of Tx4(6±1) and Pn4A to mature toxin seems to require at least two dierent proteolytic activities: one for excision of the signal peptide between Ala-16 and Ser-17 (Fig. 2) and another speci®c for Arg-34 (a supposed cleavage point between the Glu-rich segment propeptide and the major polypeptide chain). However, maturation of Pn4B may require the action of a third proteolytic enzyme involved in the removal of four amino acids residues (QNKI) at the C-terminal, which is a common step in the conversion of the precursor of neurotoxin into mature polypeptide (Diniz et al., 1993; Martin-Eauclaire et al., 1994; Becerril et al., 1996; Leisy et al., 1996; Kalapothakis et al., 1998a,b). The number and distribution of Cys residues is well conserved (100%) in these three isoforms. However, small dierences exist between the analyzed toxins, especially at the COOH-terminus and may imply dierent action of these toxins. Analysis of the alignment of these sequences with several insect toxins did not show any signi®cant level of identity; however, a similar distribution of Cys residues has been found (data not shown). In contrast, a high level of identity has been found with toxins puri®ed from the venom of the same spider (Fig. 2). Cloning constitutes an important step in characterizing new classes of insect toxins. A systematic molecular analysis of spider toxin genes can provide important information to elucidate the molecular and evolutionary mechanisms responsible to generate the great diversity of P. nigriventer neurotoxins. The expression and use of recombinant toxin may de®ne the functional signi®cance of the amino acid changes found in this work.
Acknowledgements This research was supported by FINEP, PADCT, FAPEMIG, PRONEX, PRPq-UFMG and CNPq.
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References Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403±410. Becerril, B., Corona, M., Coronas, F.I.V., Zamudio, F., Calderon-Aranda, E.S., Fletcher Jr., P.L., Martin, B.M., Possani, L.D., 1996. Toxic peptides and genes encoding toxin g of the Brazilian scorpions Tityus bahiensis and Tityus stigmurus. Biochem. J. 313, 753±760. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate±phenol±cholorofom extration. Anal. Biochem. 162, 156±159. Cordeiro, M.N., Richardson, M., Gilroy, J., Figueiredo, S.G., BeiraÄo, P.S.L., Diniz, C.R., 1995. Properties of the venom from the south American ``armed'' spider Phoneutria nigriventer (Keyserling, 1891). J. Toxicol.-Toxin Rev. 14, 309±326. Diniz, M.R.V., Paine, M.J.L., Diniz, C.R., Theakston, R.G., Crampton, J.M., 1993. Sequence of the cDNA encoding for the lethal neurotoxin Tx1 from the brazilian `armed' spider Phoneutria nigriventer predict the synthesis and processing of a preprotoxin. J. Biol. Chem. 268, 15340±15342. Feinberg, A.P., Vogelstein, B., 1984. A technique for radiolabeling DNA restriction endonuclease fragments to high speci®c activity. Anal. Biochem. 137, 266±267. Figueiredo, S.G., Garcia, M.E., Valentim, A.C., Cordeiro, M.N., Diniz, C.R., Richardson, M., 1995. Puri®cation and amino acid sequence of the insecticidal neurotoxin TX4(6±1) from the venom of the `armed' spider Phoneutria nigriventer (Keys). Toxicon 33, 83±93. Guatimosim, C., Romano-Silva, M.A., Cruz, J.S., BeiraÄo, P.S.L., Kalapothakis, E., Moraes-Santos, T., Cordeiro, M.N., Diniz, C.R., Gomez, M.V., Prado, M.A.M., 1997. A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis. Br. J. Pharmac. 122, 591± 597. Kalapothakis, E., Penaforte, C.L., BeiraÄo, P.S.L., Romano-Silva, M.A., Cruz, J.S., Prado, M.A.M., GuimaraÄes, P.E.M., Gomez, M.V., Prado, V.F., 1998a. Cloning of cDNAs encoding neurotoxic peptides from the spider Phoneutria nigriventer. Toxicon 36, 1843±1850. Kalapothakis, E., Penaforte, C.L., LeaÄo, R.M., Cruz, J.S., Prado, V.F., Cordeiro, M.N., Diniz, C.R., Romano-Silva, M.R., Prado, M.A.M., Gomez, M.V., BeiraÄo, P.S.L., 1998b. Cloning cDNA, sequence analysis and patch clamp studies of a toxin from the venom of the armed spider Phoneutria nigriventer. Toxicon 36, 1971±1980. Leisy, D.J., Mattson, J.D., Quistad, G.B., Kramer, S.J., Beek, N.V., Tsai, L.W., Enderlin, F.E., Woodworth, A.R., Digan, M.E., 1996. Molecular cloning and sequencing of cDNAs encoding insecticidal peptides from the primitive hunting spider Plectreyrys tristis (Simon). Insect Biochem. Mol. Biol. 26, 411±417. Lucas, S., 1988. Spiders in Brazil. Toxicon 26, 759±772. Martin-Eauclaire, M.F., CeÂard, B., Ribeiro, A.M., Diniz, C.R., Rochat, H., Bougis, P.E., 1994. Biochemical, pharmacological and genomic characterization of TsIV, an alpha-toxin from the venom of the south American scorpion Tityus serrulatus. FEBS Lett. 342, 181±184. Perlman, D., Halvorson, H.O., 1983. A putative signal petidase site and sequence in eukaryotic and procaryotic signal peptides. J. Mol. Biol. 167, 391±409. Prado, M.A., Guatimosim, C., Gomez, M.V., Diniz, C.R., Cordeiro, M.N., Romano-Silva, M.A., 1996. A novel tool for the investigation of glutamate release from rat cerebrocortical synaptosomes: the toxin TX3-3 from the venom of the spider Phoneutria nigriventer. Biochem. J. 314, 145±150. Sambrook J., Fritsh, E.F., Maniats, T., 1989. Molecular Cloning: A Laboratory Manual, 3 eds., vol 1± 3. Press, Cold Spring Harbor, NY. Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain termination inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463±5467. Santos, A.D., Imperial, J.S., Chaudhary, T., Beavis, R.C., Chait, B.T., Hunsperger, J.P., Oliveira, B.M., Adams, M.E., Hillyard, D.R., 1992. Heterodimeric structure of the spider toxin omegaagatoxina IA revealed by precursor analysis and mass spectrometry. J. Biol. Chem. 267, 20701± 20705. Stewart, L.M., Hirst, M., Ferber, M.L., Merryweather, A.T., Cayley, P.J., Possee, R.D., 1991.
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Construction of an improved baculovirus insecticide containing an insect-speci®c toxin gene. Nature 352, 85±88. Von Heijne, G., 1985. Signal sequences; the limits of variation. J. Mol. Biol. 184, 99±105.
Molecular cloning of cDNAs encoding insecticidal neurotoxic peptides from the spider Phoneutria nigriventer C.L. Penaforte a,b, V.F. Prado b, M.A.M. Prado a, M.A. Romano-Silva a, P.E.M. GuimaraÄes a, L. De Marco a, M.V. Gomez a, E. Kalapothakis a,* a
DivisaÄo de Biologia Celular, Departamento de Farmacologia, Instituto de CieÃncias BioloÂgicas, Universidade Federal de Minas Gerais, Av. AntoÃnio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil b Departamento de BioquõÂmica-Imunologia, Instituto de CieÃncias BioloÂgicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil Received 5 August 1999; accepted 21 October 1999
Abstract From a Phoneutria nigriventer venom gland cDNA library several clones coding for the insect speci®c neurotoxin Tx4(6±1) were isolated. cDNA analysis showed that the encoded protein contained three distinct segments, comprising a signal sequence of 16 amino acids, followed by a glutamate-rich sequence of 18 amino acids and, ®nally, the coding region for the mature toxin. The deduced amino acid sequence for the mature polypeptide was identical to the protein sequence determined chemically. In addition, two new putative toxins called Pn4A and Pn4B were characterized and their predicted complete amino acid sequence revealed approximately 78% similarity to Tx4(6±1). # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Phoneutria nigriventer; Insecticidal neurotoxin; Tx4(6±1); Pn4A; Pn4B; cDNA
* Corresponding author. Tel.: +55-31-4992695; fax: +55-31-4992713. E-mail address: [email protected] (E. Kalapothakis). 0041-0101/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 1 - 0 1 0 1 ( 9 9 ) 0 0 2 3 7 - 8
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1. Introduction The South American solitary `armed' spider Phoneutria nigriventer is probably the most aggressive species found in Brazil (Lucas, 1988). Its venom contains numerous neurotoxic polypeptides which have been used as probes in the ®eld of pharmacology and biochemistry (Prado et al., 1996; Guatimosim et al., 1997). These neurotoxins are basic polypeptides of low molecular weight which bind with nanomolar anity and high speci®city to ion channels, subsequently altering their behavior (Cordeiro et al., 1995). The speci®city of spider toxins are also very valuable to the agriculture ®eld and considerable eort has been carried out in the use of natural toxins against pests (Stewart et al., 1991). Figueiredo et al. (1995) reported the isolation of Tx4(6±1), a neurotoxic component from the fraction PhTx4, which appears to be insect-speci®c. The complete amino acid sequence of Tx4(6±1) indicates that it is a single-chain polypeptide of 48 amino acids with a molecular weight of 5251. Injection of Tx4(6±1) induced variable symptoms depending on the species and dose injected. It is highly active against house ¯ies, producing neurotoxic eects at a dose as low as 0.02 ng/mg. Additionally, Tx4(6±1) was also active against cockroaches, at doses in the range of 0.5±2.5 ng/mg. The insects are knocked down immediately, are unable to reinvert and exhibit tremors with uncoordinated movements. To further characterize insecticidal neurotoxic peptides from the spider P. nigriventer, we decided to clone the cDNA coding for Tx4(6±1) and search for new isoforms. Standard recombinant DNA techniques (DNA puri®cation, phenol extraction, ethanol precipitation, electrophoresis, etc.) were carried out as described by Sambrook et al. (1989). A P. nigriventer venom gland cDNA library was constructed using 5 mg of mRNA using the cDNA synthesis Kit (Zap-cDNA synthesis Kit) and Gigapack II Packaging Extract (Stratagene). Using the cDNA library and the methods described by Kalapothakis et al. (1998a) the cDNA encoding for Tx4(6±1) was obtained. The deduced amino acid sequence obtained by cDNA sequencing was identical to the protein sequence described for Tx4(6±1) (Figueiredo et al., 1995) (Fig. 2). The nucleotide sequence displayed an open reading frame of 249 bp encoding a precursor polypeptide of 82 amino acids and a large 3 ' untranslated region composed of 127 bp. Clones were sequenced on both strands using the chain termination method of Sanger et al. (1977) with an Automated ALF DNA Sequencer (Pharmacia). Nucleic acid sequences were analyzed using the basic local alignment search tool program for amino acids (BLASTX) comparisons (Altschul et al., 1990) and PC/GENE (nucleic acid and protein sequence analysis software system). As depicted in Fig. 1, the sequence of nucleotides 1±48 code for a signal peptide composed of 16 amino acids residues. It has all the characteristics of a typical signal peptide (Perlman and Halvorson, 1983) including a consensus cleavage point for a signal peptidase. The signal peptide is followed by an intervening propeptide with 18 amino acids (nucleotides 49±102) rich in Glu residues and ending with an Arg residue. The region corresponding to nucleotides 103 to 246 encode the mature polypeptide with 48 amino acids residues. These sequence,
Fig. 1. Comparison of the nucleotide sequence of cDNAs encoding the insecticidal toxin Tx4(6±1) and its isoforms Pn4A and Pn4B. A point (.) indicates that the nucleotide is identical to the one above, where no identity occurs, the corresponding change is indicated. Gaps (-) were introduced in the nucleotide sequence to maximize homology. The coding region is shown in upper case, with the 5' and 3' untranslated region in lower case. The predicted amino acid sequence for the Tx4(6±1), Pn4A and Pn4B toxins are shown in boldface, with the signal sequence underlined. The Glu-rich region (propeptide) is shown in square brackets. Initiation (ATG) codons, stop codon (TAA) and polyadenylation signal (AATAAA) are designated by asterisks.
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which ends with a TAA termination codon, is followed by a 3 ' untranslated region including a putative AATAAA polyadenylation sequence between the termination codon and the ®nal poly(A) tail. The search for isoforms of P. nigriventer insecticidal peptides was carried out using the isolated Tx4(6±1) cDNA to screen the original library by plaque hybridization (20,000 plaques) applying low stringency conditions (Sambrook et al., 1989). DNA was labeled by the random primer method (Feinberg and Vogelstein, 1984) using 25 ng of DNA. The sequence of the probe used for the screening (Tx4(6±1) cDNA) is shown in Fig. 1. Thirty clones were isolated, sequenced and the results showed 15 clones coding for Tx4(6±1), 14 coding for an isoform termed Pn4A and only one coding for an isoform termed Pn4B. These cDNAs with 370 bp (Pn4A) and 372 bp (Pn4B) encode precursors of 47 and 51 amino acids, respectively. The nucleotide sequence coding for the signal peptide of the three clones described showed 100% similarity. Dierences and similarities at the nucleotide and amino acid sequences are shown in Figs. 1 and 2, respectively. Total RNA from P. nigriventer venom gland was examined by Northern blot analysis using cDNAs isolated from clones coding for Tx4(6±1), Pn4A and Pn4B. A band of approximately 400 bp was observed in each case, suggesting that we isolated full length cDNAs and high levels of mRNA for each isolated toxin (data not shown). Preparation of total RNA was performed as described by Chomczynski and Sacchi (1987). Electrophoresis of RNA (10 mg) was carried out after denaturation with formaldehyde as described by Sambrook et al. (1989).
Fig. 2. Structural analysis. Gaps (dashes) were introduced to maximize sequence similarities. (A) The hydrophobic region (signal peptide), the hydrophilic region (propeptide) of Tx4(6±1), Pn4A and Pn4B are compared with Pn2-5A, Tx2-1 and Pn2-1A (Kalapothakis et al., 1998a). The residues of Pn2-1A located in the propeptide between Glu-25 and Asp-53 (SRPNAMERSANDWIPTAPSAVERSADF) are indicated by an asterisk and were omitted to improve similarities. (B) Comparison of amino acid sequences of Tx4(6±1), Pn4A, Pn4B with Pn2-5A, Tx2-1, Pn2-1A and Tx2-6, Tx2-5 (Cordeiro et al., 1995). The previously determined amino acid sequence of Tx4(6±1) is underlined.
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In this work we report the nucleotide sequences of three cDNAs encoding neurotoxins and the processing they require to generate mature toxins. We identi®ed and analyzed by direct sequencing the insecticidal toxin Tx4(6±1) and two putative neurotoxins termed Pn4A and Pn4B. The reported structure of the cDNAs have a similar overall organization (Fig. 1) showing the presence of an open reading frames encoding signal peptides with several hydrophobic residues (Von Heijne, 1985) followed by a hydrophilic region (Fig. 2), highly enriched in Glu residues with an Arg residue at the C-terminus preceding the mature toxin. The presence of such Glu-rich sequence with Arg at the C-terminus has been described in other small spiders peptide precursors, including the o-Agatoxin IA from Agelenopsis aperta (Santos et al., 1992) and several toxins from P. nigriventer venom gland (Diniz et al., 1993; Kalapothakis et al., 1998a,b). In comparison to Tx4(6±1) and Pn4A, the clone Pn4B showed four extra amino acids at the Cterminal before the stop codon (Figs. 1 and 2). Our cDNA sequencing data, together with biochemical evidence, indicate that the processing of precursors of Tx4(6±1) and Pn4A to mature toxin seems to require at least two dierent proteolytic activities: one for excision of the signal peptide between Ala-16 and Ser-17 (Fig. 2) and another speci®c for Arg-34 (a supposed cleavage point between the Glu-rich segment propeptide and the major polypeptide chain). However, maturation of Pn4B may require the action of a third proteolytic enzyme involved in the removal of four amino acids residues (QNKI) at the C-terminal, which is a common step in the conversion of the precursor of neurotoxin into mature polypeptide (Diniz et al., 1993; Martin-Eauclaire et al., 1994; Becerril et al., 1996; Leisy et al., 1996; Kalapothakis et al., 1998a,b). The number and distribution of Cys residues is well conserved (100%) in these three isoforms. However, small dierences exist between the analyzed toxins, especially at the COOH-terminus and may imply dierent action of these toxins. Analysis of the alignment of these sequences with several insect toxins did not show any signi®cant level of identity; however, a similar distribution of Cys residues has been found (data not shown). In contrast, a high level of identity has been found with toxins puri®ed from the venom of the same spider (Fig. 2). Cloning constitutes an important step in characterizing new classes of insect toxins. A systematic molecular analysis of spider toxin genes can provide important information to elucidate the molecular and evolutionary mechanisms responsible to generate the great diversity of P. nigriventer neurotoxins. The expression and use of recombinant toxin may de®ne the functional signi®cance of the amino acid changes found in this work.
Acknowledgements This research was supported by FINEP, PADCT, FAPEMIG, PRONEX, PRPq-UFMG and CNPq.
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