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Cloning and cDNA sequence analysis of Lys49 and Asp49 basic phospholipase A2 myotoxin isoforms from Bothrops asper
The International Journal of Biochemistry & Cell Biology 33 (2001) 127–132 www.elsevier.com/locate/ijbcb
Short communication
Cloning and cDNA sequence analysis of Lys49 and Asp49 basic phospholipase A2 myotoxin isoforms from Bothrops asper Sergio Lizano a,b,*, Ge´rard Lambeau c, Michel Lazdunski c a
Facultad de Microbiologı´a, Instituto Clodomiro Picado, Uni6ersidad de Costa Rica, San Jose´, Costa Rica b Departamento de Bioquı´mica, Escuela de Medicina, Uni6ersidad de Costa Rica, San Jose´, Costa Rica c Institut de Pharmacologie Mole´culaire et Cellulaire, CNRS-UPR 411, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Received 8 May 2000; accepted 23 October 2000
Abstract Snake venom myotoxic phospholipases A2 contribute to much of the tissue damage observed during envenomation by Bothrops asper, the major cause of snake bites in Central America. Several myotoxic PLA2s have been identified, but their mechanism of myotoxicity is still unclear. To aid in the molecular characterization of these venom toxins, the complete open reading frames encoding two Lys49 and one Asp49 basic PLA2 myotoxins from the Central American snake B. asper (terciopelo) were obtained by cDNA cloning from venom gland poly-adenylated RNA. The amino acid sequence deduced from the myotoxins II and III open reading frames corresponded in each case to one of the reported amino acid sequence isoforms. The sequence of a new myotoxin IV-like sequence (MT-IVa) contains conservative Val Leu18 and Ala Val23 substitutions when compared with the reported N-terminus of the native myotoxin IV, suggesting minor isoform variations among specimens of a single species. Sequence alignment studies indicated significant ( \75% sequence identity) identities with other crotalid venom Lys49 PLA2s, particularly bothropstoxin I/Ia isoforms of B. jararacussu and myotoxin II of B. asper. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Phospholipase A2; Myotoxin; cDNA cloning; Bothrops asper; Snake venom
1. Introduction * Corresponding author. Tel.: +506-2290344; fax: +5062920485. E-mail address: [email protected] (S. Lizano).
Phospholipases A2 (PLA2; EC 3.1.1.4) catalyze the hydrolysis of sn-2 linkages in phosphoglycerides in a Ca2 + -dependent manner. Animal ven-
1357-2725/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 0 ) 0 0 0 7 3 - X
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S. Lizano et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 127–132
oms contain PLA2s comprised within groups I (elapid snakes), II (viperid and crotalid snakes) and III (bees and lizards) [1]. Although not all PLA2s found in snake venoms are toxic, some are associated with a variety of toxic effects, such as muscle tissue damage (myotoxicity), neurotoxicity, anticoagulant activity, and inflammation [2]. Myotoxic group II PLA2s have been described in the venoms of viperid snake genera (family Viperidae, subfamilies Crotalinae and Viperinae) such as Vipera, Bothrops, Agkistrodon, Trimeresurus, Crotalus, and Cerrophidion [2 – 4]. Some of these toxic PLA2s contain an aspartate residue at position 49 (Asp49) which is essential for Ca2 + binding properties needed for enzymatic activity on these PLA2s, whereas others contain a Lys49 or Ser49 substitution which renders it inactive or with extremely low PLA2 [5,6]. Four isoforms of basic PLA2 myotoxins have been isolated by cation exchange chromatography from the venom of Bothrops asper (terciopelo): I, II, III, and IV [7–10]. Myotoxins I and III are Asp49 homologs, while myotoxins II and IV are Lys49 variants. Myotoxin II occurs as a dimer [8,11] and is capable of autoacylation [12]. Despite the lack of enzymatic activity of myotoxins II and IV, all isoforms exhibit myotoxic as well as edematogenic properties [4]. In light of the absence of PLA2 activity in these isoforms, the mechanism of myotoxicity may involve additional regions within the molecule which may somehow trigger the destabilization of the integrity of the phospholipid bilayer, such as a synthetic peptide comprising residues 115 –129 of B. asper myotoxin II shown to exhibit cytotoxicity, although at a lower level than the native myotoxin [13]. Although several mechanisms of myotoxicity have been proposed [4,14] including receptor mediated or direct membrane lysis (even through the interplay of intracellular PLA2s in membrane phospholipid digestion), the precise events leading to membrane damage remain unclear. The full amino acid sequences of myotoxin II Lys49 PLA2 variant [11] and an Asp49 PLA2 isoform considered to be myotoxin III have been determined [4,9], while only the first 24 N-terminal residues of myotoxin IV are known [10]. In the case of myotoxin II, two minor sequence
isoforms of this Lys49 PLA2 occur in which a leucine or phenylalanine residue is present at position 114. Myotoxin III also exhibits microheterogeneity at positions 123 (Leu or Phe), 131 (Glu or Asp), and 132 (Lys or Pro) [9]. In this study, the full cDNA open reading frames encoding myotoxic B. asper PLA2s were obtained by direct PCR amplification of cDNA synthesized from polyadenylated (poly-A+) RNA extracts of the venom gland using primers specific to the noncoding regions flanking the reading frame of group II Lys49 or Asp49 PLA2s from other crotalid snakes [15]. Poly-A+-enriched RNA was purified from fresh venom gland extracts by using the oligo(dT) RNA extraction kit from Perkin Elmer –Cetus according to the manufacturer’s instructions. Oligo(dT)-primed synthesis of cDNA was carried out using a ZAP Express cDNA synthesis kit (Stratagene, La Jolla, CA). The cDNA synthesis reaction was used as template for PCR amplification [16] using oligonucleotide primers complementary to the non-coding sequences flanking the open reading frames of reported snake venom PLA2s [15]. The following primers were used: PLA2 forward (5%-GTCTGGATTCAGGAGGATGAGG-3%) and PLA2 reverse (5%-GCCTGCAGAGACTTAGCA-3%) primers were used. PCR amplification was performed using Pwo DNA polymerase with 3%–5% proofreading capacity (Roche Molecular Biochemicals) on 50 ml reactions with approximately 0.1 mg of cDNA template, deoxynucleotide triphosphates (dNTPs, 0.2 mM each), and primer combinations (forward and reverse) at a concentration of 0.5 mg primer/reaction for each primer. PCR thermal cycling conditions were as follows: denaturing at 95°C for 5 min, followed by 25 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. Five units of Taq polymerase (Promega) were added at the end of the cycling procedure and the reactions incubated for an additional 5 min at 72°C, in order to incorporate 3% adenylyl overhangs on the blunt-ended PCR products for the cloning step (see below). Part of the PCR reaction (5ml) was analyzed by electrophoresis on 1% agarose gels with TAE (40mM Tris-acetate, 1 mM EDTA, pH 8.0), and the rest (45ml) purified on
S. Lizano et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 127–132
the Wizard PCR purification kit (Promega, Madison, WI). The purified PCR product was ligated to pGEMT (Promega) and used to transform DH10B Escherichia coli cells (Gibco BRL). DNA minipreps of these clones were made using a Wizard miniprep system (Promega) and sequenced on an ABI Prism automatic DNA sequencer model 377 (Applied Biosystems) according to manufacturer’s instructions. Each clone was sequenced in opposite directions using primers complementary to the SP6 and T7 promoter sites which flank the cloning site of pGEMT. DNA sequence data were analyzed with the SeqEd version 1.0.3 software (Applied Biosystems) and protein sequence alignments were performed on BLAST software [17] at the NCBI (National Center for Biotechnology Information)
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protein database. The nomenclature of the amino acid positions in the aligned sequences were based on the amino acid numbering scheme derived from the alignment of highly conserved regions in group I and II PLA2s with reference to the bovine pancreatic PLA2, as defined by Renetseder et al. [18]. In the case of myotoxin IV, the reported N-terminus [10] was also confirmed in this study by N-terminal sequencing-performed on an Applied Biosystems Model 477A protein sequencer. The cDNA sequences and the corresponding translated amino acid sequences for two basic PLA2 myotoxins are shown in Fig. 1. The deduced amino acid sequence for a Lys49 myotoxin (Fig. 1A) corresponds to the reported amino acid sequence of myotoxin II [9]. The cloned sequence contains phenylalanine at position 114, which in
Fig. 1. cDNA and translated amino acid sequences of basic PLA2 myotoxins. (A) Lys49 cDNA sequence aligned with the corresponding reported amino acid sequence for myotoxin II [11]. (B) Asp49 cDNA sequence aligned with the corresponding reported amino acid sequence for myotoxin III [9]. (C) cDNA and translated amino acid sequences of the Lys49 myotoxin IVa aligned with the reported N-terminal amino acid sequences for myotoxin IV [10] and confirmed in this study. The reported amino acid sequences are highlighted in bold. The amino acids in bold and italics represent additional residues determined by N-terminal sequencing in this study. The DNA sequences in bold and italics correspond to the primer sequences for the non-coding regions flanking the PLA2 reading frame [15]. The amino acid nomenclature is based on the numbering scheme devised by Renetseder et al. [18]. (*) in 1C indicate amino acids which differ between the native myotoxin IV N-terminus and the corresponding cDNA-derived sequence.
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the protein-derived sequence corresponds to either leucine or phenylalanine. Similarly, the three positions of microheterogeneity (123, 131, and 132) within the amino acid sequence corresponding to an Asp49 myotoxic PLA2 [4,9] correspond to leucine, glutamate, and lysine, respectively, in the cloned sequence of the Asp49 PLA2 (Fig. 1B). The cloning results thus indicate that the cDNA sequences of one of at least two possible variants of myotoxins II and III were obtained. The appearance of microheterogeneity within the amino acid sequences may reflect single point variations among myotoxins derived from venom samples pooled from specimens of B. asper collected from different geographical regions of Costa Rica. Venom composition is known to vary among specimens within a single species according to geographic location [19]. Since the cDNA sequences reported in this study were derived from a single specimen of B. asper, it is possible that such sequences are present or expressed only in this organism, or B. asper specimens from the same geographical location. Alternatively, it is also possible that this microheterogeneity is present within the same specimen, and that the clones obtained simply correspond to one of the minor variants for each of the myotoxins. In the case of the Asp49 cDNA sequence, it certainly corresponds to one of the isoforms described by Kaiser and colleagues [9] and considered to be myotoxin III by Gutie´rrez and Lomonte [4]. The remaining variant may correspond to a closely related form of myotoxin III or rather to myotoxin I, another Asp49 variant which has an elution profile similar to myotoxin III on carboxymethyl Sephadex (CMS) chromatography of B. asper venom, as well as similar enzymatic and pharmacological properties [4]. The first 20 N-terminal amino acid residues of the CMS fraction considered to be myotoxin I (data not shown) are identical to myotoxin III, thus supporting the hypothesis that both myotoxins are highly similar if not identical, and that the microheterogeneity observed by Kaiser and colleagues [9] may actually correspond to the two isoforms. This study also reports another open reading frame encoding a previously undescribed myotoxin sequence closely resembling myotoxin IV,
a partially characterized Lys49 PLA2 from B. asper, was determined (Fig. 1C). When compared to the reported N-terminal sequence of myotoxin IV [10], which was also confirmed in this study using the native protein, only amino acid residues Val19 and Ala24 in myotoxin IV do not coincide with the Leu19 and Val24 residues derived from the cDNA sequence (MT-IVa). Both differences, however, involve substitutions with non-polar amino acids with aliphatic side chains, and thus may not represent a critical difference in terms of structure and biological function. Interestingly, a closely related Lys49 myotoxic PLA2 from B. atrox [5] contains the same Leu19 and Val24 residues as the cloned sequence, indicating that such substitutions at these positions are likely to occur and that the discrepancies within the N-terminus of myotoxin IV may not be polymerase mistakes during PCR amplification. Finally, Met55 constitutes the only amino acid residue not present in other snake venom PLA2 sequences [4,20]. Sequence alignment of the cDNA-derived amino acid sequence of this putative myotoxin IV revealed significant sequence identities with closely related Lys49 PLA2s from crotalid venoms with myotoxic properties (Fig. 1C). The most similar of these PLA2s are bothropstoxin I/Ia isoforms from B. jararacussu, followed by the Lys49 myotoxin II from B. asper, and the T. gramineus isoform V, all of which exhibited \ 80% sequence identity with cDNA-derived myotoxin IV. In addition, myotoxin IV contains all nine amino acid residues considered to be unique to the Lys49 family of snake venom PLA2s (indicated by the asterisk in Fig. 2), according to a recent statistical analysis of aligned PLA2s sequences [20] using SequenceSpace statistical analysis software [21]. These amino acids fall within three major structural clusters comprising the active site, hydrophobic substrate binding channel, and the homodimer interface domains [20]. Although the association of such residues with these regions suggests a possible relationship to myotoxicity, it is also possible that they just represent conserved elements independent of biological activity.
S. Lizano et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 127–132
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Fig. 2. Amino acid sequence alignment of the cDNA-derived sequence of myotoxin IVa with the nine most closely related Lys49 PLA2s. The amino acid nomenclature is based on the numbering scheme devised by Renetseder et al. [18]. Asterisks (*) indicate amino acid residues which are unique (specificity-value levels \0.95) to Lys49 PLA2s, according to the SequenceSpace analysis of Ward et al. [19]. BaMT-IV, B. asper putative myotoxin IV (cDNA-derived, this study); BthTX-I, B. jararacussu bothropstoxin I [22]; BaMT-II, B. asper myotoxin II [11]; BthTX-Ia, B. jararacussu bothropstoxin Ia [23] Tgr-iso5, Trimeresurus gramineus PLA2 isotype V [24]; Agkcl PA2, Agkistrodon contortrix laticinctus PLA2 homolog [25]; GodMT-II, Cerrophidion (Bothrops) godmani myotoxin II [26]; APP-K-49, Agkistrodon pisci6orus pisci6orus pisci6orus PLA2 homolog [27]; Tfla6 BPI, Trimeresurus fla6o6iridis basic protein I [28]; Tmu bPA2, Trimeresurus mucrosquamatus basic PLA2 [29].
In conclusion, the cDNA sequences encoding the full open reading frames of myotoxins II, III, and an isoform of myotoxin IV, were obtained by PCR amplification and cloning. While the deduced amino acid sequences of myotoxins II and III confirm the reported amino acid sequences of the native toxins, the complete sequence of a myotoxin IV-related isoform is described and confirmed as a Lys49 variant. The cloning of myotoxins II and III will enable future studies of expression and mutagenesis of these isoforms to study aspects related to their structure and function.
Acknowledgements The authors wish to thank Danielle Moinier for the amino acid sequencing studies, Rodrigo Aymerich for his aid in the venom gland extrac-
tion, Dr Yamileth Angulo for providing the oligonucleotide primers, and Drs Jose´ Marı´a Gutie´rrez and Bruno Lomonte for critical reading of the manuscript. This work was supported by International Foundation for Science (IFS) grant F/2485-2, Vicerrectorı´a de Investigacio´n (Universidad de Costa Rica) grants 741-97-250 and 74198-211, and the Centre National pour la Recherche Scientifique (CNRS). SL was a postdoctoral fellow at the Institut de Pharmacologie Mole´culaire et Cellulaire (IPMC, CNRS) sponsored by the France-Central America Cooperation Program in Science and Technology (CCCAC) during part of this study.
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[2] J.B. Harris, Phospholipases in snake venoms and their effects on nerve and muscle, in: A.L. Harvey (Ed.), Snake Toxins, Pergamon Press, New York, USA, 1991, pp. 91 – 129. [3] D. Mebs, C. Ownby, Myotoxic components of snake venoms: their biochemical and biological activities, Pharmac. Ther. 48 (1990) 223–236. [4] J.M. Gutie´rrez, B. Lomonte, Phospholipase A2 myotoxins from Bothrops snake venoms, Toxicon 33 (1995) 1405– 1424. [5] J.M. Maraganore, G. Merutka, W. Cho, W. Welches, F.J. Kezdy, R.L. Heinrikson, A group of phospholipases A2 with lysine instead of aspartate 49, J. Biol. Chem. 259 (1984) 13839– 13843. [6] I. Krizaj, A.L. Bieber, A. Ritonja, F. Gubensek, The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom, Eur. J. Biochem. 202 (1991) 1165–1168. [7] J.M. Gutie´rrez, C.L. Ownby, G.V. Odel, Isolation of a myotoxin from Bothrops asper: partial characterization and action on skeletal muscle, Toxicon 22 (1984) 115– 128. [8] B. Lomonte, J.M. Gutie´rrez, A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo), Toxicon 27 (1989) 725–733. [9] I.I. Kaiser, J.M. Gutie´rrez, D. Plummer, S.D. Aird, G.V. Odell, The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper, Arch. Biochem. Biophys. 278 (1990) 319–325. [10] C. Dı´az, B. Lomonte, F. Zamudio, J.M. Gutie´rrez, Purification and characterization of myotoxin IV, a phospholipase A2 variant from Bothrops asper snake venom, Natural Toxins 3 (1995) 26–31. [11] B. Francis, J.M. Gutie´rrez, B. Lomonte, I.I. Kaiser, Myotoxin II from Bothrops asper (terciopelo) venom is a Lys49 phospholipase A2, Arch. Biochem. Biophys. 284 (1991) 352– 359. [12] J.Z. Pedersen, B. Lomonte, R. Massoud, F. Gubensek, J.M. Gutie´rrez, S. Rufini, Auto-acylation of phospholipase-like myotoxins, Biochemistry 34 (1995) 4670–4675. [13] L. Caldero´n, B. Lomonte, Immunochemical characterization and role in toxic activities of region 115-129 of myotoxin II, a Lys-49 phospholipase A2 from B. asper snake venom, Arch. Biochem. Biophys. 358 (1998) 343– 350. [14] J.E. Fletcher, M.-S. Jiang, Lys49 phospholipase A2 lyse cell cultures by two distinct mechanisms, Toxicon 36 (1998) 1549– 1555. [15] Y.-M. Wang, J.-H. Wang, F.-M. Pan, I.-H. Tsai, Lys49 phospholipase homologs from venoms of Deinagkistrodon acutus and Trimeresurus mucrosquamatus have identical protein sequences, Toxicon 34 (1996) 485–489. [16] K. Mullis, F. Faloona, S. Scharf, R. Saika, R. Horn, H. Erlich, Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction, Cold Spring Harbor Symp. Quant. Biol. 51 (1986) 263–273. .
[17] S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25 (1997) 3389– 3402. [18] R. Renetseder, S. Brunie, B.W. Dijkstra, J. Drenth, P.B. Sigler, A comparison of the crystal structures of phospholipase A2 from bovine pancreas and Crotalus atrox venom, J. Biol. Chem. 260 (1985) 11627– 11634. [19] B. Lomonte, E. Carmona, Individual expression patterns of myotoxin isoforms in the venom of the snake Bothrops asper, Comp. Biochem. Physiol. 102B (1992) 325– 329. [20] R.J. Ward, A. Rodrigues Aalves, J. Ruggiero Neto, R.K. Arni, G. Casari, A SequenceSpace analysis of Lys49 phospholipases A2: clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity, Prot. Engineering 11 (1998) 285– 294. [21] G. Casari, C. Sander, A. Valencia, A method to predict functional residues in proteins, Nature Struct. Biol. 2 (1995) 171– 178. [22] A.C. Cintra, S. Marangoni, B. Oliveira, J.R. Giglio, Bothropstoxin I: amino acid sequence and function, J. Prot. Chem. 12 (1993) 57 – 64. [23] R.J. Ward, N. Monesi, R.K. Arni, R.E. Larson, M. Paco-Larson, Sequence of a cDNA encoding bothropstoxin I, a myotoxin from the venom of Bothrops jararacussu, Gene 156 (1995) 305– 306. [24] M. Nakai, K.-I. Nakashima, T. Ogawa, Y. Shimohigashi, S. Hattori, C.-C. Chang, M. Ohno, Purification and primary structure of a myotoxic lysine-49 phospholipase A2 with low lipolytic activity from Trimeresurus gramineus venom, Toxicon 33 (1995) 1469– 1478. [25] H.S. Selistre de Araujo, S.P. White, C.L. Ownby, cDNA cloning and sequence analysis of a Lys49 phospholipase A2 myotoxin from Agkistrodon contortrix laticinctus snake venom, Arch Biochem. Biophys. 326 (1996) 21 – 30. [26] M.V. De Sousa, L. Morhy, R.K. Arni, R.J. Ward, C. Dı´az, J.M. Gutie´rrez, Amino acid sequence of a myotoxic Lys49-phospholipase A2 homologue from the venom of Cerrophidion (Bothrops) godmani, Biochem. Biophys. Acta 1384 (1998) 204– 208. [27] J.M. Maraganore, R.L. Heinrikson, The lysine-49 phospholipase A2 from the venom of Agkistrodon pisci6orus piscivorus: relation of structure and function to other phospholipases A2, J. Biol. Chem. 261 (1986) 4797– 4804. [28] K. Yoshizumi, S.Y. Liu, T. Miyata, S. Saita, M. Ohno, M.S. Iwanaga, H. Kihara, Purification and amino acid sequence of basic protein I, a lysine-49 phospholipase A2 with low activity, from the venom of Trimeresurus fla6o6iridis (habu snake), Toxicon 28 (1990) 43 – 54. [29] C.-S. Liu, J.-M. Chen, C.-H. Chang, S.-W. Chen, C.-M. Teng, I.-H. Tsai, The amino acid sequence and properties of an edema-inducing Lys49 phospholipase A2 homolog from the venom of Trimeresurus mucrosquamatus, Biochim. Biophys. Acta 1077 (1991) 362– 370.
Short communication
Cloning and cDNA sequence analysis of Lys49 and Asp49 basic phospholipase A2 myotoxin isoforms from Bothrops asper Sergio Lizano a,b,*, Ge´rard Lambeau c, Michel Lazdunski c a
Facultad de Microbiologı´a, Instituto Clodomiro Picado, Uni6ersidad de Costa Rica, San Jose´, Costa Rica b Departamento de Bioquı´mica, Escuela de Medicina, Uni6ersidad de Costa Rica, San Jose´, Costa Rica c Institut de Pharmacologie Mole´culaire et Cellulaire, CNRS-UPR 411, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Received 8 May 2000; accepted 23 October 2000
Abstract Snake venom myotoxic phospholipases A2 contribute to much of the tissue damage observed during envenomation by Bothrops asper, the major cause of snake bites in Central America. Several myotoxic PLA2s have been identified, but their mechanism of myotoxicity is still unclear. To aid in the molecular characterization of these venom toxins, the complete open reading frames encoding two Lys49 and one Asp49 basic PLA2 myotoxins from the Central American snake B. asper (terciopelo) were obtained by cDNA cloning from venom gland poly-adenylated RNA. The amino acid sequence deduced from the myotoxins II and III open reading frames corresponded in each case to one of the reported amino acid sequence isoforms. The sequence of a new myotoxin IV-like sequence (MT-IVa) contains conservative Val Leu18 and Ala Val23 substitutions when compared with the reported N-terminus of the native myotoxin IV, suggesting minor isoform variations among specimens of a single species. Sequence alignment studies indicated significant ( \75% sequence identity) identities with other crotalid venom Lys49 PLA2s, particularly bothropstoxin I/Ia isoforms of B. jararacussu and myotoxin II of B. asper. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Phospholipase A2; Myotoxin; cDNA cloning; Bothrops asper; Snake venom
1. Introduction * Corresponding author. Tel.: +506-2290344; fax: +5062920485. E-mail address: [email protected] (S. Lizano).
Phospholipases A2 (PLA2; EC 3.1.1.4) catalyze the hydrolysis of sn-2 linkages in phosphoglycerides in a Ca2 + -dependent manner. Animal ven-
1357-2725/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 0 ) 0 0 0 7 3 - X
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S. Lizano et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 127–132
oms contain PLA2s comprised within groups I (elapid snakes), II (viperid and crotalid snakes) and III (bees and lizards) [1]. Although not all PLA2s found in snake venoms are toxic, some are associated with a variety of toxic effects, such as muscle tissue damage (myotoxicity), neurotoxicity, anticoagulant activity, and inflammation [2]. Myotoxic group II PLA2s have been described in the venoms of viperid snake genera (family Viperidae, subfamilies Crotalinae and Viperinae) such as Vipera, Bothrops, Agkistrodon, Trimeresurus, Crotalus, and Cerrophidion [2 – 4]. Some of these toxic PLA2s contain an aspartate residue at position 49 (Asp49) which is essential for Ca2 + binding properties needed for enzymatic activity on these PLA2s, whereas others contain a Lys49 or Ser49 substitution which renders it inactive or with extremely low PLA2 [5,6]. Four isoforms of basic PLA2 myotoxins have been isolated by cation exchange chromatography from the venom of Bothrops asper (terciopelo): I, II, III, and IV [7–10]. Myotoxins I and III are Asp49 homologs, while myotoxins II and IV are Lys49 variants. Myotoxin II occurs as a dimer [8,11] and is capable of autoacylation [12]. Despite the lack of enzymatic activity of myotoxins II and IV, all isoforms exhibit myotoxic as well as edematogenic properties [4]. In light of the absence of PLA2 activity in these isoforms, the mechanism of myotoxicity may involve additional regions within the molecule which may somehow trigger the destabilization of the integrity of the phospholipid bilayer, such as a synthetic peptide comprising residues 115 –129 of B. asper myotoxin II shown to exhibit cytotoxicity, although at a lower level than the native myotoxin [13]. Although several mechanisms of myotoxicity have been proposed [4,14] including receptor mediated or direct membrane lysis (even through the interplay of intracellular PLA2s in membrane phospholipid digestion), the precise events leading to membrane damage remain unclear. The full amino acid sequences of myotoxin II Lys49 PLA2 variant [11] and an Asp49 PLA2 isoform considered to be myotoxin III have been determined [4,9], while only the first 24 N-terminal residues of myotoxin IV are known [10]. In the case of myotoxin II, two minor sequence
isoforms of this Lys49 PLA2 occur in which a leucine or phenylalanine residue is present at position 114. Myotoxin III also exhibits microheterogeneity at positions 123 (Leu or Phe), 131 (Glu or Asp), and 132 (Lys or Pro) [9]. In this study, the full cDNA open reading frames encoding myotoxic B. asper PLA2s were obtained by direct PCR amplification of cDNA synthesized from polyadenylated (poly-A+) RNA extracts of the venom gland using primers specific to the noncoding regions flanking the reading frame of group II Lys49 or Asp49 PLA2s from other crotalid snakes [15]. Poly-A+-enriched RNA was purified from fresh venom gland extracts by using the oligo(dT) RNA extraction kit from Perkin Elmer –Cetus according to the manufacturer’s instructions. Oligo(dT)-primed synthesis of cDNA was carried out using a ZAP Express cDNA synthesis kit (Stratagene, La Jolla, CA). The cDNA synthesis reaction was used as template for PCR amplification [16] using oligonucleotide primers complementary to the non-coding sequences flanking the open reading frames of reported snake venom PLA2s [15]. The following primers were used: PLA2 forward (5%-GTCTGGATTCAGGAGGATGAGG-3%) and PLA2 reverse (5%-GCCTGCAGAGACTTAGCA-3%) primers were used. PCR amplification was performed using Pwo DNA polymerase with 3%–5% proofreading capacity (Roche Molecular Biochemicals) on 50 ml reactions with approximately 0.1 mg of cDNA template, deoxynucleotide triphosphates (dNTPs, 0.2 mM each), and primer combinations (forward and reverse) at a concentration of 0.5 mg primer/reaction for each primer. PCR thermal cycling conditions were as follows: denaturing at 95°C for 5 min, followed by 25 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. Five units of Taq polymerase (Promega) were added at the end of the cycling procedure and the reactions incubated for an additional 5 min at 72°C, in order to incorporate 3% adenylyl overhangs on the blunt-ended PCR products for the cloning step (see below). Part of the PCR reaction (5ml) was analyzed by electrophoresis on 1% agarose gels with TAE (40mM Tris-acetate, 1 mM EDTA, pH 8.0), and the rest (45ml) purified on
S. Lizano et al. / The International Journal of Biochemistry & Cell Biology 33 (2001) 127–132
the Wizard PCR purification kit (Promega, Madison, WI). The purified PCR product was ligated to pGEMT (Promega) and used to transform DH10B Escherichia coli cells (Gibco BRL). DNA minipreps of these clones were made using a Wizard miniprep system (Promega) and sequenced on an ABI Prism automatic DNA sequencer model 377 (Applied Biosystems) according to manufacturer’s instructions. Each clone was sequenced in opposite directions using primers complementary to the SP6 and T7 promoter sites which flank the cloning site of pGEMT. DNA sequence data were analyzed with the SeqEd version 1.0.3 software (Applied Biosystems) and protein sequence alignments were performed on BLAST software [17] at the NCBI (National Center for Biotechnology Information)
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protein database. The nomenclature of the amino acid positions in the aligned sequences were based on the amino acid numbering scheme derived from the alignment of highly conserved regions in group I and II PLA2s with reference to the bovine pancreatic PLA2, as defined by Renetseder et al. [18]. In the case of myotoxin IV, the reported N-terminus [10] was also confirmed in this study by N-terminal sequencing-performed on an Applied Biosystems Model 477A protein sequencer. The cDNA sequences and the corresponding translated amino acid sequences for two basic PLA2 myotoxins are shown in Fig. 1. The deduced amino acid sequence for a Lys49 myotoxin (Fig. 1A) corresponds to the reported amino acid sequence of myotoxin II [9]. The cloned sequence contains phenylalanine at position 114, which in
Fig. 1. cDNA and translated amino acid sequences of basic PLA2 myotoxins. (A) Lys49 cDNA sequence aligned with the corresponding reported amino acid sequence for myotoxin II [11]. (B) Asp49 cDNA sequence aligned with the corresponding reported amino acid sequence for myotoxin III [9]. (C) cDNA and translated amino acid sequences of the Lys49 myotoxin IVa aligned with the reported N-terminal amino acid sequences for myotoxin IV [10] and confirmed in this study. The reported amino acid sequences are highlighted in bold. The amino acids in bold and italics represent additional residues determined by N-terminal sequencing in this study. The DNA sequences in bold and italics correspond to the primer sequences for the non-coding regions flanking the PLA2 reading frame [15]. The amino acid nomenclature is based on the numbering scheme devised by Renetseder et al. [18]. (*) in 1C indicate amino acids which differ between the native myotoxin IV N-terminus and the corresponding cDNA-derived sequence.
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the protein-derived sequence corresponds to either leucine or phenylalanine. Similarly, the three positions of microheterogeneity (123, 131, and 132) within the amino acid sequence corresponding to an Asp49 myotoxic PLA2 [4,9] correspond to leucine, glutamate, and lysine, respectively, in the cloned sequence of the Asp49 PLA2 (Fig. 1B). The cloning results thus indicate that the cDNA sequences of one of at least two possible variants of myotoxins II and III were obtained. The appearance of microheterogeneity within the amino acid sequences may reflect single point variations among myotoxins derived from venom samples pooled from specimens of B. asper collected from different geographical regions of Costa Rica. Venom composition is known to vary among specimens within a single species according to geographic location [19]. Since the cDNA sequences reported in this study were derived from a single specimen of B. asper, it is possible that such sequences are present or expressed only in this organism, or B. asper specimens from the same geographical location. Alternatively, it is also possible that this microheterogeneity is present within the same specimen, and that the clones obtained simply correspond to one of the minor variants for each of the myotoxins. In the case of the Asp49 cDNA sequence, it certainly corresponds to one of the isoforms described by Kaiser and colleagues [9] and considered to be myotoxin III by Gutie´rrez and Lomonte [4]. The remaining variant may correspond to a closely related form of myotoxin III or rather to myotoxin I, another Asp49 variant which has an elution profile similar to myotoxin III on carboxymethyl Sephadex (CMS) chromatography of B. asper venom, as well as similar enzymatic and pharmacological properties [4]. The first 20 N-terminal amino acid residues of the CMS fraction considered to be myotoxin I (data not shown) are identical to myotoxin III, thus supporting the hypothesis that both myotoxins are highly similar if not identical, and that the microheterogeneity observed by Kaiser and colleagues [9] may actually correspond to the two isoforms. This study also reports another open reading frame encoding a previously undescribed myotoxin sequence closely resembling myotoxin IV,
a partially characterized Lys49 PLA2 from B. asper, was determined (Fig. 1C). When compared to the reported N-terminal sequence of myotoxin IV [10], which was also confirmed in this study using the native protein, only amino acid residues Val19 and Ala24 in myotoxin IV do not coincide with the Leu19 and Val24 residues derived from the cDNA sequence (MT-IVa). Both differences, however, involve substitutions with non-polar amino acids with aliphatic side chains, and thus may not represent a critical difference in terms of structure and biological function. Interestingly, a closely related Lys49 myotoxic PLA2 from B. atrox [5] contains the same Leu19 and Val24 residues as the cloned sequence, indicating that such substitutions at these positions are likely to occur and that the discrepancies within the N-terminus of myotoxin IV may not be polymerase mistakes during PCR amplification. Finally, Met55 constitutes the only amino acid residue not present in other snake venom PLA2 sequences [4,20]. Sequence alignment of the cDNA-derived amino acid sequence of this putative myotoxin IV revealed significant sequence identities with closely related Lys49 PLA2s from crotalid venoms with myotoxic properties (Fig. 1C). The most similar of these PLA2s are bothropstoxin I/Ia isoforms from B. jararacussu, followed by the Lys49 myotoxin II from B. asper, and the T. gramineus isoform V, all of which exhibited \ 80% sequence identity with cDNA-derived myotoxin IV. In addition, myotoxin IV contains all nine amino acid residues considered to be unique to the Lys49 family of snake venom PLA2s (indicated by the asterisk in Fig. 2), according to a recent statistical analysis of aligned PLA2s sequences [20] using SequenceSpace statistical analysis software [21]. These amino acids fall within three major structural clusters comprising the active site, hydrophobic substrate binding channel, and the homodimer interface domains [20]. Although the association of such residues with these regions suggests a possible relationship to myotoxicity, it is also possible that they just represent conserved elements independent of biological activity.
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Fig. 2. Amino acid sequence alignment of the cDNA-derived sequence of myotoxin IVa with the nine most closely related Lys49 PLA2s. The amino acid nomenclature is based on the numbering scheme devised by Renetseder et al. [18]. Asterisks (*) indicate amino acid residues which are unique (specificity-value levels \0.95) to Lys49 PLA2s, according to the SequenceSpace analysis of Ward et al. [19]. BaMT-IV, B. asper putative myotoxin IV (cDNA-derived, this study); BthTX-I, B. jararacussu bothropstoxin I [22]; BaMT-II, B. asper myotoxin II [11]; BthTX-Ia, B. jararacussu bothropstoxin Ia [23] Tgr-iso5, Trimeresurus gramineus PLA2 isotype V [24]; Agkcl PA2, Agkistrodon contortrix laticinctus PLA2 homolog [25]; GodMT-II, Cerrophidion (Bothrops) godmani myotoxin II [26]; APP-K-49, Agkistrodon pisci6orus pisci6orus pisci6orus PLA2 homolog [27]; Tfla6 BPI, Trimeresurus fla6o6iridis basic protein I [28]; Tmu bPA2, Trimeresurus mucrosquamatus basic PLA2 [29].
In conclusion, the cDNA sequences encoding the full open reading frames of myotoxins II, III, and an isoform of myotoxin IV, were obtained by PCR amplification and cloning. While the deduced amino acid sequences of myotoxins II and III confirm the reported amino acid sequences of the native toxins, the complete sequence of a myotoxin IV-related isoform is described and confirmed as a Lys49 variant. The cloning of myotoxins II and III will enable future studies of expression and mutagenesis of these isoforms to study aspects related to their structure and function.
Acknowledgements The authors wish to thank Danielle Moinier for the amino acid sequencing studies, Rodrigo Aymerich for his aid in the venom gland extrac-
tion, Dr Yamileth Angulo for providing the oligonucleotide primers, and Drs Jose´ Marı´a Gutie´rrez and Bruno Lomonte for critical reading of the manuscript. This work was supported by International Foundation for Science (IFS) grant F/2485-2, Vicerrectorı´a de Investigacio´n (Universidad de Costa Rica) grants 741-97-250 and 74198-211, and the Centre National pour la Recherche Scientifique (CNRS). SL was a postdoctoral fellow at the Institut de Pharmacologie Mole´culaire et Cellulaire (IPMC, CNRS) sponsored by the France-Central America Cooperation Program in Science and Technology (CCCAC) during part of this study.
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