Cloning of a novel acidic phospholipase A2 from the venom gland of Crotalus durissus cascavella (Brazilian northeastern rattlesnake)

Cloning of a novel acidic phospholipase A₂ from the venom gland of Crotalus durissus cascavella (Brazilian northeastern rattlesnake)

Guarnieri MC^I; Melo ESL^I; Melo KMS^II; Albuquerque-Modesto JC^III; Prieto-da-Silva ARB^IV; Rádis-Baptista G^II,V

^IDepartment of Zoology, Federal University of Pernambuco, UFPE, Recife, Pernambuco State, Brazil

^IIDepartment of Biochemistry, Federal University of Pernambuco, UFPE, Recife, Pernambuco State, Brazil

^IIIDepartment of Biology, Federal University of Pernambuco, UFPE, Vitória de Santo Antão, Pernambuco State, Brazil

^IVLaboratory of Genetics, Butantan Institute, São Paulo, São Paulo State, Brazil

^VInstitute of Marine Sciences, Federal University of Ceará, UFC, Fortaleza, Ceará State, Brazil

^{Correspondence to}

ABSTRACT

The phospholipase A₂ superfamily encompasses 15 groups that are classified into: secreted PLA₂ (sPLA₂); cytosolic PLA₂ (cPLA₂); Ca²⁺-independent intracellular PLA₂ (iPLA₂); platelet-activating factor acetylhydrolase (PAF-AH); and lysosomal PLA₂. Currently, approximately 700 PLA₂ sequences are known, of which 200 are obtained from the venom gland of Crotalinae snakes. However, thus far, little information is available on cloning, purification and structural characterization of PLA₂ from Crotalus durisssus cascavela venom gland. In the present work, we report the molecular cloning of a novel svPLA₂ from C. d. cascavella (Cdc), a predominant rattlesnake subspecies in northeastern Brazil. The Cdc svPLA₂ cDNA precursor is 689 nucleotides long and encodes a protein of 138 amino acid residues, with a calculated molecular mass of approximately 13,847 Da and an estimated isoelectric point of 5.14. Phylogenetic analysis of Crotalinae PLA₂ reveals that Cdc PLA₂ clustered with other acidic type IIA PLA₂ homologues is also present in the venom of North American rattlesnakes. Hitherto, this study presents a novel PLA₂ cDNA precursor from C. d. cascavella and data reported herein will be useful for further steps in svPLA₂ purification and analysis.

Keywords: molecular toxinology, Crotalus durissus cascavella, snake venom gland, cDNA library, acidic PLA₂.

INTRODUCTION

The superfamily of phospholipase A₂ enzymes is currently subdivided into 15 groups based on their structures, source and localization. Distributed among these groups are the multiple forms of secreted PLA₂s (sPLA₂s – groups I, II, III, V, IX, X, XI, XII, XIII and XIV), cytosolic PLA₂s (cPLA₂ – group IV), Ca²⁺-independent intracellular PLA₂s (iPLA₂ – group VI), platelet-activating factor acetylhydrolases (PAF-AH – groups VII and VIII) and the lysosomal PLA₂s (group XV) (1).

Secreted PLA₂s are found in fungi, bacteria, plants, marine sponges, cnidarians, mollusks, starfishes, insects, reptiles and mammals (1-4). Essentially, these enzymes catalyze the hydrolysis of different membrane phospholipids at the sn-2 position, releasing free fatty acids such as arachidonic acid (AA) – a precursor of bioactive eicosanoids – and lysophospholipids (lyso-PL). Both products represent the first step in generating second messengers that play important physiological and pathological roles. Lyso-PL can be converted into lysophosphatidic acid (LPA), involved in cell proliferation, survival and migration, or into platelet activating factor (PAF), implicated specifically in inflammatory processes (5, 6). Eicosanoids affect body mechanisms including sleep regulation, immune response, inflammation and pain (7).

PLA₂s from Viperidae venoms (vPLA₂) belong to the IIsubgroup together with mammalian enzymes isolated from the spleen, mast cells, macrophages, arthritic synovial fluid and serum of patients with inflammatory diseases (8-10). This subgroup is characterized by low-molecular-mass enzymes (~14 kDa), with a rigid three-dimensional structure composed of seven disulfide bridges, whose catalytic mechanism utilizes a His-Asp dyad. These enzymes require a millimolar concentration of Ca²⁺ to exert their enzymatic action and, in contrast to cPLA₂s, they have low specifity for arachidonic acid at the sn-2 position (11).

Approximately 700 sequences from type II PLA₂s are known and compiled in databases. The diversity of snake venom PLA₂ functions includes: neurotoxicity, cardiotoxicity, myotoxicity, edema, hypotension, hyperalgesia as well as activation and inhibition of platelet aggregation (12-19). The diversity of biological and pharmacological functions of PLA₂ denotes that accelerated or positive Darwinian evolution has occurred and appears to confer a better fitness on the snake venom (10, 20). In fact, the venom PLA₂ subgroup II is further subdivided into two other smaller subgroups, vPLA₂s exhibiting enzymatic activity and a predominance of two types of amino acid residues at the catalytic site (position 49) – Asp (D49) and Ser (S49) – and non-enzymatic vPLA₂s (that is, vPLA₂ with extremely low enzymatic activity), whose residues, D49 or S49, were replaced not only with Lys (K49), but also Gln 49 (Q49), Ala (A49) and Asn (N-49) (21-26). Furthermore, D49 PLA₂ also includes acidic and basic toxic components that are found in venoms as monomers or homo- and heterodimers (27).

In this work, we report a novel PLA₂ cDNA precursor of Crotalus durissus cascavella venom, in which the predicted protein was clustered with acidic members of the type II subfamily of venom PLA₂.

MATERIALS AND METHODS

Specimens of Snake Venom Gland

For the construction of the venom gland cDNA library, a pair of glands was excised from a male adult specimen of Crotalus durissus cascavella (2 kg weight and 125 cm length – measured from rostrum to cloaca) captured in Cabaceira, Paraíba state, Brazil, and maintained from 1999 to 2006 in the Laboratory of Venomous Animals and Toxins (LAPTOX), Federal University of Pernambuco, Recife state, Brazil. The snake venom was extracted by standard procedures three days before the surgery for gland excision, with the aim of reaching the maximal level of RNA synthesis. Once surgically removed, the venom glands were kept at –80°C until the procedures for RNA purification and analysis.

Construction of C. d. cascavella Snake Venom cDNA Library

A Crotalus durissus cascavella venom cDNA library was constructed from 1 µg of total RNA, as follows: frozen venom glands were finely crushed in a mortar with a pestle under liquid nitrogen; then, total RNA was purified using Trizol® reagent (Invitrogen, USA), according to the manufacturer's instructions. The quality and yield of total RNA were verified by the integrity of 28S and 18S rRNA, through denaturing agarose gel electrophoresis using the spectrophotometric ratio 260/280 nm. Poly(A⁺)-RNA was purified from total RNA by a complex of oligo(dT)-biotin and streptavidin MagneSphere® paramagnetic particles (PolyATract® system, Promega, USA). Next, mRNA was quantified and employed for cDNA synthesis using the switching mechanism at the 5' end of RNA transcriptiom (SMART) protocol (Creator SMART cDNA Library Construction kit®, BD Biosciences, USA), which preferentially enriches the final library with full-length cDNA.

Cloning of Cdc PLA₂ cDNA and Nucleotide Sequencing

The C. durissuscascavella venom gland cDNA library was then titered and pools of approximately 10⁶ colony forming units (CFU) were used as a template in ten separate homology screening polymerase chain reactions (HR-PCR). Each reaction, in a final volume of 50 mL, in high fidelity PCR buffer (60 mM Tris-SO₄, pH 8.4, 18 mM (NH₄)₂SO₄, 2.5 mM MgSO₄), consisted of 2.5 U of Platinum® Taq DNA polymerase (Invitrogen Life Technologies, USA), 2 mM MgCl₂, 1 mM dNTPs, and 0.2 mM of each forward and reverse primer.

Of the two primers utilized to isolate the Cdc PLA₂ cDNA, one (called Cdc_PLA₂ sense primer, 5'-TGCACGACTGYTGYTAYGGA-3') anneals to the specific gene sequence, corresponding to the amino acids -FVHDCCYG-, which are conserved in most snake venom PLA₂s, and the other oligonucleotide primer to the plasmid vector (M13 reverse, 5'-AACAGCTATGACCATGTTCA- 3'), which corresponds to the flanking region of insertion in the pDNR-LIB vector.

The cloned full length Cdc PLA₂ was automatically sequenced by the dideoxy chain termination method, using the dye-terminator chemistry (DYEnamic ET Dye Terminator® kit, GE Healthcare, USA) and the MegaBACE 750 DNA Analysis System® (GE Healthcare, USA). The PLA₂ gene was in silico translated, and both nucleotide and amino acid sequences were compared against a database of genes and proteins, maintained by the NCBI (http://www.ncbi.nlm.nih.gov).

Crotalus durissus cascavella (Cdc) PLA₂ Aligment and Phylogenetic Analysis

The deduced amino acid sequence of C. d. cascavella PLA₂ cDNA precursor (present study) was compared with the GenBank (http://www.ncbi.nlm.nih.gov) by using the BLAST program (28). This search retrieved 182 protein sequences corresponding to all Crotalinae PLA₂s available in the database. The incomplete and redundant sequences were manually removed from the data set whereas the file with complete sequences was processed for alignment through the multialignment bioinformatic tool ClustalW2, available at the European Bioinformatic Institute website (http://www.ebi.ac.uk). The structural characteristics of the predicted PLA₂ precursor were manually annotated based on data from the literature. Precursors of sequences from Crotalinae PLA₂ toxins which presented higher PLA₂ member scores in comparison with Cdc PLA₂ were aligned with MUSCLE 3.6 using groups of amino acids – GA, ST, MVLI, KR, EQDN, FWYH, C and P – to determine the grade of similarity (29).

The evolutionary history was inferred using the neighbor-joining method by analyzing all sequences together, including not only higher score sequences, but also the most dissimilar PLA₂s (30). Branches corresponding to partitions reproduced in less than 50% of bootstrap replicates are defined as collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches (31). The evolutionary distances were computed using the Dayhoff matrix-based method expressed as the number of amino acid substitutions per site (32). All positions containing gaps or missing data were eliminated from the dataset (complete deletion option). There was a total of 85 positions in the final dataset. Phylogenetic analyses were conducted with MEGA4 (33).

RESULTS

A Novel PLA ₂ cDNA Precursor of Crotalus durissus cascavella Venom Gland

By a homology cloning method, a novel PLA₂ precursor, called Cdc-PLA₂, was retrieved from the venom gland cDNA library of C. d. cascavella. As indicated in Figure 1, the Cdc-PLA₂ cDNA precursor is 689 nucleotides long, with an open reading frame (ORF) of 453 nucleotides. The ORF encodes a complete precursor of 138 amino acid residues, including a signal peptide of 16 residues (MRTLWIVAVLLLGVEG). The novel Cdc-PLA₂ cDNA sequence was submitted to GenBank and received the accession number GQ466583.

Comparative Sequence Analysis of the Novel Cdc-PLA ₂

The complete amino acid sequence of Cdc-PLA₂ precursor was predicted from a cDNA sequence (Figure 1). Based on this deduced sequence, an isoeletric point of 5.14 and molecular mass of 13,846.81 Da was calculated.

The Cdc-PLA₂ conserved residues involved in Ca²⁺ binding (Tyr28, Gly30, Gly32 and Asp49) and in the catalytic network (His48), characterizing the D-49 group, and maintained conserved sequence domains common to the group IIA PLA₂, including the 14 cysteines responsible for disulfide bond formation.

Phylogenetic analysis of Cdc-PLA₂ and 54 precursors of Crotalinae PLA₂ showed that the maximum grade of parental relationship of Cdc-PLA₂ occurs with acidic PLA₂ from North American snakes (Figure 2). In this case, best similarity values (identities in the range of 60 to 86%) are observed in North American rattlesnakes, for example Crotalus v. viridis (86%).

The sequence was aligned with precursors of other Crotalinae PLA₂s, obtained by BLASTp search, which included PLA₂ with amino acid replacement at the position 49, crotapotin from C. d. terrificus and the acidic subunit of crotoxin (CA) sequence from C. d. cascavella (34). Several residues were highly conserved in the monomeric/homodimeric acidic PLA₂ analyzed in the present work as the N-terminal region (L2XXFE6), Ca²⁺ binding site (Y25GCYCGXGG33), active site (D42RCCFVHDCCYGK54) and C-terminal region (A101AXCFFDN108, Y112, Y117) (Figure 3). On the other hand, the residues A53, D79, S108 and G129 present in heterodimeric toxins (crotoxin A from C. d. terrificus,C. d. cascavella and C. s. scutulatus) are replaced in Cdc-PLA₂ and in the majority of the mono/homodimeric acid PLA₂s analyzed. Comparative analysis revealed that the hot spot of Cdc-PLA₂ mutations were found at the residues D4, I10, A34, V39, V77, K78, E85, D86, T94, G99, R118 and R128.

DISCUSSION

Although studies involving snake venom acidic PLA₂s have increased considerably in the recent years, only a few acidic PLA₂s from Brazilian snake venoms were purified and cloned (18, 21, 35-41). Up to date, nothing was known about the expression of acidic (subgroup II) PLA₂ in the venom gland of Crotalus durissus cascavella.

Cdc-PLA₂ possesses high similarity with a subgroup of acidic D49-PLA₂s which is expressed in the venom as monomers and/or as homodimers (42-45). In fact, the ability of an acidic glycosylated and phosphorylated PLA₂s to co-exist in snake venom as monomer and homodimer was recently described by Sun et al. (27).

Experimental investigations with native toxins have shown that such group of PLA₂ presents enzymatic activity and capacity of binding calcium ions for maximal catalysis, as seen by the conserved residues His48 and Asp49 in the primary sequences (39, 46-47). These acidic PLA₂s can also induce myotoxicity, platelet aggregation inhibition, hypotension, prostaglandin I2 induction or paw edema (16, 18, 21, 23, 35, 37, 39, 42, 45, 48-50). Some residues associated with antiplatelet (W21, Y113, D114) and edema-forming activities (K78 and D85) are conserved in Cdc-PLA₂ and in some very similar acidic PLA₂ isoforms from C. v. viridis venom (43, 50-51). All analyzed acidic PLA₂s presented Glu residue in the position 6, which seems an ancient condition of basic G6 and N6 PLA₂ (21, 52).

Cdc-PLA₂ possesses lower similarity with the other subgroup of acidic D49-PLA₂s (particularly, A chain crotoxin-CA) which can make high stable complexes with basic F24N6 PLA₂ (B chain crotoxin-CB) and increase the toxicity of CB in several folds (52, 53). Except for E124 residue, all amino acids that could be involved in the recognition and binding of a CA with CB (W36, E47, A53, D79, E124 and G129) are replaced in Cdc-PLA₂, what consequently suggests, at a first glance, the impossibility of this toxin to be an A chain crotoxin precursor for heterodimer formation (52). However, this point deserves more attention and further functional and structural analysis.

Acidic IIA phospholipases A₂ present multiple isoforms, generally associated with intra-specific geographic variation, as well as adaptation to prey diversity (43, 44, 54, 55). On the other hand, some PLA₂ clones apparently not translated into venom proteins has been reported, since not-expressing toxin mRNA may be a repository for snake survival under an ever-changing environment (54, 55).

In this work, we report the molecular cloning of an acidic PLA₂ type II from the venom gland of C. d. cascavella. Phylogenetic and structural analyses allowed us to make evident that the precursor, retrieved from the C. d. cascavella venom gland cDNA library, is a novel member of acidic PLA₂ subgroup. Moreover, a global analysis has shown that the most ancestral member of all PLA₂ precursors in the venom of Crotalinae snakes seems to be related to the crotoxin.

Altogether, the present data will be useful, for example, to drive steps of purification and structural analysis of such flexible and fast evolving snake venom molecule.

ACKNOWLEDGEMENTS

The authors are grateful to the National Council for Scientific and Technological Development (CNPq) from the Ministry of Science and Technology for the financial support. Part of experimental data reported herein were obtained during the course "Molecular techniques for the scientific investigation of biological and chemical diversity in terrestrial and marine organisms with potential biotechnological application", held in Recife, in 2008, in the context of the program of cooperation in biotechnology from the Brazilian and Argentinean governments (CBAB/MCT).

Received: November 22, 2008

Accepted: September 15, 2009

Abstract published online: October 9, 2009

Full paper published online: November 30, 2009

Conflicts of interest: There is no conflict.

• 1. Schaloske RH, Dennis EA. The phospholipase A₂ superfamily and its group numbering system. Biochim Biophys Acta. 2006;1761(11):1246-59.
• 2. Nevalainen TJ, Quinn RJ, Hooper JN. Phospholipase A₂ in porifera. Comp Biochem Physiol B Biochem Mol Biol. 2004;137(3):413-20.
• 3. Nevalainen TJ, Peuravuori HJ, Quinn RJ, Llewellyn LE, Benzie JAH, Fenner PJ, et al. Phospholipase A₂ in cnidaria. Comp Biochem Physiol B Biochem Mol Biol. 2004;139(4):731-5.
• 4. Ota E, Nagai H, Nagashima Y, Shiomi K. Molecular cloning of two toxic phospholipases A₂ from the crown-of-thorns starfish Acanthaster planci venom. Comp Biochem Physiol B Biochem Mol Biol. 2006;143(1):54-60.
• 5. Moolenaar WH, van Meeteren LA, Giepmans BN.The ins and outs of lysophosphatidic acid signaling. Bioessays. 2004;26(8):870-81.
• 6. Prescott SM, Zimmerman GA, Stafforini DM, McIntyre TM. Platelet-activating factor and related lipid mediators. Annu Rev Biochem. 2000;69:419-45.
• 7. Tsuboi K, Sugimoto Y, Ichikawa A. Prostanoid receptor subtypes. Prostaglandins Other Lipid Mediat. 2002;68-69:535-56.
• 8. Kudo I, Murakami M, Hara S, Inoue K. Mammalian non-pancreatic phospholipases A₂ Biochim Biophys Acta. 1993;1170(3):217-31.
• 9. Nevalainen TJ. Serum phospholipases A₂ in inflammatory diseases. Clin Chem. 1993;39(12):2453-9.
• 10. Jan VM, Guillemin I, Robbe-Vincent A, Choumet V. Phospholipase A₂ diversity and polymorphism in European viper venoms: paradoxical molecular evolution in Viperinae. Toxicon. 2007;50(8):1140-61.
• 11. Six DA, Dennis EA. The expanding superfamily of phospholipase A₂ enzymes: classification and characterization. Biochim Biophys Acta. 2000;1488(1-2):1-19.
• 12. Prijatelj P, Charnay M, Ivanovski G, Jenko Z, Pungercar J, Krizaj I, et al. The C-terminal and beta-wing regions of ammodytoxin A, a neurotoxic phospholipase A₂ from Vipera ammodytes ammodytes, are critical for binding to factor Xa and for anticoagulant effect. Biochimie. 2006;88(1):69-76.
• 13. Valentin E, Lambeau G. What can venom phospholipase A₂ tell us about the functional diversity of mammalian secreted phospholipase A₂ Biochemie. 2000;82(9-10):815-31.
• 14. Angulo Y, Olamendi-Portugal T, Possani LD, Lomonte B. Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A₂ homologue. Int J Biochem Cell Biol. 2000;32(1):63-71.
• 15. Câmara PR, Esquisatto LC, Camargo EA, Ribela MT, Toyama MH, Marangoni S, et al. Inflammatory oedema induced by phospholipases A₂ isolated from Crotalus durissus sp. in the rat dorsal skin: a role for mast cells and sensory C-fibers. Toxicon. 2003;41(7):823-9.
• 16. Adriăo-Escarso SH, Soares AM, Rodrigues VM, Angulo Y, Díaz C, Lomonte B, et al. Myotoxic phospholipases A₂ in Bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu Biochimie. 2000;82(8):755-63.
• 17. Chacur M, Milligan ED, Sloan EM, Wieseler-Frank J, Barrientos RM, Martin D, et al. Snake venom phospholipase A₂s (Asp49 and Lys49) induce mechanical allodynia upon peri-sciatic administration: involvement of spinal cord glia, proinflammatory cytokines and nitric oxide. Pain. 2004;108(1-2):180-91.
• 18. de Albuquerque-Modesto JC, Spencer PJ, Fritzen M, Valença RC, Oliva ML, da Silva MB, et al. BE-I-PLA₂, a novel acidic phospholipase A₂ from Bothrops erythromelas venom: isolation, cloning and characterization as potent anti-platelet and inductor of prostaglandin I2 release by endothelial cells. Biochem Pharmacol. 2006;72(3):377-84.
• 19. Higuchi DA, Barbosa CM, Bincoletto C, Chagas JR, Magalhaes A, Richardson M, et al. Purification and partial characterization of two phospholipases A₂ from Bothrops leucurus (white-tailed-jararaca) snake venom. Biochimie. 2007;89(3):319-28.
• 20. Ogawa T, Nakashima K, Nobuhisa I, Deshimaru M, Shimohigashi Y, Fukumaki Y, et al. Accelerated evolution of snake venom phospholipase A₂ isozymes for acquisition of diverse physiological functions. Toxicon. 1996;34(11-12):1229-36.
• 21. Cogo JC, Lilla S, Souza GH, Hyslop S, de Nucci G. Purification, sequencing and structural analysis of two acidic phospholipases A₂ from the venom of Bothrops insularis (jararaca ilhoa). Biochimie. 2006;88(12):1947-59.
• 22. Polgár J, Magnenat EM, Peitsch MC, Wells TN, Clemetson KJ. Asp-49 is not an absolute prerequisite for the enzymic activity of low-M(r) phospholipases A₂: purification, characterization and computer modeling of an enzymically active Ser-49 phospholipase A₂, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper). Biochem J. 1996;319(Pt 3):961-8.
• 23. Lomonte B, Angulo Y, Calderón L. An overview of lysine-49 phospholipase A₂ myotoxins from crotalid snake venoms and their structural determinants of myotoxic action. Toxicon. 2003;42(8):885-901.
• 24. Bao Y, Bu P, Jin L, Hongxia W, Yang Q, An L. Purification, characterization and gene cloning of a novel phospholipase A₂ from the venom of Agkistrodon blomhoffii ussurensis. Int J Biochem Cell Biol. 2005;37(3):558-65.
• 25. Liu CS, Kuo PY, Chen JM, Chen SW, Chang CH, Tseng CC, et al. Primary structure of an inactive mutant of phospholipase A₂ in the venom of Bungarus fasciatus (banded krait). J Biochem. 1992;12(5):707-13.
• 26. Wei JF, Wei XL, Chen QY, Huang T, Qiao LY, Wang WY, et al. N49 phospholipase A₂, a unique subgroup of snake venom group II phospholipase A₂ Biochim Biophys Acta. 2006;1760(3):462-71.
• 27. Sun MZ, Liu S, Yang F, Greenaway FT, Xu Y. A novel phospholipase A₂ from Agkistrodon blomhoffii ussurensis venom: purification, proteomic, functional and structural characterizations. Biochimie. 2009;91(4):558-67.
• 28. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389-402.
• 29. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004;5(1):113.
• 30. Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4(4):406-25.
• 31. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39(4):783-91.
• 32. Schwarz RM, Dayhoff MO. Matrices for detecting distant relationships. In: Dayhoff M, editor. Atlas of protein sequences and structure. Washington: National Biomedical Research Foundation; 1979. p. 353-9.
• 33. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24(8):1596-9.
• 34. de Oliveira DG, Toyama MH, Martins AM, Havt A, Nobre AC, Marangoni S, et al. Structural and biological characterization of a crotapotin isoform isolated from Crotalus durissus cascavella venom. Toxicon. 2003;42(1):53-62.
• 35. Santos-Filho NA, Silveira LB, Oliveira CZ, Bernardes CP, Menaldo DL, Fuly AL, et al. A new acidic myotoxic, anti-platelet and prostaglandin I2 inductor phospholipase A₂ isolated from Bothrops moojeni snake venom. Toxicon. 2008;52(8):908-17.
• 36. Fuly AL, de Miranda AL, Zingali RB, Guimarăes JA. Purification and characterization of a phospholipase A₂ isoenzyme isolated from Lachesis muta snake venom. Biochem Pharmacol. 2002;63(9):1589-97.
• 37. Serrano SM, Reichl AP, Mentele R, Auerswald EA, Santoro ML, Sampaio CA, et al. A novel phospholipase A₂, BJ-PLA2, from the venom of the snake Bothrops jararaca: purification, primary structure analysis, and its characterization as a platelet-aggregation-inhibiting factor. Arch Biochem Biophys. 1999;367(1):26-32.
• 38. Andriăo-Escarso SH, Soares AM, Fontes MRM, Fuly AL, Correa FMA, Rosa JC, et al. Structural and functional characterization of an acidic platelet aggregation inhibitor and hypotensive phospholipase A₂ from Bothrops jararacussu snake venom. Biochem Pharmacol. 2002;64(4):723-32.
• 39. Ketelhut DF, de Melo MH, Veronese EL, Esmeraldino LE, Murakami MT, Arni RK, et al. Isolation, characterization and biological activity of acidic phospholipase A₂ isoforms from Bothrops jararacussu snake venom. Biochimie. 2003;85(10):983-91.
• 40. Roberto PG, Kashima S, Soares AM, Chioato L, Faca VM, Fuly AL, et al. Cloning and expression of an acidic platelet aggregation inhibitor phospholipase A₂ cDNA from Bothrops jararacussu venom gland. Protein Expr Purif. 2004;37(1):102-8.
• 41. Rodrigues RS, Izidoro LF, Teixeira SS, Silveira LB, Hamaguchi A, Homsi-Brandeburgo MI, et al. Isolation and functional characterization of a new myotoxic acidic phospholipase A₂ from Bothrops pauloensis snake venom Toxicon. 2007;50(1):153-65.
• 42. Chen YC, Maraganore JM, Reardon I, Heinrikson RL. Characterization of the structure and function of three phospholipases A₂ from the venom of Agkistrodon halys pallas. Toxicon. 1987;25(4):401-9.
• 43. Tsai IH, Wang YM, Chen YH, Tu AT. Geographic variations, cloning, and functional analyses of the venom acidic phospholipases A₂ of Crotalus viridis viridis Arch Biochem Biophys. 2003;411(2):289-96.
• 44. Tsai IH, Wang YM, Chen YH, Tsai TS, Tu MC. Venom phospholipases A₂ of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries. Biochem J. 2004;377(Pt 1):215-23.
• 45. Magro AJ, Murakami MT, Marcussi S, Soares AM, Arni RK, Fontes MR. Crystal structure of an acidic platelet aggregation inhibitor and hypotensive phospholipase A₂ in the monomeric and dimeric states: insights into its oligomeric state. Biochem Biophys Res Commun. 2004;323(1):24-31.
• 46. Gu L, Zhang H, Song S, Zhou Y, Lin Z. Structure of an acidic phospholipase A₂ from the venom of Deinagkistrodon acutus Acta Crystallogr D Biol Crystallogr. 2002;58(pt 1):104-10.
• 47. Jasti J, Paramasivam M, Srinivasan A, Singh TP. Structure of an acidic phospholipase A₂ from Indian saw-scaled viper (Echis carinatus) at 2.6 Ĺ resolution reveals a novel intermolecular interaction. Acta Crystallogr D Biol Crystallogr. 2004;60(1):66-72.
• 48. Fuly AL, Calil-Elias S, Martinez AM, Melo PA, Guimarăes JA. Myotoxicity induced by an acidic Asp-49 phospholipase A₂ isolated from Lachesis muta snake venom: comparison with lysophosphatidylcholine. Int J Biochem Cell Biol. 2003;35(10):1470-81.
• 49. Fuly AL, Machado OL, Alves EW, Carlini CR. Mechanism of inhibitory action on platelet activation of a phospholipase A₂ isolated from Lachesis muta (Bushmaster) snake venom. Thromb Haemost. 1997;78(5):1372-80.
• 50. Liu X, Wu X, Zhou Y. Identification of key residues responsible for enzymatic and platelet-aggregation-inhibiting activities of acidic phospholipase A₂ from Agkistrodon halys pallas. J Nat Toxins. 2001;10(1):43-55.
• 51. Wang XQ, Yang J, Gui LL, Lin ZJ, Chen YC, Zhou YC. Crystal structure of an acidic phospholipase A₂ from the venom of Agkistrodon halys Pallas at 2.0 a resolution. J Mol Biol. 1996;255(5):669-76.
• 52. Chen YH, Wang YM, Hseu MJ, Tsai IH. Molecular evolution and structure-function relationships of crotoxin-like and asparagine-6-containing phospholipases A₂ in pit viper venoms. Biochem J. 2004;381(Pt 1):25-34.
• 53. Choumet V, Lafaye P, Demangel C, Bon C, Mazié JC. Molecular mimicry between a monoclonal antibody and one subunit of crotoxin, a heterodimeric phospholipase A₂ neurotoxin. Biol Chem. 1999;380(5):561-8.
• 54. Tsai IH, Wang YM, Au LC, Ko TP, Chen YH, Chu YF. Phospholipases A₂ from Callosellasma rhodostoma venom gland: cloning and sequencing of 10 of the cDNAs, three-dimensional modeling and chemical modification of the major isozyme. Eur J Biochem. 2000;267(22):6684-91.
• 55. Gibbs HL, Rossiter W. Rapid evolution by positive selection and gene gain and loss: PLA(2) venom genes in closely related Sistrurus rattlesnakes with divergent diets. J Mol Evol. 2008;66(2):151-66.

Publication Dates

• Publication in this collection
  27 Nov 2009
• Date of issue
  2009

History

• Accepted
  30 Nov 2009
• Reviewed
  15 Sept 2009
• Received
  22 Nov 2008