Snake Venom Phospholipase A₂

Phospholipases are a class of ubiquitous enzymes that have the common substrate, phospholipid, and they all hydrolyze the different ester linkages of phospholipid. Depending on which ester linkage hydrolyzed, phospholipases are grouped into four major categories-A, B, C and D. Phospholipase A, originally termed as lecithinase A by Contardi and Ercoli [1], was found for the first time in the venoms of cobra viper family at the end of 19^th century, and was further divided into Phospholipase A₁ (cleaves the sn-1 acyl ester of the glycerol backbone) and phospholipase A₂ (cleaves the sn-2 acyl ester of the glycerol backbone). Phospholipase B cleaves either sn-1 or sn-2 acyl ester of the glycerol backbone. Both phospholipase A and B enzyme are also known as a lysophospholipase. Phospholipase C and D are the phosphodiesterases, and they cleave before and after the phosphate, respectively [2]. Snake venom phospholipase hydrolyze the 2-acyl groups in sn-3- phosphoglycerides, thus belong to phospholipase A₂ (PLA₂). In 1994, Dennis for the first time established the systematic group numbering system for PLA₂ enzymes; afterwards, PLA₂ family has grown continuously, and now the superfamily of PLA₂ enzymes currently consists of 16 Groups [3] [4,5]. The major five types of PLA₂s in this 16 groups are Ca+2-dependent secretory PLA₂s (sPLA₂), Ca⁺²-dependent cytosolic PLA₂s (cPLA₂), Ca²⁺-independent intracellular PLA₂s (iPLA₂), platelet activation factor acetyl hydrolases (PAF-AH), and lysosomal PLA₂s [6]. Snake venom PLA₂s, part of secretory PLA₂ (sPLA₂), from old world snakes (Elapidae family) including Kraits (Bungarus species), the Indian cobra species (Naja species), tiger snake (Hemachatus haemuchatus) and the Australian tiger snake (Notechis scututus scutatus) belong to group IA, while from new world snakes (Crotalidae family) including Japanese water moccasin, rattlesnakes and vipers belong to group IIA, as well as from Viperidae family including Gaboon adder, Bitis gabonica belong to IIB [7,8]. Compared with Group I snake venom PLA₂s, the most distinguishing characteristic of Group II snake venom PLA₂ enzymes is the presence of a negative charged COOH-terminal extension of 6-7 residues [7]. Based on the amino acid composition surrounding the active site, Group II snake venom PLA₂s can be divided into, at least, two isoforms: (a) the Asp49 enzymes, having an aspartic acid residue at position 49, show catalytic activity, and (b) the Lys49 proteins, possessing a lysine residue at position 49, are catalytically inactive [9]. According to the primary structure and pI, the catalytically-active isoforms can be further subdivided into acidic and basic sub-isoforms, and the catalytically-inactive isoforms (Lys49) into only basic subisoforms [10]. In addition, two S49 PLA₂ (Serine residue at position 49) proteins [11,12], and one R49 PLA₂ (Arginine residue at positon 49) from snake (Protobothrops mucrosquamatus) [13] have been reported, suggesting the existence of more PLA₂ isoforms in the snake venoms.

Proteins and peptides (commonly referred to as toxins) constitute 90-95% of the dry weight of snake venom, and belong to relatively small stable protein families [14,15]. PLA₂s are the major component of snake venom proteomes, especially in the venoms of Agkistrodon contortrix contortrix [16], Micrurus lemniscatus [17], Agkistrodon piscivorus leucostoma [18-20], and Bungarus multicinctus [14]. Due to the pharmacological and physiopathological effects of snake venom PLA₂ in living organisms, snake venom PLA₂s have been extensively studied. Since two proteins with phospholipase A activity were purified from venom of Eastern diamondback rattlesnake (Crotus. adamanteus) [21] and crystalized [22], and one phospholipase A₂ with MW of 14.5 purified from the venom of snake (Crotalus atrox) [23], more than 500 PLA₂s isolated from snake venoms (based on NCBI and UniProt databases). The amino acid sequences of many snake venom PLA₂s have already determined, and some of their structures have been resolved by X-ray crystallography. The secreted PLA₂s including snake venom PLA₂s are characterized by a low molecular weight (13-15 kDa), and containing histidine in the catalytic site, Ca²⁺ bond in the active site, six conserved disulfide bonds with one or two variable disulfide bonds [24], and more than 50% α-helix and 10% β-sheet [25].

Antibacterial Potential of Snake Venom PLA₂

Snake venoms contain numerous medically important proteins and peptides with varied physiological activities [26,27]. Since Glaser [28] experimentally observed, for the first time, the bactericidal activity of venom from Crotalus snakes, a considerable body of work has been reported the antibacterial effects of the crude venoms as well as venom components from different snake species. The major components reported possessing antibacterial activity in snake venom to date include PLA₂ [29], L-amino acid oxidase [30], metalloproteinase [31] and lectin [32]. Among these venom molecules, PLA₂ is well documented possessing antibacterial activity. Forst et al. [33] demonstrated that PLA₂s from the venoms of snakes (Agkistrodon halys blomhoffii) and (Agkistrodon halys palas) actively hydrolyzed the phospholipids of the bactericidal/permeability-increasing protein (BPI)-treated E. coli. Since then, many researchers claimed that snake venom PLA₂s exert antibacterial effects [13,29,34-46]. However, it is interesting that Resende et al. [47] and Jia et al. [48] reported that PLA₂s isolated from cottonmouth snake venoms display no antibacterial effects, implying that not all snake venom PLA₂ possesses antibacterial effects. The precise bactericidal mechanisms of Group IIA PLA₂ such as hnpsPLA₂ isolated from human tissues and cells were clarified, and the authors demonstrated that the antibacterial activity of hnpsPLA₂ is due to a large excess of cationic residues on its surface [49]. However, it seems that the molecular mechanisms underpinning antibacterial activity of snake venom PLA₂ are varied [29,39,41] or need to be determined.

References

1. Contardi A, Ercoli A (1932) Uber die enzymatische spaltung der lecithine and lysocithine. Biochem 261: 275.
2. Waite M (1987) Phospholipases. In: Donald J Hanahan (Ed), Handbook of Lipid Research. Plenum Press, New York, NY.
3. Dennis EA (1994) Diversity of group types, regulation, and function of phospholipase A₂. J Biol Chem 269: 13057-13060.
4. Schaloske RH, Dennis EA (2006) The phospholipase A₂ superfamily and its group numbering system. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1761: 1246-1259.
5. Duncan RE, Sarkadi-Nagy E, Jaworski K, Ahmadian M, Sul HS (2008) Identification and functional characterization of adipose-specific phospholipase A₂ (AdPLA). J Biol Chem 283: 25428-25436.
6. Dennis EA, Cao J, Hsu YH, Magrioti V, Kokotos G (2011) Phospholipase A₂ enzymes: Physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chem Rev 111: 6130-6185.
7. Heinrikson RL, Krueger ET, Keim PS (1977) Amino acid sequence of phospholipase A₂-α from the venom of Crotalus adamanteus. J Biol Chem 252: 4913-4921.
8. Davidson FF, Dennis EA (1990) Evolutionary relationships and implications for the regulation of phospholipase A₂ from snake venom to human secreted forms. J Mol Evol 31: 228-238.
9. Maraganore JM, Merutka G, Cho W, Welches W, Kezdy FJ, et al. (1984) A new class of phospholipases A₂ with lysine in place of aspartate 49. Functional consequences for calcium and substrate binding. J Biol Chem 259: 13839-13843.
10. Lomonte B, Rangel J (2012) Snake venom Lys49 myotoxins: From phospholipases A₂ to non-enzymatic membrane disruptors. Toxicon 60: 520-530.
11. Krizaj I, Bieber AL, Ritonja A, Gubensek F (1991) The primary structure of ammodytin L, a myo- toxic phospholipase A₂ homologue from Vipera ammodytes venom. Eur J Biochem 202: 1165-1168.
12. Polgar J, Magnenat EM, Peitsch MC, Wells TNC, Clemetson KJ (1996) Asp-49 is not an absolute prerequisite for the enzymic activity of low-Mr phospholipases A₂: purification, characterization and computer modelling of an enzymically active Ser-49 phospholipase A₂, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper). Biochem J 319: 961-968.
13. Wei JF, Li T, Wei XL, Sun QY, Yang FM, et al. (2006) Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A₂ from the venom of Protobothrops mucrosquamatus. Biochimie 88: 1331-1342.
14. Tu AT (1988) Snake venoms: general background and composition. In: Venoms: Chemistry and Molecular Biology. John Willey and Sons, New York pp: 1-19.
15. Fry BG, Wuster W (2004) Assembling an arsenal: origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences. Mol Biol Evol 21: 870-873.
16. Bocian A, Urbanik M, Hus K, Lyskowski A, Petrilla V, et al. (2016) Proteomic analyses of Agkistrodon contortrix contortrix venom using 2D electrophoresis and MS technique. Toxins 8: 372.
17. Casais-e-Silva LL, Teixeira CFP, Lebrun I, Lomonte B, Gutiérrez JM (2016) Leminitoxin, the major component of Micrurus leminiscatus coral snake venom is a myotoxic and pro-inflammatory phospholipase A₂. Toxicol Lett 257: 60-71.
18. Lomonte B, Tsai WC, Ureña-Diaz JM, Sanz L, Mora-Obando D, et al. (2014) Venomics of New world pit vipers: genus-wide comparisons of venom proteomes across Agkistrodon. J Proteomics 96: 103-116.
19. Jia Y, Cantu BA, Sanchez EE, Perez JC (2008) Complementary DNA sequencing and identification of mRNAs from venomous gland of Agkistrodon piscivorus leucostoma. Toxicon 51: 1457-1466.
20. Jia Y, Ermolinsky B, Garza A, Provenzano D (2017) Phospholipase A₂ in the venom of three cottonmouth snakes. Toxicon 135: 84-92.
21. Saito K, Hanahan DJ (1962) A study of the purification and properties of the phospholipase A of Crotalus adamanteus venom. Biochemistry 1: 521-532.
22. Wells MA, Hanahan DJ (1969) Phospholipase A. I. Isolation and characterization of two enzymes from Crotalus adamanteus venom. Biochemistry 8: 414-424.
23. Wu TW, Tinker DO (1969) Phospholipase A₂ from Crotalus atrox venom. I. Purification and some properties. Biochemistry 8: 1558-1568.
24. Burke JE, Dennis EA (2009) Phospholipase A₂ structure⁄function, mechanism, and signaling. J Lipid Res 50: S237-S242.
25. Dufton MJ, Eaker D, Hider RC (1983) Conformational properties of phospholipase A₂ -secondary structure predication, circular dichroism and relative interface hydrophobicity. Eur J Biochem 137: 537-544.
26. Calvete JJ, Fasoli E, Sanz L, Boschetti E, Righetti PG (2009) Exploring the Venom Proteome of the Western Diamondback Rattlesnake, Crotalus atrox, via Snake Venomics and Combinatorial Peptide Ligand Library Approaches. J Proteome Research 8: 3055-3067.
27. Casewell NR, Wagstaff SC, Wűster W, Cook DAN, Bolton FMS, et al. (2014) Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms. PNAS 111: 9205-9210.
28. Glasser HR (1948) Bactericidal activity of Crotalus venom in vitro. Copeia 4: 245-247.
29. Páramo L, Lomonte B, Pizarro-Cerdá J, Bengoechea JA, Gorvel JP, et al. (1998) Bactericidal activity of Lys49 and Asp49 myotoxic phospholipase A₂ from Bothrops asper snake venom: synthetic Lys49 myotoxin II- (115-129) peptide identifies its bactericidal region. Eur J Biochem 253: 452-461.
30. Lee ML, Tan NH, Fung SY, Sekaran SD (2011) Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus Hannah) venom. Comp Biochem Physiol Toxicol Pharmacol 153: 237-242.
31. Samy RP, Gpalakrishnakone P, Chow VT, Ho B (2008a) Viper metalloproteinase (Agkistrodon halys pallas) with antibacterial activity against multi-drug resistant human pathogens. J Cell Physiol 261: 54-68.
32. Nunes Edos S, de Souza MA, Vaz AF, Santana GM, Gomes FS, et al. (2011) Purification of a lectin with antibacterial activity from Bothrops leucurus snake venom. Comparative Biochemistry and Physiology Part B 159: 57-63.
33. Forst S, Weiss J, Blackburn P, Frangione B, Goni F, et al. (1986) Amino acid sequence of a basic Agkistrodon halys blomhoffii phospholipase A₂. Possible role of NH2-terminal lysines in action on phospholipids of Escherichia coli. Biochem 25: 4309-4314.
34. Soares AM, Mancin AC, Cecchini AL, Arantes EC, França SC, et al. (2001) Effects of chemical modifications of crotoxin B, the phospholipase A₂ subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities. Int J Biochem Cell Biol 33: 877-888.
35. Rodrigues VM, Marcussi S, Cambraia RS, de Araújo AL, Malta-Neto NR, et al. (2004) Bactericidal and neurotoxic activities of two myotoxic phospholipases A₂ from Bothrops neuwiedi pauloensis snake venom. Toxicon 44: 305-314.
36. Roberto PG, Kashima S, Marcussi S, Pereira JO, Astolfi-Filho S, et al. (2004) Cloning and identification of a complete cDNA coding for a bactericidal and antitumoral Acidic phospholipase A₂ from Bothrops jararacussu venom. The Protein J 23: 273-285.
37. Santamaría C, Larios S, Angulo Y, Pizarro-Cerda J, Gorvel JP, et al. (2005a) Antimicrobial activity of myotoxic phospholipase A₂ from crotalid snake venoms and synthetic peptide variants derived from their C-terminal region. Toxicon 45: 807-815.
38. Santamaría C, Larios S, Quiró S, Pizarro-Cerda J, Gorvel JP, et al. (2005b) Bactericidal and antiendotoxic properties of short cationic peptides derived from a snake venom Lys49 phosphlipase A₂. Antimicrobial Agents and Chemotherapy 49: 1340-1345.
39. Samy RP, Gopalakrishnakone P, Ho B, Chow VTK (2008b) Purification, characterization and bactericidal activities of basic phospholipase A₂ from the venom of Agkistrodon halys (Chinese pallas). Biochimie 90: 1372-1388.
40. Samy RP, Gopalakrishnakone P, Bow H, Puspharaj PN, Chow VTK (2010) Identification and characterization of a phospholipase A₂ from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities. Biochimie 92: 1854-1866.
41. Samy RP, Gopalakrishnakone P, Stiles BG, Girish KS, Swamy SN, et al. (2012) Snake venom phospholipase A₂: a novel tool against bacterial diseases. Curr Med Chem 19: 6150-6162.
42. Samy RP, Kandasamy M, Gopalakrishnakone P, Stiles BG, Rowan EG, et al. (2014) Wound healing activity and mechanisms of action of an antibacterial protein from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). PLoS One 9: e80199.
43. Vargas LJ, Londoño M, Quintana JC, Rua C, Segura C, et al. (2012) An acidic phospholipase A with antibacterial activity from Porthidium nasutum snake venom. Comp Biochem Physiol B Biochem Mol Biol 161: 341-347.
44. Conlon JM, Attoub S, Arafat H, Mechkarska M, Casewell NR, et al. (2013) Cytotoxic activities of [Ser⁴⁹] phospholipase A₂ from the venom of the saw-scaled vipers Echis ocellatus, Echis pyramidum leakeyi, Echis carinatus sochureki, and Echis coloratus. Toxicon 71: 96-104.
45. Almeida JR, Lancellotti M, Soares AM, Calderon LA, Ramírez D, et al. (2016) CoaTx-II, a new dimeric Lys49 phospholipase A₂ from Crotalus oreganus abyssus snake venom with bactericidal potential: insights into its structure and biological roles. Toxicon 120: 147-158.
46. Bacha AB, Alonazi MA, Elshikh MS, Karray A (2018) A novel bactericidal homodimeric PLA₂ group-I from Walterinnesia aegyptia venom. Inter J Biological Macromolecules 117: 1140-1146.
47. Resende LM, Almeida JR, Schezaro-Ramos R, Collaco RCO, Simioni LR, et al. (2017) Exploring and understanding the functional role, and biochemical and structural characteristics of an acidic phospholipase A₂, AplTx-I, purified from Agkistrodon piscivorus leucostoma snake venom. Toxicon 127: 22-36.
48. Jia Y, Villarreal J (2018) Phospholipase A₂ purified from cottonmouth snake venoms display no antibacterial effect against four representative bacterial species. Toxicon 151: 1-4.
49. Beers SA, Buckland AG, Koduri RS, Cho W, Gelb MH, et al. (2002) The antibacterial properties of secreted phospholipase A₂. J Biol Chem 277: 1788-1793.