Snake Venom sPLA2s Research Articles

Bactericidal Effects of Snake Venom Phospholipases A2: A Systematic Review and Analysis of Minimum Inhibitory Concentration

Background: Infections caused by multi-drug resistance (MDR) strains are potentially fatal public health issues worldwide that need pressing attention. Previous reports suggested using snake venom fractions as an effective alternative mechanism to the already available antibacterial drugs. In this study, we conducted a systematic review to analyze the bactericidal effects of snake venom phospholipases (PLA2s). Methods: From the beginning through 30 March 2022, we searched the PubMed and Embase databases in accordance with the most recent PRISMA recommendations. We also conducted a manual search to identify relevant reports to improve literature coverage. Results: A total of 24 studies were included based on the selection criteria to compile this review. Of them, 16 studies were obtained from the abovementioned databases and eight through manual searches. The other 8 studies were obtained through the references of the included studies. According to the review, we reported that some PLA2s showed more vigorous bactericidal activity on some Gram-negative and a moderate effect on Gram-negative and Gram-positive. Furthermore, we reported that the presence of p-bromophenacyl bromide (p-BPP) showed a significant decrease in enzymatic and associated antibacterial activities. Moreover, we observed that about 80% of the PLA2s reported in our systematic review study were those from the Viperidae family, whereas 20% came from the Elapidae family. Moreover, some variations were revealed in the current study regarding the mechanism of actions of the snake venom PLA2s (svPLA2s). Conclusion: This systematic review provides a comprehensive overview of the bactericidal effect of snake venom PLA2s and the analysis of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PLA2s for bacterial strains. Varying bactericidal effects from various snake species and South American rattlesnakes were reported, presenting compelling concepts to the alternative search for therapies against bacterial resistance. Thus, further analysis of the bactericidal effects of other snake venoms PLA2s considering different strains is needed. Moreover, more data are needed to investigate other bacteria of public health priority using peptides and other purified snake toxins.

Secreted Phospholipases A₂ from Animal Venoms in Pain and Analgesia.

Animal venoms comprise a complex mixture of components that affect several biological systems. Based on the high selectivity for their molecular targets, these components are also a rich source of potential therapeutic agents. Among the main components of animal venoms are the secreted phospholipases A2 (sPLA2s). These PLA2 belong to distinct PLA2s groups. For example, snake venom sPLA2s from Elapidae and Viperidae families, the most important families when considering envenomation, belong, respectively, to the IA and IIA/IIB groups, whereas bee venom PLA2 belongs to group III of sPLA2s. It is well known that PLA2, due to its hydrolytic activity on phospholipids, takes part in many pathophysiological processes, including inflammation and pain. Therefore, secreted PLA2s obtained from animal venoms have been widely used as tools to (a) modulate inflammation and pain, uncovering molecular targets that are implicated in the control of inflammatory (including painful) and neurodegenerative diseases; (b) shed light on the pathophysiology of inflammation and pain observed in human envenomation by poisonous animals; and, (c) characterize molecular mechanisms involved in inflammatory diseases. The present review summarizes the knowledge on the nociceptive and antinociceptive actions of sPLA2s from animal venoms, particularly snake venoms.

Enzymatic action of phospholipase A2 on liposomal drug delivery systems

The overexpression of secretory phospholipase A2 (sPLA2) in tumors has opened new avenues for enzyme-triggered active unloading of liposomal antitumor drug carriers selectively at the target tumor. However, the effects of the liposome composition, drug encapsulation, and tumor microenvironment on the activity of sPLA2 are still not well understood. We carried out a physico-chemical study to characterize the sPLA2-assisted breakdown of liposomes using dye-release assays in the context of drug delivery and under physiologically relevant conditions. The influence of temperature, lipid concentration, enzyme concentration, and drug loading on the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Tm=42°C) liposomes with snake venom sPLA2 was investigated. The sensitivity of human sPLA2 to the liposome composition was checked using binary lipid mixtures of phosphatidylcholine (PC) and phosphatidylglycerol (PG) phospholipids with C14 and C16 acyl chains. Increasing temperature (36–41°C) was found to mainly shorten the enzyme lag-time, whereas the effect on lipid hydrolysis rate was modest. The enzyme lag-time was also found to be inversely dependent on the lipid-to-enzyme ratio. Drug encapsulation can alter the hydrolysis profile of the carrier liposomes. The activity of human sPLA2 was highly sensitive to the phospholipid acyl-chain length and negative surface charge density of the liposomes. We believe our work will prove useful for the optimization of sPLA2-susceptible liposomal formulations as well as will provide a solid ground for predicting the hydrolysis profile of the liposomes in vivo at the target site.

Interactions of pharmacologically active snake venom sPLA2 with different cell lines

Secreted Phospholipases A2 (sPLA2s) represent a large family of structurally related enzymes, which target different tissues and organs and induce numerous pharmacological effects based on their catalytic specificity – hydrolysis of the sn-2 ester bond of glycerophospholipids. The neurotoxin vipoxin, isolated from the venom of Vipera ammodytes meriodionalis, is a heterodimeric postsynaptic ionic complex composed of two protein subunits – a basic and toxic His48 sPLA2 enzyme and an acidic, enzymatically inactive and non-toxic component. In this paper, for the first time, we demonstrate that vipoxin sPLA2 enzyme affects cell integrity and viability of four cell types and causes different cell responses. The most dramatic local tissue effects were observed with RPE-1 (retinal pigment epithelial) cells followed by A549 (adenocarcinomic human alveolar epithelial) cells and MDCK (Madin-Darby Canine Kidney epithelial) cells. Products of the enzymatic reaction, lysophospholipids and unsaturated free fatty acids, act as lipid mediators that can induce membrane damaging or can stimulate cell proliferation. Our preliminary results on the cytotoxic effect of vipoxin sPLA2 on A549 cells are promising in searching of its eventual anticancer potential.

Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons

The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-9 toxins, an increase in free cytosolic Ca2+ and a reduction in mitochondrial transmembrane potential (ΔΨm) became evident and occurred prior to the morphological changes and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed in the electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic Ca2+ and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s.

L-type voltage-dependent calcium channel is involved in the snake venom group IA secretory phospholipase A2-induced neuronal apoptosis

Snake venom group IA secretory phospholipase A2 (sPLA2-IA) is known as a neurotoxin. Snake venom sPLA2s are neurotoxic in vivo and in vitro, causing synergistic neurotoxicity to cortical cultures when applied with toxic concentrations of glutamate. However, it has not yet been cleared sufficiently how sPLA2-IA exerts neurotoxicity. Here, we found sPLA2-IA induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6h. sPLA2-IA-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. NMDA receptor blockers suppressed the neurotoxicity of sPLA2-IA, but an AMPA receptor blocker did not. Interestingly, L-type voltage-dependent Ca2+ channel (L-VDCC) blocker significantly protected neurons from the sPLA2-IA-induced apoptosis. On the other hand, neither N-VDCC blockers nor P/Q-VDCC blocker did. In conclusion, we demonstrated that sPLA2-IA induced neuronal cell death via apoptosis. Furthermore, the present study suggests that not only NMDA receptor but also L-VDCC contributed to the neurotoxicity of snake venom sPLA2-IA.

Ammodytoxins efficiently release arachidonic acid and induce apoptosis in a motoneuronal cell line in an enzymatic activity-dependent manner

Secreted phospholipases A2 (sPLA2s) are phospholipolytic enzymes and receptor ligands whose action affects cell death and survival. We have previously shown that ammodytoxin A (AtxA), a snake venom sPLA2, is rapidly internalized into motoneuronal NSC34 cells, inducing characteristic neurotoxic sPLA2 cell damage and apoptosis. In this study, we have analyzed the role of sPLA2 enzymatic activity, including arachidonic acid (AA) release, in the induction of motoneuronal apoptosis by AtxA and homologous recombinant sPLA2s with different enzymatic properties: an AtxA mutant (V31W) with very high enzymatic activity, enzymatically inactive S49-sPLA2 (ammodytin L, AtnL), its mutant (LW) with restored enzymatic activity, and non-toxic, enzymatically active sPLA2 (AtnI2). Addition of AA, AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, to NSC34 cells resulted in caspase-3 activation, DNA fragmentation and disruption of mitochondrial membrane potential, leading to a significant and rapid decrease in motoneuronal cell viability that was not observed in C2C12 myoblasts and HEK293 cells. AtxA, AtxA-V31W and AtnL-LW, but not AtnL and AtnI2, also liberated large amounts of AA specifically from motoneuronal cells, and this ability correlated well with the ability to induce apoptotic changes and decrease cell viability. The enzymatic activity of AtxA and similar sPLA2s is thus necessary, but not sufficient, for inducing motoneuronal apoptosis. This suggests that specific binding to the motoneuronal cell surface, followed by internalization and enzymatic activity-dependent induction of apoptosis, possibly as a consequence of extensive extra- and intracellular AA release, is necessary for Atx-induced motoneuronal cell death.

Secretory phospholipases A 2 isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E 2 and D 2 and expression of cyclooxygenases

The effects of myotoxin III (MT-III), a phospholipase A 2 (sPLA 2) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA 2 from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA 2s promoted the synthesis of PGD 2 and PGE 2, with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA 2s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. Inhibition of cPLA 2 by AACOCF 3, but not of iPLA 2 by PACOCF 3 or BEL, significantly reduced PGD 2, PGE 2 and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA 2 inhibition did not modify the effects of CB, whereas iPLA 2 inhibition reduced PGD 2 and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs' synthesis are triggered by these snake venom sPLA 2s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA 2s in vivo.

Mapping the structural determinants of presynaptic neurotoxicity of snake venom phospholipases A 2

The structural features of presynaptically neurotoxic secretory phospholipases A 2 (sPLA 2s) that are responsible for their potent and specific action are still a matter of debate. To identify the residues that distinguish a highly neurotoxic sPLA 2, ammodytoxin A (AtxA), from a structurally similar but more than two orders of magnitude less toxic Russell's viper sPLA 2, VIIIa, we prepared a range of mutants and compared their properties. The results show that the structural features that confer high neurotoxicity to AtxA extend from its C-terminal part, with a central role of the residues Y115, I116, R118, N119 (the YIRN cluster) and F124, across the interfacial binding surface (IBS) in the vicinity of F24, to the N-terminal helix whose residues M7 and G11 are located on the edges of the IBS. Competition binding studies indicate that the surface of interaction with the neuronal M-type sPLA 2 receptor R180 extends over a similar region of the molecule. In addition, the YIRN cluster of AtxA is crucial for the high-affinity interaction with two intracellular binding proteins, calmodulin and R25. The concept of a single “presynaptic neurotoxic site” on the surface of snake venom sPLA 2s is not consistent with these results which suggest that different parts of the toxin molecule are involved in distinct steps of presynaptic neurotoxicity.

Secretory PLA2 inhibitor indoxam suppresses LDL modification and associated inflammatory responses in TNFα‐stimulated human endothelial cells

Secretory phospholipase A2 (sPLA2) is implicated in atherosclerosis, although the effects of specific sPLA2 inhibitors have not been studied. We investigated the effects of the indole analogue indoxam on low-density lipoprotein (LDL) modification by sPLA2 enzymes of different types and on the associated inflammatory responses in human umbilical vein endothelial cells (HUVEC). LDL modification was assessed by measuring the contents of two major molecular species of lysophosphatidylcholine (LPC) using electrospray ionization-liquid chromatography/mass spectrometry. The proinflammatory activity of the modified LDL was evaluated by determining monocyte chemoattractant protein-1 (MCP-1) mRNA expression and transcriptional factor nuclear factor-kappaB (NF-kappaB) activity in HUVEC. Indoxam dose-dependently inhibited palmitoyl- and stearoyl-LPC production in LDL incubated with snake venom sPLA2 (IC50 1.2 microM for palmitoyl-LPC, 0.8 microM for stearoyl-LPC). MCP-1 mRNA expression and NF-kappaB activity were enhanced by venom sPLA2-treated LDL, which was completely suppressed by indoxam but not by thioetheramide-PC, a competitive sPLA2 inhibitor. Indoxam also suppressed LPC production in LDL treated with human synovial type IIA sPLA2. Tumour necrosis factor alpha (TNFalpha) increased type V sPLA2 expression in HUVEC. Indoxam dose-dependently suppressed LPC production in native and glycoxidized LDL treated with TNFalpha-stimulated HUVEC. Indoxam suppressed MCP-1 mRNA expression and NF-kappaB activity in TNFalpha-stimulated HUVEC incubated with native or glycoxidized LDL. Indoxam prevented sPLA2-induced LPC production in native and glycoxidized LDL as well as LDL-induced inflammatory activity in HUVEC. Our results suggest that indoxam may be a potentially useful anti-atherogenic agent.

Crystal structure of Natratoxin, a novel snake secreted phospholipaseA2 neurotoxin from Naja atra venom inhibiting A‐type K+ currents

Snake secreted phospholipasesA2 (sPLA2s) are widely used as pharmacological tools to investigate their role in diverse pathophysiological processes. Some members of snake venom sPLA2s have been found to block voltage-activated K(+) channels (K(v) channels). However, most studies involved in their effects on ion channels were indirectly performed on motor nerve terminals while few studies were directly done on native neurons. Here, a novel snake sPLA2 peptide neurotoxin, Natratoxin, composed of 119 amino acid residues and purified from Naja atra venom was reported. It was characterized using whole-cell patch-clamp in acutely dissociated rat dorsal root ganglion (DRG) neurons. It was found to effectively inhibit A-type K(+) currents and cause alterations of channel gating characters, such as the shifts of steady-state activation and inactivation curves to hyperpolarization direction and changes of V(1/2) and slope factor. Therefore, Natratoxin was suggested to be a gating modifier of K(v) channel. In addition, this inhibitory effect was found to be independent of its enzymatic activity. These results suggested that the toxin enacted its inhibitory effect by binding to K(v) channel. To further elucidate the structural basis for this electrophysiological phenomenon, we determined the crystal structure of Natratoxin at 2.2 A resolution by molecular replacement method and refined to an R-factor of 0.190. The observed overall fold has a different structural organization from other K(+) channel inhibitors in animal toxins. Compared with other K(v) channel inhibitors, a similar putative functional surface in its C-terminal was revealed to contribute to protein-protein interaction in such a blocking effect. Our results demonstrated that the spatial distribution of key amino acid residues matters most in the recognition of this toxin towards its channel target rather than its type of fold.

Phospholipase A 2 receptor: a regulator of biological functions of secretory phospholipase A 2

The phospholipase A 2 receptor (PLA 2R) is a type I transmembrane glycoprotein related to the C-type animal lectin family that includes the mannose receptor. PLA 2R regulates a variety of biological responses elicited by specific types of secretory PLA 2s (sPLA 2s). Group IB sPLA 2 (sPLA 2-IB) acts as an endogenous PLA 2R ligand to induce cell proliferation, cell migration, and lipid mediator production. Analysis of PLA 2R-deficient mice has suggested a potential role of the sPLA 2-IB/PLA 2R pathway in the production of pro-inflammatory cytokines in endotoxic shock. PLA 2R is also involved in the clearance of sPLA 2s, including group X sPLA 2 (sPLA 2-X) and a particular type of snake venom sPLA 2, and clearance suppresses their potent enzymatic activities. In the circulation, the soluble form of PLA 2R is constitutively present as an endogenous inhibitor of sPLA 2s. This review will focus on recent findings on the roles of PLA 2R in regulating sPLA 2 functions and summarize what is known about the other binding proteins for mammalian and snake venom sPLA 2s.

Both Group IB and Group IIA Secreted Phospholipases A2 Are Natural Ligands of the Mouse 180-kDa M-type Receptor

Snake venom and mammalian secreted phospholipases A2 (sPLA2s) have been associated with toxic (neurotoxicity, myotoxicity, etc.), pathological (inflammation, cancer, etc.), and physiological (proliferation, contraction, secretion, etc.) processes. Specific membrane receptors (M and N types) for sPLA2s have been initially identified with snake venom sPLA2s as ligands, and the M-type 180-kDa receptor was cloned from different animal species. This paper addresses the problem of the endogenous ligands of the M-type receptor. Recombinant group IB and group IIA sPLA2s from human and mouse species have been prepared and analyzed for their binding properties to M-type receptors from different animal species. Both mouse group IB and group IIA sPLA2s are high affinity ligands (in the 1-10 nM range) for the mouse M-type receptor. These two sPLA2s are expressed in the mouse tissues where the M-type receptor is also expressed, making it likely that both types of sPLA2s are physiological ligands of the mouse M-type receptor. This conclusion does not hold for human group IB and IIA sPLA2s and the cloned human M-type receptor. The two mouse sPLA2s have relatively high affinities for the mouse M-type receptor, but they can have much lower affinities for receptors from other animal species, indicating that species specificity exists for sPLA2 binding to M-type receptors. Caution should thus be exerted in avoiding mixing sPLA2s, cells, or tissues from different animal species in studies of the biological roles of mammalian sPLA2s associated with an action through their membrane receptors.

Identification of the Binding Domain for Secretory Phospholipases A2 on Their M-type 180-kDa Membrane Receptor

The rabbit muscle (M)-type receptor for secretory phospholipases A2 (sPLA2s) has a large extracellular domain of 1394 amino acids, composed of an N-terminal cysteine-rich domain, a fibronectin-like type II domain, and eight carbohydrate recognition domains (CRDs). It is thought to mediate some of the physiological effects of mammalian sPLA2s, including vascular smooth muscle contraction and cell proliferation, and is able to internalize sPLA2s. Here, we show by site-directed mutagenesis that OS1, a snake venom sPLA2, binds to the receptor via its CRDs and that deletion of CRD 5 completely abolishes the binding of sPLA2s. Moreover, a receptor lacking all CRDs but CRD 5 was still able to bind OS1 although with a lower affinity. Deletion of CRDs 4 and 6, surrounding the CRD 5, slightly reduced the affinity for OS1, thus suggesting that these CRDs are also involved in the binding of OS1. The M-type sPLA2 receptor and the macrophage mannose receptor are homologous and are predicted to share the same tertiary structure. p-Aminophenyl-alpha-D-mannopyranoside bovine serum albumin, a known ligand of the macrophage mannose receptor, binds to the M-type sPLA2 receptor essentially via CRDs 3-6.

Structural Elements of Secretory Phospholipases A2 Involved in the Binding to M-type Receptors

Specific membrane receptors for secretory phospholipases A2 (sPLA2s) have been initially identified with novel snake venom sPLA2s called OS1 and OS2. One of these sPLA2 receptors (muscle (M)-type, 180 kDa) has a very high affinity for OS1 and OS2 and a high affinity for pancreatic and inflammatory-type mammalian sPLA2s, which might be the natural endogenous ligands of PLA2 receptors. Primary structures of OS1 and OS2 were determined and compared with sequences of other sPLA2s that bind less tightly or do not bind to the M-type receptor. In addition, the binding properties of pancreatic sPLA2 mutants to the M-type receptor have been analyzed. Residues within or close to the Ca(2+)-binding loop of pancreatic sPLA2 are crucially involved in the binding step, although the presence of Ca2+ that is essential for the enzymatic activity is not required for binding to the receptor. These residues include Gly-30 and Asp-49, which are conserved in all sPLA2s. Leu-31 is also essential for binding of pancreatic sPLA2 to its receptor. Many other mutations have been considered. Those occurring in the N-terminal alpha helices and the pancreatic loop do not change binding to the M-type receptor. Conversion of pancreatic prophospholipase to phospholipase is essential for the acquisition of binding properties to the M-type receptor.