ABSTRACT
Currently, the search for new alternatives to conventional antibiotics to combat bacterial resistance is an urgent task, as many microorganisms threaten human health due to increasing bacterial resistance to traditional medicines. Thus, new molecules such as antimicrobial peptides have emerged as promising alternatives because of their low induction of resistance and broad spectrum of action. In this context, in the past few years, our research group has synthesized and characterized a peptide derived from the C-terminal region of the Lys49 PLA2-like BthTX-I, named p-BthTX-I. After several studies, the peptide (p-BthTX-I)2K was proposed as the molecule with the most considerable biotechnological potential. As such, the present work aimed to evaluate whether the modifications made on the peptide (p-BthTX-I)2K can be applied to other molecules originating from the C-terminal region of PLA2-like Lys49 from snake venoms. The peptides were obtained through the solid-phase peptide synthesis technique, and biochemical and functional characterization was carried out using dichroism techniques, mass spectrometry, antimicrobial activity against ESKAPE strains, hemolytic activity, and permeabilization of lipid vesicles. The antimicrobial activity of the peptides was promising, especially for the peptides (p-AppK)2K and (p-ACL)2K, which demonstrated activity against all strains that were tested, surpassing the model molecule (p-BthTX-I)2K in most cases and maintaining low hemolytic activity. The modifications initially proposed for the (p-BthTX-I)2K peptide were shown to apply to other peptides derived from Lys49 PLA2-like from snake venoms, showing promising results for antimicrobial activity. Future assays comparing the activity of the dimers obtained through this strategy with the monomers of these peptides should be carried out.
Subject(s)
Phospholipases A2 , Phospholipases A2/pharmacology , Phospholipases A2/chemistry , Hemolysis/drug effects , Antimicrobial Peptides/pharmacology , Antimicrobial Peptides/chemistry , Antimicrobial Peptides/chemical synthesis , Animals , Microbial Sensitivity Tests , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Humans , Anti-Infective Agents/pharmacology , Anti-Infective Agents/chemistry , Anti-Infective Agents/chemical synthesis , Bacteria/drug effectsABSTRACT
Animal-derived venom, like snake venom, has been proven to be valuable natural resources for the drug development. Previously, snake venom was mainly investigated in its pharmacological activities in regulating coagulation, vasodilation, and cardiovascular function, and several marketed cardiovascular drugs were successfully developed from snake venom. In recent years, snake venom fractions have been demonstrated with anticancer properties of inducing apoptotic and autophagic cell death, restraining proliferation, suppressing angiogenesis, inhibiting cell adhesion and migration, improving immunity, and so on. A number of active anticancer enzymes and peptides have been identified from snake venom toxins, such as L-amino acid oxidases (LAAOs), phospholipase A2 (PLA2), metalloproteinases (MPs), three-finger toxins (3FTxs), serine proteinases (SPs), disintegrins, C-type lectin-like proteins (CTLPs), cell-penetrating peptides, cysteine-rich secretory proteins (CRISPs). In this review, we focus on summarizing these snake venom-derived anticancer components on their anticancer activities and underlying mechanisms. We will also discuss their potential to be developed as anticancer drugs in the future.
Subject(s)
Antineoplastic Agents , Snake Venoms , Humans , Snake Venoms/chemistry , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Animals , Neoplasms/drug therapy , L-Amino Acid Oxidase/chemistry , L-Amino Acid Oxidase/pharmacology , Apoptosis/drug effects , Phospholipases A2/metabolism , Phospholipases A2/chemistry , Toxins, Biological/chemistry , Toxins, Biological/pharmacologyABSTRACT
Endogenous phospholipase A2 (PLA2) plays an important role in phospholipids degradation during cured meat products manufacturing. The present study was undertaken to reveal more information about the endogenous PLA2 in muscles and its role in degradation of intramuscular phospholipids. With the catalytic domain of pork calcium-independent PLA2 (iPLA2cd), impacts of physic-chemical factors on the activity were investigated and substrate specificity of the enzyme were tested respectively. The optimum temperature and pH of pork iPLA2cd were 40 °C and 7.5, respectively. The iPLA2cd could be stimulated by adequate contents of NaCl and ATP, and inhibited by CaCl2 and NaNO2. For native phospholipids, the iPLA2cd was of a little higher affinity towards phosphatidylcholine (PC) than phosphatidylethanolamine (PE), phosphoserine (PS) and phosphatidylinositol (PI). The iPLA2cd could preferentially hydrolyze peroxidized PC over the native PC. The results would help better understand the degradation of phospholipids and the role played by endogenous enzymes during meat products manufacturing.
Subject(s)
Catalytic Domain , Phosphatidylcholines , Phospholipases A2 , Animals , Hydrolysis , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Swine , Phospholipases A2/metabolism , Phospholipases A2/chemistry , Hydrogen-Ion Concentration , Substrate Specificity , Temperature , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/chemistryABSTRACT
Catuneragam nilotica has been used in ethnomedicine to treat snakebite, inflammation, and diarrhea among others. The aim of this research is to isolate, and characterize potential potential phospholipase A2 (PLA2) inhibitors from the roots of C. nilotica. The plant material was collected, authenticated, and sequentially extracted using solvents of increasing polarity starting from n-hexane, ethyl acetate, and methanol. The extracts as reported in our previous work, were screened in vitro for their inhibitory activity against PLA2 enzyme from N. nigricollis venom using acidimetric assay. In line with the bio-activity guided isolation, methanol extract (being the most active) was subjected to chromatographic separation using silica gel and sephadex LH-20 which resulted in the isolation and characterization of scopoletin, and scopolin; the compounds were able to inhibit the hydrolytic actions of PLA2 enzyme with percentage inhibition ranging from 67.82 to 100.00 % and 65.76-93.15 %, respectively while the standard Antisnake Venom (ASV) had 74.96-85.04 % after 10 min incubation at 37 °C. The molecular docking of the compounds against PLA2 enzyme was performed using Auto Dock Vina while ADME-Tox analysis was evaluated using swissADME and ProTox-II online servers; The findings indicated that both compounds were able to bind to the active site of PLA2 enzyme with high affinity (-6.5 to -6.2 kcal/mol) and they exhibited favorable drug-likeness and pharmacokinetic properties, and according to toxicity predictions, scopolin was found to be non-toxic (LD50 of 5000 mg/kg) while scopoletin has a slight chance of being toxic (LD50 of 3800 mg/kg). In conclusion, the findings of the research revealed that the roots of C. nilotica contains phytoconstituents with anti-PLA2 enzyme activity and thus, validates the ethnomedicinal claim of the use of the plant as herbal therapy against N. nigricollis envenomation.
Subject(s)
Molecular Docking Simulation , Phospholipase A2 Inhibitors , Phospholipases A2 , Plant Roots , Scopoletin , Animals , Elapid Venoms/enzymology , Elapid Venoms/chemistry , Naja , Phospholipase A2 Inhibitors/pharmacology , Phospholipases A2/chemistry , Plant Extracts/pharmacology , Plant Extracts/chemistry , Plant Roots/chemistry , Scopoletin/pharmacology , Coumarins/chemistry , Coumarins/pharmacologyABSTRACT
The phospholipase A2 (PLA2) superfamily consists of lipolytic enzymes that hydrolyze specific cell membrane phospholipids and have long been considered a central hub of biosynthetic pathways, where their lipid metabolites exert a variety of physiological roles. A misregulated PLA2 activity is associated with mainly inflammatory-derived pathologies and thus has shown relevant therapeutic potential. Many natural and synthetic anti-inflammatory drugs (AIDs) have been proposed as direct modulators of PLA2 activity. However, despite the specific chemical properties that these drugs share in common, little is known about the indirect modulation able to finely tune membrane structural changes at the precise lipid-binding site. Here, we use a novel experimental strategy based on differential scanning calorimetry to systematically study the structural properties of lipid membrane systems during PLA2 cleavage and under the influence of several AIDs. For a better understanding of the AIDs-membrane interaction, we present a comprehensive and comparative set of molecular dynamics (MD) simulations. Our thermodynamic results clearly demonstrate that PLA2 cleavage is hindered by those AIDs that significantly reduce the lipid membrane cooperativity, while the rest of the AIDs oppositely tend to catalyze PLA2 activity to different extents. On the other hand, our MD simulations support experimental results by providing atomistic details on the binding, insertion, and dynamics of each AID on a pure lipid system; the drug efficacy to impact membrane cooperativity is related to the lipid order perturbation. This work suggests a membrane-based mechanism of action for diverse AIDs against PLA2 activity and provides relevant clues that must be considered in its modulation.
Subject(s)
Molecular Dynamics Simulation , Phospholipids , Phospholipases A2/chemistry , Phospholipids/chemistry , Cell Membrane/metabolism , Biophysical PhenomenaABSTRACT
Snake venoms are known to contain toxins capable of interfering with normal physiological processes of victims. Specificity of toxins from snake venoms give scope to identify new molecules with therapeutic action and/or help to understand different cellular mechanisms. Russell's viper venom (RVV) is a mixture of many bioactive molecules with enzymatic and non-enzymatic proteins. The present article describes Daboialipase (DLP), an enzymatic phospholipase A2 with molecular mass of 14.3 kDa isolated from RVV. DLP was obtained after cation exchange chromatography followed by size-exclusion high performance liquid chromatography (SE-HPLC). The isolated DLP presented strong inhibition of adenosine di-phosphate (ADP) and collagen induced platelet aggregation. It also showed anti-thrombin properties by significantly extending thrombin time in human blood samples. Trypan blue and resazurin cell viability assays confirmed time-dependent cytotoxic and cytostatic activities of DLP on MCF7 breast cancer cells, in vitro. DLP caused morphological changes and nuclear damage in MCF7 cells. However, DLP did not cause cytotoxic effects on non-cancer HaCaT cells. Peptide sequences of DLP obtained by O-HRLCMS analysis showed similarity with a previously reported PLA2 (Uniprot ID: PA2B_DABRR/PDB ID: 1VIP_A). An active Asp at 49th position, calcium ion binding site and anticoagulant activity sites were identified in 1 VIP_A. These findings are expected to contribute to designing new anti-platelet, anticoagulant and anti-cancer molecules.
Subject(s)
Anticoagulants , Phospholipases A2 , Vipera , Animals , Humans , Anticoagulants/chemistry , Anticoagulants/isolation & purification , Anticoagulants/pharmacology , Phospholipases A2/chemistry , Phospholipases A2/isolation & purification , Phospholipases A2/pharmacology , Thrombin/antagonists & inhibitors , Viper Venoms/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacologyABSTRACT
Viper venom phospholipase A2 enzymes (vvPLA2s) and phospholipase A2-like (PLA2-like) proteins are two of the principal toxins in viper venom that are responsible for the severe myotoxic and neurotoxic effects caused by snakebite envenoming, among other pathologies. As snakebite envenoming is the deadliest neglected tropical disease, a complete understanding of these proteins' properties and their mechanisms of action is urgently needed. Therefore, we created a database comprising information on the holo-form, cofactor-bound 3D structure of 217 vvPLA2 and PLA2-like proteins in their physiologic environment, as well as 79 membrane-bound viper species from 24 genera, which we have made available to the scientific community to accelerate the development of new anti-snakebite drugs. In addition, the analysis of the sequenced, 3D structure of the database proteins reveals essential aspects of the anatomy of the proteins, their toxicity mechanisms, and the conserved binding site areas that may anchor universal interspecific inhibitors. Moreover, it pinpoints hypotheses for the molecular origin of the myotoxicity of the PLA2-like proteins. Altogether, this study provides an understanding of the diversity of these toxins and how they are conserved, and it indicates how to develop broad, interspecies, efficient small-molecule inhibitors to target the toxin's many mechanisms of action.
Subject(s)
Snake Bites , Viper Venoms , Humans , Viper Venoms/chemistry , Phospholipases A2/chemistry , Myotoxicity , Binding SitesABSTRACT
INTRODUCTION: Parvovirus B19 (B19V) is a human pathogen, and the minor capsid protein of B19V possesses a unique N terminus called VP1u that plays a crucial role in the life cycle of the virus. OBJECTIVES: The objective of this study was to develop a method for domain segmentation of B19 VP1u using intein technology, particularly its receptor binding domain (RBD) and phospholipase A2 (PLA2) domain. METHODS: RBD and PLA2 domains of VP1u were each fused to the DnaE split inteins derived from the Nostoc punctiforme. Each of these precursor proteins was expressed in E. coli. Combining the purified precursors in equal molar ratios resulted in the formation of full-length VP1u. Furthermore, Circular Dichroism (CD) spectroscopy and PLA2 assays were used to probe the structure and activity of the newly formed protein. RESULTS: The CD spectrum of the full length VP1u confirmed the secondary structure of protein, while the PLA2 assay indicated minimal disruption in enzymatic activity. CONCLUSION: This method would allow for the selective incorporation of NMR-active isotopes into either of the VP1u domains, which can reduce signal overlap in NMR structural determination studies.
Subject(s)
Capsid Proteins , Escherichia coli , Inteins , Inteins/genetics , Capsid Proteins/chemistry , Capsid Proteins/genetics , Capsid Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Protein Domains , Parvovirus B19, Human/genetics , Parvovirus B19, Human/chemistry , Nostoc/genetics , Nostoc/enzymology , Nostoc/chemistry , Phospholipases A2/chemistry , Phospholipases A2/genetics , Phospholipases A2/metabolism , Circular Dichroism , HumansABSTRACT
Viperids of the genus Lachesis, also known as bushmasters, are capable of injecting great amounts of venom that cause severe envenomation incidents. Since phospholipases type A2 are mainly involved in edema and myonecrosis within the snakebite sites, in this work, the isolation, amino acid sequence and biochemical characterization of the first phospholipase type A2 from the venom of Lachesis acrochorda, named Lacro_PLA2, is described. Lacro_PLA2 is an acidic aspartic 49 calcium-dependent phospholipase A2 with 93% similarity to the L. stenophrys phospholipase. Lacro_PLA2 has a molecular mass of 13,969.7 Da and an experimental isoelectric point around 5.3. A combination of N-terminal Edman degradation and MS/MS spectrometry analyses revealed that Lacro_PLA2 contains 122 residues including 14 cysteines that form 7 disulfide bridges. A predicted 3D model shows a high resemblance to other viperid phospholipases. Nevertheless, immunochemical and phospholipase neutralization tests revealed a notorious level of immunorecognition of the isolated protein by two polyclonal antibodies from viperids from different genus, which suggest that Lacro_PLA2 resembles more to bothropic phospholipases. Lacro_PLA2 also showed significantly high edema activity when was injected into mice; so, it could be an alternative antigen in the development of antibodies against toxins of this group of viperids, seeking to improve commercial polyclonal antivenoms.
Subject(s)
Crotalinae , Viperidae , Animals , Mice , Viperidae/metabolism , Tandem Mass Spectrometry , Phospholipases A2/chemistry , Viper Venoms/toxicity , Edema/chemically inducedABSTRACT
In accidents involving Crotalus snakes, the crotoxin complex (CTX) plays lethal action due to its neurotoxic activity. On the other hand, CTX have potential biotechnological application due to its anti-tumoral, anti-inflammatory, antimicrobial, analgesic and immunomodulatory properties. CTX is a heterodimer composed of Crotoxin A (CA or crotapotin), the acidic nontoxic and non-enzymatic component and; Crotoxin B (CB), a basic, toxic and catalytic PLA2. Currently, there are two classes of CTX isoforms, whose differences in their biological activities have been attributed to features presented in CB isoforms. Here, we present the crystal structure of CB isolated from the Crotalus durissus collilineatus venom. It amino acid sequence was assigned using the SEQUENCE SLIDER software, which revealed that the crystal structure is a heterodimer composed of two new CB isoforms (colCB-A and colCB-B). Bioinformatic and biophysical analyses showed that the toxin forms a tetrameric assembly in solution similar to CB from Crotalus durissus terrificus venom, despite some differences observed at the dimeric interface. By the previously proposed classification, the colCB-B presents features of the class I isoforms while colCB-A cannot be classified into classes I and II based on its amino acid sequence. Due to similar features observed for other CB isoforms found in the NCBI database and the results obtained for colCB-A, we suggest that there are more than two classes of CTX and CB isoforms in crotalic venoms.
Subject(s)
Crotalid Venoms , Crotoxin , Venomous Snakes , Animals , Crotoxin/chemistry , Phospholipases A2/chemistry , Crotalus/metabolism , Crotalid Venoms/chemistry , Protein Isoforms/metabolismABSTRACT
Snakebite is a significant health concern in tropical and subtropical regions, particularly in Africa, Asia, and Latin America, resulting in more than 2.7 million envenomations and an estimated one hundred thousand fatalities annually. The Bothrops genus is responsible for the majority of snakebite envenomings in Latin America and Caribbean countries. Accidents involving snakes from this genus are characterized by local symptoms that often lead to permanent sequelae and death. However, specific antivenoms exhibit limited effectiveness in inhibiting local tissue damage. Phospholipase A2-like (PLA2-like) toxins emerge as significant contributors to local myotoxicity in accidents involving Bothrops species. As a result, they represent a crucial target for prospective treatments. Some natural and synthetic compounds have shown the ability to reduce or abolish the myotoxic effects of PLA2-like proteins. In this study, we employed a combination approach involving myographic, morphological, biophysical and bioinformatic techniques to investigate the interaction between chlorogenic acid (CGA) and BthTX-I, a PLA2-like toxin. CGA provided a protection of 71.8% on muscle damage in a pre-incubation treatment. Microscale thermophoresis and circular dichroism experiments revealed that CGA interacted with the BthTX-I while preserving its secondary structure. CGA exhibited an affinity to the toxin that ranks among the highest observed for a natural compound. Bioinformatics simulations indicated that CGA inhibitor binds to the toxin's hydrophobic channel in a manner similar to other phenolic compounds previously investigated. These findings suggest that CGA interferes with the allosteric transition of the non-activated toxin, and the stability of the dimeric assembly of its activated state.
Subject(s)
Chlorogenic Acid , Cinnamates , Chlorogenic Acid/pharmacology , Phospholipases A2/chemistry , Phospholipases A2/metabolism , Phospholipases A2/toxicityABSTRACT
Little is known of the biochemical composition and functional features of the venoms of poorly known Colombian coral snakes. Here, we provide a preliminary characterization of the venom of two Colombian endemic coral snake species, Micrurus medemi and M. sangilensis, as well as Colombian populations of M. helleri. Electrophoresis and RP-HPLC techniques were used to identify venom components, and assays were conducted to detect enzyme activities, including phospholipase A2, hyaluronidase, and protease activities. The median lethal dose was determined using murine models. Cytotoxic activities in primary cultures from hippocampal neurons and cancer cell lines were evaluated. The venom profiles revealed similarities in electrophoretic separation among proteins under 20 kDa. The differences in chromatographic profiles were significant, mainly between the fractions containing medium-/large-sized and hydrophobic proteins; this was corroborated by a proteomic analysis which showed the expected composition of neurotoxins from the PLA2 (~38%) and 3FTx (~17%) families; however, a considerable quantity of metalloproteinases (~12%) was detected. PLA2 activity and protease activity were higher in M. helleri venom according to qualitative and quantitative assays. M. medemi venom had the highest lethality. All venoms decreased cell viability when tested on tumoral cell cultures, and M. helleri venom had the highest activity in neuronal primary culture. These preliminary studies shed light on the venoms of understudied coral snakes and broaden the range of sources that could be used for subsequent investigations of components with applications to specific diseases. Our findings also have implications for the clinical manifestations of snake envenoming and improvements in its medical management.
Subject(s)
Coral Snakes , Snake Bites , Humans , Animals , Mice , Coral Snakes/metabolism , Elapid Venoms/chemistry , Antivenins/metabolism , Colombia , Proteomics , Snake Venoms/metabolism , Phospholipases A2/chemistry , Peptide Hydrolases/metabolism , Elapidae/metabolismABSTRACT
Secreted phospholipases A2 are snake-venom proteins with many biological activities, notably anti-tumor activity. Phospholipases from the same snake type but different geographical locations have shown similar biochemical and biological activities with minor differences in protein sequences. Thus, the discovery of a new phospholipase A2 with unique characteristics identified in a previously studied venom could suggest the origins of these differences. Here, a new Group II secreted phospholipase A2 (Cc-PLA2-II) from the snake venom of Saudi Cerastes cerastes gasperetti was isolated and characterized. The purified enzyme had a molecular weight of 13.945 kDa and showed high specific activity on emulsified phosphatidylcholine of 1560 U/mg at pH 9.5 and 50 °C with strict calcium dependence. Interestingly, stability in extreme pH and high temperatures was observed after enzyme incubation at several pH levels and temperatures. Moreover, a significant dose-dependent cytotoxic anti-tumor effect against six human cancer cell lines was observed with concentrations of Cc-PLA2 ranging from 2.5 to 8 µM. No cytotoxic effect on normal human umbilical-vein endothelial cells was noted. These results suggest that Cc-PLA2-II potentially has angiogenic activity of besides cytotoxicity as part of its anti-tumor mechanism. This study justifies the inclusion of this enzyme in many applications for anticancer drug development.
Subject(s)
Antineoplastic Agents , Viperidae , Animals , Humans , Group II Phospholipases A2 , Saudi Arabia , Phospholipases A2/pharmacology , Phospholipases A2/chemistry , Phospholipases , Viper Venoms/pharmacology , Viper Venoms/chemistry , Antineoplastic Agents/pharmacologyABSTRACT
Trimeresurus gracilis is an endemic alpine pitviper in Taiwan with controversial phylogeny, and its venom proteome remains unknown. In this study, we conducted a proteomic analysis of T. gracilis venom using high-performance liquid chromatography-tandem mass spectrometry and identified 155 toxin proteoforms that belong to 13 viperid venom toxin families. By searching the sequences of trypsin-digested peptides of the separated HPLC fractions against the NCBI database, T. gracilis venom was found to contain 40.3% metalloproteases (SVMPs), 15.3% serine proteases, 6.6% phospholipases A2, 5.0% L-amino acid oxidase, 4.6% Cys-rich secretory proteins (CRISPs), 3.2% disintegrins, 2.9% vascular endothelial growth factors (VEGFs), 1.9% C-type lectin-like proteins, and 20.2% of minor toxins, nontoxins, and unidentified peptides or compounds. Sixteen of these proteoforms matched the toxins whose full amino-acid sequences have been deduced from T. gracilis venom gland cDNA sequences. The hemorrhagic venom of T. gracilis appears to be especially rich in PI-class SVMPs and lacks basic phospholipase A2. We also cloned and sequenced the cDNAs encoding two CRISP and three VEGF variants from T. gracilis venom glands. Sequence alignments and comparison revealed that the PI-SVMP, kallikrein-like proteases, CRISPs, and VEGF-F of T. gracilis and Ovophis okinavensis are structurally most similar, consistent with their close phylogenetic relationship. However, the expression levels of some of their toxins were rather different, possibly due to their distinct ecological and prey conditions.
Subject(s)
Crotalid Venoms , Trimeresurus , Animals , Proteome/analysis , Vascular Endothelial Growth Factor A/genetics , Phylogeny , Taiwan , Proteomics/methods , Crotalid Venoms/chemistry , Phospholipases A2/genetics , Phospholipases A2/chemistry , Peptides/geneticsABSTRACT
In this work, we report the design, synthesis, and application of a bis-pyrene phospholipid probe for detection of phospholipase A2 action through changes in pyrene monomer and excimer fluorescence intensities. Continuous fluorometric assays enabled detection of the activities of multiple PLA2 enzymes as well as the decrease in catalysis by PLA2 from honey bee venom caused by the inhibitor p-bromo phenacylbromide. Thin-layer chromatography and mass spectrometry analysis were also used to validate probe hydrolysis by PLA2. Mass spectrometry data also supported cleavage of the probe by phospholipase C and D enzymes, although changes in fluorescence were not observed in these cases. Nevertheless, the bis-pyrene phospholipid probe developed in this work is effective for detection of PLA2 enzyme activity through an assay that enables screening for inhibitor development.
Subject(s)
Phospholipases , Phospholipids , Hydrolysis , Phospholipases/analysis , Phospholipases A2/chemistry , PyrenesABSTRACT
Snake venom-secreted phospholipase A2 (svPLA2) enzymes, both catalytically active and inactive, are a central component in envenoming. These are responsible for disrupting the cell membrane's integrity, inducing a wide range of pharmacological effects, such as the necrosis of the bitten limb, cardiorespiratory arrest, edema, and anticoagulation. Although extensively characterized, the reaction mechanisms of enzymatic svPLA2 are still to be thoroughly understood. This review presents and analyses the most plausible reaction mechanisms for svPLA2, such as the "single-water mechanism" or the "assisted-water mechanism" initially proposed for the homologous human PLA2. All of the mechanistic possibilities are characterized by a highly conserved Asp/His/water triad and a Ca2+ cofactor. The extraordinary increase in activity induced by binding to a lipid-water interface, known as "interfacial activation," critical for the PLA2s activity, is also discussed. Finally, a potential catalytic mechanism for the postulated noncatalytic PLA2-like proteins is anticipated.
Subject(s)
Crotalid Venoms , Snake Venoms , Humans , Phospholipases A2/chemistry , Phospholipases A2/metabolism , WaterABSTRACT
Phospholipases have diverse roles in lipid and cell membrane biology. In animal venoms, they can have roles as neurotoxins or myotoxins that disrupt the integrity of cell membranes. In this work, we describe a temperature-controlled, continuous electrospray ionization mass spectrometry (ESI-MS) assay for measuring phospholipase A2 activity against liposomes. The enzyme used in this assay was paradoxin, which is a neurotoxic trimeric phospholipase A2 from inland taipan snake venom. Previously developed ESI-MS-based phospholipase assays have been discontinuous and analyzed hydrolysis of single lipid molecules by liquid chromatography ESI-MS. In this work, a continuous assay was developed against liposomes, a more complex substrate that more closely reflects the natural substrate for paradoxin. The assay confirmed the requirement for Ca2+ and allowed measurement of Michaelis-Menten-type parameters. The use of ESI-MS for lipid detection enabled nuanced insights into the effect of changing assay conditions not only on the enzyme but also on the liposome substrate. Changing the metal ion concentrations did not significantly change the liposomes but did affect enzymatic activity. Increasing temperature did not substantially affect the secondary structure of paradoxin but affected liposome size, resulting in increased enzymatic activity consistent with the disruption of the phosphatidylcholine membrane, increasing accessibility of sn-2 ester bonds. The continuous ESI-MS method described herein can be applied to other enzyme reactions, particularly those which utilize complex lipid substrates.
Subject(s)
Liposomes , Spectrometry, Mass, Electrospray Ionization , Animals , Liposomes/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Phospholipases A2/chemistry , Phospholipases , PhosphatidylcholinesABSTRACT
The Crotalus intermedius group is a clade of rattlesnakes consisting of several species adapted to a high elevation habitat, primarily in México. Crotalus tancitarensis was previously classified as C. intermedius, until individuals occurring on Cerro Tancítaro in Michoacán, México, were reevaluated and classified as a new species (C. tancitarensis) based on scale pattern and geographic location. This study aimed to characterize the venom of C. tancitarensis and compare the venom profile to those of other species within the Crotalus intermedius group using gel electrophoresis, biochemical assays, reverse-phase high performance liquid chromatography, mass spectrometry, and lethal toxicity (LD50) assays. Results show that the venom profiles of species within the Crotalus intermedius group are similar, but with distinct differences in phospholipase A2 (PLA2), metalloproteinase PI (SVMP PI), and kallikrein-like serine proteinase (SVSP) activity and relative abundance. Proteomic analysis indicated that the highland forms produce venoms with 50-60 protein isoforms and a composition typical of type I rattlesnake venoms (abundant SVMPs, lack of presynaptic PLA2-based neurotoxins), as well as a diversity of typical Crotalus venom components such as serine proteinases, PLA2s, C-type lectins, and less abundant toxins (LAAOs, CRiSPs, etc.). The overall venom profile of C. tancitarensis appears most similar to C. transversus, which is consistent with a previous mitochondrial DNA analysis of the Crotalus intermedius group. These rattlesnakes of the Mexican highlands represent a radiation of high elevation specialists, and in spite of divergence of species in these Sky Island habitats, venom composition of species analyzed here has remained relatively conserved. The majority of protein family isoforms are conserved in all members of the clade, and as seen in other more broadly distributed rattlesnake species, differences in their venoms are largely due to relative concentrations of specific components.
Subject(s)
Crotalid Venoms , Crotalus , Humans , Animals , Mexico , Crotalus/genetics , Proteomics , Crotalid Venoms/chemistry , Metalloproteases/metabolism , Phospholipases A2/chemistryABSTRACT
Varespladib (LY315920) is a potent inhibitor of human group IIA phospholipase A2 (PLA2) originally developed to control inflammatory cascades of diseases associated with high or dysregulated levels of endogenous PLA2. Recently, varespladib was also found to inhibit snake venom PLA2 and PLA2-like toxins. Herein, ex vivo neuromuscular blocking activity assays were used to test the inhibitory activity of varespladib. The binding affinity between varespladib and a PLA2-like toxin was quantified and compared with other potential inhibitors for this class of proteins. Crystallographic and bioinformatic studies showed that varespladib binds to PrTX-I and BthTX-I into their hydrophobic channels, similarly to other previously characterized PLA2-like myotoxins. However, a new finding is that an additional varespladib binds to the MDiS region, a particular site that is related to muscle cell disruption by these toxins. The present results further advance the characterization of the molecular interactions of varespladib with PLA2-like myotoxins and provide additional evidence for this compound as a promising inhibitor candidate for different PLA2 and PLA2-like toxins.
Subject(s)
Bothrops , Crotalid Venoms , Toxins, Biological , Animals , Humans , Bothrops/metabolism , Neurotoxins , Keto Acids , Crotalid Venoms/toxicity , Crotalid Venoms/chemistry , Phospholipases A2/chemistryABSTRACT
Snake envenomation is an ongoing global health problem and tropical neglected disease that afflicts millions of people each year. The only specific treatment, antivenom, has several limitations that affects its proper distribution to the victims and its efficacy against local effects, such as myonecrosis. The main responsible for this consequence are the phospholipases A2 (PLA2) and PLA2-like proteins, such as BthTX-I from Bothrops jararacussu. Folk medicine resorts to plants such as Tabernaemontana catharinensis to palliate these and other snakebite effects. Here, we evaluated the effect of its root bark extract and one of its isolated compounds, 12-methoxy-4-methyl-voachalotine (MMV), against the in vitro paralysis and muscle damage induced by BthTX-I. Secondary and quaternary structures of BthTX-I were not modified by the interaction with MMV. Instead, this compound interacted in an unprecedented way with the region inside the toxin hydrophobic channel and promoted a structural change in Val31, loop 58-71 and Membrane Disruption Site. Thus, we hypothesize that MMV inhibits PLA2-like proteins by preventing entrance of fatty acid into the hydrophobic channel. These data may explain the traditional use of T. catharinensis extract and confirm MMV as a promising candidate to complement antivenom or a structural guide to develop more effective inhibitors.