Development and Optimization of a Bromothymol Blue-Based PLA2 Assay Involving POPC-Based Self-Assemblies.

Phospholipase A2 (PLA2) is a superfamily of phospholipase enzymes that dock at the water/oil interface of phospholipid assemblies, hydrolyzing the ester bond at the sn-2 position. The enzymatic activity of these enzymes differs based on the nature of the substrate, its supramolecular assemblies (micelle, liposomes), and their composition, reflecting the interfacial nature of the PLA2s and requiring assays able to directly quantify this interaction of the enzyme(s) with these supramolecular assemblies. We developed and optimized a simple, universal assay method employing the pH-sensitive indicator dye bromothymol blue (BTB), in which different POPC (3-palmitoyl-2-oleoyl-sn-glycero-1-phosphocholine) self-assemblies (liposomes or mixed micelles with Triton X-100 at different molar ratios) were used to assess the enzymatic activity. We used this assay to perform a comparative analysis of PLA2 kinetics on these supramolecular assemblies and to determine the kinetic parameters of PLA2 isozymes IB and IIA for each supramolecular POPC assembly. This assay is suitable for assessing the inhibition of PLA2s with great accuracy using UV-VIS spectrophotometry, being thus amenable for screening of PLA2 enzymes and their substrates and inhibitors in conditions very similar to physiologic ones.

Phospholipase-catalyzed degradation drives domain morphology and rheology transitions in model lung surfactant monolayers.

Lung surfactant is inactivated in acute respiratory distress syndrome (ARDS) by a mechanism that remains unclear. Phospholipase (PLA2) plays an essential role in the normal lipid recycling processes, but is present in elevated levels in ARDS, suggesting it plays a role in ARDS pathophysiology. PLA2 hydrolyzes lipids such as DPPC-the primary component of lung surfactant-into palmitic acid (PA) and lyso-PC (LPC). Because PA co-crystallizes with DPPC to form rigid, elastic domains, we hypothesize that PLA2-catalyzed degradation establishes a stiff, heterogeneous rheology in the monolayer, and suggests a potential mechanical role in disrupting lung surfactant function during ARDS. Here we study the morphological and rheological changes of DPPC monolayers as they are degraded by PLA2 using interfacial microbutton microrheometry coupled with fluorescence microscopy. While degrading, domain morphology passes through qualitatively distinct transitions: compactification, coarsening, solidification, aggregation, network percolation, network erosion, and nucleation of PLA2-rich domains. Initially, condensed domains relax to more compact shapes, and coarsen via Ostwald ripening and coalescence up until the domains solidify, marked by a distinct roughening of domain boundaries that does not relax. Domains aggregate and eventually form a percolated network, whose elements then erode and whose connections are broken as degradation continues. The relative enzymatic activity of PLA2, set by the age of the sample, impacts the order and the duration of morphology transitions. The fresher the PLA2, the faster the overall degradation, and the earlier the onset of domain solidification: domains solidify before aggregating with fresh PLA2 samples, but aggregate and percolate before solidification with aged PLA2. Irrespective of the activity of the PLA2, all measured linear viscoelastic surface shear moduli obey the same dependence on condensed phase area fraction (log|G*| ∝ ϕ) throughout monolayer degradation. Moreover, the onset of domain solidification coincides with the time when the relative surface elasticity begins to increase.

Proteomic Analysis and Immunoprofiling of Persian Horned Viper Venom, Pseudocerastes Persicus, from Central Part of Iran.

Numerous species of venomous snakes of medical importance exist in Iran. Pseudocerastes persicus (P. persicus), one of the medically important snakes, also called the Persian horned viper, has a geographical spread that extends to the east, southwest, and central areas of Iran and is endemic across the wider region. As a result, this species is responsible for many snakebite occurrences. Venom from P. persicus found in the central province of Semnan contains phospholipase A2 and L-amino acid oxidase activities, and high toxic potency. The venom was fractionated by reverse-phase high-performance liquid chromatography (HPLC) and analyzed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and two-dimensional electrophoresis. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), a range of components were identified, consistent with the biochemical and toxicological properties of the venom. Proteins identified from 2D electrophoresis and shotgun methods included metallo- and serine proteases, phospholipases, oxidases, and Kunitz trypsin inhibitors, along with many other components at lower qualitative abundance. This study provides a more detailed understanding of the protein profile of Iranian P. persicus venom, which can be effective in the production of an effective antidote against it. The analysis of the resulting data shows that there is a wide range of proteins in the venom of the Persian horned viper. This information can provide a better understanding of how venom is neutralized by polyclonal antivenom. Considering the wide presence of this snake and its related species in Iran and surrounding countries, knowing the venom protein profile of this family can be of great support to antivenom producers such as Razi Vaccine & Serum Research Institute in the preparation of regional antivenoms.

Comparative in vitro and in silico analysis of the ability of basic Asp49 phospholipase A2 and Lys49-phospholipase A2-like myotoxins from Bothrops diporus venom to inhibit the metastatic potential of murine mammary tumor cells and endothelial cell tubulogenesis: Asp49 vs Lys49 phospholipases A2: Inhibition of metastasis and angiogenesis.

Snake venoms are a complex mixture of proteins and polypeptides that represent a valuable source of potential molecular tools for understanding physiological processes for the development of new drugs. In this study two major PLA2s, named PLA2-I (Asp49) and PLA2-II (Lys49), isolated from the venom of Bothrops diporus from Northeastern Argentina, have shown cytotoxic effects on LM3 murine mammary tumor cells, with PLA2-II-like exhibiting a stronger effect compared to PLA2-I. At sub-cytotoxic levels, both PLA2s inhibited adhesion, migration, and invasion of these adenocarcinoma cells. Moreover, these toxins hindered tubulogenesis in endothelial cells, implicating a potential role in inhibiting tumor angiogenesis. All these inhibitory effects were more pronounced for the catalytically-inactive toxin. Additionally, in silico studies strongly suggest that this PLA2-II-like myotoxin could effectively block fibronectin binding to the integrin receptor, offering a dual advantage over PLA2-I in interacting with the αVß3 integrin. In conclusion, this study reports for the first time, integrating both in vitro and in silico approaches, a comparative analysis of the antimetastatic and antiangiogenic potential effects of two isoforms, an Asp49 PLA2-I and a Lys49 PLA2-II-like, both isolated from Bothrops diporus venom.

Preparation of microgel particles from egg yolk components by combining phospholipase A2 with high-pressure homogenization: Physicochemical, structural properties and their effects on foaming, processing stability of egg white protein.

In this study, two types of microgel particles from egg yolk components were prepared by combining enzymatic hydrolysis with high-pressure homogenization (HPH), and their differences in physicochemical properties, foaming properties, and microstructure were compared. Results showed that the particle size of both types of microgel particles had decreased from 2744.07 ± 408.26 nm (egg yolk, EY) to 144.97 ± 3.19 nm (PLA2 hydrolyzed egg yolk microgel particles, PYM) and 535.07 ± 46.07 nm (egg yolk microgel particles hydrolyzed by PLA2, YMP), from 736.24 ± 34.61 nm (EG) to 182.76 ± 4.12 nm (PLA2 hydrolyzed egg yolk granules microgel particles, PGM) and 443.98 ± 27.09 nm (egg yolk granules microgel particles hydrolyzed by PLA2, GMP). Besides, their interfacial adsorption abilities were significantly improved, reflected in the increase values in overrun, from161.90 % ± 9.84 % (EY) to 269.64 % ± 16.73 % (PMY) and 307.20 % ± 16.09 % (YMP), from 189.21 % ± 5.02 % (EG) to 280.38 % ± 36.05 % (PGM) and 261.91 % ± 34.03 % (GMP). Their structural properties showed higher stabilities after treatments. When the microgel particles are applied to cakes, the specific volume was increased from 2.05 ± 0.1 mL/g (EY) to 2.25 ± 0.13 mL/g (PYM) and 2.45 ± 0.03 mL/g (YPM), and from 2.00 ± 0.09 mL/g (EG) to 2.51 ± 0.13 mL/g (PGM) and 2.75 ± 0.21 mL/g (GMP), respectively. The hardness and chewiness were reduced with both types of microgel particles from egg yolk components, which indicated their potential value as edible foam stabilizers in the baking industry.

A novel sustainable photo/chemical magnetic nanomaterial effectively mitigates the allergenicity of phospholipase A2.

Reducing the allergenicity of edible insects is crucial for the comprehensive utilization of insect resources. Phospholipase A2 (PLA2) exists in various edible insects and mammalian tissues, which can cause serious allergic reactions. Herein, we constructed a magnetic nanocomposite with photo/chemical synergistic capability to mitigate the allergenicity of PLA2. The formation of prepared nanocomposite was systematically confirmed using various techniques. The nanocomposite exhibited uniform diameters, abundant functional groups, excellent magnetic capabilities. An effective photo/chemical method was established to reduce the allergenicity of PLA2 in vitro. The feasibility of the method was demonstrated through circular dichroism, fluorescence spectrum and IgE-binding analysis. The allergenicity and IgE-binding effect of PLA2 were significantly reduced due to conformational changes after nanomaterial treatment. These results demonstrate the sensitivity and effectiveness a strategy for reducing PLA2 allergenicity, providing a basis for development of nanomaterials to reduce the risk of novel food allergies in response to edible insect products.

Conjugation with the Carrier Helped to Reveal acidification-Induced Structural Shift in the Peptide from Phospholipase Domain of Parvovirus B19.

Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144-159; 164; 171-183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).

Synthesis, Characterization, and Study of the Antimicrobial Potential of Dimeric Peptides Derived from the C-Terminal Region of Lys49 Phospholipase A2 Homologs.

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.

Emerging anticancer potential and mechanisms of snake venom toxins: A review.

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.

A study on the catalytic domain of pork phospholipase A2: Enzymatic properties and hydrolysis characteristics of phosphatidylcholine and its hydroperoxide.

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.

Exploring venom diversity in Mixcoatlus browni and Mixcoatlus barbouri: A comparative analysis of two rare Mexican snake species with crotoxin-like presence.

The genus Mixcoatlus is composed of three species: Mixcoatlus barbouri, M. browni, and M. melanurus, of which the venom composition of M. melanurus, the most common species of the three, has only recently been described. However, very little is known about the natural history of M. barbouri and M. browni, and the venom composition of these two species has remained thus far unexplored. In this study we characterize the proteomic profiles and the main biochemical and toxic activities of these two venoms. Proteomic data obtained by shotgun analysis of whole venom identified 12 protein families for M. barbouri, and 13 for M. browni. The latter venom was further characterized by using a quantitative 'venomics' protocol, which revealed that it is mainly composed of 51.1 % phospholipases A2 (PLA2), 25.5 % snake venom serine proteases (SVSP), 4.6 % l-amino oxidases (LAO), and 3.6 % snake venom metalloproteases (SVMP), with lower percentages other six protein families. Both venoms contained homologs of the basic and acidic subunits of crotoxin. However, due to limitations in M. barbouri venom availability, we could only characterize the crotoxin-like protein of M. browni venom, which we have named Mixcoatlutoxin. It exhibited a lethal potency in mice like that described for classical rattlesnake crotoxins. These findings expand knowledge on the distribution of crotoxin-like heterodimeric proteins in viper snake species. Further investigation of the bioactivities of the venom of M. barbouri, on the other hand, remains necessary.

Deciphering the interactions of scopoletin and scopolin from Catunaregam nilotica roots against Naja nigricollis phospholipase A2 enzyme.

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.

Modulation of Phospholipase A2 Membrane Activity by Anti-inflammatory Drugs.

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.

Viper Venom Phospholipase A2 Database: The Structural and Functional Anatomy of a Primary Toxin in Envenomation.

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.

Daboialipase, a phospholipase A2 from Vipera russelli russelli venom posesses anti-platelet, anti-thrombin and anti-cancer properties.

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.

Feasibility of Domain Segmentation of B19V VP1u Using Intein Technology for Structural Studies.

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.

Structural studies with crotoxin B from Crotalus durissus collilineatus venom suggest a heterodimeric assembly formed by two new isoforms.

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.

Structural, biochemical and immunochemical characterization of an acidic phospholipase A2 from Lachesis acrochorda (Viperidae: Crotalinae) venom.

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.

BthTX-I, a phospholipase A2-like toxin, is inhibited by the plant cinnamic acid derivative: chlorogenic acid.

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.

Unveiling the Venom Composition of the Colombian Coral Snakes Micrurus helleri, M. medemi, and M. sangilensis.

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.