Venom diversity in Naja mossambica: Insights from proteomic and immunochemical analyses reveal intraspecific differences.

BACKGROUND: Intraspecific variations in snake venom composition have been extensively documented, contributing to the diverse clinical effects observed in envenomed patients. Understanding these variations is essential for developing effective snakebite management strategies and targeted antivenom therapies. We aimed to comprehensively investigate venoms from three distinct populations of N. mossambica from Eswatini, Limpopo, and KwaZulu-Natal regions in Africa in terms of their protein composition and reactivity with three commercial antivenoms (SAIMR polyvalent, EchiTAb+ICP, and Antivipmyn Africa). METHODOLOGY/PRINCIPAL FINDINGS: Naja mossambica venoms from Eswatini region exhibited the highest content of neurotoxic proteins, constituting 20.70% of all venom proteins, compared to Limpopo (13.91%) and KwaZulu-Natal (12.80%), and was characterized by the highest diversity of neurotoxic proteins, including neurotoxic 3FTxs, Kunitz-type inhibitors, vespryns, and mamba intestinal toxin 1. KwaZulu-Natal population exhibited considerably lower cytotoxic 3FTx, higher PLA2 content, and significant diversity in low-abundant proteins. Conversely, Limpopo venoms demonstrated the least diversity as demonstrated by electrophoretic and mass spectrometry analyses. Immunochemical assessments unveiled differences in venom-antivenom reactivity, particularly concerning low-abundance proteins. EchiTAb+ICP antivenom demonstrated superior reactivity in serial dilution ELISA assays compared to SAIMR polyvalent. CONCLUSIONS/SIGNIFICANCE: Our findings reveal a substantial presence of neurotoxic proteins in N. mossambica venoms, challenging previous understandings of their composition. Additionally, the detection of numerous peptides aligning to uncharacterized proteins or proteins with unknown functions underscores a critical issue with existing venom protein databases, emphasizing the substantial gaps in our knowledge of snake venom protein components. This underscores the need for enhanced research in this domain. Moreover, our in vitro immunological assays suggest EchiTAb+ICP's potential as an alternative to SAIMR antivenom, requiring confirmation through prospective in vivo neutralization studies.

Development and Characterization of Anti-Naja ashei Three-Finger Toxins (3FTxs)-Specific Monoclonal Antibodies and Evaluation of Their In Vitro Inhibition Activity.

Antivenom immunotherapy is the mainstay of treatment for snakebite envenoming. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific antibodies production hold much promise in future therapeutic strategies of snakebite envenoming. We report anti-3FTxs monoclonal antibodies developed against N. ashei venom in mice. All the three test mAbs (P4G6a, P6D9a, and P6D9b) were found to be IgG antibodies, isotyped as IgG1. SDS-PAGE analysis of the test mAbs showed two major bands at approximately 55 and 29 kDa, suggestive of immunoglobulin heavy and light chain composition, respectively. The immunoaffinity-purified test mAbs demonstrated higher binding efficacy to the target antigen compared to negative control. Similarly, a cocktail of the test mAbs was found to induce a significantly higher inhibition (p-value < 0.0001) compared to two leading commercial brands of antivenoms on the Kenyan market, implying a higher specificity for the target antigen. Both the test mAbs and 3FTxs polyclonal antibodies induced comparable inhibition (p-value = 0.9029). The inhibition induced by the 3FTxs polyclonal antibodies was significantly different from the two antivenoms (p-value < 0.0001). Our results demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.

Development of an Inhibition Enzyme-Linked Immunosorbent Assay (ELISA) Prototype for Detecting Cytotoxic Three-Finger Toxins (3FTxs) in African Spitting Cobra Venoms.

The administration of toxin-specific therapy in snake envenoming is predicated on improved diagnostic techniques capable of detecting specific venom toxins. Various serological tests have been used in detecting snakebite envenoming. Comparatively, enzyme-linked immunosorbent assay (ELISA) has been shown to offer a wider practical application. We report an inhibition ELISA for detecting three-finger toxin (3FTx) proteins in venoms of African spitting cobras. The optimized assay detected 3FTxs in N. ashei (including other Naja sp.) venoms, spiked samples, and venom-challenged mice samples. In venoms of Naja sp., the assay showed inhibition, implying the detection of 3FTxs, but showed little or no inhibition in non-Naja sp. In mice-spiked samples, one-way ANOVA results showed that the observed inhibition was not statistically significant between spiked samples and negative control (p-value = 0.164). Similarly, the observed differences in inhibition between venom-challenged and negative control samples were not statistically significant (p-value = 0.9109). At an LOD of 0.01 µg/mL, the assay was able to confirm the presence of 3FTxs in the samples. Our results show a proof of concept for the use of an inhibition ELISA model as a tool for detecting 3FTxs in the venoms of African spitting cobra snakes.

Effects of 3FTx Protein Fraction from Naja ashei Venom on the Model and Native Membranes: Recognition and Implications for the Mechanisms of Toxicity.

Three-finger toxins are naturally occurring proteins in Elapidae snake venoms. Nowadays, they are gaining popularity because of their therapeutic potential. On the other hand, these proteins may cause undesirable reactions inside the body's cells. A full assessment of the safety of Naja ashei venom components for human cell application is still unknown. The aim of the study was to determine the effect of the exogenous application of three-finger toxins on the cells of monocytes (U-937) and promyelocytes (HL-60), with particular emphasis on the modification of their membranes under the influence of various doses of 3FTx protein fraction (0-120 ng/mL). The fraction exhibiting the highest proportion of 3FTx proteins after size exclusion chromatography (SEC) separation was used in the experiments. The structural response of cell membranes was described on the basis of single-component and multi-component Langmuir monolayers that mimicked the native membranes. The results show that the mechanism of protein-lipid interactions depends on both the presence of lipid polar parts (especially zwitterionic type of lipids) and the degree of membrane saturation (the greatest-for unsaturated lipids). The biochemical indicators reflecting the tested cells (MDA, LDH, cell survival, induction of inflammation, LD50) proved the results that were obtained for the model.

Different Research Approaches in Unraveling the Venom Proteome of Naja ashei.

The dynamic development of venomics in recent years has resulted in a significant increase in publicly available proteomic data. The information contained therein is often used for comparisons between different datasets and to draw biological conclusions therefrom. In this article, we aimed to show the possible differences that can arise, in the final results of the proteomic experiment, while using different research workflows. We applied two software solutions (PeptideShaker and MaxQuant) to process data from shotgun LC-MS/MS analysis of Naja ashei venom and collate it with the previous report concerning this species. We were able to provide new information regarding the protein composition of this venom but also present the qualitative and quantitative limitations of currently used proteomic methods. Moreover, we reported a rapid and straightforward technique for the separation of the fraction of proteins from the three-finger toxin family. Our results underline the necessary caution in the interpretation of data based on a comparative analysis of data derived from different studies.

Evaluation of Antifungal Activity of Naja pallida and Naja mossambica Venoms against Three Candida Species.

In contrast to comprehensively investigated antibacterial activity of snake venoms, namely crude venoms and their selected components, little is known about antifungal properties of elapid snake venoms. In the present study, the proteome of two venoms of red spitting cobra Naja pallida (NPV) and Mozambique spitting cobra Naja mossambica (NMV) was characterized using LC-MS/MS approach, and the antifungal activity of crude venoms against three Candida species was established. A complex response to venom treatment was revealed. NPV and NMV, when used at relatively high concentrations, decreased cell viability of C. albicans and C. tropicalis, affected cell cycle of C. albicans, inhibited C. tropicalis-based biofilm formation and promoted oxidative stress in C. albicans, C. glabrata and C. tropicalis cells. NPV and NMV also modulated ammonia pulses during colony development and aging in three Candida species. All these observations provide evidence that NPV and NMV may diminish selected pathogenic features of Candida species. However, NPV and NMV also promoted the secretion of extracellular phospholipases that may facilitate Candida pathogenicity and limit their usefulness as anti-candidal agents. In conclusion, antifungal activity of snake venoms should be studied with great caution and a plethora of pathogenic biomarkers should be considered in the future experiments.

Comparison of Methods for Measuring Protein Concentration in Venom Samples.

Snake venom is an extremely interesting natural mixture of proteins and peptides, characterized by both high diversity and high pharmacological potential. Much attention has been paid to the study of venom composition of different species and also detailed analysis of the properties of individual components. Since proteins and peptides are the active ingredients in venom, rapidly developing proteomic techniques are used to analyze them. During such analyses, one of the routine operations is to measure the protein concentration in the sample. The aim of this study was to compare five methods used to measure protein content in venoms of two snake species: the Viperids representative, Agkistrodon contortrix, and the Elapids representative, Naja ashei. The study showed that for A. contortrix venom, the concentration of venom protein measured by four methods is very similar and only the NanoDrop method clearly stands out from the rest. However, in the case of N. ashei venom, each technique yields significantly different results. We hope that this report will help to draw attention to the problem of measuring protein concentration, especially in such a complex mixture as animal venoms.

Antimicrobial Activity of Protein Fraction from Naja ashei Venom Against Staphylococcus epidermidis.

One of the key problems of modern infectious disease medicine is the growing number of drug-resistant and multi-drug-resistant bacterial strains. For this reason, many studies are devoted to the search for highly active antimicrobial substances that could be used in therapy against bacterial infections. As it turns out, snake venoms are a rich source of proteins that exert a strong antibacterial effect, and therefore they have become an interesting research material. We analyzed Naja ashei venom for such antibacterial properties, and we found that a specific composition of proteins can act to eliminate individual bacterial cells, as well as the entire biofilm of Staphylococcus epidermidis. In general, we used ion exchange chromatography (IEX) to obtain 10 protein fractions with different levels of complexity, which were then tested against certified and clinical strains of S. epidermidis. One of the fractions (F2) showed exceptional antimicrobial effects both alone and in combination with antibiotics. The protein composition of the obtained fractions was determined using mass spectrometry techniques, indicating a high proportion of phospholipases A2, three-finger toxins, and L-amino acids oxidases in F2 fraction, which are most likely responsible for the unique properties of this fraction. Moreover, we were able to identify a new group of low abundant proteins containing the Ig-like domain that have not been previously described in snake venoms.

An Effective Method of Isolating Honey Proteins.

Honey is a natural sweetener composed mostly of sugars, but it contains also pollen grains, proteins, free amino acids, and minerals. The amounts and proportions of these components depend on the honey type and bee species. Despite the low content of honey protein, they are becoming a popular study object, and have recently been used as markers of the authenticity and quality of honey. Currently, the most popular methods of protein isolation from honey are dialysis against distilled water, lyophilization of dialysate, or various precipitation protocols. In this work, we propose a new method based on saturated phenol. We tested it on three popular polish honey types and we proved its compatibility with both 1D and 2D polyacrylamide gel electrophoresis (PAGE) and MS (mass spectrometry) techniques. The elaborated technique is also potentially less expensive and less time-consuming than other previously described methods, while being equally effective.

Snake venoms promote stress-induced senescence in human fibroblasts.

Snake venoms are widely studied in terms of their systemic toxicity and proteolytic, hemotoxic, neurotoxic, and cytotoxic activities. However, little is known about snake-venom-mediated effects when used at low, noncytotoxic concentrations. In the current study, two human fibroblast cell lines of different origin, namely WI-38 fetal lung fibroblasts and BJ foreskin fibroblasts were used to investigate snake-venom-induced adaptive response at a relatively noncytotoxic concentration (0.01 µg/ml). The venoms of Indochinese spitting cobra ( Naja siamensis), western green mamba ( Dendroaspis viridis), forest cobra ( Naja melanoleuca), and southern copperhead ( Agkistrodon contortrix) were considered. Snake venoms promoted FOXO3a-mediated oxidative stress response and to a lesser extent DNA damage response, which lead to changes in cell cycle regulators both at messenger RNA and protein levels, limited cell proliferation and migration, and induced cellular senescence. Taken together, we have shown for the first time that selected snake venoms may also exert adverse effects when used at relatively noncytotoxic concentrations.

First Look at the Venom of Naja ashei.

Naja ashei is an African spitting cobra species closely related to N. mossambica and N. nigricollis. It is known that the venom of N. ashei, like that of other African spitting cobras, mainly has cytotoxic effects, however data about its specific protein composition are not yet available. Thus, an attempt was made to determine the venom proteome of N. ashei with the use of 2-D electrophoresis and MALDI ToF/ToF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry techniques. Our investigation revealed that the main components of analysed venom are 3FTxs (Three-Finger Toxins) and PLA2s (Phospholipases A2). Additionally the presence of cysteine-rich venom proteins, 5'-nucleotidase and metalloproteinases has also been confirmed. The most interesting fact derived from this study is that the venom of N. ashei includes proteins not described previously in other African spitting cobras-cobra venom factor and venom nerve growth factor. To our knowledge, there are currently no other reports concerning this venom composition and we believe that our results will significantly increase interest in research of this species.

Proteomic Analyses of Agkistrodon contortrix contortrix Venom Using 2D Electrophoresis and MS Techniques.

Snake venom is a complex mixture of proteins and peptides which in the Viperidae is mainly hemotoxic. The diversity of these components causes the venom to be an extremely interesting object of study. Discovered components can be used in search for new pharmaceuticals used primarily in the treatment of diseases of the cardiovascular system. In order to determine the protein composition of the southern copperhead venom, we have used high resolution two dimensional electrophoresis and MALDI ToF/ToF MS-based identification. We have identified 10 groups of proteins present in the venom, of which phospholipase A2 and metalloprotease and serine proteases constitute the largest groups. For the first time presence of 5'-nucleotidase in venom was found in this group of snakes. Three peptides present in the venom were also identified. Two of them as bradykinin-potentiating agents and one as an inhibitor.

Proteome and Peptidome of Vipera berus berus Venom.

Snake venom is a rich source of peptides and proteins with a wide range of actions. Many of the venom components are currently being tested for their usefulness in the treatment of many diseases ranging from neurological and cardiovascular to cancer. It is also important to constantly search for new proteins and peptides with properties not yet described. The venom of Vipera berus berus has hemolytic, proteolytic and cytotoxic properties, but its exact composition and the factors responsible for these properties are not known. Therefore, an attempt was made to identify proteins and peptides derived from this species venom by using high resolution two-dimensional electrophoresis and MALDI ToF/ToF mass spectrometry. A total of 11 protein classes have been identified mainly proteases but also l-amino acid oxidases, C-type lectin like proteins, cysteine-rich venom proteins and phospholipases A2 and 4 peptides of molecular weight less than 1500 Da. Most of the identified proteins are responsible for the highly hemotoxic properties of the venom. Presence of venom phospholipases A2 and l-amino acid oxidases cause moderate neuro-, myo- and cytotoxicity. All successfully identified peptides belong to the bradykinin-potentiating peptides family. The mass spectrometry data are available via ProteomeXchange with identifier PXD004958.