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Toxins
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New Trends in Identification and Characterization of Venom Components
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New Trends in Identification and Characterization of Venom Components

A special issue of Toxins (ISSN 2072-6651). This special issue belongs to the section "Animal Venoms".

Deadline for manuscript submissions: closed (31 March 2023) | Viewed by 3192

Special Issue Editors

Dr. Montamas Suntravat

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Guest Editor
National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX 78363, USA
Interests: snake venom toxins; pathophysiology; recombinant proteins; molecular mechanisms of action; envenomation; proteomics; vascular permeability; extracellular vesicle; inflammatory responses; signaling pathways
Special Issues, Collections and Topics in MDPI journals
Dr. Emelyn Salazar

E-Mail Website
Guest Editor
National Natural Toxins Research Center, Texas A&M University-Kingsville, Kingsville, TX, USA
Interests: snake venom; phospholipases A2; inflammation; immune response; coagulation; hemostasis; pathophysiology; antivenoms; recombinant protein; molecular mechanisms of action; envenomation; proteomics

Special Issue Information

Dear Colleagues,

Animal venoms are rich sources of bioactive molecules, displaying a variety of molecular targets and functions. Many toxins have been identified and characterized by venomous animals such as scorpions, spiders, honeybees, and snails, and most of these are from snakes. Each component in the venom has different targets and interferes with the normal biological functions of the target cells, tissues, organs, and physiological systems and can cause severe consequences to human health. Due to their pharmacological activities, several venom components are extensively studied and can be used as diagnostic tools and therapeutic agents. Over the years, more species of venomous animals have been reported, leading to the enormous diversity of unstudied venoms. The advent of more sophisticated identification and characterization techniques has accelerated a more comprehensive knowledge of the venom composition and unveiled the pharmacological complexity of venoms. The exploration of new venom components contributes not only to understanding the pathophysiological changes observed after envenomation but also offers an exciting new avenue for studying venom evolution and toxicology, as well as the discovery of novel pharmacological tools and drug candidates. This Special Issue of Toxins welcomes contributions to the development of innovative approaches, advanced instrumental techniques, and methods to identify and characterize the venom components.

Dr. Montamas Suntravat
Dr. Emelyn Salazar
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a double-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Toxins is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • identification and characterization
  • venomous animals
  • venom components
  • toxin
  • mechanism of action
  • venom secretions

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Published Papers (1 paper)

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Research

30 pages, 5095 KiB  
Article
A Combined Bioassay and Nanofractionation Approach to Investigate the Anticoagulant Toxins of Mamba and Cobra Venoms and Their Inhibition by Varespladib
by Arif Arrahman, Taline D. Kazandjian, Kristina B. M. Still, Julien Slagboom, Govert W. Somsen, Freek J. Vonk, Nicholas R. Casewell and Jeroen Kool
Toxins 2022, 14(11), 736; https://doi.org/10.3390/toxins14110736 - 27 Oct 2022
Cited by 6 | Viewed by 2711
Abstract
Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However, mounting evidence suggests these venoms can additionally cause coagulopathic symptoms, as demonstrated by some Asian elapids and African spitting cobras. This [...] Read more.
Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However, mounting evidence suggests these venoms can additionally cause coagulopathic symptoms, as demonstrated by some Asian elapids and African spitting cobras. This study sought to investigate the coagulopathic potential of venoms from medically important elapids of the genera Naja (true cobras), Hemachatus (rinkhals), and Dendroaspis (mambas). Crude venoms were bioassayed for coagulant effects using a plasma coagulation assay before RPLC/MS was used to separate and identify venom toxins in parallel with a nanofractionation module. Subsequently, coagulation bioassays were performed on the nanofractionated toxins, along with in-solution tryptic digestion and proteomics analysis. These experiments were then repeated on both crude venoms and on the nanofractionated venom toxins with the addition of either the phospholipase A2 (PLA2) inhibitor varespladib or the snake venom metalloproteinase (SVMP) inhibitor marimastat. Our results demonstrate that various African elapid venoms have an anticoagulant effect, and that this activity is significantly reduced for cobra venoms by the addition of varespladib, though this inhibitor had no effect against anticoagulation caused by mamba venoms. Marimastat showed limited capacity to reduce anticoagulation in elapids, affecting only N. haje and H. haemachatus venom at higher doses. Proteomic analysis of nanofractionated toxins revealed that the anticoagulant toxins in cobra venoms were both acidic and basic PLA2s, while the causative toxins in mamba venoms remain uncertain. This implies that while PLA2 inhibitors such as varespladib and metalloproteinase inhibitors such as marimastat are viable candidates for novel snakebite treatments, they are not likely to be effective against mamba envenomings. Full article
(This article belongs to the Special Issue New Trends in Identification and Characterization of Venom Components)
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Figure 1

Figure 1
<p>A schematic overview of the complete analytical and biochemical workflow. There are two main experiments shown in <a href="#toxins-14-00736-f001" class="html-fig">Figure 1</a> that run simultaneously in this study: (i) crude snake venom plate reader-based bioassaying (for assessing inhibition potential of anticoagulant activity by the small molecule inhibitors varespladib and marimastat), (ii) venom separation (using reversed-phase high-performance liquid chromatography; RP-HPLC or LC in short) coupled to UV detection followed by mass spectrometry (MS) with parallel nanofractionation for high-resolution fraction collection of separated toxins onto 384-well plates. Well plates with nanofractionated venom toxins are then vacuum centrifuged to dryness overnight, followed by bioassaying to assess anticoagulant activity. This data obtained is then processed to deliver bioassay chromatograms. Dried well plates can also be used for subsequent toxin identification by proteomics. Key: TIMS-TOF-MS, trapped ion mobility spectrometry time of flight mass spectrometry; HRMS, high-resolution mass spectrometry (HRMS); RP-nanoLC, reversed-phase nanoflow liquid chromatography.</p> Full article ">Figure 2
<p>Venoms from elapids demonstrate anticoagulant activity on bovine plasma. Crude venom activity was measured as the mean area under the curve (AUC) of each venom concentration in the relevant clotting period (24–71 min), standardised by the mean AUC of the negative control in the same period. Lines were generated using a linear model and error bars represent the standard error of the mean (SEM) of 3–7 replicates (wells containing excess bubbles caused reading errors and had to be removed from analysis).</p> Full article ">Figure 3
<p>Anticoagulant venom activity of cobra venoms are inhibited by the PLA<sub>2</sub> inhibitor varespladib. <span class="html-italic">H. haemachatus</span>, <span class="html-italic">N. haje</span>, and <span class="html-italic">N. naja</span> additionally show significant inhibition by some marimastat doses. Clotting activity was measured as the mean area under the curve (AUC) of each sample type in the relevant clotting period (22–71 min), standardised by the mean AUC of the negative control in the same period. Error bars represent the standard error of the mean (SEM) of 3–4 measurements (some sample wells needed to be removed due to disruption in readings caused by bubbles). Asterisks represent significant increases in coagulant activity as measured by one-way ANOVA and Dunnett’s Multiple Comparison post-hoc testing; *—<span class="html-italic">p</span> &lt; 0.05, **—<span class="html-italic">p</span> &lt; 0.01, ***—<span class="html-italic">p</span> &lt; 0.0001.</p> Full article ">Figure 4
<p>Correlation of LC-UV and MS chromatogram and bioassay chromatogram of <span class="html-italic">D. polylepis</span> venom. (<b>A</b>) UV chromatogram of <span class="html-italic">D. polylepis</span> venom (1 mg/mL, 50 μL injection volume, post-column split into 1:9 ratio. The smaller portion went to UV (220 and 254 nm recorded), and then to MS detection. Superimposed bioassay chromatograms resulting from analyses of <span class="html-italic">D. polylepis</span> venom (1 mg/mL, 50 µL injection volume) in the presence of different concentrations of (<b>B</b>) varespladib and (<b>C</b>) marimastat. (<b>D</b>) Superimposed anticoagulation bioassay chromatograms resulting from analyses of several diluted <span class="html-italic">D. polylepis</span> venom samples ranging from 1 mg/mL to 0.2 mg/mL (50 µL per injection). (<b>E</b>) Base peak chromatogram (BPC) and extracted ion currents (XIC) mass spectrometry. Bioassay chromatograms are correlated to UV and MS. Proteomic annotation of <span class="html-italic">D. polylepis</span> venom using Mascot software against (<b>F</b>) the Swiss-Prot database and (<b>G</b>) species-specific venom gland transcriptomic-derived database. The protein score represents the probability that designated proteins, defined by elution time, are present in the sample.</p> Full article ">Figure 5
<p>Correlation of LC-UV and MS chromatogram and bioassay chromatogram of <span class="html-italic">D. angusticeps</span> venom. (<b>A</b>) UV chromatogram of <span class="html-italic">D. angusticeps</span> venom (1 mg/mL, 50 μL injection volume, post-column split into 1:9 ratio. The smaller portion went to UV (220 and 254 nm recorded); and then to MS detection. Superimposed bioassay chromatograms resulting from analyses of <span class="html-italic">D. angusticeps</span> venom (1 mg/mL, 50 µL injection volume) in the presence of different concentrations of (<b>B</b>) varespladib and (<b>C</b>) marimastat. (<b>D</b>) Superimposed anticoagulation bioassay chromatograms resulting from analyses of several diluted <span class="html-italic">D. angusticeps</span> venom samples ranging from 1 mg/mL to 0.2 mg/mL (50 µL per injection). (<b>E</b>) Base peak chromatogram (BPC) and extracted ion currents (XIC) mass spectrometry. Bioassay chromatograms are correlated to UV and MS. Proteomic annotation of <span class="html-italic">D. angusticeps</span> venom using Mascot software against (<b>F</b>) the Swiss-Prot database and (<b>G</b>) species-specific venom gland transcriptomic-derived database. The protein score represents the probability that designated proteins, defined by elution time, are present in the sample.</p> Full article ">Figure 6
<p>Correlation of LC-UV and MS chromatogram and bioassay chromatogram of <span class="html-italic">N. naja</span> venom. (<b>A</b>) UV chromatogram of <span class="html-italic">N. naja</span> venom (1 mg/mL, 50 μL injection volume, post-column split into 1:9 ratio of which the smaller portion went to UV (220 and 254 nm recorded); and then to MS detection. Superimposed bioassay chromatograms resulting from analyses of <span class="html-italic">N. naja</span> venom (0.2 mg/mL, 50 µL injection volume) in the presence of different concentrations of (<b>B</b>) varespladib. (<b>C</b>) Superimposed anticoagulation bioassay chromatograms resulting from analyses of several diluted <span class="html-italic">N. naja</span> venom samples ranging from 1 mg/mL to 0.008 mg/mL (50 µL per injection). (<b>D</b>) Base peak chromatogram (BPC) and extracted ion currents (XIC) mass spectrometry. Bioassay chromatograms are correlated to UV and MS. Proteomic annotation of <span class="html-italic">N. naja</span> venom using Mascot software against (<b>E</b>) the Swiss-Prot database and (<b>F</b>) species-specific venom gland transcriptomic-derived database.</p> Full article ">Figure 7
<p>Correlation of LC-UV and MS chromatogram and bioassay chromatogram of <span class="html-italic">N. pallida</span> venom. (<b>A</b>) UV chromatogram of <span class="html-italic">N. pallida</span> venom (1 mg/mL, 50 μL injection volume, post-column split into 1:9 ratio of which the smaller portion went to UV (220 and 254 nm recorded); and then to MS detection. Superimposed bioassay chromatograms resulting from analyses of <span class="html-italic">N. pallida</span> venom (0.2 mg/mL, 50 µL injection volume) in the presence of different concentrations of (<b>B</b>) varespladib. (<b>C</b>) Superimposed anticoagulation bioassay chromatograms resulting from analyses of several diluted <span class="html-italic">N. pallida</span> venom samples ranging from 1 mg/mL to 0.008 mg/mL (50 µL per injection). (<b>D</b>) Base peak chromatogram (BPC) and extracted ion currents (XIC) mass spectrometry. Bioassay chromatograms are correlated to UV and MS. Proteomic annotation of <span class="html-italic">N. pallida</span> venom using Mascot software against (<b>E</b>) the Swiss-Prot database and (<b>F</b>) species-specific venom gland transcriptomic-derived database. The protein score represents the probability that designated proteins, defined by elution time, are present in the sample.</p> Full article ">Figure 8
<p>Correlation of LC-UV and MS chromatogram and bioassay chromatogram of <span class="html-italic">N. nigricollis</span> venom. (<b>A</b>) UV chromatogram of <span class="html-italic">N. nigricollis</span> venom (1 mg/mL, 50 μL injection volume, post-column split into 1:9 ratio and a smaller portion went to UV (220 and 254 nm recorded); and then to MS detection. Superimposed bioassay chromatograms resulting from analyses of <span class="html-italic">N. nigricollis</span> venom (0.2 mg/mL, 50 µL injection volume) in the presence of different concentrations of (<b>B</b>) varespladib. (<b>C</b>) Superimposed anticoagulation bioassay chromatograms resulting from analyses of several diluted <span class="html-italic">N. nigricollis</span> venom samples ranging from 1 mg/mL to 0.008 mg/mL (50 µL per injection). (<b>D</b>) Base peak chromatogram (BPC) and extracted ion currents (XIC) mass spectrometry. Bioassay chromatograms are correlated to UV and MS. Proteomic annotation of <span class="html-italic">N. nigricollis</span> venom using Mascot software against (<b>E</b>) the Swiss-Prot database and (<b>F</b>) species-specific venom gland transcriptomic-derived database. The protein score represents the probability that designated proteins, defined by elution time, are present in the sample.</p> Full article ">Figure 9
<p>Correlation of LC-UV and MS chromatogram and bioassay chromatogram of <span class="html-italic">N. haje</span> venom. (<b>A</b>) UV chromatogram of <span class="html-italic">N. haje</span> venom (1 mg/mL, 50 μL injection volume, post-column split into 1:9 ratio of which the smaller portion went to UV (220 and 254 nm recorded); and then to MS detection. Superimposed bioassay chromatograms resulting from analyses of <span class="html-italic">N. haje</span> venom (0.2 mg/mL, 50 µL injection volume) in the presence of different concentrations of (<b>B</b>) varespladib. (<b>C</b>) Superimposed anticoagulation bioassay chromatograms resulting from analyses of several diluted <span class="html-italic">N. haje</span> venom samples ranging from 1 mg/mL to 0.008 mg/mL (50 µL per injection). (<b>D</b>) Base peak chromatogram (BPC) and extracted ion currents (XIC) mass spectrometry. Bioassay chromatograms are correlated to UV and MS. Proteomic annotation of <span class="html-italic">N. haje</span> venom using Mascot software against (<b>E</b>) the Swiss-Prot database and (<b>F</b>) species-specific venom gland transcriptomic-derived database. The protein score represents the probability that designated proteins, defined by elution time, are present in the sample.</p> Full article ">Figure 10
<p>Correlation of LC-UV and MS chromatogram and bioassay chromatogram of <span class="html-italic">H. Haemachatus</span> venom. (<b>A</b>) UV chromatogram of <span class="html-italic">H. haemachatus</span> venom (1 mg/mL, 50 μL injection volume, post-column split into 1:9 ratio. The smaller portion went to UV (220 and 254 nm recorded), and then to MS detection. Superimposed bioassay chromatograms resulting from analyses of <span class="html-italic">H. haemachatus</span> venom (0.2 mg/mL, 50 µL injection volume) in the presence of different concentrations of (<b>B</b>) varespladib. (<b>C</b>) Superimposed anticoagulation bioassay chromatograms resulting from analyses of several diluted <span class="html-italic">H. haemachatus</span> venom samples ranging from 1 mg/mL to 0.008 mg/mL (50 µL per injection). (<b>D</b>) Base peak chromatogram (BPC) and extracted ion currents (XIC) mass spectrometry. Bioassay chromatograms are correlated to UV and MS. Proteomic annotation of <span class="html-italic">H. haemachatus</span> venom using Mascot software against (<b>E</b>) the Swiss-Prot database and (<b>F</b>) species-specific venom gland transcriptomic-derived database. The protein score represents the probability that designated proteins, defined by elution time, are present in the sample. Note: after nanofractionation analytics, no procoagulant toxin peaks were observed (not shown in the figure). SVSPs and other mainly larger toxins can denature during the chromatographic separation thereby losing their biological activity (which we have observed in other ongoing research projects).</p> Full article ">
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