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.

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Animal Venoms have Potential to Treat Cancer

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666181221120817 ◽

2019 ◽

Vol 18 (30) ◽

pp. 2555-2566 ◽

Cited By ~ 6

Author(s):

Bhaswati Chatterjee

Keyword(s):

Cancer Therapeutics ◽

Sea Anemones ◽

Anticancer Activities ◽

Inhibition Of Proliferation ◽

Cycle Arrest ◽

Venomous Animals ◽

Anti Cancer ◽

Cancerous Cells ◽

Animal Venoms ◽

Proteins And Peptides

The resistance to chemotherapeutics by the cancerous cells has made its treatment more complicated. Animal venoms have emerged as an alternative strategy for anti-cancer therapeutics. Animal venoms are cocktails of complex bioactive chemicals mainly disulfide-rich proteins and peptides with diverse pharmacological actions. The components of venoms are specific, stable, and potent and have the ability to modify their molecular targets thus making them good therapeutics candidates. The isolation of cancer-specific components from animal venoms is one of the exciting strategies in anti-cancer research. This review highlights the identified venom peptides and proteins from different venomous animals like snakes, scorpions, spiders, bees, wasps, snails, toads, frogs and sea anemones and their anticancer activities including inhibition of proliferation of cancer cells, their invasion, cell cycle arrest, induction of apoptosis and the identification of involved signaling pathways.

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Rapid and differential evolution of the venom composition of a parasitoid wasp depending on the host strain

10.1101/796649 ◽

2019 ◽

Author(s):

Fanny Cavigliasso ◽

Hugo Mathé-Hubert ◽

Laurent Kremmer ◽

Christian Rebuf ◽

Jean-Luc Gatti ◽

...

Keyword(s):

Differential Evolution ◽

Rapid Evolution ◽

Intraspecific Variability ◽

Protein Composition ◽

Host Strain ◽

Parasitoid Wasps ◽

Evolutionary Potential ◽

Venom Protein ◽

Venom Composition ◽

Venom Proteins

AbstractParasitoid wasps rely primarily on venom to suppress the immune response and regulate the physiology of their host. Intraspecific variability of venom protein composition has been documented in some species, but its evolutionary potential is poorly understood. We performed an experimental evolution initiated with crosses of two lines of Leptopilina boulardi of different venom composition to generate variability and create new combinations of venom factors. The offspring were maintained for 10 generations on two strains of Drosophila melanogaster differing in resistance / susceptibility to the parasitoid lines. The venom composition of individuals was characterized by a semi-automatic analysis of 1D SDS-PAGE protein profiles whose accuracy was checked by Western blot analysis of well-characterized venom proteins. Results evidenced a rapid and differential evolution of the venom composition on both hosts and showed that the proteins beneficial on one host can be costly on the other. Overall, we demonstrated the capacity of rapid evolution of the venom composition in parasitoid wasps, important regulators of arthropod populations, suggesting a potential for adaptation to new hosts. Our approach also proved relevant in identifying, among the diversity of venom proteins, those possibly involved in parasitism success and whose role deserves to be deepened.Key ContributionThe venom protein composition of parasitoid wasps can evolve rapidly and differently depending on the host strain. Studying this evolution can help identify new venom proteins possibly involved in parasitism success on a given host.

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Parasitic success and venom composition evolve upon specialization of parasitoid wasps to different host species

10.1101/2020.10.24.353417 ◽

2020 ◽

Author(s):

Fanny Cavigliasso ◽

Hugo Mathé-Hubert ◽

Jean-Luc Gatti ◽

Dominique Colinet ◽

Marylène Poirié

Keyword(s):

Biological Control ◽

Immune Response ◽

Host Species ◽

Experimental Evolution ◽

Parasitoid Wasps ◽

New Host ◽

Venom Protein ◽

Leptopilina Boulardi ◽

Venom Composition ◽

Sds Page

AbstractFemale endoparasitoid wasps usually inject venom into hosts to suppress their immune response and ensure offspring development. However, the parasitoid’s ability to evolve towards increased success on a given host simultaneously with the evolution of the composition of its venom has never been demonstrated. Here, we designed an experimental evolution to address this question. We crossed two parasitoid lines of Leptopilina boulardi differing both in parasitic success on different Drosophila hosts and venom composition. F2 descendants were reared on three different Drosophila species for nine generations. We tested for evolution of parasitic success over the generations and for the capacity of parasitoids selected on a given host to succeed on another host. We also tested whether the venom composition - analyzed on the basis of the variation in intensity of the venom protein bands on SDS-PAGE 1D - evolved in response to different host species. Results showed a specialization of the parasitoids on their selection host and a rapid and differential evolution of the venom composition according to the host. Overall, data suggest a high potential for parasitoids to adapt to a new host, which may have important consequences in the field as well in the context of biological control.

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Gradual and Discrete Ontogenetic Shifts in Rattlesnake Venom Composition and Assessment of Hormonal and Ecological Correlates

Toxins ◽

10.3390/toxins12100659 ◽

2020 ◽

Vol 12 (10) ◽

pp. 659 ◽

Cited By ~ 1

Author(s):

Richard B. Schonour ◽

Emma M. Huff ◽

Matthew L. Holding ◽

Natalie M. Claunch ◽

Schyler A. Ellsworth ◽

...

Keyword(s):

Protein Composition ◽

Adult Size ◽

Venom Protein ◽

Ontogenetic Shifts ◽

Venom Composition ◽

Serial Sampling ◽

Body Sizes ◽

Dietary Shifts ◽

Chromatographic Peaks ◽

The Relationship

Ontogenetic shifts in venom occur in many snakes but establishing their nature as gradual or discrete processes required additional study. We profiled shifts in venom expression from the neonate to adult sizes of two rattlesnake species, the eastern diamondback and the timber rattlesnake. We used serial sampling and venom chromatographic profiling to test if ontogenetic change occurs gradually or discretely. We found evidence for gradual shifts in overall venom composition in six of eight snakes, which sometimes spanned more than two years. Most chromatographic peaks shift gradually, but one quarter shift in a discrete fashion. Analysis of published diet data showed gradual shifts in overall diet composition across the range of body sizes attained by our eight study animals, while the shifts in abundance of different prey classes varied in form from gradual to discrete. Testosterone concentrations were correlated with the change in venom protein composition, but the relationship is not strong enough to suggest causation. Venom research employing simple juvenile versus adult size thresholds may be failing to account for continuous variation in venom composition lifespan. Our results imply that venom shifts represent adaptive matches to dietary shifts and highlight venom for studies of alternative gene regulatory mechanisms.

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Proteomic characterization of two snake venoms: Naja naja atra and Agkistrodon halys

Biochemical Journal ◽

10.1042/bj20040354 ◽

2004 ◽

Vol 384 (1) ◽

pp. 119-127 ◽

Cited By ~ 80

Author(s):

Shuting LI ◽

Jingqiang WANG ◽

Xumin ZHANG ◽

Yan REN ◽

Ning WANG ◽

...

Keyword(s):

Southern China ◽

Gel Filtration ◽

Complex Mixture ◽

Tandem Ms ◽

Post Translational Modification ◽

Combination Strategy ◽

Naja Naja ◽

Maldi Tof ◽

Proteins And Peptides ◽

Naja Atra

Snake venom is a complex mixture of proteins and peptides, and a number of studies have described the biological properties of several venomous proteins. Nevertheless, a complete proteomic profile of venom from any of the many species of snake is not available. Proteomics now makes it possible to globally identify proteins from a complex mixture. To assess the venom proteomic profiles from Naja naja atra and Agkistrodon halys, snakes common to southern China, we used a combination strategy, which included the following four different approaches: (i) shotgun digestion plus HPLC with ion-trap tandem MS, (ii) one-dimensional SDS/PAGE plus HPLC with tandem MS, (iii) gel filtration plus HPLC with tandem MS and (iv) gel filtration and 2DE (two-dimensional gel electrophoresis) plus MALDI–TOF (matrix-assisted laser desorption ionization–time-of-flight) MS. In the present paper, we report the novel identification of 124 and 74 proteins and peptides in cobra and viper venom respectively. Functional analysis based upon toxin categories reveals that, as expected, cobra venom has a high abundance of cardio- and neurotoxins, whereas viper venom contains a significant amount of haemotoxins and metalloproteinases. Although approx. 80% of gel spots from 2DE displayed high-quality MALDI-TOF-MS spectra, only 50% of these spots were confirmed to be venom proteins, which is more than likely to be a result of incomplete protein databases. Interestingly, these data suggest that post-translational modification may be a significant characteristic of venomous proteins.

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PURIFICATION AND CHARACTERIZATION OF A PROTEIN C ACTIVATOR FROM THE VENOM OF AGKISTRODON CONTORTRIX CONTORTRIX

10.1055/s-0038-1643813 ◽

1987 ◽

Author(s):

Carolyn L Orthner ◽

Prabir Bhattacharya ◽

Dudley K Strikland

Keyword(s):

Protein C ◽

Protein Concentration ◽

Gel Filtration ◽

Purification And Characterization ◽

Agkistrodon Contortrix ◽

Purification And Properties ◽

Sds Page ◽

Single Protein ◽

Agkistrodon Contortrix Contortrix

There are two recent reports on the purification and properties of a protein C activator (PCA) from the venom of the Southern copperhead snalce. The purification of a 37,000 Mr nonenzymatic PCA (Martinoli and Stocker, Thrcmb. Res. 43, 253, 1976) as well as of a 20,000 Mr thrombin-like enzyme (Klein and Walker, Biochem. ,25, 4175, 1986) have been described. The purpose of this investigation was to purify and further characterize the PCA(s) from this vencm. A PCA has been isolated by sulphopropyl-Sephadex followed by gel filtration chromatography resulting in approximately a 100-fold purification with a 50% yield. PCA appeared as a single band on SDS-PAGE with an estimated Mr of 32,000 or 37,000 in the absence or presence of β-mercaptoethanol, respectively. High pressure gel permeation cinematography of PCA in Tris-buffered saline, pH 7.5 resulted in a single protein peak with a Mr of 39,000 which was coincident with activity. PCA was a potent activator of human protein C (PC) with a Km for PC of 0.6uM and a Vm of 0.02 sec-1. In addition, PCA catalyzed the arnidolysis of Tosyl-gly-pro-arg-p-nitroanilide (TGPRpNA) with a Km of 1.1 irM and a Vim of 66 sec-1. The rate of arnidolysis of five other pept idyl-arginyl-pNA substrates each tested at 1.0 mM was < 10% that of TGPRpNA. PCA was inhibited by nitrophenylguanidi-nobenzoate (NPGB), phenylmethylsulphonylflouride, D-phe-pro-arg-chloromethyi_ketone (PPACK) and soybean trypsin inhibitor indicating that PCA is a serine protease. The active site concentration of PCA as measured by NPGB titration was 90% that of the protein concentration. Measurement of the rate of PCA inhibition at varying levels of PPACK indicated that it had a Ki of 34uM .and an aUcylation rate constant of 0.09 min-1. PCA activation of PC was completely inhibited by CaC12 with an apparent Ki of 99uM. Since neither PCA arnidolysis of TGPRpNA nor inhibition by PPACK was affected by Ca2+, the effect of this metal was likely on the substrate PC. In summary, a PCA has been purified to homogeneity and has properties which are distinct from those reported. PCA premises to be a useful enzyme in studies of PC and its activation.

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Malaysian Cobra Venom: A Potential Source of Anti-Cancer Therapeutic Agents

Toxins ◽

10.3390/toxins11020075 ◽

2019 ◽

Vol 11 (2) ◽

pp. 75 ◽

Cited By ~ 4

Author(s):

Syafiq Zainal Abidin ◽

Yee Lee ◽

Iekhsan Othman ◽

Rakesh Naidu

Keyword(s):

Snake Venom ◽

Anticancer Agents ◽

Complex Mixture ◽

Therapeutic Agents ◽

Venom Protein ◽

Potential Source ◽

Anti Cancer ◽

Naja Kaouthia ◽

Ophiophagus Hannah

Cancer is a deadly disease and there is an urgent need for the development of effective and safe therapeutic agents to treat it. Snake venom is a complex mixture of bioactive proteins that represents an attractive source of novel and naturally-derived anticancer agents. Malaysia is one of the world’s most biodiverse countries and is home to various venomous snake species, including cobras. Naja kaouthia, Naja sumatrana, and Ophiophagus hannah are three of the most common cobra species in Malaysia and are of medical importance. Over the past decades, snake venom has been identified as a potential source of therapeutic agents, including anti-cancer agents. This present review highlights the potential anticancer activity of the venom and purified venom protein of N. kaouthia, N. sumatrana, and O. hannah. In conclusion, this review highlights the important role of the venom from Malaysian cobras as an important resource that researchers can exploit to further investigate its potential in cancer treatment.

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Proteomic Characterization of the Venom Of Five Bombus (Thoracobombus) Species

10.1101/193524 ◽

2017 ◽

Author(s):

Nezahat Pinar Barkan ◽

Mustafa Bilal Bayazit ◽

Duygu Demiralp Özel

Keyword(s):

Laser Desorption ◽

Bumble Bee ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Flight Mass Spectrometry ◽

Venomous Animals ◽

Venom Composition ◽

Proteomic Techniques ◽

Proteins And Peptides

AbstractVenomous animals use venom; a complex biofluid composed of unique mixtures of proteins and peptides, to act on vital systems of the prey or predator. In bees, venom is solely used for defense against predators. However, the venom composition of bumble bees (Bombus sp.) is largely unknown. Thoracobombus subgenus of Bombus sp. is a diverse subgenus represented by 14 members across Turkey. In this study, we sought out to proteomically characterize the venom of five Thoracobombus species by using bottom-up proteomic techniques. We have obtained two-dimensional polyacrylamide gel (2D-PAGE) images of each venom sample. We have subsequently identified the protein spots by using matrix assisted laser desorption ionization / time of flight mass spectrometry (MALDI-TOF MS). We have identified 47 proteins for Bombus humilis; 32 for B. pascuorum, 60 for B. ruderarius; 39 for B. sylvarum and 35 for B. zonatus. Our analyses provide the primary proteomic characterization of five bumble bee species’ venom composition.

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Bottom-Up Proteomic Analysis of Polypeptide Venom Components of the Giant Ant Dinoponera Quadriceps

Toxins ◽

10.3390/toxins11080448 ◽

2019 ◽

Vol 11 (8) ◽

pp. 448 ◽

Cited By ~ 7

Author(s):

Douglas Oscar Ceolin Mariano ◽

Úrsula Castro de Oliveira ◽

André Junqueira Zaharenko ◽

Daniel Carvalho Pimenta ◽

Gandhi Rádis-Baptista ◽

...

Keyword(s):

Biological Effects ◽

Glucose Dehydrogenase ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Crude Venom ◽

Venom Protein ◽

Bottom Up ◽

Venom Composition ◽

Proteomics Approach ◽

Analytical Approaches

Ant species have specialized venom systems developed to sting and inoculate a biological cocktail of organic compounds, including peptide and polypeptide toxins, for the purpose of predation and defense. The genus Dinoponera comprises predatory giant ants that inoculate venom capable of causing long-lasting local pain, involuntary shaking, lymphadenopathy, and cardiac arrhythmias, among other symptoms. To deepen our knowledge about venom composition with regard to protein toxins and their roles in the chemical–ecological relationship and human health, we performed a bottom-up proteomics analysis of the crude venom of the giant ant D. quadriceps, popularly known as the “false” tocandiras. For this purpose, we used two different analytical approaches: (i) gel-based proteomics approach, wherein the crude venom was resolved by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and all protein bands were excised for analysis; (ii) solution-based proteomics approach, wherein the crude venom protein components were directly fragmented into tryptic peptides in solution for analysis. The proteomic data that resulted from these two methodologies were compared against a previously annotated transcriptomic database of D. quadriceps, and subsequently, a homology search was performed for all identified transcript products. The gel-based proteomics approach unequivocally identified nine toxins of high molecular mass in the venom, as for example, enzymes [hyaluronidase, phospholipase A1, dipeptidyl peptidase and glucose dehydrogenase/flavin adenine dinucleotide (FAD) quinone] and diverse venom allergens (homologous of the red fire ant Selenopsis invicta) and venom-related proteins (major royal jelly-like). Moreover, the solution-based proteomics revealed and confirmed the presence of several hydrolases, oxidoreductases, proteases, Kunitz-like polypeptides, and the less abundant inhibitor cysteine knot (ICK)-like (knottin) neurotoxins and insect defensin. Our results showed that the major components of the D. quadriceps venom are toxins that are highly likely to damage cell membranes and tissue, to cause neurotoxicity, and to induce allergic reactions, thus, expanding the knowledge about D. quadriceps venom composition and its potential biological effects on prey and victims.

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Rapid and Differential Evolution of the Venom Composition of a Parasitoid Wasp Depending on the Host Strain

Toxins ◽

10.3390/toxins11110629 ◽

2019 ◽

Vol 11 (11) ◽

pp. 629 ◽

Cited By ~ 9

Author(s):

Cavigliasso ◽

Mathé-Hubert ◽

Kremmer ◽

Rebuf ◽

Gatti ◽

...

Keyword(s):

Differential Evolution ◽

Rapid Evolution ◽

Intraspecific Variability ◽

Protein Composition ◽

Parasitoid Wasp ◽

Parasitoid Wasps ◽

Evolutionary Potential ◽

Venom Protein ◽

Venom Composition ◽

Venom Proteins

Parasitoid wasps rely primarily on venom to suppress the immune response and regulate the physiology of their host. Intraspecific variability of venom protein composition has been documented in some species, but its evolutionary potential is poorly understood. We performed an experimental evolution initiated with the crosses of two lines of Leptopilina boulardi of different venom composition to generate variability and create new combinations of venom factors. The offspring were maintained for 10 generations on two strains of Drosophila melanogaster differing in resistance/susceptibility to the parental parasitoid lines. The venom composition of individuals was characterized by a semi-automatic analysis of 1D SDS-PAGE electrophoresis protein profiles whose accuracy was checked by Western blot analysis of well-characterized venom proteins. Results made evident a rapid and differential evolution of the venom composition on both hosts and showed that the proteins beneficial on one host can be costly on the other. Overall, we demonstrated the capacity of rapid evolution of the venom composition in parasitoid wasps, important regulators of arthropod populations, suggesting a potential for adaptation to new hosts. Our approach also proved relevant in identifying, among the diversity of venom proteins, those possibly involved in parasitism success and whose role deserves to be deepened.

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