Influence of the collection methodology on the Apis mellifera venom composition: Peptide analysis

Differences of venom peptide composition as function of two collection methodologies, electrical stimulation (ES) and reservoir disruption (RD), were analyzed by reverse-phase HPLC in Apis mellifera races - A. m. adansonii, A. m. ligustica and Africanized honeybee. The analyses were performed through determination of the relative number and percentage of each molecular form associated to the peaks eluted by chromatography. Comparison of these profiles revealed qualitative and quantitative differences related to the venom collection methodology as well to the three races analyzed. In contrast to data usually found for venom proteins, the three races presented a major number of peaks or molecular forms when venom was collected by ES. Besides, in general, the relative concentration of each peak was higher for ES in relation to RD. That indicates the presence of molecular precursors in the venom obtained by RD. The presence/absence pattern of the peaks, such as their relative concentrations showed a closer similarity between A. m. adansonii and the Africanized honeybees than that observed between these and A. m. ligustica. The obtained data allowed a discussion about the differences in the relative concentration of each venom component according to the collection methodology, and finally the biological action of the venom in different races. So, these results, apart from being useful to establish a peptide profile for each bee race as a function of the venom collection methodology, pointed out once more that the chromatographic techniques are a great tool for the identification of A. mellifera subspecies.

The venom composition of the parasitic wasp Chelonus inanitus resolved by combined expressed sequence tags analysis and proteomic approach

Background Parasitic wasps constitute one of the largest group of venomous animals. Although some physiological effects of their venoms are well documented, relatively little is known at the molecular level on the protein composition of these secretions. To identify the majority of the venom proteins of the endoparasitoid wasp Chelonus inanitus (Hymenoptera: Braconidae), we have randomly sequenced 2111 expressed sequence tags (ESTs) from a cDNA library of venom gland. In parallel, proteins from pure venom were separated by gel electrophoresis and individually submitted to a nano-LC-MS/MS analysis allowing comparison of peptides and ESTs sequences. Results About 60% of sequenced ESTs encoded proteins whose presence in venom was attested by mass spectrometry. Most of the remaining ESTs corresponded to gene products likely involved in the transcriptional and translational machinery of venom gland cells. In addition, a small number of transcripts were found to encode proteins that share sequence similarity with well-known venom constituents of social hymenopteran species, such as hyaluronidase-like proteins and an Allergen-5 protein. An overall number of 29 venom proteins could be identified through the combination of ESTs sequencing and proteomic analyses. The most highly redundant set of ESTs encoded a protein that shared sequence similarity with a venom protein of unknown function potentially specific of the Chelonus lineage. Venom components specific to C. inanitus included a C-type lectin domain containing protein, a chemosensory protein-like protein, a protein related to yellow-e3 and ten new proteins which shared no significant sequence similarity with known sequences. In addition, several venom proteins potentially able to interact with chitin were also identified including a chitinase, an imaginal disc growth factor-like protein and two putative mucin-like peritrophins. Conclusions The use of the combined approaches has allowed to discriminate between cellular and truly venom proteins. The venom of C. inanitus appears as a mixture of conserved venom components and of potentially lineage-specific proteins. These new molecular data enrich our knowledge on parasitoid venoms and more generally, might contribute to a better understanding of the evolution and functional diversity of venom proteins within Hymenoptera.

Venom composition and strategies in spiders: is everything possible?

This review on all spider venom components known by the end of 2010 bases on 1618 records for venom compounds from 174 spider species (= 0.41% of all known species) belonging to 32 families (= 29% of all existing spider families). Spiders investigated for venom research are either big (many mygalomorph species, Nephilidae, Ctenidae and Sparassidae) or medically important for humans (e.g. Loxosceles or Latrodectus species). Venom research widely ignored so far the two most species-rich families (Salticidae and Linyphiidae) and strongly neglected several other very abundant families (Araneidae, Lycosidae, Theridiidae, Thomisidae and Gnaphosidae). We grouped the known 1618 records for venom compounds into six categories: low molecular mass compounds (16 % of all compounds), acylpolyamines (11 %), linear peptides (6 %), cysteine-knotted mini-proteins (60 %), neurotoxic proteins (1 %) and enzymes (6 %). Low molecular mass compounds are known from many spider families and contain organic acids, nucleosides, nucleotides, amino acids, amines, polyamines, and some further substances, many of them acting as neurotransmitters. Acylpolyamines contain amino acids (Araneidae and Nephilidae) or not (several other families) and show a very high diversity within one species. Linear peptides, also called cytolytic, membranolytic or antimicrobial, exert a highly specific structure and are so far only known from Ctenidae, Lycosidae, Oxyopidae and Zodariidae. Cysteine-knotted mini-proteins represent the majority of venom compounds because research so far focused on them. They probably occur in most but not all spider families. Neurotoxic proteins so far are only known from theridiid spiders. Enzymes had been neglected for some time but meanwhile it becomes obvious that they play an important role in spider venoms. Sixteen enzymes either cleave polymers in the extracellular matrix or target phospholipids and related compounds in membranes. The overall structure of these compounds is given and the function, as far as it is known, is described. Since several of these component groups are presented in one average spider venom, we discuss the known interactions and synergisms and give reasons for such a functional redundancy. We also discuss main evolutionary pathways for spider venom compounds such as high variability among components of one group, synergistic interactions between cysteine-knotted mini-proteins and other components (low molecular mass compounds and linear peptides), change of function from ion-channel acting mini-proteins to cytolytic effects and replacement of mini-proteins by linear peptides, acylpolyamines, large proteins or enzymes. We also add first phylogenetic considerations.

Crotalus durissus collilineatus venom gland transcriptome: Analysis of gene expression profile

Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil. Crotalus durissus collilineatus subspecies is related to a great number of accidents in Southeast and Central West regions, but few studies on its venom composition have been carried out to date. In an attempt to describe the transcriptional profile of the C. durissus collilineatus venom gland, we generated a cDNA library and the sequences obtained could be identified by similarity searches on existing databases. Out of 673 expressed sequence tags (ESTs) 489 produced readable sequences comprising 201 singletons and 47 clusters of two or more ESTs. One hundred and fifty reads (60.5%) produced significant hits to known sequences. The results showed a predominance of toxin-coding ESTs instead of transcripts coding for proteins involved in all cellular functions. The most frequent toxin was crotoxin, comprising 88% of toxin-coding sequences. Crotoxin B, a basic phospholipase A(2) (PLA(2)) subunit of crotoxin, was represented in more variable forms comparing to the non-enzymatic subunit (crotoxin A), and most sequences coding this molecule were identified as CB1 isoform from Crotalus durissus terrificus venom. Four percent of toxin-related sequences in this study were identified as growth factors, comprising five sequences for vascular endothelial growth factor (VEGF) and one for nerve growth factor (NGF) that showed 100% of identity with C. durissus terrificus NGF. We also identified two clusters for metalloprotease from PII class comprising 3% of the toxins, and two for serine proteases, including gyroxin (2.5%). The remaining 2.5% of toxin-coding ESTs represent singletons identified as homologue sequences to cardiotoxin, convulxin, angiotensin-converting enzyme inhibitor and C-type natriuretic peptide, Ohanin, crotamin and PLA(2) inhibitor. These results allowed the identification of the most common classes of toxins in C. durissus collilineatus snake venom, also showing some unknown classes for this subspecies and even for C. durissus species, such as cardiotoxins and VEGF. (C) 2009 Published by Elsevier Masson SAS.

Africanized honey bee (Apis mellifera) venom profiling: Seasonal variation of melittin and phospholipase A(2) levels

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of a novel melittin isoform from Africanized Apis mellifera venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Combined snake venomics and venom gland transcriptomic analysis of Bothropoides pauloensis

Unraveling the repertoire of venom toxins of Bothropoides pauloensis was assessed by snake venomics and venom gland transcriptomic surveys. Both approaches yielded converging overall figures, pointing to metalloproteinases (similar to 37%), PLA(2)s (26-32%), and vasoactive (bradykinin-potentiating) peptides (12-17%) as the major toxin classes. The high occurrence of SVMPs, PLA(2) molecules, vasoactive peptides, along with serine proteinases, explains the local and systemic effects observed in envenomations by B. pauloensis. Minor (<3%) C-type lectin, serine proteinase, L-amino acid oxidase, nerve growth factor, and CRISP molecules were also identified in the transcriptome and the proteome. Low abundance (0.3%) EST singletons coding for vascular endothelial growth factor (svVEGF), ohanin, hyaluronidase, and 5' nucleotidase were found only in the venom gland cDNA library. At the molecular level, the transcriptomic and proteomic datasets display low compositional concordance. In particular, although there is good agreement between transcriptome and proteome in the identity of BPPs, PLA(2) molecules and L-amino acid oxidase, both datasets strongly depart in their C-type lectin and SVMP complements. These data support the view that venom composition is influenced by transcriptional and translational mechanisms and emphasize the value of combining proteomic and transcriptomic approaches to acquire a more complete understanding of the toxinological profile and natural history of the snake venom. (C) 2012 Elsevier B.V. All rights reserved.

Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management

We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4-115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20-25 main toxins belonging to the following protein families: disintegrin, PLA(2), serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), L-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA2 crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA(2)s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for similar to 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted (similar to 60%) recognition of PLA(2) molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA(2) molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed. (C) 2010 Elsevier B.V. All rights reserved.

Species and regional variations in the effectiveness of antivenom against the in Vitro neurotoxicity of death adder (Acanthophis) venoms

Although viperlike in appearance and habit, death adders belong to the Elapidae family of snakes. Systemic envenomation represents a serious medical problem with antivenom, which is raised against Acanthophis antarcticus venom, representing the primary treatment. This study focused on the major Acanthophis variants from Australia and islands in the Indo-Pacific region. Venoms were profiled using liquid chromatography-mass spectrometry, and analyzed for in vitro neurotoxicity (0.3-10 mug/ml), as well as the effectiveness of antivenom. (1-5 units/ml; 10 min prior to the addition of 10 mug/ml venom). The following death adder venoms were examined: A. antarcticus (from separate populations in New South Wales, Queensland, South Australia, and Western Australia), A. hawkei, A. praelongus, A. pyrrhus, A. rugosus, A. wellsi, and venom from an unnamed species from the Indonesian island of Seram. All venoms abolished indirect twitches of the chick isolated biventer cervicis nerve-muscle preparation in a dose-dependent manner. In addition, all venoms blocked responses to exogenous acetylcholine (1 m-M) and carbachol (20 muM), but not KCl (40 mM), suggesting postsynaptic neurotoxicity. Death adder antivenom (1 unit/ml) prevented the neurotoxic effects of A. pyrrhus, A. praelongus, and A. hawkei venoms, although it was markedly less effective against venoms from A. antarcticus (NSW, SA, WA), A. rugosus, A. wellsi, and A. sp. Scram. However, at 5 units/ml, antivenom was effective against all venoms tested. Death adder venoms, including those from A. antarcticus geographic variants, differed not only in their venom composition but also in their neurotoxic activity and susceptibility to antivenom. For the first time toxicological aspects of A. hawkei, A. wellsi, A. rugosus, and A. sp. Seram venoms were studied. (C) 2001 Academic Press.

Electrospray liquid chromatography/mass spectrometry fingerprinting of Acanthophis (death adder) venoms: taxonomic and toxinological implications

Death adders (genus Acanthophis) are unique among elapid snakes in both morphology and venom composition. Despite this genus being among the most divergent of all elapids, the venom has been historically regarded as relatively quite simple. In this study, liquid chromatography/mass spectrometry (LC/MS) analysis has revealed a. much greater diversity in venom composition, including the presence of molecules of novel molecular weights that may represent a new class of venom component. Furthermore, significant variation exists between species and populations,, which allow for the LC/MS fingerprinting of each species. Mass profiling of Acanthophis venoms clearly demonstrates the effectiveness of this technique which underpins fundamental studies ranging from chemotaxonomy to drug design. Copyright (C) 2002 John Wiley Sons, Ltd.

Bothrops insularis venomics: A proteomic analysis supported by transcriptomic-generated sequence data

A joint transcriptomic and proteomic approach employing two-dimensional electrophoresis, liquid chromatography and mass spectrometry was carried out to identify peptides and proteins expressed by the venom gland of the snake Bothrops insularis, an endemic species of Queimada Grande Island, Brazil. Four protein families were mainly represented in processed spots, namely metalloproteinase, serine proteinase, phospholipase A(2) and lectin. Other represented families were growth factors, the developmental protein G10, a disintegrin and putative novel bradykinin-potentiating peptides. The enzymes were present in several isoforms. Most of the experimental data agreed with predicted values for isoelectric point and M(r) of proteins found in the transcriptome of the venom gland. The results also support the existence of posttranslational modifications and of proteolytic processing of precursor molecules which could lead to diverse multifunctional proteins. This study provides a preliminary reference map for proteins and peptides present in Bothrops insularis whole venom establishing the basis for comparative studies of other venom proteomes which could help the search for new drugs and the improvement of venom therapeutics. Altogether, our data point to the influence of transcriptional and post-translational events on the final venom composition and stress the need for a multivariate approach to snake venomics studies. (c) 2009 Elsevier B.V. All rights reserved.

Comparative structural studies on Lys49-phospholipases A(2) from Bothrops genus reveal their myotoxic site

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A new scenario of bioprospecting of Hymenoptera venoms through proteomic approach

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pseudechis australis Venomics: Adaptation for a Defense against Microbial Pathogens and Recruitment of Body Transferrin

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Variação sexual, ontogenética e ambiental do veneno de Bothrops jararaca da icro-região de Botucatu-SP: caracterização enzimática, bioquímica e farmacológica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)