Protein Profile Analysis of Two Australian Snake Venoms by One- Dimensional Gel Electrophoresis and MS/MS Experiments.

The Pseudechis colletti and Pseudechis butleri venoms were analyzed by 1-D gel electrophoresis, followed by mass spectrometric analysis of tryptic peptides obtained from the protein bands. Both venoms contain highly potent pharmacologically active components, which were assigned to the following protein families: basic and acidic phospholipases A2 (PLA2s), L-amino acid oxidases (LAAOs), P-III metalloproteinases (P-III SVMPs), 5'- nucleotidases (5'-NTDs), cysteine-rich secretory proteins (CRISPs), venom nerve growth factors (VNGFs) and post-synaptic neurotoxins. Considerable predominance of PLA2s over other toxins is a characteristic feature of both venoms.

Elapid snake venom analyses show the specificity of the peptide composition at the level of genera Naja and Notechis.

Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms-that of the African cobra, N. m.

Structure of the polypeptide crotamine from the Brazilian rattlesnake Crotalus durissus terrificus.

The crystal structure of the myotoxic, cell-penetrating, basic polypeptide crotamine isolated from the venom of Crotalus durissus terrificus has been determined by single-wavelength anomalous dispersion techniques and refined at 1.7 Å resolution. The structure reveals distinct cationic and hydrophobic surface regions that are located on opposite sides of the molecule.

Purification, crystallization and preliminary X-ray diffraction analysis of crotamine, a myotoxic polypeptide from the Brazilian snake Crotalus durissus terrificus.

Crotamine, a highly basic myotoxic polypeptide (molecular mass 4881 Da) isolated from the venom of the Brazilian rattlesnake Crotalus durissus terrificus, causes skeletal muscle contraction and spasms, affects the functioning of voltage-sensitive sodium channels by inducing sodium influx and possesses antitumour activity, suggesting potential pharmaceutical applications. Crotamine was purified from C. durissus terrificus venom; the crystals diffracted to 1.

Crystal structure of a dimeric Ser49- PLA₂-like myotoxic component of the Vipera ammodytes meridionalis venomics reveals determinants of myotoxicity and membrane damaging activity.

Myotoxicity and membrane damage play a central role in the life-threatening effects of the viper envenomation. Myotoxins are an important part of the viper venomics. A Ser49 PLA₂-like myotoxin from the venom of Vipera ammodytes meridionalis, the most venomous snake in Europe, was crystallized and its three-dimensional structure determined.

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae.

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10,000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms.

Three-dimensional modelling of honeybee venom allergenic proteases: relation to allergenicity.

Api SI and Api SII are serine proteases of the honeybee venom containing allergenic determinants. Each protease consists of two structural modules: an N-terminal CUB (Api SI) or a clip domain (Api SII) and a C-terminal serine protease-like (SPL) domain. Both domains are connected with a linker peptide.

Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis.

Snake venoms are cocktails of enzymes and non-enzymatic proteins used for both the immobilization and digestion of prey. The most common snake venom enzymes include acetylcholinesterases, l-amino acid oxidases, serine proteinases, metalloproteinases and phospholipases A(2) . Higher catalytic efficiency, thermal stability and resistance to proteolysis make these enzymes attractive models for biochemists, enzymologists and structural biologists.

Pseudechis australis venomics: adaptation for a defense against microbial pathogens and recruitment of body transferrin.

The venom composition of Pseudechis australis, a widely distributed in Australia reptile, was analyzed by 2-DE and mass spectrometric analysis. In total, 102 protein spots were identified as venom toxins. The gel is dominated by horizontal trains of spots with identical or very similar molecular masses but differing in the pI values.

The structure of a native l-amino acid oxidase, the major component of the Vipera ammodytes ammodytes venomic, reveals dynamic active site and quaternary structure stabilization by divalent ions.

The crystal structure of the major component of the Vipera ammodytes ammodytes venomic, a flavotoxin, member of the l-amino acid oxidase (LAAO) family, has been determined and refined at 2.6 Å resolution. The asymmetric unit consists of four molecules, each bound to oxidized FAD, representing a dimer of dimers.

Three-dimensional model of the honeybee venom allergen Api m 7: structural and functional insights.

Api m 7 is one of the major protease allergens of the honeybee venom. It consists of a serine protease-like (SPL) and a CUB domain. The knowledge about the structure and function of Api m 7 is limited mainly to its amino acid sequence.

The venomics of Bothrops alternatus is a pool of acidic proteins with predominant hemorrhagic and coagulopathic activities.

The venom proteome of Bothrops alternatus, a venomous snake widespread in South America, was analyzed by 2-D electrophoresis followed by mass spectrometric analysis and determination of enzymatic activities. The venomic composition revealed that metallo- and serine proteinases play primary roles in the pathogenesis of the envenomation by this pitviper. The identified 100 venom components with molecular masses from 10 to 100 kDa belong to six protein families: metalloproteinases, serine/thrombin-like proteinases, phospholipases A(2), L-amino acid oxidases, disintegrins and thrombin inhibitors.

Snake venomic of Crotalus durissus terrificus--correlation with pharmacological activities.

The snake venomic of Crotalus durissus terrificus was analyzed by 2-D and 1-D electrophoresis and subsequent MS/MS and enzymatic assays. The venomic of the South American rattlesnake comprises toxins from seven protein families: phospholipases A(2), serine proteinases, ecto-5'-nucleotidases, metalloproteinases, nerve growth factors, phosphodiesterases, and glutaminyl cyclase. The venom toxin composition correlates with the clinical manifestation of the crotalinae snake bites and explains pathological effects of the venom such as neurotoxicity, systemic myonecrosis, hemostatic disorders, myoglobinuria, and acute renal failure.

Interactions of ginkgolides A and B with a recombinant human prion protein.

In the presence of ginkgolides A and B a recombinant human prion protein (90-231) is more susceptible to proteolysis, which is characteristic for the non-infective form of prion proteins and more thermostable than in the absence of ginkgolides. Probably, ginkgolides exert a neuroprotective effect through a rearrangement of the prion structure.

Snake venomics of the Siamese Russell's viper (Daboia russelli siamensis) -- relation to pharmacological activities.

The venom proteome of Daboia russelli siamensis, a snake of medical importance in several Asian countries, was analysed by 2-D electrophoresis, subsequent MS/MS and enzymatic assays. The proteome comprises toxins from six protein families: serine proteinases, metalloproteinases, phospholipases A(2), L-amino acid oxidases, vascular endothelial growth factors and C-type lectin-like proteins. The venom toxin composition correlates with the clinical manifestation of the Russell's viper bite and explains pathological effects of the venom such as coagulopathy, oedema, hypotensive, necrotic and tissue damaging effects.

Proteome analysis of snake venom toxins: pharmacological insights.

Snake venoms are an extremely rich source of pharmacologically active proteins with a considerable clinical and medical potential. To date, this potential has not been fully explored, mainly because of our incomplete knowledge of the venom proteome and the pharmacological properties of its components, in particular those devoid of enzymatic activity. This review summarizes the latest achievements in the determination of snake venom proteome, based primarily on the development of new strategies and techniques.

3-D Model of the bee venom acid phosphatase: insights into allergenicity.

The acid phosphatase Api m 3 is the major allergen of the honeybee venom. Except for the amino acid sequence, no other structural information for the enzyme is available. We applied homology modeling to assign the three-dimensional structure of Api m 3.

Isolation, crystallization and preliminary X-ray diffraction analysis of L-amino-acid oxidase from Vipera ammodytes ammodytes venom.

L-Amino-acid oxidase from the venom of Vipera ammodytes ammodytes, the most venomous snake in Europe, was isolated and crystallized using the sitting-drop vapour-diffusion method. The solution conditions under which the protein sample was monodisperse were optimized using dynamic light scattering prior to crystallization. The crystals belonged to space group C2, with unit-cell parameters a = 198.

Comparative analysis of the venom proteomes of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis.

The venom proteomics of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, snakes of public health significance and the most poisonous reptiles in Europe, were analyzed by FPLC, 2-D electrophoresis, sequence analysis, and MS/MS. FPLC analysis showed the presence of l-amino acid oxidase, monomeric and heterodimeric phospholipases A2, C-type lectin protein, and proteinases in the venom of V. a.

Structural characterization of beta-sheeted oligomers formed on the pathway of oxidative prion protein aggregation in vitro.

The pathology of transmissible spongiform encephalopathies (TSEs) is strongly associated with the structural conversion of the cellular prion protein (PrPC) into a misfolded isoform (PrPSc) that assembles into amyloid fibrils. Since increased levels of oxidative stress have been linked to prion diseases, we investigated the metal-induced oxidation of human PrP (90-231). A novel in vitro conversion assay based on aerobic incubation of PrP in the presence of elemental copper pellets at pH 5 was established, resulting in aggregation of highly beta-sheeted prion proteins.

Catalytic efficiencies of alkaline proteinases from microorganisms.

Catalytic efficiencies of proteinase K and mesentericopeptidase were determined using series of peptide-4-nitroanilide substrates and compared with those of subtilisin DY, savinase and esperase. For each enzyme the subsites S1-S4 were characterized. The data for the enzyme specificities were related to our high resolution X-ray models of the five enzymes and their complexes with peptides.

Interactions of recombinant prions with compounds of therapeutical significance.

The transformation of the cellular prion protein (PrP(C)) into the infectious form (PrP(Sc)) is implicated in the invariably fatal transmissible spongiform encephalopathies. To identify a mechanism to prevent the undesired PrP(C)-->PrP(Sc) transformation, we investigated the interactions of recombinant prion proteins with a number of potential therapeutic agents which inhibit the PrP(Sc) formation, infectivity, and the accumulation of the misfolded form. We show that the prion aggregates formed in the presence of six compounds have no beta-structure, which is typical of the infectious form, and possess considerably higher alpha-helical content than the normal PrP(C).

Bacillus licheniformis variant DY proteinase: specificity in relation to the geometry of the substrate recognition site.

S1-S4 specificity of the Bacillus licheniformis variant DY proteinase (subtilisin DY) was determined by a series of peptide nitroanilides. The broad S1 specificity is due to the relative flexibility of the binding loop, which exhibits a preference for phenylalanine and accepts poorly the side chains of alanine, valine, lysine, and especially that of glutamic acid, due probably to a steric repulsion by Asn 155 and the narrow entrance of the "pocket." Alanine in position P2 of the substrate is more favorable for the catalysis than glycine.

Comparative analysis of the human and chicken prion protein copper binding regions at pH 6.5.

Recent experimental evidence supports the hypothesis that prion proteins (PrPs) are involved in the Cu(II) metabolism. Moreover, the copper binding region has been implicated in transmissible spongiform encephalopathies, which are caused by the infectious isoform of prion proteins (PrP(Sc)). In contrast to mammalian PrP, avian prion proteins have a considerably different N-terminal copper binding region and, most interestingly, are not able to undergo the conversion process into an infectious isoform.

Synthetic human prion protein octapeptide repeat binds to the proteinase K active site.

Proteinase K is widely used in tests for the presence of infectious prion protein causing fatal spongiform encephalopathies. To investigate possible interactions between the enzyme and the functionally important N-terminal prion domain, we crystallized mercury-inhibited proteinase K in the presence of the synthetic peptides GGGWGQPH and HGGGW. The octapeptide sequence is identical to that of a single octapeptide repeat (OPR) from the physiologically important OPR region.