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. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (S[combining low line]QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.

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. This suggests possible post-translational modifications of toxins, changing their electrostatic charge. The results demonstrate a highly specialized biosynthesis of toxins destroying the hemostasis (P-III metalloproteases, SVMPs), antimicrobial proteins (L-amino acid oxidases, LAAOs, and transferrin-like proteins, TFLPs), and myotoxins (phospholipase A(2)s, PLA(2)s). The three transferrin isoforms of the Australian P. australis (Elapidae snake) venom are highly homologous to the body transferrin of the African Lamprophis fuliginosus (Colubridae), an indication for the recruitment of body transferrin. The venomic composition suggests an adaptation for a defense against microbial pathogens from the prey. Transferrins have not previously been reported as components of elapid or other snake venoms. Ecto-5'-nucleotidases (5'-NTDs), nerve growth factors (VNGFs), and a serine proteinase inhibitor (SPI) were also identified. The venom composition and enzymatic activities explain the clinical manifestation of the king brown snakebite. The results can be used for medical, scientific, and biotechnological purposes.

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. Metalloproteinases predominate and belong exclusively to the P-III class including the most potent hemorrhagic toxins. They represent 50% of all identified proteins. Two isoforms were identified: homologous to jararhagin, a hemorrhagic toxin, and to beritractivase, a nonhemorrhagic and pro-coagulant metalloproteinase. The B. alternatus venom is a rich source of proteins influencing the blood coagulation system with a potential for medical application. The isoelectric points of the components are distributed in the acidic pH range (the pI values are between 4 and 7) and no basic proteins were detected.

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. The vast majority of toxins are potentially involved in neurotoxicity, myotoxicity, and coagulopathy. The predominant venom components are neurotoxic phospholipases A(2) and serine proteinases. The venom is a rich source of 5'-nucleotidases (7.8% of the identified toxins) inducing hemostatic disorders. Analysis of the venom protein composition provided a catalogue for secreted toxins. The venomic composition of Crotalus d. terrificus and venom gland transcriptome of the synonymous subspecies Crotalus d. collilineatus show differences in the occurrence of protein families and in the abundance of toxins. Some of the venom components identified by the proteomic analysis were not reported in the transcriptome of the Crotalus d. collilineatus venom gland. Enzymatic activities of the Crotalus d. terrificus venom were determined and correlated with the proteomic composition.

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. The vast majority of toxins are potentially involved in coagulopathy and neurotoxic effects. The predominant venom components are proteinases capable of activating blood coagulation factors and promoting a rapid clotting of the blood, and neurotoxic phospholipase A(2)s. The analysis of the venom protein composition provides a catalogue of secreted toxins. The proteome of D. r. siamensis exhibits a lower level of toxin diversity than the proteomes of other viperid snakes. In comparison to the venoms of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, the venom from D. r. siamensis showed quantitative differences in the proteolytic, phospholipase A(2), L-amino acid oxidase and alkaline phosphatase activities.

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. The structure of the homodimeric human prostatic acid phosphatase was used to model the Api m 3 tertiary structure. IgE epitopes and antigenic sites were predicted using programs based on the structure of known epitopes and analysis of the 3-D model. The model of Api m 3 revealed an active site similar to those of the histidine-type acid phosphatases with conservation of the catalytically important residues. The observed substitutions in the phosphate ion binding site suggest differences in the substrate specificity in comparison to other acid phosphatases. The analysis of the Api m 3 three-dimensional model revealed a very likely mechanism of enzyme action.

Spectroscopic properties and conformational stability of Concholepas concholepas hemocyanin.

The structure in solution and conformational stability of the hemocyanin from the Chilean gastropod mollusk Concholepas concholepas (CCH) and its structural subunits, CCH-A and CCH-B, were studied using fluorescence spectroscopy and differential scanning calorimetry (DSC). The fluorescence properties of the oxygenated and apo-form (copper-deprived) of the didecamer and its subunits were characterized. Besides tryptophan residues buried in the hydrophobic interior of the protein molecule also exposed fluorophores determine the fluorescence emission of the oxy- and apo-forms of the investigated hemocyanins. The copper-dioxygen system at the binuclear active site quenches the tryptophan emission of the oxy-forms of CCH and its subunits. The removal of this system increases the fluorescence quantum yield and causes structural rearrangement of the microenvironment of the emitting tryptophan residues in the respective apo-forms. Time-resolved fluorescence measurements show that the oxygenated and copper-deprived forms of the CCH and its subunits exist in different conformations. The thermal denaturation of the hemocyanin is an irreversible process, under kinetic control. A successive annealing procedure was applied to obtain the experimental deconvolution of the irreversible thermal transitions. Arrhenius equation parameter for the two-state irreversible model of the thermal denaturation of oxy-CCH at pH 7.2 was estimated. Both factors, oligomerization and the copper-dioxygen system at the active site, are important for stabilizing the structure of the hemocyanin molecule.

First experimental evidence for the preferential stabilization of the natural D- over the nonnatural L-configuration in nucleic acids.

The homochirality of biomolecules is a prerequisite for the origin and evolution of terrestrial life. The unique selection of D-monosaccharides, in particular, D-ribose in RNA and D-deoxyribose in DNA, leads to the construction of proteins by L-amino acids. This points to the exclusive role of stereoselectivity in the most important physiological processes. So far, there is no experimental confirmation for the theoretical calculations of the energy differences between enantiomers used for the explanation of the stereoselection of biomolecules. Therefore, the question of why nature prefers one configuration over the other still lacks a definitive answer. Here, we present the first experimental evidence that the D-enantiomer of RNA has a different electronic structure compared to the corresponding L-enantiomer. When varying the incident photon energy of the ultraviolet Raman probe across 5 eV, D- and L-isomers of the RNA duplex with the sequence [r(CUGGGCGG).r(CCGCCUGG)] show differences in the intensity of the vibrational modes with energies of 124.0 meV to 210.8 meV. The intensity difference of these vibrational modes can be traced back to energy differences in the electronic levels of D- and L-RNA leading to the preferential stabilization of the naturally occurring D-configuration of RNA over the L-configuration.

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. We show for the first time that two discrete oligomeric species of elongated shape, approx. 25 mers and 100 mers, are formed on the pathway of oxidative PrP aggregation in vitro, which are well characterized regarding shape and size using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and electron microscopy (EM). Considering that small oligomers of highly similar size have recently been reported to show the highest specific infectivity within TSE-infected brain tissues of hamsters, the novel oligomers observed in this study are interesting candidates as agent causing neurodegenerative and/or self-propagating effects. Moreover, our results significantly strengthen the theory that oxidative stress might be an influence that leads to substantial structural conversions of PrP in vivo.

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. The catalytic efficiencies of the alkaline proteinases are modulated by the hydrophobicity, solvent accessibility, flexibility and electrostatic effects in the substrate binding sites. The longer and nonpolar S1 loop offers more possibilities for hydrophobic interactions and increases the enzyme efficiency. S2 is a small narrow cleft which limits the possibilities for effective substitutions in P2. The wide specificity of S3 is due to its location on the protein surface of all investigated proteinases. The affinity of S4 for aromatic groups depends on the nature of the residues building the hydrophobic cavity.

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). The investigated compounds stimulate the formation of alpha-helices and the destruction of beta-structure. They prevent the transformation of alpha-helical structure into beta-sheets. Probably, this is the reason for the resistance to PrP(C)-->PrP(Sc) transformation in the presence of these compounds. The results may be useful for the future therapy of neurodegenerative diseases.

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. S3 is located on the protein surface. It is more open than the other subsites and can accept a variety of residues. S4 exhibits an extremely high affinity for the aromatic group of phenylalanine. Evidently, hydrophobic forces predominate in the S4--P4 interactions. The results characterize subtilisin DY as a bacterial proteinase with a broad specificity due to the specific geometry and flexibility of the substrate recognition site, which can accommodate different types of amino acid side chains.

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. Therefore, we applied x-ray absorption spectroscopy to analyze in detail the Cu(II) geometry of selected synthetic human PrP Cu(II) octapeptide complexes in comparison with the corresponding chicken PrP hexapeptide complexes at pH 6.5, which mimics the conditions in the endocytic compartments of neuronal cells. Our results revealed that structure and coordination of the human PrP copper binding sites are highly conserved in the pH 6.5-7.4 range, indicating that the reported pH dependence of copper binding to PrP becomes significant at lower pH values. Furthermore, the different chicken PrP hexarepeat motifs display homologous Cu(II) coordination at sub-stoichiometric copper concentrations. Regarding the fully cation-saturated prion proteins, however, a reduced copper coordination capability is supposed for the chicken prion protein based on the observation that chicken PrP is not able to form an intra-repeat Cu(II) binding site. These results provide new insights into the prion protein structure-function relationship and the conversion process of PrP.

Conformational states of the Rapana thomasiana hemocyanin and its substructures studied by dynamic light scattering and time-resolved fluorescence spectroscopy.

Hemocyanins are dioxygen-transporting proteins freely dissolved in the hemolymph of mollusks and arthropods. Dynamic light scattering and time-resolved fluorescence measurements show that the oxygenated and apo-forms of the Rapana thomasiana hemocyanin, its structural subunits RtH1 and RtH2, and those of the functional unit RtH2e, exist in different conformations. The oxygenated respiratory proteins are less compact and more asymmetric than the respective apo-forms. Different conformational states were also observed for the R. thomasiana hemocyanin in the absence and presence of an allosteric regulator. The results are in agreement with a molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins including transfer of conformational changes from one functional unit to another.

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. Here, we present the first direct evidence for the complex formation between a proteolytic enzyme and a segment of human prion molecule. The X-ray structures of the complexes at 1.4 and 1.8A resolution, respectively, revealed that in both cases the segment GGG is strongly bound as a real substrate at the substrate recognition site of the proteinase forming an antiparallel beta-strand between the two parallel strands of Asn99-Tyr104 and Ser132-Gly136. The complex is stabilized through an extended H-bonding network.

Oligomerization of the proteolytic products is an intrinsic property of prion proteins.

In the present study we show that the oligomerization of the proteolytic products is an intrinsic property of prion proteins. No such oligomerization was observed for the proteolytic products of other proteins after identical treatment. The rate of enzymatic hydrolysis of recombinant human (rhPrP) (23-231) and golden hamster (rmaPrP) (23-231) prion proteins as well as that of rmaPrP (90-231), corresponding to the infectious fragment of the scrapie form, drastically increases in the presence of chemical chaperones like dimethyl sulphoxide and glycerol as well as in 20% ethanol. A bacterial proteinase, termed "prionase," has a superior efficiency towards prion proteins in comparison to proteinase K and subtilisin DY. The early steps in the proteolysis by the latter enzymes have been identified. The results have potential impact on the treatment of scrapie-infected materials.

Contribution of disulfide bonds and calcium to Molluscan hemocyanin stability.

Disulfide bonds and calcium ions contribute significantly to the stability of the hemocyanin from the mollusc Rapana thomasiana grosse (gastropod). An extremely powerful protective effect of Ca2+ at a concentration of 100 mM (100% protection) against the destructive effect of reductants like dithiothreitol was observed. This is important for the practical application of molluscan hemocyanins in experimental biochemistry, immunology and medicine. The reduction of the disulfide bonds in the Rapana hemocyanin leads to a 20% decrease of the a-helical structure. The S-S bonds contribute significantly to the free energy of stabilization in water increasing delta G(D)H2O by 6.9 kJ mol (-1) The data are related to the X-ray model of the Rapana hemocyanin functional unit RtH2e. The results of this study can be of common validity for related respiratory proteins because the cysteine residues are conserved in all sequences of molluscan hemocyanins published so far.

Asp49 phospholipase A(2)-elaidoylamide complex: a new mode of inhibition.

The inhibition of phospholipase A(2)s (PLA(2)s) is of pharmacological and therapeutic interest because these enzymes are involved in several inflammatory diseases. Elaidoylamide is a powerful inhibitor of a neurotoxic PLA(2) from the Vipera ammodytes meridionalis venom. The X-ray structure of the enzyme-inhibitor complex reveals a new mode of Asp49 PLA(2) inhibition by a fatty acid hydrocarbon chain. The structure contains two identical homodimers in the asymmetric unit. In each dimer one subunit is rotated by 180 degrees with respect to the other and the two molecules are oriented head-to-tail. One molecule of elaidoylamide is bound simultaneously to the substrate binding sites of two associated neurotoxic phospholipase A(2) molecules. The inhibitor binds symmetrically to the hydrophobic channels of the two monomers. The structure can be used to design anti-inflammatory drugs.

The X-ray structure of a snake venom Gln48 phospholipase A2 at 1.9A resolution reveals anion-binding sites.

Phospholipase A2 is an "interfacial" enzyme and its binding to negatively charged surfaces is an important step during catalysis. The Gln48 phospholipase A2 from the venom of Vipera ammodytes meridionalis plays the role of chaperone and directs a toxic His48 PLA2 onto its acceptor. In the venom the two phospholipases A2 exist as a postsynaptic neurotoxic complex, Vipoxin. The X-ray structure of Gln48 PLA2, complexed to sulphate ions, which mimic the negatively charged groups of anionic membranes, has been determined by the molecular replacement method and refined to 1.9A resolution. The protein forms a homodimer stabilized by ionic, hydrophobic, and hydrogen-bond interactions. The structure reveals two anion-binding sites per subunit. These sites are probably involved in interactions with the negatively charged membrane surface and, in this way, in the "targeting" of the toxic component to the receptors of the postsynaptic membranes. In the absence of the chaperone subunit the toxin changes the target of the physiological attack. A comparison of the homodimeric Gln48 PLA2 structure with that of the heterodimeric Vipoxin reveals differences in regions involved in the pharmacological activity of the toxin. This fact, except the active site histidine substitution, can explain the absence of toxicity in the Gln48 protein in comparison to the His48 phospholipase A2.