A comparative investigation of snake venom neurotoxins and their triplet-state tryptophan-disulfide interactions using phosphorescence and optically detected magnetic resonance.

We have investigated the luminescence and optically detected magnetic resonance (ODMR) of the highly homologous snake venom neurotoxins alpha-bungarotoxin (BgTX), alpha-cobratoxin (CbTX), and cobrotoxin (CoTX) in frozen aqueous glasses. The phosphorescence intensity and lifetime of the single invariant tryptophan, Trp29, are found to be diminished in BgTX and CbTx relative to CoTX both at 77 K and at 4.2 K. Selective reduction of the Cys30-Cys34 disulfide proximal to Trp29 in BgTX and CbTX, that is absent in CoTX, results in the enhancement of the phosphorescence to fluorescence intensity ratio of Trp29 and identifies this disulfide as the source of the triplet-state quenching. Variations of the phosphorescence parameters are observed for differently frozen BgTX and CbTX samples. We argue that this observation is consistent with conformational flexibility in the region of Trp29. For BgTX and CbTX, changing the wavelength of excitation from 285 to 300 nm results in a small bathochromic phosphorescence shift of 0.4 nm, an average decrease in the lifetime, and a change in the polarity of the normally positive D-E ODMR signal. From the small excitation-dependent emission shift, we infer that Trp29 is in a relatively hydrophobic environment. The excitation-dependent changes in lifetime and ODMR signal parameters arise from subtle heterogeneity in the disposition of Trp29 with respect to Cys30-Cys34. We discuss the mechanism of disulfide-induced quenching of the Trp29 triplet state in BgTX and CbTX and argue that it most probably is due to electron transfer.     

Amino acid sequence of kappa-flavitoxin: establishment of a new family of snake venom neurotoxins

The complete amino acid sequence of kappa-flavitoxin, a neurotoxin isolated from the venom of Bungarus flaviceps, has been determined by automated Edman analysis of the intact protein and of peptides derived from digests with trypsin and chymotrypsin. kappa-Flavitoxin consists of a single 66-residue polypeptide chain which is completely devoid of methionine. The amino acid sequence of kappa-flavitoxin demonstrates that although the toxin is related to the alpha-neurotoxin family, it displays a much higher degree of homology with kappa-bungarotoxin. The conserved structural features of the kappa-neurotoxins and their pharmacological profiles, which are distinct from those of all known alpha-neurotoxins, provide evidence for a new, structurally and functionally unique family of snake venom neurotoxins.     

A monoclonal antibody which recognized the functional site of snake neurotoxins and which neutralizes all short-chain variants

We isolated a neurotoxin-specific monoclonal antibody (Mab) which is capable of recognizing and neutralizing all short-chain toxin variants that have been tested, including those with widely divergent sequences. The epitope incorporates the three invariant residues Lys-27, Trp-29 and Lys-47 which form part of the site by which the toxins bind to the nicotinic acetylcholine receptor. To our knowledge, this is the first Mab which possesses the universal capacity of neutralizing all natural variants of a toxic protein.     

Molecular interactions between p-coumaric acid and snake venom toxins

A large number of natural compounds, such as phenolic compounds, have been scientifically evaluated in the search for enzyme inhibitors. The interactions between the phenolic compound p-coumaric acid and the enzymes present in snake venoms (used as research tools) were evaluated in vitro and in silico. The p-coumaric acid was able to inhibit 31% of the phospholipase activity induced by Bothrops alternatus venom, 27% of the hemolytic activity induced by B. moojeni, 62.5% of the thrombolytic activity induced by B. jararacussu, and approximately 27% of the activity thrombosis induced by Crotalus durissus terrificus. Previous incubation of p-coumaric acid with the venoms of B. atrox and B. jararacussu increased the coagulation time by 2.18 and 2.16-fold, respectively. The activity of serine proteases in B. atrox and B. jararacussu venoms was reduced by 60% and 66.34%, respectively. Computational chemistry analyses suggests the specific binding of p-coumaric acid to the active site of proteases through hydrogen and hydrophobic interactions. The phenolic compound evaluated in this work has great potential in therapeutic use to both prevent and treat hemostatic alterations, because the venom proteins inhibited by the p-coumaric acid have high homology with human proteins that have a fundamental role in several pathologies.     

Functional importance of short-range binding and long-range solvent interactions in helical antifreeze peptides

Short-range ice binding and long-range solvent perturbation both have been implicated in the activity of antifreeze proteins and antifreeze glycoproteins. We study these two mechanisms for activity of winter flounder antifreeze peptide. Four mutants are characterized by freezing point hysteresis (activity), circular dichroism (secondary structure), Förster resonance energy transfer (end-to-end rigidity), molecular dynamics simulation (structure), and terahertz spectroscopy (long-range solvent perturbation). Our results show that the short-range model is sufficient to explain the activity of our mutants, but the long-range model provides a necessary condition for activity: the most active peptides in our data set all have an extended dynamical hydration shell. It appears that antifreeze proteins and antifreeze glycoproteins have reached different evolutionary solutions to the antifreeze problem, utilizing either a few precisely positioned OH groups or a large quantity of OH groups for ice binding, assisted by long-range solvent perturbation.     

State of functionally essential Trp-29 in snake venom neurotoxins: A proton nuclear magnetic resonance study

Proton nuclear magnetic resonance (NMR) spectra have been recorded of various neurotoxins from snake venoms.pH dependence of the chemical shifts and resonance intensity has been followed for the functionally essential Trp-29. The indole N-1 proton of Trp-29 in -bungarotoxin, toxin B, and cobrotoxin exhibits appreciably large upfield shifts as thepH is lowered and the suppressed exchange with the solvent hydrogen atpH 3–4, but not inNaja haje annulifera 10 where Asp-31 is replaced with Gly-31. This observation strongly suggests the presence of a hydrogen bond between Trp-29 and Asp-31 that is probably important in stabilizing the arrangement of the functionally essential residues to form a distinct binding region for the receptor.     

Amino acid sequences of two novel long-chain neurotoxins from the venom of the sea snake Laticauda colubrina.

From the venom of a population of the sea snake Laticauda colubrina from the Solomon Islands, a neurotoxic component, Laticauda colubrina a (toxin Lc a), was isolated in 16.6% (A280) yield. Similarly, from the venom of a population of L. colubrina from the Philippines, a neurotoxic component, Laticauda colubrina b (toxin Lc b), was obtained in 10.0% (A280) yield. The LD50 values of these toxins were 0.12 microgram/g body wt. on intramuscular injection in mice. Toxins Lc a and Lc b were each composed of molecules containing 69 amino acid residues with eight half-cystine residues. The complete amino acid sequences of these two toxins were elucidated. Toxins Lc a and Lc b are different from each other at five positions of their sequences, namely at positions 31 (Phe/Ser), 32 (Leu/Ile), 33 (Lys/Arg), 50 (Pro/Arg) and 53 (Asp/His) (residues in parentheses give the residues in toxins Lc a and Lc b respectively). Toxins Lc a and Lc b have a novel structure in that they have only four disulphide bridges, although the whole amino acid sequences are homologous to those of other known long-chain neurotoxins. It is remarkable that toxins Lc a and Lc b are not coexistent at the detection error of 6% of the other toxin. Populations of Laticauda colubrina from the Solomon Islands and from the Philippines have either toxin Lc a or toxin Lc b and not both of them.     

Synthetic peptides corresponding to sequences of snake venom neurotoxins and rabies virus glycoprotein bind to the nicotinic acetylcholine receptor

Peptides corresponding to portions of loop 2 of snake venom curare-mimetic neurotoxins and to a structurally similar region of rabies virus glycoprotein were synthesized. Interaction of these peptides with purified Torpedo electric organ acetylcholine receptor was tested by measuring their ability to block the binding of 125I-labeled alpha-bungarotoxin to the receptor. In addition, inhibition of alpha-bungarotoxin binding to a 32-residue synthetic peptide corresponding to positions 173-204 of the alpha-subunit was determined. Neurotoxin and glycoprotein peptides corresponding to toxin loop 2 inhibited labeled toxin binding to the receptor with IC50 values comparable to those of nicotine and the competitive antagonist d-tubocurarine and to the alpha-subunit peptides with apparent affinities between those of d-tubocurarine and alpha-cobratoxin. Substitution of neurotoxin residue Arg37, the proposed counterpart of the quaternary ammonium of acetylcholine, with a negatively charged Glu residue reduced the apparent affinity about 10-fold. Peptides containing the neurotoxin invariant residue Trp29 and 10- to 100-fold higher affinities than peptides lacking this residue. These results demonstrate that relatively short synthetic peptides retain some of the binding ability of the native protein from which they are derived, indicating that such peptides are useful in the study of protein-protein interactions. The ability of the peptides to compete alpha-bungarotoxin binding to the receptor with apparent affinities comparable to those of other cholinergic ligands indicates that loop 2 of the neurotoxins and the structurally similar segment of the rabies virus glycoprotein act as recognition sites for the acetylcholine receptor. Invariant toxin residues Arg37 and Trp29 and their viral homologs play important, although not essential, roles in binding, possibly by interaction with complementary anionic and hydrophobic subsites on the acetylcholine receptor. The alpha-subunit peptide most likely contains all of the determinants for binding of the toxin and glycoprotein peptides present on the alpha-subunit, because these peptides bind to the 32-residue alpha-subunit peptide with the same or greater affinity as to the intact subunit.     

Structure and pharmacology of spider venom neurotoxins

Spider venoms are complex mixtures of neurotoxic peptides, proteins and low molecular mass organic molecules. Their neurotoxic activity is due to the interaction of the venom components with cellular receptors, in particular ion channels. Spider venoms have proven to be a rich source of highly specific peptide ligands for selected subtypes of potassium, sodium and calcium channels, and these toxins have been used to elucidate the structure and physiological roles of the channels in excitable and non-excitable cells. Spider peptides show great variability in their pharmacological activity and primary structure but relative homogeneity in their secondary structure. Following diverse molecular evolution mechanisms, and in particular selective hypermutation, short spider peptides appear to have functionally diversified while retaining a conserved molecular scaffold. This paper reviews the composition and pharmacology of spider venoms with emphasis on polypeptide toxin structure, mode of action and molecular evolution.     

On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors

Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors alpha-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors.     

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.     

Cellular mechanisms of netrin function: long-range and short-range actions.

Netrins are secreted proteins that direct axon extension and cell migration during neural development. They are bifunctional cues that act as an attractant for some cell types and as a repellent for others. Several lines of evidence suggest that two classes of receptors, the deleted in colorectal cancer (DCC) family and the UNC-5 family, mediate the attractant and repellent response to netrin. Although netrins were first identified as diffusible long-range cues for developing axons, recent findings provide evidence that they also function as short-range cues close to the surface of the cells that produce them. This short-range function of netrin contributes to guiding neurite outgrowth and mediating cell-cell interactions during development and perhaps also in adults.     

Isolation, properties and amino acid sequences of three neurotoxins from the venom of a sea snake, Aipysurus laevis.

Aipysurus laevis venom was chromatographed on CM-cellulose and Bio-Rex 70 columns. Three neurotoxic components, toxins Aipysurus laevis a, b and c, were isolated. The toxins a, b and c corresponded to 22, 33 and 21% respectively of the proteins in the original venom, and accounted for almost all the lethal activity of the venom. The three toxins a, b and c were monodisperse on disc electrophoresis at pH4; toxins a and b moved at the same velocity and c a little faster. They were monodisperse also on sodium dodecyl sulphate-polyacrylamide-disc-gel electrophoresis, giving a molecular weight of 7600. The molecular weight of toxin b estimated by gel filtration was 7000. The amino acid sequence analyses of these toxins revealed that they consisted of 60 amino acid residues and that Aipysurus laevis b was [25-methionine, 28-arginine] Aipysurus laevis a. Aipysurus laevis c was [28-lysine] Aipysurus laevis a, the tryptic peptide sequence relying on homology. The LD50 values of these toxins for 20g mice were 0.076 mug/g body wt. They inhibited the acetylcholine-induced contracture but did not affect the CKl-induced contracture of the isolated muscle.     

The neurotoxins of the sea snake Laticauda schistorhynchus.

Erabutoxins a and b, the major neurotoxins in the venom of the sea snake Laticauda semifasciata, were detected in the venom of Laticauda schistorhynchus. The identity of the toxins was confirmed on the basis of elution position on CM-cellulose column chromatography, disc electrophoretic mobility, amino acid analysis and toxicity measurement.     

NMR analysis of structure and function of snake neurotoxins

The 270-MHz proton-nmr spectra of short neurotoxins (erabutoxins from Laticauda semifasciata and cobrotoxin from Naja naja atra) and long neurotoxins (toxin B from Naja naja and α-bungarotoxin from Bungarus multicinctus) have been analyzed. The conformation of erabutoxin b in solution is largely consistent with the x-ray crystal analysis, although the environment of His-7 in solution is definitely different from that in the crystal. The pH-dependent transition has been found for toxin B, indicating that the conformation in neutral solution is different from that in the crystal as grown from acidic solution. The deuterium-exchange rates of the amide protons for the four neurotoxins have been measured. The order of structural rigidity is the same as the order of the irreversibility of neuromuscular block by neurotoxins.