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Crotalus durissus terrificus snakes possess a protein in their blood, named crotoxin inhibitor from Crotalus serum (CICS), which protects them against crotoxin, the main toxin of their venom. CICS neutralizes the lethal potency of crotoxin and inhibits its phospholipase A2 (PLA2) activity. The aim of the present study is to investigate the specificity of CICS towards snake venom neurotoxic PLA2s (beta-neurotoxins) and nontoxic mammalian PLA2s. This investigation shows that CICS does not affect the enzymatic activity of pancreatic and nonpancreatic PLA2s, bee venom PLA2 and Elapidae beta-neurotoxins but strongly inhibits the PLA2 activity of Viperidae beta-neurotoxins. Surface plasmon resonance and PAGE studies further demonstrated that CICS makes complexes with monomeric and multimeric Viperidae beta-neurotoxins but does not interact with nontoxic PLA2s. In the case of dimeric beta-neurotoxins from Viperidae venoms (crotoxin, Mojave toxin and CbICbII), which are made by the noncovalent association of a PLA2 with a nonenzymatic subunit, CICS does not react with the noncatalytic subunit, instead it binds tightly to the PLA2 subunit and induces the dissociation of the heterocomplex. In vitro assays performed with Torpedo synaptosomes showed a protective action of CICS against Viperidae beta-neurotoxins but not against other PLA2 neurotoxins, on primary and evoked liberation of acetylcholine. In conclusion, CICS is a specific PLA2 inhibitor of the beta-neurotoxins from the Viperidae family.

Nigroxins A and B, two myotoxic phospholipases A2 (PLA2s) from the venom of the American elapid Micrurus nigrocinctus, belong to a new PLA2 subclass. Their primary structures were established and compared with those of PLA2s that have already been studied with respect to myotoxic activity. The combination of amino acid residues Arg15, Ala100, Asn108 and a hydrophobic residue at position 109 is present exclusively in class I PLA2s that display myotoxic activity. These residues cluster within a surface region rich in positive charges and are suggested to play a role in the interaction with the target membrane of the muscle fibers. It is concluded that the myotoxic PLA2s resulted from recruitment of an ancient scaffold. Dendrotoxins and alpha-neurotoxins are similarly derived from other old structures, which are, however, now also present in nontoxic proteins that are widely distributed throughout the animal kingdom. The evolutionary pathways by which elapid PLA2s acquired myotoxicity and dendrotoxins acquired K+-channel blocker activity are traced. They demonstrate how existing scaffolds were adapted stepwise to serve toxic functions by exchange of a few surface-exposed residues.

Mammalian group-II phospholipases A2 (PLA2) of inflammatory fluids display bactericidal properties, which are dependent on their enzymatic activity. This study shows that myotoxins II (Lys49) and III (Asp49), two group-II PLA2 isoforms from the venom of Bothrops asper, are lethal to a broad spectrum of bacteria. Since the catalytically inactive Lys49 myotoxin II isoform has similar bactericidal effects to its catalytically active Asp49 counterpart, a bactericidal mechanism that is independent of an intrinsic PLA2 activity is demonstrated. Moreover, a synthetic 13-residue peptide of myotoxin II, comprising residues 115-129 (common numbering system) near the C-terminal loop, reproduced the bactericidal effect of the intact protein. Following exposure to the peptide or the protein, accelerated uptake of the hydrophobic probe N-phenyl-N-naphthylamine was observed in susceptible but not in resistant bacteria, indicating that the lethal effect was initiated on the bacterial membrane. The outer membrane, isolated lipopolysaccharide (LPS), and lipid A of susceptible bacteria showed higher binding to the myotoxin II-(115-129)-peptide than the corresponding moieties of resistant strains. Bacterial LPS chimeras indicated that LPS is a relevant target for myotoxin II-(115-129)-peptide. When heterologous LPS of the resistant strain was present in the context of susceptible bacteria, the chimera became resistant, and vice versa. Myotoxin II represents a group-II PLA2 with a direct bactericidal effect that is independent of an intrinsic enzymatic activity, but adscribed to the presence of a short cluster of basic/hydrophobic amino acids near its C-terminal loop.

Three new toxins acting on muscarinic receptors were isolated from the venom of the black mamba Dendroaspis polylepis. They were called muscarinic toxins alpha, beta, and gamma (MT alpha, MT beta, and MT gamma). All of the toxins have four disulphide bonds and 65 or 66 amino acids. The sequences of MT alpha and MT beta were determined. The muscarinic toxins, of which about 12 have been isolated from venoms of green and black mambas, have 60-98% sequence identity with each other, and are similar to many (about 180) other snake venom components, such as alpha-neurotoxins, cardiotoxins, and fasciculins. In contrast to the alpha-neurotoxins, muscarinic toxins do not bind to nicotinic acetylcholine receptors. The binding constants of MT alpha and MT beta were determined for human muscarinic receptors of subtypes m1-m5 stably expressed in Chinese hamster ovary cells. The toxins are less selective than the earlier discovered muscarinic toxins from the green mamba Dendroaspis angusticeps. MT alpha and the muscarinic toxin MT4 from D. angusticeps differ only in a region of three amino acids (residues 31-33), which are Leu-Asn-His in MT alpha and Ile-Val-Pro in MT4. This difference causes a pronounced shift in subtype selectivity. MT alpha has high affinity to all subtypes, with Ki (inhibition constant) values of 23 nM (m1; pKi = 7.64 +/- 0.10), 44 nM (m2; pKi = 7.36 +/- 0.06), 3 nM (m3; pKi = 8.46 +/- 0.14), 5 nM (m4; pKi = 8.32 +/- 0.07), and 8 nM (m5; pKi = 8.09 +/- 0.07). MT4 has high affinity only to m1 (Ki = 62 nM) and m4 (87 nM) receptors, and low (Ki > 1 microM) affinity to m2, m3, and m5. The region at positions 31-33 evidently plays an important role in the toxin-receptor interaction. MT beta has low affinity for m1 and m2 receptors (Ki > 1 microM) and intermediate affinity for m3 (140 nM; pKi = 6.85 +/- 0.03), m4 (120 nM; pKi = 6.90 +/- 0.06), and m5 (350 nM; pKi = 6.46 +/- 0.01). The low affinity of MT beta may reflect a tendency for spontaneous inactivation.

An antivenom protein has been identified in the blood of the snake Crotalus durissus terrificus and proved to act by specifically neutralizing crotoxin, the main lethal component of rattlesnake venoms. The aim of this study was to purify the crotoxin inhibitor from Crotalus serum (CICS), and to analyze its mechanism of action. CICS has been purified from blood serum of the Crotalus snake by gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephacel, and FPLC gel filtration on a Superose 12 column. It is an oligomeric glycoprotein of 130 kDa, made by the non-covalent association of 23-25-kDa subunits. Two different subunit peptides were identified by SDS/PAGE, however, their N-terminal sequences are identical. They are characterized by the absence of methionine residues and a high content of acidic, hydrophobic and cysteine residues. The neutralizing effect of purified CICS towards the neurotoxic effects of crotoxin has been demonstrated in vivo by lethality assays. CICS binds to the phospholipase subunit CB of crotoxin, but not to the acidic chaperon subunit CA; it efficiently inhibits the phospholipase activity of crotoxin and its isolated CB subunit and evokes the dissociation of the crotoxin complex. The molecular mechanism of the interaction between CICS and crotoxin seems to be very similar to that of crotoxin with its acceptor. It is, therefore, tempting to suggest that CICS acts physiologically as a false crotoxin acceptor that would retain the toxin in the vascular system, thus preventing its action on the neuromuscular system.

The binding properties of the coagulation factor IX/factor X-binding anticoagulant protein (IX/X-bp) isolated from the venom of Trimeresurus flavoviridis (habu snake) were investigated with an enzyme-linked immunosorbent assay. The half-maximal binding and maximal binding of IX/X-bp to both factors IX and X were observed at concentrations of Ca2+ ions of 0.4 mM and 1 mM, respectively. Concentration of IX/X-bp at half-maximal binding to solid-phase bovine factor IX and solid-phase bovine factor X were 0.4 +/- 0.1 nM and 1.1 +/- 0.4 nM, respectively, in the presence of 1 mM Ca2+ ions. The kinetics of binding activity of IX/X-bp to bovine factors IXa and Xa and to human factors IX and X resembled those of the binding to bovine factors IX and X. IX/X-bp did not bind to solid-phase coagulation factors other than factor IX/IXa and factor X/Xa, for example, prothrombin, factor VII, protein C, and protein Z, under the conditions of the experiment. To localize the binding sites of IX/X-bp on the coagulation factors, the ability of IX/X-bp to bind to various fragments derived from factors IX and X was examined. The binding of IX/X-bp to solid-phase factor IX was inhibited by a peptide containing the 4-carboxyglutamic acid (Gla) domain derived from factor IXa beta' (residues 1-42) in the liquid phase, but the binding was not inhibited by Gla-domainless factor IXa beta'. Half-maximal binding of IX/X-bp to solid-phase Gla-domain peptide of factor IX occurred at 9.2 +/- 1.9 nM. Factor X was partially reduced and the S-carboxymethylated light and heavy chains of factor X were prepared. IX/X-bp bound to the S-carboxymethylated light chain of factor X but not to the heavy chain. The binding of IX/X-bp to solid-phase factor X was inhibited by the Gla-domain peptide of factor X (residues 1-44) but not by Gla-domainless factor X. IX/X-bp bound to PCGFX, a recombinant human protein C whose Gla-domain region (residues 1-43) had been replaced by residues 1-43 of human factor X. The affinity of binding was about one tenth of that to intact human factor X. IX/X-bp was unable to bind at all to human protein C. These data indicate that IX/X-bp is a protein that binds to the Gla-domain regions of factors IX and X in the presence of Ca2+ ions.

THe characteristic feature of the crystal structure of erabutoxin b, a short neurotoxin from Laticauda semifasciata, and alpha-cobratoxin, a long neurotoxin from Naja naja siamensis, is the presence of a triple-stranded antiparallel pleated beta-sheet structure formed by the central and the third peptide loops. In the present study, we have studied the assignment of slowly exchangeable amide protons of Laticauda semifasciata III from L. semifasciata, using nuclear Overhauser effects (NOE) and spin-decoupling methods. The results show that nearly all of the slowly exchangeable amide protons are to be assigned to the back-bone amide protons, involved in the triple-stranded antiparallel pleated beta-sheet structure, indicating that this sheet is stable in 2H2O solution. In contrast, the amide protons in short neurotoxins are readily exchangeable under the same experimental condition, suggesting that long neurotoxins have a more rigid sheet structure than short ones. This rigidity may come from the hydrophobic and hydrogen bond interaction between the central loop and the tail, which is not present in short neurotoxins. Since the functionally important residues are located on this beta-sheet, the different kinetic properties of the neurotoxins are well correlated with the difference in the rigidity of the beta-sheet.

The 300-MHz and 500-MHz NMR spectra of a long neurotoxin laticauda semifasciata III (LS III) from Laticauda semifasciata have ben analysed. Comparison with the NMR spectra of alpha-cobratoxin from Naja naja siamensis, a homologous long neurotoxin to laticauda semifasciata III, allowed the assignment of all the aromatic protein resonances to specific amino acid residues. All the methyl proton resonances have been assigned to specific types of amino acid residues. The pH dependences of the aromatic and methyl proton chemical shifts were analyzed by the non-linear least-square method to give the pKa values and protonation shifts. The interproton nuclear Overhauser effect enhancements were measured in order to elucidate the spatial proximity of the methyl-bearing residues and aromatic residues. On the basis of these NMR data and using the crystal structure of alpha-cobratoxin by Walkinshaw et al., more than half of the methyl proton resonances have been assigned to specific amino acid residues. A hydrophobic core comprising the first loop, the central loop and the tail part of the molecule has been defined. This hydrophobic core may be common to all long neurotoxins and may protect the three-stranded antiparallel pleated beta-sheet structure, thus making the backbone structure of long neurotoxins more rigid than that of short neurotoxins. The positively charged surface of laticauda semifasciata III, which is responsible for binding to the acetylcholine receptor protein, is confirmed as the concave surface formed by the central and the third loop. The arrangement of the amino acid residues on this surface is similar to that of all other neurotoxins. Accordingly, the slow on-off rates of association of long neurotoxins with receptor is considered to arise from the rigid backbone structure. A small conformation change is thought to be associated with binding to the receptor protein.