Delineating residues for haemolytic activities of snake venom cardiotoxin 1 from Naja naja as probed by molecular dynamics simulations and in vitro validations.

Cardiotoxins (CTXs) are single polypeptide chain consisting of 59-62 amino acids with four disulfide bridges and globular proteins of simple ß-sheet folds. The CTXs are one of principal toxic components causing haemolysis and damaging various cells and belong to three-finger toxin (TFT) superfamily of snake venoms. However, there is no natural or synthetic small molecular inhibitor to the protein toxins to date. In the present study, modes of interaction of cardiotoxin 1 (CTX1) from Indian cobra (Naja naja) with heterogeneous erythrocyte membrane (EM) model system have been extensively examined by using all-atom molecular dynamics (MD) simulations in near physiological conditions and comprehensive analyses of the MD data revealed two distinct principal regions ('head groove' and 'loop groove') of the protein toxin for establishing structural interactions with the EM system. Moreover, combined analyses of data from high-throughput virtual screening of NCI small molecular database, in vitro haemolytic assays for top-hits of the chemical compounds against crude venom of Naja naja and as well CTXs purified from the venom and pharmacokinetic examinations on the chemical compounds retarding haemolytic activities of CTXs suggested that Etidronic acid and Zoledronic acid are promising prototypic chemical inhibitors to CTXs of snake venoms.

Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins.

The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a 'toxicity score', were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening.

Development of specific inhibitors for heparin-binding proteins based on the cobra cardiotoxin structure: an effective synthetic strategy for rationally modified heparin-like disaccharides and a trisaccharide.

Recently, a new heparin disaccharide-binding site on the convex side of cobra cardiotoxin (CTX) was identified by NMR spectroscopy and molecular modeling. To further characterize this site two heparin-like disaccharides were synthesized for binding studies with CTX, and a trisaccharide was synthesized for testing the sequence of the disaccharide binding to CTX. Thus six differentially protected monosaccharide building blocks (three l-iduronic acids and three d-glucosamines) were prepared. These include a l-iduronic acid elongation building block namely methyl 2-O-acetyl-4-O-levulinoyl-3-O-pivaloyl-alpha-l-idopyranosyluronate trichloroacetimidate for which a single-crystal X-ray structure was determined to have M(r)=576.79, a=9.3098(11)A alpha=90 degrees , b=10.3967(12)A beta=90 degrees , c=28.026(3)A gamma=90 degrees , V=2712.7(6)A(3), P2(1)2(1)2(1), Z=4, mu=0.71073A, and R=0.0378 for 7586 observed reflections. It shows that the molecular structure of the donor is in the (1)C(4) conformation with significant 1,3-diaxial interactions between O-1 and O-3 as well as O-2 and O-4. The disaccharides and trisaccharide vary in the degree and position of O- and N-sulfation. The pivaloyl group was used as permanent protecting group of hydroxyl. The levulinoyl group was used as the temporary protecting group to protect the hydroxyl for elongation.

Conformational change and inactivation of membrane phospholipid-related activity of cardiotoxin V from Taiwan cobra venom at acidic pH.

The phospholipid binding activity of cardiotoxin V from Naja naja atra (CTX A5) was studied by use of Langmuir monolayers and found to exhibit pH-dependence in binding to phosphatidylcholine membrane with an apparent pKa around 6.0. Proton NMR investigation of the CTX A5 molecule in the presence of phosphatidylcholine micelles reveals a decrease in association of CTX A5 with membranes at low pH as a result of the protonation of His-4 near the membrane binding site of loop I region of CTX. The pH-dependent binding can be attributed mainly, but not solely, to the change in charge content of the CTX molecule upon His-4 protonation at the membrane/water interface. This is shown by analyzing the pH- and ionic strength dependence of binding of CTXs to phospholipid monolayers according to Gouy-Chapman theory. The protonation of the His-4 residue also results in a local conformational change in the loop I region since the chemical shifts of amide protons for the amino acid residues from Cys-3 to Thr-14 are all found to vary as a function of pH with an apparent pKa similar to that of His-4. Interestingly, the effect is relayed to other amino acid residues in the structural core of the protein such as those in C-terminal (Lys-60, Cys-61, and Asn-62) and triple-stranded antiparallel beta-sheet (Cys-22, Lys-24, Ala-25, Arg-38, and Ala-41) regions. An additional local conformational change in the molecule results around pH 5 as evidenced by circular dichroism spectroscopic studies, although this change does not affect the characteristic beta-sheet and three-finger loop structure of CTX molecule as revealed by two-dimensional NOESY 1H NMR study. The latter conformational change at acidic pH, however, completely inactivates CTX-induced aggregation/fusion activity of sphingomyelin vesicles. The results suggest that deciphering the functional sites of CTXs on the basis of structure and dynamics determined at low pH should be done with caution. Since 19 out of 44 CTX homologues with known amino acid sequence contain His-4, the effect of His-4 on the structure and function of CTX molecules is important and is discussed in terms of the diverse membrane targets of CTX subtypes. Also discussed is the pH-induced activation of snake venom proteins in the victim.

The role of acidic amino acid residues in the structural stability of snake cardiotoxins.

We have recently shown that membrane-related activities of cardiotoxin V from Naja naja atra (CTX A5) are diminished at acidic pH although the overall beta-sheet structure of the molecule is maintained. In order to understand more about the mechanism of inactivation of CTX at acidic pH, we studied the effect of pH and denaturing reagents on the structural stability of CTX. We found, first, pH-induced structural transitions occurred in CTX A5 at two pH values as judged by the CD ellipticity around 195 nm: an increase in the beta-sheet content occurred around pH 4 and followed by a decrease, therein, around pH 2. The pKa of three acidic amino acid residues in CTX A5, i.e., Glu-17, Asp-42, and Asp-59, were determined to be 4.0, 3.2, and below 2.3, respectively, by NMR spectroscopy. The low pKa value of Asp-59 implies salt bridge formation between Lys-2 and Asp-59. Thus, electrostatic interaction may stabilize the three loop structure in addition to the hydrogen bonds between N- and C-termini of CTX molecule. Second, 2,2,2-trifluoroethanol (TFE) and guanidinium chloride (GdmHCI) were found to induce alpha-helical and random coil formation, respectively, in CTX A5 and eight other beta-sheet CTXs. Comparison of the relative potencies of TFE and GdmHCI to induce structural changes suggests that the amino acid residue located at position 17 plays a role in the structural stability. Specifically, CTXs containing negatively charged Glu-17 are least stable. It is suggested that Glu-17 may perturb the interaction between Lys-2 and Asp-59, and thus the overall stability of beta-sheet, in the presence of denaturing reagent. In conclusion, the perturbed structural stability of CTXs may partially explain the lower activity CTX exhibits at acidic pH. A structural model to account for the unfolding and refolding of CTX molecules without the breaking of disulfide bonds is also proposed.

Effects of different antagonists on depolarization of cultured chick myotubes by cobra venom cardiotoxins and Pyrularia thionin from the plant Pyrularia pubera.

Cardiotoxins (3.12 and 3.12.1) purified from cobra venom (Naja naja siamensis) are basic single-chain polypeptides of about 60 residues. Although they depolarize nerve and muscle cells and have cytolytic effects, their mechanism of action is still unknown. Pyrularia thionin (P-thionin) isolated from nuts of the parasitic plant Pyrularia pubera is a strongly basic, single-chain polypeptide containing 47 residues. It is known to be haemolytic and cytotoxic, and to depolarize muscle cells, but its mechanism of action is unclear. The present studies explored the possible similarities between P-thionin and cobra venom cardiotoxins by comparing their effects on depolarization of cultured chick skeletal muscle cells in the presence and absence of possible antagonists. Cardiotoxins and P-thionin depolarized cultured chick skeletal muscle cells, but with P-thionin showing a steeper concentration-dependence. Ca2+ was more effective at reducing cardiotoxin action than P-thionin, while the Ca(2+)-channel blockers Ni2+ (100 microM) and verapamil (100 microM) had no blocking effects on the toxins. Ca2+ may block the binding of both toxins. Indomethacin (100 microM, an inhibitor of cyclooxygenase), quinacrine and dexamethasone (100 microM, inhibitors of phospholipase A2) did not block the effects of the toxins, implying that the actions on cultured chick skeletal muscle cells are not due to activation of endogenous phospholipase A2.

Effect of anti-inflammatory drugs on the cardiotoxin-induced hind-paw oedema in rats.

Cardiotoxin, isolated from Naja naja atra venom, induced rat hind-paw oedema. This effect was suppressed by the pretreatment with dexamethasone or BW 755C, or subplantar co-injection with FPL 55712. Pretreatment with aspirin alone did not affect this response, while a significant reduction of cardiotoxin-induced paw oedema was achieved with aspirin in combination with diphenhydramine and methysergide. Subplantar co-injection of PAF antagonist, BN 52021 or L 652731, with cardiotoxin had no effect on paw oedema, whereas superoxide dismutase/catalase reduced this oedematous response. Cardiotoxin-induced paw oedema was also suppressed by pretreating the rats with isoprenaline. Pretreatment with rat anti-platelet plasma, which greatly reduced peripheral platelet count, did not affect cardiotoxin-induced paw oedema. Cardiotoxin did not trigger platelet aggregation or release reaction either in platelet-rich plasma or in washed platelet suspension. The oedematous response after subplantar co-injection of cardiotoxin with basic or acidic phospholipase A2 appeared to be only an additive effect. These results suggest that arachidonate metabolites, in which leukotrienes are most important, participated in cardiotoxin-induced paw oedema. Superoxide radical was also involved, while PAF and platelets showed little influence in this oedema effect.

Effects of divalent cations on snake venom cardiotoxin-induced hemolysis and 3H-deoxyglucose-6-phosphate release from human red blood cells.

At a low concentration of Naja naja kaouthia cardiotoxin (3 microM) Ca2+, Sr2+ and Ba2+ (2 mM), had little to no effect on 3H-deoxyglucose-6-phosphate (3H-dGlu-6-p) or hemoglobin release. At higher concentrations of N. n. kaouthia cardiotoxin (greater than or equal to 10 microM), Ca2+ (2 mM), but not Sr2+ or Ba2+, significantly enhanced 3H-dGlu-6-p and hemoglobin release. Mn2+ (2 mM) almost completely inhibited 3H-dGlu-6-p release and hemolysis at both the 3 microM and 10 microM concentrations of cardiotoxin. At a fixed concentration of N. n. kaouthia cardiotoxin (3 microM). Ca2+ at low concentrations (0.5 mM) enhanced 3H-dGlu-6-p and hemoglobin release, but at higher concentrations caused a dose-dependent inhibition of cardiotoxin action. The cardiotoxin from N. n. kaouthia venom (3 microM) induced 3H-dGlu-6-p release and hemolysis release with similar time courses and to similar extents. 3H-dGlu-6-p release induced by cardiotoxin was greatly enhanced as the pH of the medium was increased from 7.0 to 8.5. Similarities between 3H-dGlu-6-p and hemoglobin release do not support opening of pores in the plasmalemma of all red blood cells as the mode of action of cardiotoxins, but suggests that complete lysis of a subpopulation of cells occurs. Cardiotoxins have two components of lysis, only one of which is Ca2+-dependent. The Ca2+-dependent lysis is only evident at higher cardiotoxin concentrations and is likely due to trace phospholipase A2 contamination in the toxin fraction. Mn2+ is an effective antagonist of cardiotoxin action.