Comparative Study of Structure and Activity of Cytotoxins from Venom of the Cobras Naja oxiana, Naja kaouthia, and Naja haje

Cytotoxins are positively charged polypeptides that constitute about 60% of all proteins in cobra venom; they have a wide spectrum of biological activities. By CD spectroscopy, cytotoxins CT1 and CT2 Naja oxiana, CT3 Naja kaouthia, and CT1 and CT2 Naja haje were shown to have similar secondary structure in an aqueous environment, with dominating beta-sheet structure, and to vary in the twisting angle of the beta-sheet and the conformation of disulfide groups. Using dodecylphosphocholine micelles and liposomes, CT1 and CT2 Naja oxiana were shown to incorporate into lipid structures without changes in the secondary structure of the peptides. The binding of CT1 and CT2 Naja oxiana with liposomes was associated with an increase in the beta-sheet twisting and a sign change of the dihedral angle of one disulfide group. The cytotoxins were considerably different in cytotoxicity and cooperativity of the effect on human promyelocytic leukemia cells HL60, mouse myelomonocytic cells WEHI-3, and human erythroleukemic cells K562. The most toxic CT2 Naja oxiana and CT3 Naja kaouthia possessed low cooperativity of interaction (Hill coefficient h = 0.6-0.8), unlike 10-20-fold less toxic CT1 and CT2 Naja haje (h = 1.2-1.7). CT1 Naja oxiana has an intermediate position on the cytotoxicity scale and is characterized by h = 0.5-0.8. The cytotoxins under study induced necrosis of HL60 cells and failed to activate apoptosis. The differences in cytotoxicity are supposed to be related not with features of the secondary structure of the peptides, but with interactions of side chains of variable amino acid residues with lipids and/or membrane proteins.     

Effect of iodination on the activity of cytotoxin P4 of Naja nigricollis nigricollis.

Iodination of cytotoxin P4, isolated from the venom of Naja nigricollis nigricollis, develops gradually and depends on the molar ratio between the free iodine and the cytotoxin reaching a maximum of two equivalents at a molar ratio of 250 or higher. The cytotoxic activity was also gradually decreased and was totally abolished when one equivalent of iodination was achieved. However, antigenic properties of the cytotoxin were preserved in the iodinated form. When the iodination of the cytotoxin was carried out with a carrier free radiolabeled iodide, the molar ratio was 0.05 resulting in labelling of only 2% of the cytotoxin molecules, which explains the cytotoxicity of the radiolabeled mixture.     

Experimental study on developing rat kidney after Naja haje envenomation

In the present experiment 164 pregnant white Wistar rats were used to study the effect of Naja haje (Egyptian cobra) venom on the developing kidney. The rats were divided into 3 groups; a control group, a group receiving one LD50 of N. haje venom and the third injected with 1/8 of LD50. The injection was performed at different stages of gestation. After birth, the neonates of group I and III and embryos of group II were examined histologically, histochemically and electron-microscopically. Both lethal and sublethal doses of N. haje venom produced haemorrhages and vascular congestion of the developing kidney. The lethal dose had degenerative effects on the podocytes and endothelium. Tubular damage appeared mainly as mitochondrial degeneration and bud-like extension, protrusions of cytoplasm and vacuolization. The succinic dehydrogenase enzyme showed decreased activity. The sublethal dose had an effect on the glomerular basement membrane in the form of splitting, increased mesangial cells and matrix, mitochondrial degeneration and fusion of podocyte processes. Tubulization of the parietal epithelium, vacuolization of the proximal tubules, mitochondrial degeneration and apical budding were evident.     

Amino-acid sequence of toxin III of Naja haje.

The complete amino acid sequence of toxin III of Naja haje (72 residues) has been established mainly by use of a protein sequenator (identification of 70 residues). The two C-terminal residues have been determined by digestion with carboxypeptidases A and B. Addition of succinylated protein or peptide greatly improved the performance of the sequenator for the Edman degradation of peptides: on one peptide (39 residues) degradation went to step 34 with a protein program and on two peptides (10 and 13 residues) degradation reached the last amino acid with a peptide program (use of dimethylbenzylamine). Amino acid analysis of tryptic peptides obtained by digestion of the C-terminal cyanogen bromide peptide are in full agreement with the sequence established by automatic degradation. The sequence of toxin III of Naja haje is unique and is very similar to that of Naja nivea alpha (although there are 9 differences), of Naja melanoleuca b (11 differences) and also to that of Naja naja A (18 differences).     

Identification of a novel family of proteins in snake venoms. Purification and structural characterization of nawaprin from Naja nigricollis snake venom

The three-dimensional structure of nawaprin has been determined by nuclear magnetic resonance spectroscopy. This 51-amino acid residue peptide was isolated from the venom of the spitting cobra, Naja nigricollis, and is the first member of a new family of snake venom proteins referred to as waprins. Nawaprin is relatively flat and disc-like in shape, characterized by a spiral backbone configuration that forms outer and inner circular segments. The two circular segments are held together by four disulfide bonds, three of which are clustered at the base of the molecule. The inner segment contains a short antiparallel beta-sheet, whereas the outer segment is devoid of secondary structures except for a small turn or 310 helix. The structure of nawaprin is very similar to elafin, a human leukocyte elastase-specific inhibitor. Although substantial parts of the nawaprin molecule are well defined, the tips of the outer and inner circular segments, which are hypothesized to be critical for binding interactions, are apparently disordered, similar to that found in elafin. The amino acid residues in these important regions in nawaprin are different from those in elafin, suggesting that nawaprin is not an elastase-specific inhibitor and therefore has a different function in the snake venom.     

Basic phospholipase from the venom of Naja nigricollis is cytotoxic

The basic phospholipase A2 purified from the venom of Naja nigricollis (Institut Pasteur), possesses an intense cytotoxic activity toward fetal cells from FL strain. At a concentration equal to 1.6 x 10(-6) M, the PLA2 lyses 50% of the cells present in a suspension containing 3.5 x 10(6) cells per millilitre. Other PLA2 from various origins do not exhibit such cytotoxic activity.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca ²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity. Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

Isolation and characterization of a cytotoxin P4 from the venom of Naja nigricollis nigricollis preferentially active on tumor cells.

Cytotoxin P4 was isolated from the venom of Naja nigricollis nigricollis in three steps and contained 55% of the crude cytotoxic activity. It had a molecular weight of 8 KD, was stable over a pH range of 1-11 and in boiling water for at least 15 min. It had no measurable enzymatic activities, but was destroyed by proteases. Concentrations of 0.8, 1, 1.2, 25. 20 and 45 ug/ml, were needed to destroy murine melanoma B16 and WEHI 3B leukemia, rat chondrosarcoma, mouse erythrocytes and spleen cells, and human erythrocytes, respectively, thereby showing preferential cytotoxicity to the examined tumor cells. It also prevented the development of the melanoma, leukemia and chondrosarcoma tumors in vivo when mixed with the cells prior to the injection into the animal.     

Structure and chemical modifications of neurotoxin from Naja nigricollis studied by Raman spectroscopy

Raman spectroscopy was used to determine structural features of the native toxin alpha from Naja nigricollis, which contains only one Trp and one Tyr, and of chemically modified toxins having chromophores added to these two conserved aromatic amino acids. The percentages of secondary structure were determined by using amide I polypeptidic vibration analysis and are in agreement with X-ray structure [Low et al. (1976) Proc. Natl. Acad Sci. U.S.A. 73, 2991-2994] as well as with the geometry of the disulfide bridges estimated by using the v(S-S) vibrations. In the native toxin alpha, the single invariant tyrosine 25 appears to be buried in the structure and involved in a strong hydrogen bond. We have chemically modified these two invariant aromatic side chains by addition of chromophores. The presence of a (nitrophenyl)sulfenyl (NPS) chromophore bound to the Trp does not perturb the secondary structure of the toxin as shown by the analysis of the polypeptidic amide I vibrations; however, the environment of this Trp and the geometry of a disulfide bridge seem to be modified. The secondary structure is not affected by the presence of the NPS chromophore; therefore, the decrease in binding affinity observed after modification of Trp-29 by the reagent NPS-Cl [Faure et al. (1983) Biochemistry 22, 2068-2076] is due to an alteration of the environment of this aromatic amino acid and/or a steric hindrance and not to an overall modification of the toxin structure. The binding assays of [nitrotyrosyl]toxin show that after nitration the affinity toward the monoclonal antibody M alpha 1 is unchanged and that the affinity toward the cholinergic receptor (AcChR) from Torpedo marmorata remains high. We concluded that the structure of toxin alpha after adding the NO2 chromophore to Tyr-25 is the same as it is in native toxin.     

Differential action of proteases from Trimeresurus malabaricus, Naja naja and Daboia russellii venoms on hemostasis

The action of venom proteases and their role in hemostasis has been compared in the venoms of Trimeresurus malabaricus, Daboia russellii and Naja naja from the Southern region of Western Ghats, India. These venoms exhibit varying amounts of proteolytic activity and also influence hemostasis differently. Casein hydrolyzing activity of T. malabaricus venoms was 16 and 24 fold higher than those of N. naja and D. russellii venoms, respectively. With the synthetic substrate TAME, the highest activity was observed in T. malabaricus venom. N. naja venom did not hydrolyze TAME even at higher concentrations. These variations in proteolytic activity also influenced the coagulation process. T. malabaricus and D. russellii venoms are strongly procoagulant and reduce the re-calcification time from 148 to 14 and 12 s, respectively. Similarly, both T. malabaricus and D. russellii venoms reduce the prothrombin time from 12.5 to 6.0 s. On the other hand, N. naja venom is anticoagulant and prolongs re-calcification time to 600 s and prothrombin time to 42 s. In spite of varied effects on hemostasis, all the venoms hydrolyze fibrinogen. T. malabaricus venom hydrolyses both Aalpha and Bbeta subunits. While D. russellii and N. naja venoms hydrolyse only Aalpha. None of these venoms hydrolyze the gamma subunit of fibrinogen. Inhibition studies with specific protease inhibitors revealed that both N. naja and T. malabaricus venoms contain only metalloproteases. D. russellii venom contained both serine and metalloproteases. Only, T. malabaricus venom exhibited thrombin-like activity and induces fibrin clot formation with purified fibrinogen within 58 s. Even though D. russellii venom exhibits procoagulant activity, it did not show thrombin-like activity and may act on other coagulation factors.     

Non-enzymatic inhibitors of coagulation and platelet aggregation from Naja nigricollis venom are cardiotoxins

Four non-enzymatic polypeptides from Naja nigricollis crawshawii venom were recently isolated and shown to inhibit plasma coagulation and platelet aggregation. We have now determined the amino acid compositions, amino terminal sequences and direct lytic activity of these anticoagulants. The results of these studies allow us to identify the anticoagulants as cardiotoxins. The anticoagulant activity of these cardiotoxins is far more potent than that of other cardiotoxins previously reported to have anticoagulant activity.     

The effect of maternal Naja nigricollis envenomation on the placenta. An experimental study

The effect of maternal envenomation with N. nigricollis venom on the placental tissues was studied in mice. The venom seemed to exert a direct toxic effect on the constituent parts of the placenta. Glycogen appeared to advantage in the cells of the trophospongeal part of the placenta. Giant cells and basophilic cells were encountered around such glycogen cells. However, some giant cells showed vacuolation of the cytoplasm and pyknosis of the nuclei. The labyrinthine part of the placenta showed large and multiple small thrombi and congestion of the blood spaces. Again glycogen appeared to advantage in the syncytial cells. Non cellular exudate was also met with in such cases. The possible mechanisms of these changes were discussed.     

Binding of cytotoxin P4 from Naja nigricollis nigricollis to B16F10 melanoma and WEHI-3B leukemia cells

Abstract Cobra venoms cause irreversible destruction of cells cultured in vitro [1,2]. The venom of Naja nigricollis nigricollis possessed the most potent cytotoxic activity towards B16F10 melanoma cells among various examined venoms [2]. The main cytotoxic factor (P4) isolated from this venom showed preferential activity on tumor cell lines and caused lysis at concentrations of 10⁻⁷ M (0.8–1 μg/ml) [3]. The present study examined the binding of cytotoxin P4 to melanoma B16F10 and WEHI-3B leukemia cell lines and found that, like cytotoxicity, it depended on concentration, temperature and incubation time. Cytotoxin concentrations that elicited no apparent damage to cells during the first hour of incubation caused lysis after a longer period of incubation, suggesting that a critical number of bound molecules is required in order to cause cell death. Bivalent ions, such as Mg²⁺, Ca²⁺ or Sr²⁺, which decreased binding to the cells also inhibited cytotoxicity. Competition experiments as well as the displacement of 75% of the bound radiolabelled cytotoxin with 'cold' cytotoxin, suggest the presence of specific binding sites for the toxin in the examined tumor cells. The non-specific binding of the cytotoxin P4 to sea urchin ova and sperm cells without affecting their fertility, even at high concentrations of 10⁻⁵ M, indicates that the specific binding to cells is probably a necessary condition for cell lysis.     

Snake alpha-neurotoxin binding site on the Egyptian cobra (Naja haje) nicotinic acetylcholine receptor Is conserved

Evolutionary success requires that animal venoms are targeted against phylogenetically conserved molecular structures of fundamental physiological processes. Species producing venoms must be resistant to their action. Venoms of Elapidae snakes (e.g., cobras, kraits) contain alpha-neurotoxins, represented by alpha-bungarotoxin (alpha-BTX) targeted against the nicotinic acetylcholine receptor (nAChR) of the neuromuscular junction. The model which presumes that cobras (Naja spp., Elapidae) have lost their binding site for conspecific alpha-neurotoxins because of the unique amino acid substitutions in their nAChR polypeptide backbone per se is incompatible with the evolutionary theory that (1) the molecular motifs forming the alpha-neurotoxin target site on the nAChR are fundamental for receptor structure and/or function, and (2) the alpha-neurotoxin target site is conserved among Chordata lineages. To test the hypothesis that the alpha-neurotoxin binding site is conserved in Elapidae snakes and to identify the mechanism of resistance against conspecific alpha-neurotoxins, we cloned the ligand binding domain of the Egyptian cobra (Naja haje) nAChR alpha subunit. When expressed as part of a functional Naja/mouse chimeric nAChR in Xenopus oocytes, this domain confers resistance against alpha-BTX but does not alter responses induced by the natural ligand acetylcholine. Further mutational analysis of the Naja/mouse nAChR demonstrated that an N-glycosylation signal in the ligand binding domain that is unique to N. haje is responsible for alpha-BTX resistance. However, when the N-glycosylation signal is eliminated, the nAChR containing the N. haje sequence is inhibited by alpha-BTX with a potency that is comparable to that in mammals. We conclude that the binding site for conspecific alpha-neurotoxin in Elapidae snakes is conserved in the nAChR ligand binding domain polypeptide backbone per se. This conclusion supports the hypothesis that animal toxins are targeted against evolutionarily conserved molecular motifs. Such conservation also calls for a revision of the present model of the alpha-BTX binding site. The approach described here can be used to identify the mechanism of resistance against conspecific venoms in other species and to characterize toxin-receptor coevolution.