Snake venom toxins. The amino acid sequence of three toxins (CM-2e, CM-4a and CM-) from Naja haje annulifera (Egyptian cobra) venom.

Three toxins (CM-2e, CM-4a and CM-7) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex and by ion-exchange chromatography on CM-cellulose. They comprise 60 amino acid residues and are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of the three toxins have been elucidated. The toxicities, the serological properties, the sequences and the invariant amino acid residues of toxin CM-2e, CM-4a and CM-7 resemble the corresponding properties of the cytotoxin group.     

The purification and amino acid sequence of toxin CM-13b from Naja haje annulifera (Egyptian cobra) venom.

Toxin CM-13b was purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. The toxin comprises 65 amino acid residues and is cross-linked by five disulphide bridges. The complete amino acid sequence of toxin CM-13b was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides purified by DEAE-cellulose chromatography and chromatography or electrophoresis on paper. The amino acid sequences of the intact toxin and its constituent peptides were determined by the Edman-Begg procedure, either through the use of the automatic sequenator or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The primary structure of toxin CM-13b shows a high degree of homology with that of protein S4C11 from Naja melanoleuca venom[1], but their toxicities are very different.     

The amino acid sequence of toxin V II 2, a cytotoxin homologue from banded Egyptian cobra (Naja haje annulifera) venom.

Toxin V II 2 comprises 60 amino acid residues and is cross-linked by four disulphide bridges. The complete amino acid sequence of this toxin was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides were purified by ion-exchange chromatography and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequenator or by manual manipulation, was employed to obtain the sequence of the intact toxin and the pure peptides. The chymotryptic digest provided the necessary overlapping peptides which allowed the alignment of tryptic peptides. The amino acid sequence of Naja haje annulifera toxin V II 2 shows a high degree of homology with cytotoxin V II 1 of the same venom.     

Snake venom toxins. The amino-acid sequences of three toxins (9B, 11 and 12A) from Hemachatus haemachatus (Ringhals) venom.

Three toxins (9B, 11 and 12A) were purified from the venom of Hemachtus haemachatus as described previously. Whereas toxin 11 and 12A comprise 61 amino acid residues, toxin 9B contains 63 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have elucidated. The properties of the toxins were compared with those of the cytotoxin group. The toxicities, the sequences and some of the invariant residues of toxin 11 and 12A resemble the corresponding properties of the cytotoxin group. However their immunochemical properties indicate that they are distinct from both the cytotoxin and neurotoxin groups. The sequence of toxin 9B shows that it is related to the cytotoxins, but its toxicity is much lower than those encountered among members of this group.     

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.     

Snake venom toxins. The primary structure of protein S4C11. A neurotoxin homologue from the venom of forest cobra (Naja melanoleuca).

Six minor protein constituents (S4C10-S4C15) have been isolated from the venom of Naja melanoleuca. The complete amino acid sequence of S4C11 has been established and indicates that it is a homologue of the neurotoxins which are found in elapid venoms. The other proteins appear from the amino acid compositions to be homologues of the cyto- or cardiotoxins found in cobra venoms. Protein S4C11 has a low toxicity, failing to kill mice at an intravenous dose of 20 mug/g body weight. The sequence of the first 25 residues out of the total of 65, was determined using the automatic sequenator. The remainder of the sequence was derived with the aid of tryptic and chymotryptic peptides. The sequence showed the unusual feature of having 65 amino acid residues including 10 half-cystine residues.     

The embryonic development of the Egyptian cobra Naja h. haje (Squamata: Serpentes: Elapidae)

The Egyptian cobra, Naja h. haje, is the largest of the African cobras and is a member of a successful and medically important species complex found throughout Africa, north and south of the Sahara, as well as across the Arabian Peninsula to Oman. Although its phylogenetic position and venom characteristics have been well studied, its development has not. Here, we present a normal staging table for N. h. haje, based on external features. Comparison with firstly the Asian monocled cobra, Naja kaouthia, and then with the small number of other oviparous snake species, allowed us to examine whether differences between two species in the same genus were of the same type and magnitude as those between unrelated genera. In fact, at least with respect to external features, we found a similar level of disparity. N. h. haje embryos lagged behind those of N. kaouthia in body and head scale development, size in ovo and hatchling length, despite having a slightly shorter incubation period and a somewhat larger adult size. Some of these differences may have been the result of differing incubation temperatures. Nonetheless, there does appear to be a broadly conserved pattern of in ovo development in at least macrostomatan snakes.     

The first representative of glycosylated three-fingered toxins. Cytotoxin from the Naja kaouthia cobra venom.

There are different glycosylated proteins in snake venoms, but no glycosylated representatives of a large family of three-fingered toxins have previously been detected. A new glycoprotein was isolated from the venom of the Thai cobra Naja kaouthia. MALDI MS of the glycoprotein contained an array of peaks in the range from approximately 8900 to approximately 9400 Da indicating its microheterogeneity. Carbohydrate analysis showed the presence of mannose, galactose, N-acetylglucosamine, fucose and neuraminic acid. The N-terminal sequence of the glycoprotein was identical to that of cytotoxin 3 (CX3) from N. kaouthia, and CD spectra of the glycoprotein and CX3 were almost the same. Cleavage of a glycan moiety by N-glycosidase F gave a protein of molecular mass practically coinciding with that of CX3. MALDI MS of the tryptic digest of reduced glycoprotein S-pyridylethylated at cysteine residues, contained peaks corresponding to all tryptic fragments of CX3, with the exception of fragment 24-30. The peak corresponding to this peptide appeared in the mass-spectrum of similarly treated deglycosylated glycoprotein. These data show that the potential N-glycosylation site at Asn29 in CX3 is utilized for glycan attachment and that the glycoprotein is glycosylated CX3. In vivo toxicity of the glycoprotein to the cricket Gryllus assimilis was twofold lower than that of CX3. The cytotoxic activity of the glycoprotein towards HL60 cells was about two orders of magnitude lower than that of CX3, but could be made equal to the CX3 cytotoxicity by deglycosylation. Thus for the first time we have isolated a glycosylated three-fingered snake venom toxin wherein glycosylation appears to modulate its biological activity.     

Comparison of the structural stability of two homologous toxins isolated from the Taiwan cobra (Naja naja atra) venom

Cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX) isolated from the Taiwan cobra venom (Naja naja atra) are structurally homologous, small molecular weight, all-beta-sheet proteins, cross-linked by four disulfide bonds at identical positions. The conformational stabilities of these toxins are compared based on temperature-dependent chemical shifts and amide proton exchange kinetics using two-dimensional NMR spectroscopy. The structure of CTX III is found to be significantly more stable than that of CBTX. In both the toxins, beta-strand III appears to constitute the stability core. In CTX III, the stability of the triple-stranded beta-sheet domain is observed to be markedly higher than the double-stranded beta-sheet segment. In contrast, in CBTX, both structural domains (double- and triple-stranded beta-sheet domains) appear to contribute equally to the stability of the protein. Estimation of the free energy of exchange (Delta G(ex)) of residues in CBTX and CTX III reveals that the enhanced stability of the structure of CTX III stems from the strong interactions among the beta-strands constituting the triple-stranded beta-sheet domain and also the molecular forces bridging the residues at the N- and C-terminal ends of the molecule.