erabutoxin-a and alpha-cobratoxin

Snake neurotoxins (NTX) have proven to be valuable tools for the characterisation of muscular nicotinic acetylcholine receptor structure and function. It is very likely that they could also be utilised to identify subtypes of neuronal nicotinic receptors controlling specific functions within the central nervous system. In this study we examined the effects of long alpha NTX (alpha-bungarotoxin, alpha-Bgt, and alpha-cobratoxin, alpha-Cbt) and short alpha NTX (alpha-erabutoxin a, alpha-Ebt) as well as the anticholinesterase toxin fasciculin-2 (FAS), on the nicotine-evoked release of dopamine (DA) in the striatum, using the in vivo push-pull technique. The short toxins alpha-Ebt and FAS blocked the extracellular increase of DA evoked by nicotine at 4.2 microM concentrations and alpha-Ebt was more potent, as reflected by the blockade at the lower dose of 0.42 microM. In contrast, the long toxins showed a different profile of action. Alpha-Cbt did not show any blockade of the nicotine-evoked release of DA at the doses studied while alpha-Bgt did block it only at the higher dose (4.2 microM) These results indicate that short neurotoxins show a stronger interaction with striatal nicotinic receptors subtypes controlling DA release when compared to the long ones. This interaction of short neurotoxin polypeptides and presynaptic receptors may permit the further elucidation of the particular nicotinic receptor populations responsible for the modulation of striatal DA release.

Topics: Animals; Bungarotoxins; Cobra Neurotoxin Proteins; Corpus Striatum; Dopamine; Dose-Response Relationship, Drug; Elapid Venoms; Erabutoxins; Male; Neurotoxins; Nicotine; Osmolar Concentration; Rats; Rats, Inbred Strains; Snakes

Structural features associated with the ability of a monoclonal antibody (mAb) to discriminate between protein variants are identified and engineered. The variants are the curaremimetic toxin alpha from Naja nigricollis and erabutoxin a or b from Laticauda semifasciata, which differ from each other by 16 substitutions and one insertion. The neutralizing mAb M alpha 1 recognizes with high affinity a topographical epitope on the surface of toxin alpha, but fails to recognize the erabutoxins although they possess most of the residues forming the presumed epitope. Examinations of the toxin alpha and erabutoxin 3-D structures and molecular dynamics simulations reveal several differences between the variants. In particular, the region involving the beta-turn 17-24 is organized differently. Analysis of the differences found in this region suggest that the insertion (or deletion) at position 18 of the variant amino acid sequences is particularly important in determining the differential cross-reactivity. To test this proposal, residue 18 was deleted in one erabutoxin using site-directed mutagenesis, and the biological properties of the resulting mutant were examined. We found that full antigenicity was restored in the previously unrecognized variant. The implications of this finding are discussed.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Antigen-Antibody Reactions; Cholinergic Antagonists; Cobra Neurotoxin Proteins; Computer Simulation; Cross Reactions; Epitopes; Erabutoxins; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Protein Engineering; Recombinant Fusion Proteins; Sequence Deletion

The positive charge of Lys27 was suppressed by chemical means in two short-chain curaremimetic toxins, namely erabutoxin a (Ea) from Laticauda semifasciata and toxin alpha from Naja nigricollis. This modification leads to a decrease in the binding affinity of the toxins for the nicotinic acetylcholine receptor, which range 6-15-fold, as judged from both the data reported here and those previously described in the literature. A negatively charged glutamate residue has been introduced at position 27 of erabutoxin a by site-directed mutagenesis. This change provokes a 120-fold decrease in the affinity, which reflects a major alteration of toxin-receptor cognate events. Using toxin-alpha derivative harbouring a photoactive group at Lys27, we probed the toxin local environment in a receptor-bound state by photocoupling experiments. The delta chain was the predominant coupling target, in contrast to previous observations indicating that a photoactive probe on Lys47 predominantly labelled the alpha chain. The toxin derivative weakly labelled the alpha and gamma chains but not the beta chain. The toxin may therefore interact with subunits other than the alpha chain, at least in the vicinity of Lys27.

Topics: Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Cell Membrane; Cobra Neurotoxin Proteins; Cross-Linking Reagents; Erabutoxins; Lysine; Mutagenesis, Site-Directed; Neurotoxins; Receptors, Nicotinic; Recombinant Proteins; Structure-Activity Relationship