High-level production and isotope labeling of snake neurotoxins, disulfide-rich proteins.

The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B. Nilsson et al., 1987, Protein Eng. 1, 107-113), thus preventing degradation in the bacterial cytoplasm and providing a simple affinity-purification method on IgG Sepharose. A soluble fusion protein was obtained with a yield of 60 mg/L, corresponding to 20 mg/L toxin. The toxin moiety was folded on the column while the hybrid was still bound. The oxidoreducing conditions for the refolding were optimized and were found to be oxidative but with a need for reducing molecules. The concentration of the hybrid bound to the column could be increased up to 3.3 mg/ml without significantly altering the folding process. CNBr cleavage of the fusion protein followed by a purification step yielded about 2 mg of biologically active toxin mutant per gram of dry cell weight. This procedure was applied to produce 55 mg of a toxin uniformly labeled with 15N.

[Genetic origin of venom variability: impact on the preparation of antivenin serums]. / L'origine génétique de la variabilité des venins: impact sur la préparation des sérums antivenimeux.

One of the main possible origin of the biochemical variations of venoms could be genetic. We studied the venom of members of litters born in a snake farm (12 Crotalus atrox and 21 Naja haje). We first used the electrophoresis in cellulose acetate (AE). Then, variations were confirmed by immunoelectrophoresis (AIE) using an antivenom (IPSER Africa, Pasteur Mérieux Sérums & Vaccines) and immunsera prepared on rabbit from i) venom presenting the maximum of bands in electrophoresis (complete venom) and ii) pure toxins (neurotoxin-alpha and cardiotoxin-gamma). At last, the toxicity of some samples was measured and the ability of SAV to neutralise the corresponding sample was measured. The AE of C. atrox venoms showed a good homogeneity, probably due to a good genetic stability of the investigated group. On the other hand, N. haje venoms have revealed a great heterogeneity. The 13 samples were allocated to five groups according to the absence of some fractions compared to the complete venom. The AIE showed that the neurotoxin-alpha is present in every sample, but variable in quantity, even when it did not appear on AE. We suggest that these pattern variations are due either to relative variations of protein fractions in samples or to modifications of the chemical composition of the neurotoxin-alpha. However, the variation of toxicity between the different samples questioned the neutralisation ability of antivenoms. We propose that venom sample choice for SAV production should be based on biochemical criteria and toxicity of samples rather than random pooling.

Mimicry between receptors and antibodies. Identification of snake toxin determinants recognized by the acetylcholine receptor and an acetylcholine receptor-mimicking monoclonal antibody.

In several instances, a monoclonal antibody raised against a receptor ligand has been claimed to mimic the ligand receptor. Thus, a specific monoclonal antibody (Malpha2-3) raised against a short-chain toxin from snake was proposed to mimic the nicotinic acetylcholine receptor (AChR) (). Further confirming this mimicry, we show that (i) like AChR, Malpha2-3 elicits anti-AChR antibodies, which in turn elicit anti-toxin antibodies; and (ii) the region 106-122 of the alpha-chain of AChR shares 66% primary structure identity with complementarity-determining regions of Malpha2-3. Also, a mutational analysis of erabutoxin a reveals that the epitope recognized by Malpha2-3 consists of 10 residues, distributed within the three toxin loops. Eight of these residues also belong to the 10-residue epitope recognized by AChR, a result that offers an explanation as to the functional similarities between the receptor and the antibody. Strikingly, however, most of the residues common to the two epitopes contribute differentially to the energetic formation of the antibody-toxin and the receptor-toxin complexes. Together, the data suggest that the mimicry between AChR and Malpha2-3 is partial only.

Genetic engineering of snake toxins. The functional site of Erabutoxin a, as delineated by site-directed mutagenesis, includes variant residues.

Using site-directed mutagenesis, we previously identified some residues that probably belong to the site by which Erabutoxin a (Ea), a sea snake toxin, recognizes the nicotinic acetylcholine receptor (AcChoR) (Pillet, L., Trémeau, O., Ducancel, F. Drevet, P., Zinn-Justin, S., Pinkasfeld, S., Boulain, J.-C., and Ménez, A. (1993) J. Biol. Chem. 268, 909-916). We have now studied the effect of mutating 26 new positions on the affinity of Ea for AcChoR. The mutations are F4A, N5V, H6A, Q7L, S9G, Q10A, P11N, Q12A, T13V, T14A, K15A, T16A, delta S18, E21A, Y25F, Q28A, S30A, T35A, I36R, P44V, T45A, V46A, K47A, P48Q, I50Q, and S53A. Binding affinity decreases upon mutation at Gln-7, Gln-10 and to a lesser extent at His-6, Ser-9 and Tyr-25 whereas it increases upon mutation at Ile-36. Other mutations have no effect on Ea affinity. In addition, new mutations of the previously explored Ser-8, Asp-31, Arg-33, and Glu-38 better explain the functional role of these residues in Ea. The previous and present mutational analysis suggest that the "functional" site of Ea covers a homogeneous surface of at least 680 A2, encompassing the three toxin loops, and includes both conserved and variant residues. The variable residues might contribute to the selectivity of Ea for some AcChoRs, including those from fish, the prey of sea snakes.

Aggregation ofNaja nigricollis cardiotoxin: Characterization and quantitative estimate by time-resolved polarized fluorescence.

After purification to homogeneity by Bio-Rex 70 ion exchange chromatography, micromolar solutions ofNaja nigricollis cardiotoxin were found to contain significant amounts of aggregates, as detected by time-resolved polarized fluorescence of its single tryptophan residue. The level of cardiotoxin aggregation depends strongly and reversibly on the protein concentration and pH. However, supplementary reverse-phase HPLC completely suppresses this aggregation, resulting in all cases in fluorescence anisotropy decays characteristic of the pure cardiotoxin monomer. The self-association properties of cardiotoxin, in the presence of a possible cofactor eliminated by the HPLC step, may be functionally relevant, and would deserve further investigation. The physical heterogeneity of the cardiotoxin samples required an appropriate model for the analysis of fluorescence depolarization, which was iteratively improved by comparison with experimental results. In this way, an approximate molar fraction of 10-15% aggregated cardiotoxin at a 90ΜM total protein concentration, pH 7, was determined. The fluorescence of the partly aggregated samples is significantly perturbed as compared to the HPLC-treated monomer, indicating that the cardiotoxin aggregate must have an increased average fluorescence lifetime and a strongly decreased initial anisotropy. The decrease in initial anisotropy suggests either an increased mobility of the tryptophan residue upon aggregation or fast energy transfers between residues of different cardiotoxin molecules brought within a short distance in the aggregate. This study illustrates the high sensitivity of the time-resolved fluorescence technique, through both total fluorescence and anisotropy parameters, to low levels of physical or chemical heterogeneity in a protein sample.

Proton NMR studies of the structural and dynamical effect of chemical modification of a single aromatic side-chain in a snake cardiotoxin. Relation to the structure of the putative binding site and the cytolytic activity of the toxin.

This paper presents the comparative comprehensive analysis of NMR structural parameters (NOEs, scalar coupling, chemical shifts) of toxin gamma, a cardiotoxin isolated from the venom of Naja nigricollis, and three chemical derivatives, i.e. the 2-nitrophenylsulphonyl (NPS)-Trp11, 3-nitro-Tyr22 and 3-nitro-Tyr51 derivatives. In previous work, the chemical modifications of single side chains have suggested that these aromatic residues, in association with several lysine residues, contributed to the cytotoxicity of toxin gamma. Analysis of these results based on the refined solution structure of the toxin has resulted in the proposal of a conserved phospholipid binding site through which cardiotoxins are likely to interact with the membrane of target cells. The present work shows that modifications of either the tryptophan residue or the tyrosine residues, which are within or near the proposed binding site, have no influence on the three-dimensional structure of the protein. On the other hand, the proton exchange study of the backbone amides indicates that the structural core of the protein is destabilized in the three derivatives. This corresponds to a decrease of the overall stability of the protein as indicated by the comparative solvent denaturation study of the unmodified toxin gamma and the Trp11 derivative. More specifically, the dynamics of the three-stranded beta sheet, a part of the structural core, are highly perturbed by the chemical modifications. This sheet was previously proposed as a part of the phospholipid binding site of cardiotoxins. The dynamical perturbation of this site appears to be correlated with the decrease in toxicity of the chemical derivatives.

Dynamics of the active loop of snake toxins as probed by time-resolved polarized tryptophan fluorescence.

The local environment and dynamics of the single tryptophan residue in the respective active loops of cardiotoxin and alpha-neurotoxin from Naja nigricollis and of erabutoxin b from Laticauda semifasciata have been studied by steady-state and time-resolved polarized fluorescence and analyzed with distributions of decay times. Trp11 in loop I of cardiotoxin exhibits a very broad and complex distribution of fluorescence lifetimes at 20 degrees C. Despite its relatively external location in the toxin, the residue appears to be partly shielded from water and shows restricted but significant conformational fluctuations on the picosecond and nanosecond time scales. The thermal stability of cardiotoxin allowed a study of its static and dynamic fluorescence properties over a large range of temperatures. Interconversions in the intermediate nanosecond range lead to a thorough reorganization of the cardiotoxin fluorescence lifetime distribution with temperature. On the contrary, the fluorescence kinetics of Trp29 in loop II of the two neurotoxins is dominated by about 80% of a major decay time, which suggests that a nearly unique local conformation of the toxin is maintained over all time scales above the sub-nanosecond range. The fluorescence anisotropy decays show that the residue also has extremely limited rotational freedom down to the picosecond time scale. These findings are in good agreement with structural and dynamic information previously reported on the different toxins from NMR and X-ray crystallographic studies. The different dynamic properties around the tryptophan residue of the cardiotoxin and neurotoxin active loops can be analyzed within the frame of their different respective mechanisms of toxicity.

Genetic engineering of snake toxins. Role of invariant residues in the structural and functional properties of a curaremimetic toxin, as probed by site-directed mutagenesis.

To study the site by which erabutoxin a (Ea) from Laticauda semifasciata binds to the nicotinic acetylcholine receptor, we mutated most residues that are shared with other curaremimetic toxins and studied the structural and biological consequences of introduced mutations. By site-directed mutagenesis, we changed Ser-8 into Gly (EaS8G), Lys-27 into Glu (EaK27E), Trp-29 into Phe (EaW29F) and His (EaW29H), Asp-31 into His (EaD31H), Phe-32 into Leu (EaF32L), Arg-33 into Lys (EaR33K) and Glu (EaR33E), Gly-34 into Ser (EaG34S), Glu-38 into Gln (EaE38Q) and Lys (EaE38K), Gly-49 into Val (EaG49V), and Leu-52 into Ala (EaL52A). All mutants were homogeneous as judged by various analytical procedures. EaE38Q, EaG49V, and EaL52A bound the nicotinic acetylcholine receptor with apparent Kd values close to 10(-10) M, virtually identical to wild Ea. Therefore, Glu-38, Gly-49, and Leu-52 are not important elements in the expression of curaremimetic function in Ea. Mutations of Phe-32 and Gly-34 provoked a 7-fold affinity decrease, suggesting that these residues moderately contribute to function. The 176-fold affinity decrease due to mutation of Ser-8 may reflect some structural change that operates in the polypeptide chain of the mutant, as detected by circular dichroism. Decreases in affinity by a factor of 175, 67, 46, and 318 were seen upon mutations of Lys-27 into Glu, Trp-29 into Phe, Asp-31 into His, and Arg-33 into Glu, with no concomitant change in secondary structure. These residues appear to be important elements of the curaremimetic function of Ea. Thus, a picture of the contribution of conserved residues to the function of a curaremimetic toxin is proposed on the basis of experimental evidence.

Role and environment of the conserved Lys27 of snake curaremimetic toxins as probed by chemical modifications, site-directed mutagenesis and photolabelling experiments.

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.

Notechis 11'2, a non-toxic phospholipase A2 from the venom of Notechis scutatus scutatus.

Previously, we deduced the amino acid sequence of a novel phospholipase-A2-like protein (PLA2) from the nucleotide sequence of a cDNA isolated from a library prepared from the venom gland of the Australian elapid Notechis scutatus scutatus. The corresponding protein has now been identified, purified from the venom and named Notechis 11'2. Its complete amino acid sequence has been determined by automated Edman degradation of both the whole protein and peptides generated by Staphylococcus aureus protease digestion and chemical cleavage at a tryptophan residue. As predicted from its sequence which contains all the residues putatively required for PLA2 activity, Notechis 11'2 exhibits an esterase activity, preferentially against neutral phospholipids. However, despite its sequence homology with other highly toxic PLA2 present in the venom of Notechis scutatus scutatus, notechis 11'2 has no lethal activity. This observation further supports the view that the lethal activity of PLA2 from Notechis scutatus scutatus is not due to the esterasic activity only.

A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties.

We previously reported the production of a fused snake neurotoxin composed of protein A and erabutoxin a in E. coli. The hybrid had much lower toxicity and affinity for the acetylcholine nicotinic receptor than natural erabutoxin. By treating the hybrid with cyanogen bromide we generated a toxin which was purified in a single step by RP-HPLC. This compound, produced in a good yield, recovered all properties of native erabutoxin a, implying that the lower toxic activities of the hybrid were due to the bulky protein A and not to an incorrect folding of the toxin. This work serves as a basis for future studies of toxin-receptor interactions using engineered toxin mutants.

Topography of toxin-acetylcholine receptor complexes by using photoactivatable toxin derivatives.

We have defined the molecular environment of a snake neurotoxin interacting with the high- and low-affinity binding sites of the nicotinic acetylcholine receptor (AcChoR). This was done by photocoupling reactions using three toxin derivatives with photoactivatable moieties on Lys-15, Lys-47, and Lys-51. Competition data showed that Lys-47 belongs to the toxin-AcChoR interacting domain whereas the other two residues are excluded from it. We first tentatively determined the threshold of covalent coupling, indicative of the proximity between the photoactivatable probes and subunits, by quantifying the coupling occurring between the same derivatives and a model compound (i.e., a toxin-specific monoclonal antibody). We then (i) quantified the coupling yields occurring when both binding sites of AcChoR were occupied by the toxin derivatives, (ii) discriminately quantified the coupling yields at the high-affinity binding site, and (iii) deduced the coupling yields at the low-affinity binding site. In the high-affinity site, the probes on Lys-15 and Lys-47 predominantly reacted with the high-affinity site of the AcChoR alpha subunit whereas the probe on Lys-51 reacted with the delta subunit. In the low-affinity site, the probe on Lys-47 predominantly reacted with the low-affinity site of the alpha chain and the beta chain whereas those on Lys-15 and Lys-51 reacted with the gamma and delta chains, respectively. A three-dimensional model showing a unique organization of AcChoR bound to two toxin molecules is presented.

Direct expression in E. coli of a functionally active protein A--snake toxin fusion protein.

We constructed a recombinant expression plasmid encoding a protein A--neurotoxin fusion protein. The fused toxin is directly expressed in the periplasmic space of Escherichia coli and can be purified in the milligram range by a single immuno-affinity step. The LD50 values of the fused toxin and native toxin are 130 and 20 nmol/kg mouse respectively. The Kd values characterizing their binding to the nicotinic acetylcholine receptor (AcChoR) are respectively 4.8 +/- 0.8 and 0.07 +/- 0.03 nM. In contrast, the fused and native toxins are equally well recognized by a toxin-specific monoclonal antibody which recognizes the AcChoR binding site. The lower toxicity of the fused toxin might result, therefore, from a steric hindrance, due to the presence of the bulky protein A moiety (mol. wt = 31 kd) rather than to a direct alteration of the 'toxic' site. The fused toxin is more immunogenic than native toxin, since 1 nmol of hybrid toxin and 14 nmol of native toxin give rise to comparable titers of antitoxin antibodies which, furthermore, are equally potent at neutralizing neurotoxicity. The work described in this paper shows that the use of fused toxins may be of paramount importance for future development of serotherapy against envenomation by snake bites.

A monoclonal antibody which recognized the functional site of snake neurotoxins and which neutralizes all short-chain variants.

We isolated a neurotoxin-specific monoclonal antibody (Mab) which is capable of recognizing and neutralizing all short-chain toxin variants that have been tested, including those with widely divergent sequences. The epitope incorporates the three invariant residues Lys-27, Trp-29 and Lys-47 which form part of the site by which the toxins bind to the nicotinic acetylcholine receptor. To our knowledge, this is the first Mab which possesses the universal capacity of neutralizing all natural variants of a toxic protein.

On the molecular mechanisms of neutralization of a cobra neurotoxin by specific antibodies.

Examination of 76 homologous neurotoxin sequences suggested that the "toxic" domain of these compounds consists of twelve highly conserved residues. Five of these, namely Lys-27, Trp-29, Asp-31, Arg-33 and Glu-38, together with a variant residue at position 36 are organized into a pattern which resembles that of d-tubocurarine. Two lines of experimental evidence are in agreement with the proposed topology of the "toxic" site in Naja nigricollis toxin alpha--Three highly conserved residues (Lys-27, Trp-29 and Lys-47) have been modified individually in toxin alpha. These modifications induce a decrease in binding affinity of toxin alpha for its target, the nicotinic acetylcholine receptor. In contrast, modifications of three residues (Leu-1, Lys-15 and Lys-51) excluded from the "toxic" domain, do not alter the binding properties of toxin alpha.--Five toxin derivatives carrying a nitroxide group at residues 1, 15, 27, 47 or 51 have been prepared. ESR spectra have been recorded for each derivative in both the free state and bound to the receptor. Mobility of the probes of the residues excluded from the "toxic" site is not altered upon receptor binding. In contrast mobility of the nitroxide of the presumed "toxic" Lys-47 becomes markedly reduced after toxin receptor complex formation. Lys-27 nitroxide is immobilized in both the free and bound state. The antigenic structure of N. nigricollis toxin alpha has been partially clarified using two different approaches. --Fifteen antigenically important residues of toxin alpha have been identified by analyzing cross-reactions between toxin alpha and eleven homologous neurotoxins, using polyclonal antibodies.--- One monoclonal antibody (M alpha 1) specific for toxin alpha has been prepared. Competition experiments, made with (3H) toxin alpha, six mono modified toxin derivatives or alpha three homologous neurotoxins, showed that the binding site of (M alpha 1) comprises the N-terminal group, Lys-15, Pro-18 and probably Thr-16. This site is topographically different from the "toxic" domain. (M alpha 1) inhibits the toxicity of toxin alpha under both in vivo and in vitro conditions. In addition, (M alpha 1) is capable of "removing" toxin molecules bound to the receptor, allowing a rapid recovery of the functional properties of the receptor.

[Binding of a tritiated enkephalinergic analog (FK 33-824) to a mitochondrial fraction of rat brain]. / Liaison d'un analogue enképhalinergique [FK 33-824] tritié à une fraction mitochondriale de cerveau de rat.

FK-33-824 (Try-D-Ala-Gly-MePhe-Met(O)ol) is a potent enkephalin analog which has been tritium labelled with a high specific radioactivity (41 Ci/mmole). The labelled drug exhibits specific and saturable binding to rat brain crude mitochondrial fraction. Specific binding is inhibited by low concentrations of morphine, levallorphan and beta-endorphin, suggesting that FK 33-824 [3H] binds preferentially to mu opiate sites. Binding studies at equilibrium and kinetics of formation and dissociation of the labelled ligand-receptor complex indicate that FK 33-824 [3H] binds to two classes of specific sites. Their affinities are distinguishable at 0 degree (KD = 1.3 and 5.8 nM) and very close to each other at 37 degree (KD = 1.9 nM).

[Visualization of opiate receptors in the locus coeruleus of rats: high resolution radioautography after administration of a tritiated met-enkephalin analog]. / Visualisation des récepteurs aux opiacés dans le locus coeruleus du rat: étude radioautographique à haute résolution après administration d'un analogue tritié de la met-enképhaline.

Opiate receptor sites were visualized by high resolution radioautography in the locus coeruleus of the Rat following intraventricular injection of the met-enkephalin analogue FK 33-824 tritiated with a high specific activity (3H-FK). After administration of a tracer dose of 3H-FK, more than 75% of the radioactivity detected in the locus coeruleus is specifically bound to opiate receptor sites. These are distributed both between and inside neuronal perikarya. After administration of 3H-FK at high concentration, electron microscope radioautography shows the presence of specific opiate binding sites at the level of axo-somatic and axo-dendritic synaptic junctions. These synaptic binding sites could correspond to receptor sites for endogenous enkephalins.