André Menez

LOCALIZATION OF THE CHOLINERGIC RECEPTOR PROTEIN IN ELECTROPHORUS ELECTROPLAX 'BY HIGH RESOLUTION AUTORADIOGRAPHY

We first confirm that the [“HI o-toxin binds exclusively to the innervated (caudal) side of the eel electroplax and further show that the o-toxin binds both between the synapses and under the nerve terminals. However, taking into account the particular stereology of the cytoplasmic membrane, it appears that the density of a-toxin molecules bound per unit area of actual membrane surface is approx. 100 times larger under the synapses than outside the synapses. Absolute values of this density and of the total number of receptor sites per cell are given.

Snake Neurotoxins that Interact with Nicotinic Acetylcholine Receptors

Snakes produce a diversity of toxins, the α-neurotoxins or curaremimetic toxins, which act on nicotinic acetylcholine receptors (AChRs). As shown in Table 1, these toxins can be divided into four categories. First, there are the a-neurotoxins, which bind with high affinity to muscular AChRs only. These include a large family of short-chain, three-fingered toxins from Elapidae (elapids and hydrophiids), and the waglerins from Viperidae (Trimeresurus wagleri) (1–3). Second, there are the α/k neurotoxins, which bind with high affinities to both muscular and some neuronal receptors (α7, α8, and α9) (4–6). These toxins correspond to the family of long-chain, three-finger toxins found in venoms from Elapidae, which, until recently (7), were systematically associated with the family of short-chain three-fingered toxins (1). Third, there are the K-neurotoxins, which bind with high affinity to neuronal receptors only. So far, only four toxins of this category have been described and all of them are long-chain, three-fingered toxins from elapid snakes (8,9). Fourth, there are nonconventional neurotoxins with an additional disulfide bond in the first loop. These toxins, also called weak neurotoxins, interact with low affinities (their Kds are in the µM range) on muscular-type AChRs (9a, 9b, 9c, 9d).

Relative Spatial Position of a Snake Neurotoxin and the Reduced Disulfide Bond α(Cys192-Cys193) at the αγ Interface of the Nicotinic Acetylcholine Receptor

We determined the distances separating five functionally important residues (Gln10, Lys27, Trp29, Arg33, and Lys47) of a three-fingered snake neurotoxin from the reduced disulfide bond α(Cys192-Cys193) located at the αγ interface of the Torpedo nicotinic acetylcholine receptor. Each toxin position was substituted individually for a cysteine, which was then linked to a maleimido moiety through three different spacers, varying in length from 10 to 22 Å. We estimated the coupling efficiency between the 15 toxin derivatives and the reduced cystine α(192–193) by gel densitometry of Coomassie Blue-stained gels. A nearly quantitative coupling was observed between αCys192 and/or αCys193 and all probes introduced at the tip of the first (position 10) and second (position 33) loops of Naja nigricollis α-neurotoxin. These data sufficed to locate the reactive thiolate in a “croissant-shaped” volume comprised between the first two loops of the toxin. The volume was further restrained by taking into account the absence or partial coupling of the other derivatives. Altogether, the data suggest that αCys192and/or αCys193, at the αγ interface of a muscular-type acetylcholine receptor, is (are) located in a volume located between 11.5 and 15.5 Å from the α-carbons at positions 10 and 33 of the toxin, under the tip of the toxin first loop and close to the second one.

CHAPTER 60 – Engineering of bacterial toxins for research and medicine

It is tempting for the biochemist, the protein engineer, the biotechnologist, or the medical scientist to exploit the sophisticated properties of bacterial toxins to design new toxin-derived molecules for research, biotechnology, or medical treatments. Some toxins or toxin subunits are used in their natural form as biochemical and cell biology tools or for the treatment of specific diseases. Besides the use of native toxins as tools, therapeutics, or vaccines, toxins or toxin fragments can be engineered or combined with other protein domains to build rationally new proteins with new defined activities. This chapter focuses on the vast possibilities offered by toxin engineering for the design of new tools and therapeutics. The FDA has approved one engineered toxin targeted to an endogenous human receptor for the treatment of a lymphoma. New indications are under study and the number of patients who will benefit from this molecule should increase in the future. Many fusion toxins built on the same model and targeted to a variety of receptors are developed against a series of malignancies. A few are now being evaluated in patients. Toxins used as delivery vehicles for vaccines are starting to reach clinical trials. Many other engineered toxins are still in the test tubes. Some will remain as laboratory curiosities, while others may be future drugs. Some may become important biological tools for diagnostics, detectors, nano-technologies, or bioelectronics.

Sarafotoxins:Cloning of mRNAs Encoding Sarafotoxin Precursors

Whe applied to animal toxins, molecular biology technologies offer several interests, including the possibility to elucidate the structure of toxin precursors(Ducancel et al., 1991). In General, purified messenger RNAs (mRNAs) are converted into stable complementary copies of DNA (cDNAs) which are then cloned info recombinant vectors and used to transform bacteria. Two screening strategies can be considered to identify clones containing the precursor nucleic information. One of them consist is performing in situ hybridizations (Sambrook et al., 1989), using labeled nucleic acid probes that are complementary to a portion of the precursor sequence. The other strategy consist in using cloning/expression vectors such as λgt11 or pBluescript, and in screening with precursor-specific antibodies (Mierendorf et al., 1987). The nucleic informations contained in the screened clones are then elucidated by DNA sequencing (Sanger et al., 1977)

“Three-Fingered” Toxins from Hydrophid and Elapid Snakes: Artificial Procedures to Overproduce Wild-Type and Mutated Curaremimetic Toxins

Toxic proteins from animal venoms usually act on molecular targets that are critically involved in the function of a physiological system of a prey. With the ultimate view of designing new pharmacological tools as well as original drugs acting on these targets, animal toxins are the subject of extensive molecular analyses. In particular, the topographies by which toxins bind to their targets and hence affect the function of the associated physiological system clearly need to be identified.