Literature Reference for Α-Conotoxin

Biochem. J. (1997) 328, 245–250 (Printed in Great Britain) 245 Characterization of α-conotoxin interactions with the nicotinic acetylcholine receptor and monoclonal antibodies James D. ASHCOM and Bradley G. STILES1 Toxinology Division, USAMRIID, 1425 Porter St., Fort Detrick, MD 21702-5011, U.S.A. The venoms of predatory marine cone snails, Conus species, nicotinic acetylcholine receptor (nAchR) and monoclonal anti contain numerous peptides and proteins with remarkably diverse bodies developed against the toxin. The binding of FGI to pharmacological properties. One group of peptides are the α nAchR or antibody had apparent dissociation constants of conotoxins, which consist of 13–19 amino acids constrained by 10–100 nM. Structure–function studies with α-conotoxin GI two disulphide bonds. A biologically active fluorescein derivative analogues composed of a single disulphide loop revealed that of Conus geographus α-conotoxin GI (FGI) was used in novel different conformational restraints are necessary for effective solution-phase-binding assays with purified Torpedo californica toxin interactions with nAchR or antibodies. INTRODUCTION of neuronal and muscle nAchR [13,14]. The natural variations During the last 50 million years, predatory marine cone snails among α-conotoxin sequences and use of these molecules for (Conus species) have developed a wide array of biologically studying receptor subtypes may result in pharmaceutically useful active peptides which are found in their venom and possess compounds [3,10,11,15–17]. exquisite specificities for many different receptors [1–3]. Some This study describes the use of non-radioactive solution venoms contain over a 100 different peptides [2], thereby giving phase-binding assays to quickly measure specific receptor–ligand these animals a substantial arsenal for subjugating various prey. and antibody–antigen interactions of small peptides such as the The α-conotoxins represent just one group of structurally related α-conotoxins. These novel methods enabled us to investigate the neurotoxic peptides [1,2]. These toxins, the smallest known structure–function properties of these toxins by using single-loop polypeptide inhibitors of nicotinic acetylcholine receptor α-conotoxin GI analogues. Finally, we characterized the binding (nAchR), compete with acetylcholine and block signal trans specificity and epitopes recognized by two unique monoclonal mission at the neural synapse [4]. The nAchR isolated from antibodies developed against α-conotoxin GI. Torpedo californica electric organ and skeletal muscle consists of five protein subunits (α# βγδ) which form a cylindrical ion channel EXPERIMENTAL within the postsynaptic membrane [5]. Acetylcholine and other Toxins, peptides and nAchR ligands interact with the extracellular domain of both α-subunits, but adjacent subunits contribute significantly to some unique Synthetic α-conotoxins GI (ECCNPACGRHYSC-NH# ), MI binding properties [6,7]. For example, the α}δ and α}γ regions of (GRCCHPACGKNYSC-NH# ), SI (ICCNPACGPKYSC-NH# ) nAchR have strikingly different binding affinities for the α and µ-conotoxins GIIIB (RDCCTP*P*RKCKDRRCKP* conotoxins [8–10]. MKCCA-NH# ;P*¯ hydroxyproline) and GIIIA (RDCCTP*P* The α-conotoxins have potent biological activities that cor KKCKDRQCKP*QRCCA-NH# ) were purchased from Sigma relate well with their binding affinities for T. californica nAchR Chemical Co. (St. Louis, MO, U.S.A.). Homologues of α [8,9,11]. α-Conotoxins GI and MI, isolated from the respective conotoxin GI containing the natural C-terminal amide group venoms of Conus geographus and Conus magus, cause a rapidly and various cysteine to serine substitutions, GIp1 (ESSNPAS lethal muscle paralysis in mice at submicrogram quantities. In GRHYSS-NH# ), GIp2 (ECSNPACGRHYSS-NH# ) and GIp3 contrast, no signs of intoxication are observed in mice given (ESCNPASGRHYSC-NH# ), were obtained from Quality Con relatively large doses of Conus striatus α-conotoxin SI [11], which trolled Biochemicals (Hopkinton, MA, U.S.A.). These peptides also binds very weakly to the T. californica nAchR [8]. Selective were characterized by the manufacturer using HPLC plus MS. binding of α-neurotoxins to the nAchR from different animal The primary sequence of each α-conotoxin GI homologue was species is also evident for α-bungarotoxin, a snake postsynaptic kindly verified by Dr. James Schmidt (USAMRIID) with an neurotoxin purified from Bungarus multicinctus venom [12]. Applied Biosystems 470A sequencer (Foster City, CA, U.S.A.). The α-conotoxins are structurally simpler than α-bungarotoxin Affinity-purified nAchR was prepared from frozen T. californica and other snake venom α-neurotoxins, which usually consist of electric organ (Pacific Biomarine, Venice, CA, U.S.A.) as pre 60–70 residues plus four or five disulphide bridges. Subsequently, viously described [18,19]. the α-conotoxins are likely to have fewer contact sites necessary for efficient binding to nAchR, thus making these molecules an Fluoresceinated α-conotoxin GI (FGI) invaluable tool for analysing ligand–nAchR interactions. Recent crystallographic studies with α-conotoxins provide further in A 1 ml solution containing 0±35 mg of α-conotoxin GI in 0±1M formation on toxin structure and may lead to a better under phosphate buffer, pH 8±0, was mixed for 30 min with 100 µlof standing of how these toxins differentially interact with subtypes DMSO containing 0±5 mg of FITC (Molecular Probes, Eugene, Abbreviations used: FGI, fluoresceinated α-conotoxin GI; mAb, monoclonal antibody; nAchR, nicotinic acetylcholine receptor. 1 To whom correspondence should be addressed. 246 J. D. Ashcom and B. G. Stiles OR, U.S.A.). FGI was purified by reverse-phase HPLC with a unlabelled α-conotoxins MI or GI in each experiment verified PepRPC HR5}5 column (Pharmacia, Piscataway, NJ, U.S.A.) that quenching caused by the addition of nAchR was specifically and represented 82% of the initial peptide used for conjugation. reversible. After a dilution correction, the binding reaction was The N-terminal glutamic acid was modified [20], as evidenced by analysed with a modified form of eqn. (1) where the observed unsuccessful Edman degradation of FGI (500 pmol). After fluorescence change (Fobs ® Fmin ) was normalized relative to the lyophilization, FGI was dissolved in sterile PBS and stored in a initial unquenched fluorescence value (Fobs ) and the terms for LT foil-covered tube at 4 �C. There was no detectable decrease in and PT were interchanged because FGI was titrated with receptor. fluorescence or receptor-binding activity of FGI over several The specificity of FGI binding to receptor and relative dis months when stored at 4 �C. An absorption coefficient of placement efficiencies of several competitive ligands were also " " 66800 cm− [M− at λ ¯ 495 nm [21] was used to determine the measured with a reverse fluorescence quenching assay. FGI was FGI concentration. The fluorescence spectra, obtained from a incubated with excess nAchR, so that the initial ligand was Perkin–Elmer model 650-40 spectrophotometer (Norwalk, CT, predominantly bound to receptor. Preformed complexes were U.S.A.), showed characteristic excitation and emission maxima titrated with multiple aliquots of unlabelled ligand, and the at λ ¯ 492 nm and λ ¯ 517 nm respectively. With 2 nm slits, the fluorescence measured at 5 min intervals. The data were corrected detection sensitivity in PBS containing 0±1% Triton X-100 for dilution and analysed using the curve-fitting routines of (PBST) was 20 fluorescence units}nmol of FGI. SigmaPlot and eqn. (2) [26]: (Fobs ® Fmin )}(F max ®Fmin ) ¯ L}(LC" /#) (2) Spin-column assays L represents the total molar concentration of unlabelled ligand, FGI binding to T. californica nAchR or monoclonal antibody Fobs is the observed fluorescence, Fmin is the initial quenched (mAb) was determined by spin-column experiments at room fluorescence value, Fmax is the maximum fluorescence corre temperature. Affinity-purified nAchR or mAb, with protein sponding to free ligand at final titration, and C" /# is the best-fit concentrations measured by a microBCA assay (Pierce Chemical midpoint in the titration curve. Co., Rockford, IL, U.S.A.) using BSA standards, were equi librated with PBST on a PD-10 column (Pharmacia). Direct binding studies employed various concentrations of FGI incu Characterization of single disulphide-loop analogues of α bated for 60 min with the nAchR or mAb. For competitive conotoxin GI ligand-displacement assays, FGI was premixed with various Freshly prepared solutions of GIp2 and GIp3 contained 2 mol of dilutions of the unlabelled ligands and then incubated with cysteine}mol of tyrosine, as shown by reaction with excess 5,5« nAchR or mAb. An aliquot (200 µl) of the incubation mixture dithiobis-(2-nitrobenzoic acid). The peptides were diluted to was applied to a 1 ml bed of Bio-Gel P10 (Bio-Rad Laboratories, 50 µM with PBS and oxidized slowly at room temperature for Richmond, CA, U.S.A.), and the protein-bound fraction of FGI 48 h with continuous mixing. Analysis with 5,5«-dithiobis-(2 was rapidly separated using centrifugal flow methods [22,23]. nitrobenzoic acid) revealed that the free cysteine concentration The void-volume samples from receptor-binding assays were gradually decreased to the limit of thiol detection. After oxi diluted with PBST containing 10 mM carbamoylcholine to dation, the peptides were concentrated tenfold by lyophilization dissociate the ligand–receptor complex. Fluorescence was meas and a 120 l aliquot of each was added to a calibrated Bio-Gel # µ ured in

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