Eur. J. Biochem. 204,281 - 292 (1992) 0 FEBS 1992

Amino acid sequence and immunological characterization with monoclonal antibodies of two toxins from the venom of the scorpion Centruroides noxius Hoffmann Fernando ZAMUDIO ', Rafael SAAVEDRA', Brian Michael MARTIN3, Georgina GURROLA-BRIONES ', Pascal HERION and Lourival Domingos POSSANI'

Departamento de Bioquimica, Tnstituto de Biotecnologia, Universidad Nacional Autbnoma de Mkxico, Cuernavaca, Mkxico Departamento de Inmunologia, Instituto de Investigaciones Biomedicas, Universidad Nacional Autbnoma de Mixico, Ciudad Universitaria, Mexico City, Mtxico National Institute of Mental Health, Section on Molecular Neurogenetics, Clinical Neuroscience Branch, National Institutes of Health, Bethesda, USA

(Received August 7, 1991) - EJR 91 1071

Two toxins, which we propose to call toxins 2 and 3, were purified to homogeneity from the venom of the scorpion Centruroicles noxius Hoffmann. The full primary structures of both peptides (66 amino acid residues each) was determined. Sequence comparison indicates that the two new toxins display 79% identity and present a high similarity to previously characterized Centruroides toxins, the most similar toxins being Centruroides suffusus toxin 2 and Centruroides limpidus tecomanus toxin 1. Six monoclonal antibodies (mAb) directed against purified fraction I1 - 9.2 (which contains toxins 2 and 3 ) were isolated in order to carry out the immunochemical characterization of these toxins. mAb BCF2, BCF3, BCF7 and BCF9 reacted only with toxin 2, whereas BCFI and BCF8 reacted with both toxins 2 and 3 with the same affinity. Simultaneous binding of mAb pairs to the toxin and cross-reactivity of the venoms of different scorpions with the mAb were examined. The results of these experiments showed that the mAb define four different epitopes (A- D). Epitope A (BCF8) is topographically unrelated to epitopes B (BCFZ and BCF7), C (BCF3) and D (BCF9) but the latter three appear to be more closely related or in close proximity to each other. Epitope A was found in all Centruroides venoms tested as well as on four different purified toxins of C. noxius, and thus seems to correspond to a highly conserved structure. Based on the cross-reactivity of their venoms with the mAb, Centruroides species could be classified in the following order: Centruroides elegans, Centruroides su~uusus suflisus = Centruroides infamatus infamatus, Centruroides limpidus tecornanus, Centruroides limpidus limpidus, and Centruroides limpidus acatlanensis, according to increasing immunochemical relatedness of their toxins to those of Centruroides noxius. All six mAb inhibited the binding of toxin 2 to rat brain synaptosomal membranes, but only mAb BCF2, which belongs to the IgGza subclass, displayed a clear neutralizing activity in vivo.

Scorpion venoms constitute a rich source of low-molecular mass peptides toxic to a variety of organisms, including man (Miranda et al., 1970; reviews by Zlotkin et al., 1978, and Possani, 1984). Most scorpion venoms, so far studied, have been shown to contain two kinds of toxins: long-chain poly- peptides of 60-70 amino acid residues, which block Na' channels of excitable cells (Catterall, 1977; Couraud et al., 1982); short-chain peptides of 37 - 39 amino acid residues affecting K' channels (Carbone et al., 1982; Possani et al., 1982; Miller et al., 1985; Gimenez-Gallego et al., 1988; Strong et al., 1989). Toxins modifying Na' channels were classified

Correspondence to L. D. Possani, Instituto de Biotechnologia - UNAM, Apartado Postal 510-3, Cuernavaca, Mtxico 62271

Abbreviations. BCF, hybridomas producing monoclonal anti- bodies against scorpion toxins; Cm, carboxymethyl; Cn, Centrurniks noxius; ID50, median inhibitory-dose; LD50, median lethal dose; mAb, monoclonal antibody.

Enzymes. Proteasc V8 from Staphylococcus aureus (EC 3.4.21.19).

as c1 and /3 toxins (Couraud et al., 1982; Wheeler et al., 1983). The former type was originally described using peptides purified from the venom of Old World scorpions, such as Androctonus australis and Leiurus quinquestriatus, while the latter type was initially purified from New World scorpions, the model peptide being toxin I1 from C. suffsus sliffusus (Jover et al., 1980; Couraud et al., 1982). The a toxins modify mainly the inactivation mechanism of the channels (Nonner, 1979) while the f l toxins modify preferentially the activation mechanism of Na+ channels (reviewed by Meves et at., 1986, and Strichartz et al., 1987). The existence of two different binding sites for scorpion toxins at Na' channels (Jover et al., 1980; Thomsen and Catterall, 1989) and the a and f l classification regarding the gating mechanism of the Na' channels is widely accepted (Strichartz et al,, 1987). Recently, toxins that modify both the activation and inactivation mech- anisms of Na' channels have been shown to coexist in venom from the same scorpion Tityus srrrulatus (Yatani et al., 1988;

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Kirsch et al., 1989). Comparison of the three-dimensional structure of a and fl toxins showed that the two molecules have a very similar dense core formed by an a-helical and an anti-parallel P-sheet moiety, but they differ markedly in the orientation of loops protruding from the core (Fontecilla- Camps et al., 1988).

The a toxin, Androctonus australis Hector toxin 11, has been extensively characterized by means of synthetic peptides containing predicted antigenic sites of the toxin (for a revLew see Granier et al., 1989) and by means of mAb (Bahraoui et al., 1988). Antibodies against a toxins of North African scorpions have also been used to classify them into immunologically cross-reactive groups which are in good agreement with the proposed structural groups (Delori et al., 1981).

In previous communications (Possani et al., 1981a, b; Sitges et al., 1987), we have described the purification and partial characterization of toxin 2 [component C. noxius (Cn)II-9.2.21 from C. noxius venom. Toxin 2, a p toxin, is the major toxic component in the venom of C. noxius, the most dangerous scorpion in Mexico, with a median lethal dose (LDS0) equal to 5 l g venom/20 g mouse (Dent et al., 1980). In the present communication, we report a new purification procedure that led to the isolation of two toxins from C. noxius venom: the previously reported toxin 2 and a new related toxin, toxin 3, in homogeneous form, allowing the determination of their full amino acid sequence. In order to carry out the immunochemical characterization of these toxins, mAb were produced against the toxin Cn-11-9.2. The relationships between the epitopes defined by these mAb were studied, and cross-reactivity of several purified toxins and venoms of different scorpions with the mAb was examined. In vitro and in vivo neutralizing activities of the mAb are also reported.

MATERIALS AND METHODS

Source of venoms and chemicals

The venom from various species of Centruroides scorpion used in this work was obtained by electrical stimulation of living animals, in the laboratory, as previously reported (Dent ct al., 1980). T. srrrulatus venom was a gift from Instituto Butantan (S5o Paulo, Brazil). All solvents and chemicals used were analytical grade and obtained from the companies re- ported in Martin et al. (1988). Double-distilled water (over quartz) was used throughout. Porcine thyroglobulin, l-ethyl- 3-(3-dimethylaminopropyl)-carbodiimide, Tris, Coom.assie brilliant blue G250, o-phenylenediamine, Tween 20 and bovine serum albumin were obtained from Sigma (St. Louis, MO, USA). Freund’s complete adjuvant was from Difco (Detroit, MI, USA), acrylamide and N,W-methylene-bis- acrylamide were from Bio-Rad Laboratories (Richmond, CA, USA), urea hydroperoxide was from BDH (Poole, England). Ovalbumin was purified according to Alexander et al. (1966).

Toxin purification procedure

CnII-9.2 from C. noxius venom was obtained following in part the strategy earlier described (Possani et al., 1981 a, b). Briefly, soluble venom was separated by molecular-mass sieving through Sephadex (3-50, followed by carboxymethyl- cellulose (Cm-cellulose) column chromatography in 20 mM ammonium acetate buffer, pH 4.7. CnII-9 obtained in this way was further separated in the same column run at

50 mM sodium phosphate buffer, pH 6.0 yielding CnII-9.2. Highly purified toxins 2 and 3 (CnII-9.2.2 and CnII-9.2.3) were finally obtained in homogeneous form by column chromatography on Cm-cellulose, equilibrated and run in 50 mM sodium phosphate buffer, pH 8.0. (See details in the legends of the corresponding figures.) CnII-10 and CnII-14 and toxin 1 from C. limpidus tecomanus were obtained as previously described (Possani et al., 1981a; Ramirez et al., 1988).

Sequence determination

N-terminal amino acid sequence of pure toxin was performed by automatic Edman degradation (Edman and Begg, 1967) in a model 890M microsequencer (Beckman, Palo Alto, CA, USA), using the chemicals and procedures pre- viously described (Martin et al., 1988). The same procedures were used for reduction, alkylation, enzymatic digestion of toxins and subsequent separation of the resulting peptides, by HPLC.

Preparation of CnII-9.2 - thyroglobulin conjugate

CnI1-9.2 (20 mg) and 10 mg porcine thyroglobulin were dissolved in 0.5 ml water. To this mixture, 150 mg l-ethyl-3- (3-dimethyl-aminopropyl)carbodiimide dissolved in 0.25 ml water were added with constant stirring. The reaction was allowed to proceed for 2 h at room temperature with constant stirring and the product was then dialysed against water.

Mouse immunization

Female Balb/c mice (bred at the Instituto de Investigaciones Biomkdicas) were immunized by subcutaneous injection of 200 pg conjugate emulsified in Freund’s complete adjuvant on days 1,15 and 36. The mice were boosted by intraperitoneal injection of 1 pg conjugate in saline on day 96 and 50 - 200 pg conjugate in saline on day 99, and by intravenous injection of 50 - 200 pg conjugate in saline on day 100. Mice were sacrified on day 103 and their spleens used for cell fusion.

mAb production

Cells and media were prepared as previously described (Franssen et al., 1981). The mouse spleen cells were fused with a selected subclone of the SpyO-Agl4 myeloma line, as described by Franssen et al. (1981). After fusion, cells were plated in hypoxanthine/aminopterin/thymidine selection me- dium in 96-well microplates (at a density corresponding to 1.5 x lo5 spleen cells/well) on a feeder layer consisting of 5 x lo3 mouse peritoneal macrophages. Hybridomas were cloned in soft agar and immunoglobulin-secreting clones were detected in situ as previously described (Herion et al., 1981).

PAGE mAb samples were electrophoresed in a continuous 7.5%

acrylamide, 0.2% N,N-methylene-bisacry1amide, 0.37 M Tris/HCl, pH 8.9, slab gel. Electrode buffer consisted of 5 mM Tris and 40 mM glycine, pH 8.3. The gel was stained with Coomassie brilliant blue G250 in perchloric acid, as described by Reisner et al. (1975). For purity control of toxins, the p- alanine/acetate/urea gel system of Reisfeld et al. (1962) was used.

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ELISA

Binding experiments

96-well vinyl ELISA plates (COSTAR, Cambridge, MA, USA) were coated by incubation (overnight, 4°C) with CnII- 9.2 (100 pl of a 3-mg/l solution in 20 mM NaHC03 pH 9.2). After saturation of the remaining sites with ovalbumin [I O h in 0.15 M NaC1/0.02 M Pi pH 7.8 (NaC1/Pi), 2 h, at room temperature], antibodies in dilution buffer [NaCI/Pi, 0.05% Tween 20, 10% (by vol.) horse serum, 1 % bovine serum albumin] were incubated in the wells overnight at 4°C. After extensive washing with NaC1/Pi and 0.05% Tween 20, bound antibodies were revealed using peroxidase-labelled rabbit anti- (mouse IgG) antibodies (4 h, at room temperature) and the chromogenic substrate (o-phenylenediamine 0.4 mg/ml and urea hydroperoxide 0.2 mg/ml in 0.1 M NaH2P04, pH 5).

injected intraperitoneally into five mice (strain CDI). Mean survival times were recorded.

Competition

C. noxius-toxin-2-coated plates were prepared as described above. The wells were then filled with 50 pl of a dilution of the inhibitor peptide or venom and 50 p1 of a suitable dilution of mAb. After overnight incubation at 4"C, bound antibodies were revealed as described above.

Sandwich ELISA

Purified mAb were labelled with horseradish peroxidase according to Nakane (1 979), with the modifications described by Htrion et al. (1983). Vinyl ELISA plates were coated by overnight incubation at 4°C with purified mAb (4 mg/l in NaCI/P,). After saturation of the remaining sites with ovalbumin (1 YO in NaC1/Pi), Cn11-9.2 (6 mg/l in dilution buffer) was added and incubated overnight at 4°C . Excess CnII-9.2 was then washed out and '251-labelled mAb was added (8 mg/l in dilution buffer). After incubation (4 h, at room temperature), bound antibodies were revealed using the chromogenic substrate.

Toxin binding to rat brain membranes

Rat brain membranes were obtained according to Catterall et al. (1979). C. noxius toxin 2 was labelled with lZ5I by the lactoperoxidase method (Morrison and Bayse, 3 970) and the '251-labelled toxin (33.2 Ci/mmol) was purified on a 0.7 cm x 20 cm Sephadex G-10 column. The reaction buffer for the binding assay consisted of choline chloride (140 mM), KCI (5 mM), CaC12 (2.5 mM), MgS04 (1.8 mM), Tris/HCl (20 mM) and bovine serum albumin (0.1%), pH 7.4. '"I- labelled C . noxius toxin 2 (1.25 pmol) was incubated with serial dilutions of purified mAb for 1 h at room temperature in a total volume of 480 11.20 p1 rat brain membranes (500 pg protein) was then added and the incubation was allowed to proceed for 1 h more at room temperature. Free toxin was then separated by filtration on glass-fiber filters (GF/B, Whatman, Clifton, NJ, USA), previously soaked in buffer containing 1 % bovine serum albumin. The filters were quickly washed three times with 5 ml of the same buffer, dried, and the radioactivity was measured in a Beckman y counter.

Toxin neutralization by mAb

For each 20 g mouse, 7.5 times the LDS0 (3 pg) of C. noxius toxin 2 were incubated with 650 pg mAb in a final volume of 250 - 650 pI for 1 h at 37 "C. This mixture was then

RESULTS

Purification and amino acid sequence of toxins

CnI1-9.2 was isolated from C . noxius venom as described in Materials and Methods. Fig. l a shows the profile of the chromatographic separation of this component. The overall recovery was 90% and fraction Cnll-9.2 corresponded to 80% of all the material absorbing at 280 nm. CnII-9.2 was further purified by a third ion-exchange chromatographic step (see Materials and Methods), yielding two well-resolved major peaks (Fig. 1 b). Component CnII-9.2.2 (toxin 2) correspond- ed to 74% and component CnII-9.2.3 (toxin 3) to 24% of the material recovered in this purification. Both toxins ran as a single component in the PAGE system of Reisfeld et al. (1 962) (data not shown). Lethality-dose tests showed that the LD5,, value (in Balb/c mice) was in the order of 16 kg/kg mouse for toxin 2 and 32 pg/kg mouse for toxin 3.

Toxins 2 and 3 were reduced, alkylated, purified by gel filtration on Bio-Gel P-30 and subjected to Edman degradati- on for N-terminal amino acid determination. Toxin 2 loaded into the automatic sequencer gave the identity of the first 60 amino acid residues (Fig. 2), confirming the 48 first amino acid residues already reported for the N-terminal part of the molecule (Possani et al., 1981a). Toxin 3, in the same con- ditions, gave unequivocal determinations for the first 37 resi- dues. In order to obtain the full sequences, both reduced and alkylated toxins were cleaved with protease V8 of Staphylococ- cus aureus; the resulting peptides were separated by HPLC (Fig. S 1) and loaded into the sequencer. The peptide eluting at 35.50 min for toxin 2 had the following amino acid sequence: QAIVWPLPNKRCS. This sequence overlaps with residues 54-60 of the N-terminal sequence, and thus provides the full primary structure (Fig. 2) of this 66-residue toxin, also in agreement with the amino acid composition previously deter- mined for this peptide (Possani et al., 1981b). Similarly for toxin 3, purified fragments from the enzymatic cleavage al- lowed the complete alignment of the full amino acid sequence, as shown in Fig. 2. Amino acid analysis of purified toxin 3 (data not shown) gave results in agreement with the sequencing data.

Production of anti-(CnII-9.2)mAb

Spleen cells of three Balb/c mice immunized with a conju- gate of CnII-9.2 with porcine thyroglobulin were fused with cells of the myeloma line Sp2/0-Ag14. Hybridomas producing antibodies against CnII-9.2 were detected by ELlSA (see Ma- terials and Methods) and cloned once in soft agar. Six indepen- dent hybridoma clones named BCF1, BCF2, BCF3, BCF7, BCFX and BCF9 were obtained and grown in the peritoneal cavity of pristane-primed Balb/c mice. mAb purified from the ascitic fluids by DEAE-cellulose ion-exchange chromatog- raphy were analysed by non-denaturating gel electrophoresis. A single monoclonal Ig band was observed in each prep- aration. Moreover, except for BCFl and BCF3, the mAb had distinct electrophoretic mobilities (data not shown).

Immunoglobulin class determination

Analysis of hybridoma spent culture medium by Ouchterlony double-diffusion test (Ouchterlony, 1948) with

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It-9.2.2

L G Elution volume (ml Elution volume (ml 1

Fig. 1. Purification of C. noxius toxins 2 (11-9.2.2) and (11-9.2.3) 3. (a) Lethal fraction CnII-9 (11-9; 58 mg) was applied and run in a Cm- cellulose column (0.9 cm x 35 cm) in 50 mM potassium phosphate buffer, pH 6.0, at a flow rate of 30 ml/h. A linear gradient of salt from zero (250 ml) to 0.4 M NaCl(250 ml) in the equilibration buffer was applied to the column. Fractions of 2.5 rnl were collected. (0) Protein eluted. (b) Tubes from fraction CnI1-9.2 were pooled (indicated by the horizontal bar) and dialysed against 50 mM potassium phosphate buffer, pH 8.0. A total of 42 mg of toxin CnII-9.2 was applied to a Cm-cellulose column (0.9 cm x 35 cm) equilibrated in 50 mM potassium phosphate buffer, pH 8.0, and eluted with a salt gradient from zero (250 ml) to 0.3 M NaCl (250 ml) at a flow rate of 30 ml/h. (0) Protein eluted. L, loading of the column; G, start of gradient. Conductivity measurements were converted to NaCl molarities and are represented by (m).

Cn2 Cn3 Cn2 Cn3

Cn2 Cn3 Cn2 Cn3 Cn3

Cn2 Cn3 Cn2 Cn2 Cn3

Cn2 Cn3 Cn2 Gn3

1 5 10 15 20 Lys-Glu-Gly-Tyr-Leu-Val-Asp-Lys-Asn-T~-Gly-Cys-Lys-Tyr-Glu-Cys-Leu-Lys-Leu-Gly- Lys-Glu-Gly-Tyr-Leu-Val-Glu-Leu-Gly-Thr-Gly-Cys-Lys-Tyr-Glu-Cys-Phe-Lys-Leu-Gly- D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D.......................................D.. D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...........................................

21 25 30 35 40 A s p - A s n - A s p - T y r - C y s - L e u - A r g - G l u - C y s - L J r s s - G l y - A l a - G l y - G l y - T y r - Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-LJrs-Ala-Arg-Tyr-Gly-Lys-G1y-Ala-Gly-Gly-Tyr- D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D.......................................D.. D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................... D

va-----------------------------------------~a--

Fig. 2. Complete amino acid sequence of C. noxius toxins 2 and 3. D followed by dots under the amino acid sequences means that the sequence was obtained by direct Edman degradation; V8 followed by dashes, also under the amino acid sequences, means that the amino acid sequence of peptides was obtained after cleavage with protease V8 and scparation by HPLC according to Fig. S1. Invariant residucs are in bold.

class-specific and subclass-specific antisera (Meloy Labora- tories, Spring Field, VA, USA) showed that most mAb (BCF1, BCF3, BCF7, BCF8 and BCF9) belong to the IgG, subclass. BCF2 belongs to the IgGz, subclass.

Characterization of mAb by E L S A and RIA

The six purified mAb were able to bind in a dose-depen- dent manner to ELISA plates coated with CnIl-9.2, whereas

285

no binding was observed with normal mouse IgG (Fig. S2). The antibody concentrations required to obtain 50% of the maximal binding varied over 0.03 -0.1 mg/l. The specificity of the mAb for toxin 2 was studied in competition experiments (Figs S3 and S4). Fig. S3a shows that toxin 2 inhibited the binding of the six mAb to the solid phase coated with CnII-9.2. The toxin 2 concentration required to produce 50% inhibition (IDSO) was very similar for all mAb (0.15-0.24 mg/l) except for BCFl (2.4mg/l). Toxin 3 was also able to inhibit the binding of BCFl and BCF8, but not of the other mAb against CnII-9.2 (Fig. S3b). Furthermore, for BCFl and BCF8, the IDSo of toxins 2 and 3 were very similar. These results suggested that BCFl and BCF8 recognize an epitope shared by toxins 2 and 3. This was further confirmed by the results of the experiment shown in Fig. S4: binding of BCFl and BCF8 to solid phase coated with toxin 2 was equally and completely inhibited by toxins 2 and 3. In addition, the six mAb were able to bind '251-labelled toxin 2 in a classical double-precipitation RIA, and this binding was inhibited by cold toxin 2 (data not shown).

Cross-reaction of other scorpion toxins with BCFS

In order to explore further possible immunochemical re- lationships between C. noxius toxin 2 and other Centruroides toxins, we carried out competition experiments with BCF8. Fig. S 5 shows that this mAb bound to purified toxins CnII- 10, CnII-14 (now proposed to be called C. noxius toxin 1, see Discussion), C. limpidus tecomanus toxin 1 and C. noxius toxin 2 with very similar affinity. These results indicate that the epitope defined by BCF8 is conserved on different Centru- roides toxins.

Simultaneous binding of mAb pairs to CnII-9.2

To examine whether the six mAb bound to distinct or overlapping epitopes of CnII-9.2, we used a sandwich ELISA in which one member of each antibody pair was immobilized on the ELISA plate and the other was labelled with peroxidase and used to detect the complex of CnII-9.2 with the immobilized antibody. All possible mAb pairs were investi- gated and the results (Fig. 3) showed that the mAb could be classified into two groups: group 1 included BCFl and BCF8, and group 2 included BCF2, BCF3, BCF7 and BCF9. A pair of mAb belonging to the same group could not bind simultaneously to the toxin, indicating that the epitopes they define are identical, overlapping or in close proximity. On the other hand, a mAb belonging to one group could bind to the toxin simultaneously with a mAb of the other group, indicating that they recognize non-overlapping epitopes.

Presence of cross-reacting components in the venom of other scorpion species

An ELISA competition experiment was carried out in which the binding of BCF2, BCF3, BCF7, BCF8 and BCF9 to CnlI-9.2 was assayed in the presence of venoms of various scorpions species (Fig. S6). With BCF2 and BCF7, we ob- served the presence of cross-reacting components in the venoms of C . limpidus limpidus, C . limpidus tecomanus and C . limpidus acatlanensis and to a lesser extent in the venoms of C. sujfusus suffusus and C. infamatus infamatus. No cross- reacting component was detected in the venoms of C. elegans and T. serrulatus (Fig. S6a and c). With BCF3, cross-reacting

Ag or A b bound to the ELISA plate nmIqG BCFl BCFZ BCF3 BCF7 BCF8 BCFS C.n.II9.2

Fig. 3. Simultaneous binding of mAb (MAb) pairs to CnI1-9.2 assessed by sandwich ELISA. ELISA plates were coated with normal mouse IgG, BCF1, BCF2, BCF3, BCF7, BCF8, BCF9 or CnIT-9.2. After saturation with ovalbumin, CnII-9.2 was incubated in the wells coated with antibody. After washing, bound CnII-9.2 was revealed using peroxidase-labelled BCF1, BCF2, BCF3, BCF7 or BCF8. A492 in wells coated with normal mouse IgG was used as background values. For a given pair of antibodies, when A492 < background, the result is represented by a blank square (0); when the ratio A492/ background > 0.2, the result is represented by a dashed square (B). Results are means of duplicates. Ag, antigen.

2 and the difference A a g 2 - background

r 100 -

h

$ v

71) c 3 0

cu .C 50- x 0

c 0 I

n

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11 10 9 7

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Fig.4. Inhibition of binding of 1251-labelled C. noxius toxin 2 to rat brain synaptosomal membranes by anti-(C. noxius toxin 2) mAb. Serial dilutions of purified BCFl (O), BCF2 (O), BCF3 (n), BCF7 (m), BCF8 (A) , BCF9 (A) or normal mouse IgG (V) were incubated with 'Z51-labelled C. noxius toxin 2 for 1 h at room temperature; then rat brain membranes were added and the incubation was allowed to proceed for 1 h more at room temperature. Results are expressed as percentage binding of C. noxius toxin 2 to brain membranes in thc presence of mAb and are mean5 or duplicates. Non-specific binding of 1z51-labelled toxin, in the presence of an excess of unlabclled C. noxius toxin 2 (1 pM) was 20%. Saturation experiments showed that the binding of '251-labelledC. noxius toxin 2 was saturable and the Scatchard plot indicated an affinity of the order of 10 nM (data not

components were found in the venoms of all Centruroides shown).

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Table 1. In vivo neutralization test. 7.50 times the LDso of C. noxius toxin 2 were incubated with mAb at a molar ratio of 1 : lO (toxin/ antibody) and injected intraperitoneally into each mouse (see hla- lcrials and Methods for details). BNTX16 is a mAb directed against an unrelated toxin.

lnjected mixture Mouse survival Mean -

(survivors/injected) survival time -

h Cn toxin 2 0: 5 0.33 Cn toxin 2/BNTX16 0: 5 0.33

Cn toxin 2/BCF2 2:5" 60 Cn toxin 2/BCF3 0:5 15

Cn toxin 2/BCFl 0 3 1

Cn toxin 2/BCF7 0:5 1 Cn toxin 2/BCF8 0: 5 1 Cn toxin 2/BCF9 0:5 1 mAb alone 5:5 - -

The two mice survived the three-wcck observation period.

species assayed except C. eleguns. T. serrulutus venom gave no inhibition (Fig. S6 b). With BCF9, C. limpidus ucutlunerzsis venom reacted as well as C. noxius venom; C. limpidus tecomunus and C. limpidus limpidus were about three times less potent and C. infumntus infamatus and C. suflusus sujfusus, 30- 100 times less potent. C. eleguns and T. serrulutus gave no significant inhibition (Fig. S6e). Finally, venoms of all Centruroides species contained very similar levels of components cross-reacting with BCF8, whereas no inhibilion was observed with T. serrulutus venom (Fig. S6d).

The mAb inhibit the binding of C. noxius toxin 2 to rat brain membranes

The six mAb were tested for their ability to inhibit the binding of 1251-labelled toxin 2 to brain synaptosomal mem- branes in vifro. As shown in Fig. 4, all the mAb were able to inhibit the binding of the toxin to the synaptosomal mem- branes in a dose-dependent manner. The mAb IDSo varied over 0.16-5 nM for BCFS, BCF7, BCF2, BCF3 and BCF9; BCFl was a weaker inhibitor (IDso > 0.1 pM) probably be- cause of its lower affinity for the toxin. N o inhibition was observed with normal mouse IgG.

In vivo neutralization test

In order to test the in vivo neutralizing activity of the mAb, 7.5 times the LDS0 of C. noxius toxin 2 was incubated with each mAb before being injected in groups of five mice. The results of this experiment (Table 1) showed that the six mAbs had some protective activity, as demonstrated by delayed mouse death (1 - 15 h in comparison to 20 min for control mice). BCF2 had a strong neutralizing activity, since death was delayed for 60 h for three mice and the other two mice survived the challenge for the three-week observation period.

DISCUSSION

In this communication, we report the strategy used to purify to homogeneity two toxins from thc venom of the Mexican scorpion C. noxius Hoffmann, the determination of the full primary structures of these two toxins and their immunochemical characterization with mAb. This is the first

report on the immunochemical characterization of a scorpion p toxin.

The purification procedure herein described differs slightly from the original communications, which allowed the purifi- cation of toxin CnII-9.2.2 and the determination of its N- terminal amino acid sequence (Possani et al., 1981 a, b). In the previous publications, the third step of purification consisted of a rechromatography of CnII-9 under conditions similar to those in the second step (Cm-cellulose column, in 20mM ammonium acetate buffer, pH 4.7), which did not add much to the purification efficiency of the procedure, and in which the third fraction obtained was not further characterized. The fourth step (Possani et al., 1981 b) consisted of a Bio-Rex 60 column at pH 6.5, resulting in a relatively poor resolution. With the present strategy (Fig. l a and b), we can observe a considerable improvement of the method, which allowed the complete separation and sequencing of CnII-9.2.2 and CnII- 9.2.3, now named C. noxius toxins 2 and 3, respectively. The first Na' channel-blocking toxin completely sequenced from C. noxius venom was CnII-14 (Possani et al., 1985), which we now propose to call toxin 3 , to avoid cumbersome or mislead- ing nomenclatures. It is our opinion that until the complete primary structure of a toxin or venom component is known, it is advisable to identify it according to the elution pattern in chromatographic steps, but when the complete amino acid sequence becomes available, the nomenclature should be sim- plified, as we now propose for the first three long-chain toxins purified and sequenced from C. noxius scorpion venom.

When the two new sequences were compared to the se- quences of scorpion toxins present in data banks, the highest similarity was found with other Centruroides toxins. In Fig. 5 , we have listed and aligned these sequences and also, the se- quences of two toxins from another American scorpion, T. serrulutus. If we compare the two new toxins with these other elucidated toxins, we can observe that C. noxius toxin 2 dis- plays the highest similarity with c. s ~ f u s u s suffusus toxin 2 and C. limpidus tecomanus toxin I (86%) and 85% identity respectively) whereas the identity is 79% with C. noxius toxin 3 and 55% with C. noxius toxin 1 . C. noxius toxin 3 displays respectively 86%, 82% and 56% identity with C. limpidus tecomunus toxin 1, C. sujjusus suffusus toxin 2 and C. noxius toxin 1. Thus, there may be more sequence similarity between toxins of different species than between different toxins of the same species. This suggests that during evolution, the different toxins appeared before species differentiation. Also worthwhile mentioning from the analysis of Fig. 5 is that the overall identity is 39% if we compare all the known Centruroides toxins, whereas it drops to 20% if we include the two toxins of the Brazilian scorpion T. serrulutus in the comparison.

Using the data of Fig. 5 , we have computed a variability index for the nine known Centruroides toxins, according to Wu and Kabat (1970). Examination of the variability index profile (Fig. 6 ) indicates that not only the residues in 27 pos- itions (including the cysteine residues) are absolutely in- variant, but also that the variability is not uniformly distrib- uted and occurs mainly within two regions: residues 7-10 and 31 - 34. It is noteworthy that these regions correspond to areas of maximum accessibility and thus constitute potential antigenic sites (Novotny et al., 1987). Moreover, half of the 14 amino acid differences between C. noxius toxins 2 and 3 occur in patches (residues 7 - 9 and 31 - 34) corresponding to these high-variability regions. This observation probably explains our experimental finding that four of our six mAb reacted with C. noxius toxin 2 , but not with C. noxius toxin 3, although the two toxins display 79% identity. On the other

287 .............................................................................. Toxin Amino Acid Sequence Reference

1 10 20 30 4 0 50 60 66 ..............................................................................

Cn2 KEGYLVDKNTGCKYECLKLGDNDYCLRECKQQGYKGAGGYKGAGGYCYAFACWCTHLYEQAI~PLPNKRCS ( a )

Cltl KEGYLVNHSTGCKYECFKLGDNDYCLRECRQQYGKGAGGYCYAFGCWCTHLYEQAWWPLPNKTCS (c) Cn3 KEGYLVELGTGCKYECFKLGDNDYCLRECKARYGKGAGGYCYAFGCWCTQLYEQAVWVPLKNKTCR (a)

CSSII KEGYLVSKSTGCKYECLKLGDNDYCLRECKQQYGKSSGGYCYAFACWCTHLYEQAVVWPLPNKTCN (b)

CsEl KEGYLVKKSDGCKYDCFWLGKNEHNTCECKAKNQGGSYGYCYAFA~CEGLPESTPTYPLPNK-CS (d) CsE2 KEGYLVNKSTGCKYGCLKLGENEGNKCECKAKNQGGSYGYCYAFACWCEGLPESTPTYPLPNK-CSS (d) CsE3 KEGYLVKKSDGCKYGCLKLGENEGCDTECKAKNQGGSYGYCYAFA~CEGLPESTPTYPLPNKSC- (d) CSEI KDGYLVEK-TGCKKTCYKLGENDFCNRECKWKHIGGSYGYCYGFGCYCEGLPDSTQTWPLPNK-CT ( e )

T S l O KEGYLMDHE-GCKLSCF-IRPSGYCGRECGIK-KGSS-GYCAWPACYCYGLP~KWVD~TNKC- ( f ) TS0 KEGYAElDHE-GCICFSCF-IRPAGFCDGYCKTHLKASS-GYCAWPACYCYGVPDHIKWVDYATNKC- (g)

* **** *** * ** * ** **** * * * * ** ** *

Cnl WGYLVDA-KGCKKNCYKLGKNDYCNRECRMKHRGGSYGYCYGFGCYCEGLSDSTPTWPLTNKTC- (f)

Fig. 5. Comparison of amino acid sequences of American scorpion toxins. The sequences of 1 1 American scorpion toxins have been aligned. Cltl, is toxin 1 from C . limpidus tecomnnus. CssII is toxin I1 from C. su~usus sufJusus. CsEl, CsE2, CsE3 and CsET are toxins from Centruroides scuIpturufus Ewing. Ts8 and TslO are toxins 111-8 and 111-10 from T. serrulutus. Gaps (-) werc introduced for alignment purposes. Identical amino acids in all sequences are represented by bold letters. Stars (*) designate positions perfectly conserved among Centruroides toxins. (a) This study; (b) Garcia (1976); (c) Martin et al. (1988); (d) Babin et al. (1974); (e) Babin et al. (1975); (0 Possani et al. (1985); (g) Possani et al. (1991).

25

20

2 15 U C

a

13

.-

+ .- - .-

.; 10

5

5

0

I

10 20 30 40 50 60 70

Amino acid position Fig. 6. Plot of amino acid variability in Centvuvoides scorpion toxins. The variability index was computed for the nine Centruroides sequcnces shown in Fig. 5 according to the formula used by Wu and Kabat-(l970). Positions corresponding to maxima in the large-probe accessibility profile (Novotny et al., 1987) are indicated by an arrow.

hand, the most conserved stretches of amino acid sequences (allowing for conservative substitutions, i. e. glutamic acid for aspartic acid, lysine for arginine, hydrophobic residues for similar ones) correspond to the regions of the molecule in- volved in cr-helix or fl-pleated sheet, as shown by X-ray diffrac- tion data (Fontecilla-Camps et al., 1980). In this respect, the high similarity of all the Centruroides toxins shown in Fig. 5 is remarkable and certainly has a physiological meaning, since all these toxins, so far studied, exert an action on Na’ channels and are classified as fl toxins (Couraud et al., 1982; Wheeler et al., 1983; Meves et al., 1986; Martin et al., 1988; Yatani et al., 1988).

By examining whether pairs of mAb could simultaneously bind to the toxin and by measuring the cross-reactivity of the venoms of different scorpions with the mAb, we could tentatively define at least four epitopes (A-D) on C. noxius toxin 2 as schematically depicted in Fig. 7. Epitope A, defined by BCF8, is present in all Centruroides venoms tested (C. noxius, C. limpidus limpidus, C. limpidus tecomanus, C. lim- pidus acatlanensis, C . sufJsus suJfusus, C . infamatus injamatus and C. elegans) but not in T. serrulatus venom. Moreover, this epitope is present on several purified toxins of C . noxius (C. noxius toxins 1, 2 and 3 and CnII-10) and on C. limpidus tecomunus toxin 1. The highly conserved nature of this epitope

288

Fig.7. Proposed pattern of phylogenetic relationships between Centruruides scorpions. The degree of cross-reaction of each scorpion venom with each mAb is depicted in the box representing the corre- sponding epitope on the right side of the figure. Based on the cross- reaction pattern of their venom (ID,o differences), the scorpions were ordered relatively to C. noxius in the phylogenetic tree shown or1 the left side.

suggests that it corresponds to a structure essential for the biological activity of the Centruroides scorpions toxins. BCFl seems to react with the same or an overlapping epitope as demonstrated by its inability to bind to C. noxius toxin 2 simultaneously with BCF8. Epitopes B -D are defined by mAb BCF2 and BCF7, BCF3 and BCF9 respectively, on the basis of the three distinct patterns of cross-reactivity of the different venoms for these three mAb groups (Fig. S6). Epitopes B - D appear to be overlapping or in close proximity because mAb recognizing these epitopes are unable to bind simultaneously to the toxin (Fig. 3). On the contrary, epitope A is topographically unrelated to epitopes B - D as shown by the simultaneous binding to the toxin of epitope-A-reactive mAb and epitope-B - D-reactive mAb (Fig. 3).

The assay of components cross-reacting with the set of mAb in the venom of several scorpion species also suggests a pattern of phylogenetic relationships between the Centruroides species. Indeed, IDs0 differences between venoms reflect either variations in the relative concentration of the common epitope or affinity change due to sequence variation. Thus, ID,,] dif- ferences reflect phylogenetic distances. According to this cri- teria, the presence of epitope A establishes a link between all Centruroides species and distinguishes them from the South American scorpion T. serrulatus (Fig. 7). The assays with mAb BCF2 and BCF7 suggest that C. limpidus limpidus, C. limpidus ucatlanensis and C . limpidus tecomanus are almost equidistant from C. noxius. C. su[fusus suffusus and C. infamatus infarnutus are also nearly equidistant from C. noxius but much more

distant than the three C. limpidus subspecies. Although there are some quantitative differences in the inhibition patterns with BCF3 and BCF9 with respect to BCF2 and BCF7, the results with the former also suggest that the three C. limpidus subspecies are more related to C . noxius than C. infamatus infamatus and C. suffusus suffusus are. Altogether, these results suggest the phylogenetic relationships among Centruroides species shown in Fig. 7. In this figure, one can observe that C . limpidus acatlanensis, C . limpidus limpidus, and C . limpidus tecomanus which all belong to the same species are immunologically closely related. Immunological relatedness also reflects the geographic proximity between C. suffu~us suffusus and C. infamatus infamatus. C. elegans, which is mor- phologically very different from the other Centruroides, is also immunologically the most distant.

In vitro neutralization experiments showed that all six mAb could inhibit the binding of C. noxius toxin 2 to its receptor on rat brain synaptosomal membranes. Possible interpreta- tions of this observation are that either the epitopes defined by the mAb are overlapping or close to the active site of the toxin, or binding of the mAb induces a conformational change in the toxin reducing its affinity for the receptor as has been observed for a neutralizing mAb against a snake toxin (Boulain and Menez, 1982). However, the physiological mean- ing of this in vitro inhibitory effect is not clear since there is no apparent relationship between the results of the in vitro and the in vivo neutralization experiments. In vivo, all the mAb increased the survival time of mice when incubated with the toxin before intraperitoneal inoculation, but only BCF2 can be considered as a neutralizing antibody. Indeed, it was the only mAb able to protect definitively some of the mice injected with 7.5 times the LDSo of the toxin (two out of five mice). Perhaps, the protective activity of BCF2 is related to the fact that it belongs to the IgG,, subclass, since BCF7, which has an indistinguishable epitope specificity and the same affinity for the toxin, but belongs to the IgGl subclass, does not display the same protective activity. Immune complexes of toxin and antibody of the IgGza subclass could be much more efficiently removed from the circulation by peritoneal macrophages or other components of the reticuloendothelial system, due to the presence on these cells of high-affinity receptors specific for this IgG subclass (Spiegelberg, 1974; Unkeless et al., 1988). Moreover, mouse IgGza bind comp- lement much more efficiently than mouse IgGI, and comp- lement binding can further increase the opsonizing function of the IgGza subclass (Spiegelberg, 1974). If, as suggested by this study, immunoglobulin subclass is a major parameter for the in vivo neutralizing activity of anti-toxin antibodies, this observation could have important implications for serotherapy of scorpion sting intoxication.

The authors are greatly indebted to Mr. Fredy Coronas and Timoteo Olamendi for technical assistance. This work was supported in part by National Council of Science and Technology (CONACyT- Mexico), Direccitjn General de Asuntos del Personal Acadimico- UNAM, grant nos IN202689 and IN300991 and United Nations for Industrial Development, grant no PNUD/UNESCO/ONUDI/DP/ RLA/83/003. LPD is a Howard Hughes Medical Institute scholar.

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290

Supplementary material to :

Amino acid sequence and immunological characterization with monoclonal antibodies of two toxins from the venom of the scorpion Centruroides noxius Hoffmann Fernando ZAMUDIO ', Rafael SAAVEDRA', Brian Michael MARTIN ', Georgina GURROLA-BRIONES ', Pascal HERION' and Lourival Domingos POSSANT

~.

Time (min 1

. .

0 10 20 30 40 60 Time (min)

Fig. S1. HPLC separation of peptides obtained by protease V8 cleavage of the reduced and alkylated C. noxius toxins 2 and 3. (a) 300 p1 of toxin 2 (5 nmol) treated with protease V8 were applied to the columri (Cls reverse phase, 4.6 mm (internal diameter) x 25 cm) and eluted with a linear gradient gencrated by mixing solution A (0.12% trifluoroacetic acid in water) and solution B (acetonitrile with 0.1 "10 trifluoroacetic acid). Peptide eluted at 35.50 min gave the sequence QAIVWPL,PNKRCS, corresponding to the C-terminal portion of toxin 2, as shown in Fig. 2. (b) 200 p1 of toxin 3 (7 nmol) after cleavage with protease V8 were separated in the same conditions as in (a). The peptide eluting at 32.03 min gave the sequence CKARYGKGAGGYCYAFGCWCTQLYE, and the peptide at 25.03 min gave the sequence QAVVWPLKNKTCR of the C-terminal part of the molecule, as shown in Fig. 2.

(\I (T, d - 1 a

0.03 0.3 3 30

MAb concentration ( m g / l ) Fig.% Titration of anti-(CnI1-9.2) mAb (MAb) by ELISA. Serial dilutions of purified BCFl (0), BCF2 (O), BCF3 (Ll), BCF7 (m), BCF8 (A) , BCF9 (A) or normal mouse IgG ( V ) were incubated in ELISA trays coated with Cn 11-9.2. Bound antibodies were revealed using rabbit anti-(mouse IgG) antibodies conjugated with pcroxidase and the chromogcnic substrate (see Materials and Methods for de- tails). Rcsults are means of triplicates.

29 1

100

h

$

a

v

N cn * 5 0

0 0.03 0.3 3 30

u

100

50

0 0.03 0.3 3 30

Cn toxin 2 concentration (mg/l ) Cn toxin 3 concentration (mg/ l )

Fig.S3. mAb reactivity with C. noxius toxins 2 and 3. ELISA trays were coated with Cn 11-9.2. Purified BCFl (0, 3 mg/l), BCF2 (0 , 3 mg/ I), BCF3 (0, 3 nig/l), BCF7 ( ,1 mg/i), BCFS ( A , 1.5 mg/l) and BCF9 (A, 1 mg/l) were incubated in the wells together with serial dilutions of C. noxius toxin 2 (Fig. S3a) or C. noxius toxin 3 (Fig. S3 b). Bound antibodies were then revealed using rabbit anti-(mouse IgG) antibodies conjugated with pcroxidase and the chromogenic substrate. The ratio between the absorbance in the presence of competing toxin and the absorbance in its absence was plotted against toxin Concentration. Results are means of triplicates. The A492 (100%) in the absence of inhibitor were 1.94 for BCFl, 2.0 for BCF2, BCF3, BCF7, BCF8 and BCF9 in Fig. S3a, 1.34 for BCF1, 1.45 for BCFZ, 1.53 for BCF3, 1.43 for BCF7, 1.78 for BCF8 and 1.63 for BCF9 in Fig. S3b.

0.1 1 10 100

Toxin concentration (rng/l) Fig. S4. C. noxius toxin 2 and C. norius toxin 3 share epitope(s) iden- tified by BCFl and BCFI. ELISA trays were coated with C. noxius toxin 2. Purified BCFl (3 mg/l; 0, 0 ) or BCF8 (3 mg/l; 0, I) were incubated in the wells together with serial dilutions of C. noxius toxin 2 (0, 0) or C. noxius toxin 3 (0 , I). Plates were then processed and the results were plotted as in Fig. S3. Kesults are means of duplicates. The A49Z values (100%) in the absence ofcompeting toxins were 0.79 for BCFl and 1.71 for BCFR.

100

- 8 v

N

d- 50

a

0 0.03 0.3 3 30

Toxin concentration ( mg/\ 1 Fig. S5. Cross-reaction of BCF8 with other scorpion toxins. The binding of BCF8 (1.5 mg/l) to immobilized Cn 11-9.2 was assayed in the presence of serial dilutions of Cn 11-9.2 ( O ) , Cn 11-10 (O) , Cn toxin 1 ( A ) or C. limpidus tecomanus toxin 1 (V) . Bound antibodies were revealed using rabbit anti-(mouse IgG) antibodies conjugated with peroxidasc and the chromogenic substrate. The ratio between the A492 in the presence of competing toxin and A492 in its absence was plotted against competing toxin concentration. Results are means of triplicates. The A492 (100%) in the absence of competing toxin was 0.44.

292

100

h

$! v

P a

C

a

I I - ' 1 c

a3 3 30 300 1 A 0.3 3 30 300

Venom concentration ( m g / l )

Venom concentration ( m g / l ) Fig. S6. Presence of cross-reacting components in the venom of other scorpion species. The binding of BCF2, BCF3, BCF7, BCF8 and BCF9 to immobilized Cn 11-9.2 was assayed in the presence of serial dilutions of venoms from C. noxius (O) , C. limpidus tecomunus (m), C. limpidus limpidus ( O ) , C. limpidus acatlanensis (A), C. elegans (O), C. in$zmatus infamatus (a), C. suffusus suffusus ( A ) and T. serrulatus (0). Bound antibodies were revealed and results were plotted as described in Fig. S5. Results are means of triplicates.