Two neutralizing monoclonal antibodies specific for Naja nigricollis cardiotoxin: Preparation, characterization and localization of the epitopes

Molecular Immunology,Vol. 23, No. 12, pp. 1329-1337,1986 Printed in Great Britain.

0161-5890/86$3.00+O.OO 0 1986Pergamon Journals Ltd

TWO NEUTRALIZING MONOCLONAL ANTIBODIES SPECIFIC FOR NAJA NIGRICULLIS CARDIOTOXIN: PREPARATION, CHARACTERIZATION AND LOCALIZATION OF THE EPITOPES JEAN-MARC GROGNET,* ERIC GATINEAU,* PIERRE BOUGIS,~ ALAN L. HARVEY,~ JACQUES COUDERC,~ PIERRE FROMAGEOT* and ANDRE MBNEz*II *Service de Biochimie, CEA Saclay, 91191 Gif/Yvette, Cedex, France; TLaboratoire de Biochimie Facultt de MCdecine nord de Marseille, 13326, Marseille Cedex 15, France; IUniversity of Strathclyde, Department of Physiology and Pharmacology, Glasgow, U.K. and sUnit& INSERM 20, HBpital Broussais, 75014 Paris, France (Firs? received 2 February 1986; accepted in revised form 26 April 1986)

Abstract-Two monoclonal antibodies have been raised against the native form of the potent cardiotoxin isolated from the venom of Nuju nigricollis. The toxic action to mice as well as the depolarizing effect on muscle fibres in culture of the cardiotoxin are neutralized by the two immunoglobulins. Binding studies revealed that the radiolabelled toxin has a high affinity for both antibodies, the equilibrium dissociation constant values being equal to 0.2 and 0.4 nM. The epitopes that are recognized by the antibodies have been localized on the basis of competition experiments between the labelled toxin and a series of variants or a Trp-11 modified derivative, toward both antibodies. The data obtained indicate that the antibodies bind at topographically different antigenic sites. Knowing that the toxin is a single polypeptide chain folded in a structure that contains three adjacent loops emerging from a small globular region, it appears that one of the two antibodies binds on loop I, at a site which involves Trp-1 1 whereas the other binds at a site which involves one or both of loops II and III. Possible mechanisms of neutralization of the toxin by the antibodies are discussed.

INTRODUCI’ION It is well known that injection of a toxic protein into

an animal induces the production of specific antibodies which are generally neutralizing (Calmette, 1907; Boquet, 1979). This observation implies that one or more of the immunoglobulins present in the antisera bind to critical epitopes on the toxin surface. Clearly, identification of such epitopes constitutes an important step in the understanding of the molecular mechanisms that are associated with the inactivation of a toxin by its specific immunoglobulins. The advent of hybridoma technology to prepare monoclonal antibodies certainly provides exceptional tools for probing a single epitope at a time (Kiihler and Milstein, 1975). One approach for localizing an epitope on a protein surface consists of identifying the amino acid residues that are critical for the binding capacity of the antibody. This can be achieved, for example, by analyzing the cross-reactivities between the reference antigen and a series of variants or of chemically modified derivatives, toward the antibody (Boulain et al., 1982; Berzofsky et al., 1982). Without doubt, toxic proteins isolated from snake venoms are wellsuited antigens for such investigation because (i) these immunogenic substances (Boquet, 1979) are //Author to whom correspondence

should be addressed.

readily amenable to monoderivatization (see review by Karlsson, 1979) and (ii) a large set of homologous toxins is available (Dufton and Hider, 1983). Cardiotoxins constitute the major toxic components of the venoms of a number of Elapids. They are small proteins with a 60 amino acid single chain cross-linked by four invariant disulfide bridges (Karlsson, 1979). Evidence based on (i) secondary structure prediction studies (Dufton and Hider, 1977; MCnez et aI., 1978), (ii) circular dichroism spectra analysis (Visser and Louw, 1978; Drake et al., 1980) and (iii) NMR spectroscopy (Lauterwein et al., 1977; Steinmetz et al., 1981), indicates that the structure of cardiotoxins is rich in b-pleated sheets and /?-turns with no helicity. This was definitely confirmed by Rees et al. (1983) who reported the preliminary data of the X-ray studies of cardiotoxin V”4 from N. m. mossambica. The toxin contains three adjacent loops rich in b-sheet structure, emerging from a small globular region containing the four disulfide bridges. The mode of action of cardiotoxins is still obscure (Chang, 1979; Condrea, 1979; Lee and Lee, 1979; Harvey, 1985). Without doubt, however, they alter the state of the membranes from a variety of excitable cells (Condrea, 1974) including those from skeletal muscle fibres (Harvey et al., 1983; Harvey, 1985). Toxin y is the single cardiotoxin that has been found in the venom of Naja nigricollis (Fryklund and Eaker, 1975). This substance is responsible for the

1330

JEAN-MARC GROGNET et 01.

lethal effect of the venom (Cheymol et al., 1966). In the present paper the preparation of two monoclonal antibodies which neutralize toxin y under both in vivo and in vitro conditions is described. The epitopes that are recognized by the antibodies have been localized on the basis of competition experiments using one chemically modified toxin and a number of different natural variants. Possible mechanisms of neutralization by the antibodies are discussed. MATERIALS

AND METHODS

Naja nigricollis toxin y was purified from venom (Institut Pasteur, Paris), according to Fryklund and Eaker (1975). Naja mossumbica mossambica cardiotoxins were purified as described previously (Bougis et al., 1983; Joubert, 1974; Louw, 1974). Nuja kaje ~nul~~ra CM1 1 was kindly donated by Dr Joubert (South Africa). Bio-Rex 70 resin was from Bio-Rad (Richmond, CA). N-succinimidyl (propionate2,3,3H) was from New England Nuclear (Boston, MA). 2-Nitrophenylsulfenyl chloride was obtained from Fluka (Buchs, Switzerland). Cn p-Bondapack column was used for high-pressure liquid chromatography (HPLC) (Waters Associates, Milford, MA). Freund’s adjuvant was purchased from Difco Laboratories (Detroit, MI) and BALB/c mice were from IFFA CREDO (Lyon, France). RPM1 medium, HAT mixture, fetal calf serum and normal horse serum were purchased from GIBCO Europe (Glasgow, U.K.). Poly(ethylene glycol) (PEG) MR 4000 or 6000 (for radioimmunoassay only) was obtained from BDH Chemical Co. (Poole, U.K.) The 96-well Linbroplates were from Flow Laboratories. Lipoluma and Lumagel were obtained from Lumac, The Netherlands. Preparation of radioactive toxin y Naja nigri&o~li~toxin y (Fig. 1) contains two tyrosines. However in pilot experiments iodination proved difficult (data not shown). Consequently, we incorporated [3H]propionyl groups on lysine residues. The expe~mental conditions were designed to favour monoderivatized toxin populations: 700 ~1 (700 PCi) of a benzene solution of N-succinimidyl propionate (= 0.01 pmole) was evaporated under nitrogen and dissolved in tetrahydrofuran (100 @I). Two milligrams of toxin y (0.3ymole) in 100~1 water was added and the solution was stirred at room temp overnight. The product was filtered on a Biogel P, column (1s x 1 cm) eq~librated with acetic acid (10% w/v) and the protein which eluted with the void vol was freeze-dried. The molecular species corresponding to monopropionylated cardiotoxin were subsequently separated from unreacted material as well as from polypropionylated species in the following manner: the mixture was dissolved in 0.1 M ammonium acetate (pH 7.0) and applied to a Bio-Rex 70 column (5 x 1 cm) equilibrated in the same buffer. Elution was made with a linear gradient of 0.1-0.6 M

Fig. 1. Amino acid sequence of toxin y (Fryklund and Baker, 197.5). The backbone of the toxin has been ~hematic~ly folded in a manner which is similar to that of Baja mossambieamossambicatoxin Vi’4 as deduced from X-ray crystallographic data (Rees B., Moras D. and Thiery J. C., preliminary results kindly communicated to us prior to publication). The amino acid residues are represented by the one-letter code (IUPAC). Numbers I, II and III indicate the three main loops of the toxin.

ammonium acetate, pH 7.0. Unreacted and monopropionylated toxin y eluted approx. at 0.4 and 0.2 M ammonium acetate, respectively. Radioactive fractions were pooled and freeze-dried. The specific radioactivity of the labelled toxin was 36 Ci/mmole. Control experiments made with non-radioactive reagents indicate that the structure and toxicity of monopropionylated toxin y are identical to those of the parent molecule. Preparation of Trp - I 1 modi~ed toxin

2-Nitrophenylsulfenyl chloride (0.4 mg) in 2 ml of pure acetic acid was added to 14 mg of toxin in 2 ml of the same solvent and the mixture was left at room temp overnight. The reaction mixture was filtered through a Bio-Gel P2 column equilibrated in 10% acetic acid. The protein which eluted in the void vol was chromatographed on Cn p-Bondapak column ~uilibrated in 0.1 ~-t~ethylamine adjusted to pH 5.6 with pure formic acid. Elution was made using acetonitrile as the secondary solvent (Faure et al., 1983). Production of pur@ed mono~~onal antibodies

Immunization of mice was achieved by S.C.injecting 40~8 of native toxin y three times at 10 day intervals in complete Freund’s adjuvant {total vol 0.2 ml). One week after the last injection, 12 pg of

Two neutralizing monoclonal antibodies for Naja nigricolliscardiotoxin

native toxin y in 9% NaCl (0.2 ml) was injected i.p. Four days after this fourth injection, the mice were killed and their spleens removed. The fusion procedure was carried out according to Kohler and Milstein (1975). Spleen cells (lo*) were mixed with 10’ myeloma cells (NSl) in RPM1 medium and centrifuged at 600g for 5 min. The pellet was slowly resuspended at 37°C over a period of exactly 1 min in PEG 4000-RPM1 (1 ml, 50% w/v) with stirring and further incubated for 1 min. The cell suspension was then diluted with 20 ml of RPM1 and centrifuged at 600 g for 5 min. The pellet was resuspended in 1.5 ml of RPM1 containing 2 mM glutamine and antibiotic (gentamycin 50 pgg/ml) and supplemented with 20% (w/v) fetal calf serum for 30 min at 37°C. The mixture was diluted with the same medium supplemented with HAT (hypoxanthine, aminopteridin and thymidine) and 10’ macrophages obtained from mice, thus enabling the cells to be plated into 100 wells (1 ml well). Antitoxin occurrence was tested 2-3 weeks later by radiobinding assays. Positive wells were cloned by limiting dilution into 96-well microtiter plates containing lo6 thymocytes per well. Positive wells were further recloned according to the same methods. Monoclonal hybridoma cells (5 x 106) producing antitoxin y were injected i.p. (0.2ml) into one BALB/c mouse. One to two weeks later, ascitic fluid was withdrawn and centrifuged for 5 min at 1OOOg. The supernatant was allowed to stand at 4°C overnight, centrifuged at 3000g for 20 min and filtered through a toxin y Sepharose 4B column preequilibrated in 0.2 M Tris-HCl buffer, pH 8.0, containing 0.5 A4 NaCl. The column was extensively washed with the same buffer and bound antibodies were finally eluted by using a 0.2 M HCl-glycine buffer, pH 2. The eluate was immediately adjusted to pH 8.0 with 2 M Tris-HCl buffer. Purified antibodies were cont. with immersible Millipore CX 30 ultrafilters. The immunoglobulin subclass was characterized by double diffusion in plates (Ouchterlony’s method) using specific rabbit antimouse immunoglobulin subclass antisera (Nordic, The Netherlands). Binding assays

Radioimmunoassays were carried out by using poly(ethylene glycol) 6000 (PEG) at a final concn of 12.5% (w/v) in order to precipitate the antigenantibody complexes. The plate (Flow Laboratories) culture supernatants (0.1-0.2 ml) were incubated at 4°C overnight with 0.1 ml (0.7 pmole) of 3H-labelled toxin y (36 Ci/mmole) in 0.05 M phosphate buffer and 0.45% NaCl, 1% (w/v) bovine serum albumin pH 7.0. Normal horse serum (0.02 ml) and PEG (0.5 ml) were then added to the solution which was stirred and centrifuged at 2000g in a Sorvall HS4 rotor at 4°C for 30 min. The pellets were dissolved in 0.75 ml of 0.01 M NaOH and placed in lOm1 of Lumagel solution. The radioactivity was estimated

1331

using a liquid scintillation counter (Intertechnique). The background was less than 10% of the total radioactivity. The precipitation method by poly(ethylene glycol) is extremely reproducible with low mean standard deviation. Competition assays

Monoclonal antibodies (0.17 pmole/O. 1 ml expressed in toxin binding sites) were incubated overnight at 4°C in the presence of 23000 dpm 3H toxin y (0.35 pmole/O.l ml) with various amounts of toxin analogs or derivatives. Precipitation of antigenantibody complexes and counting of radioactivity were achieved as in binding assays. Neutralization by purt$ed monoclonal antibodies

(1) In vivo. Native toxin y (30 pg/mouse) was injected iv. in the tail of BALBjC mice (weight 20 g f 2 g) in the presence and absence of 1 mg of each of the two purified monoclonal antibodies. A final vol of 0.4 ml of 0.9% NaCl solution was injected into each mouse. The toxin-antibody complexes were incubated overnight at 4°C prior to injection. Injected mice were examined 30 min, 24 hr and 1 week after injection. (2) In vitro. The depolarizing effect of toxin y on skeletal muscle fibres grown in cell culture from chick embryo muscle was determined as previously described (Harvey et al., 1983). The two monoclonal antibodies (10 PM) were tested for their ability to neutralize this effect by preincubation with toxin y (1 PM).

RESULTS

AND DISCUSSION

Toxin y (Fig. 1) is a toxic protein [LD,, N 55 pg i.p. in 20 g mice] and, as a result, the amount of injected antigen was carefully limited during the immunization procedure. Under these conditions, high titers of specific antitoxin y antibodies (-l/1000) were nevertheless obtained in mice sera after the third injection. A similar successful immunization has also been reported for the even more toxic homologous N. nigricoflis toxin tl (Boulain et al., 1982). Preparation of monoclonal antibodies

Six fusion experiments using spleen cells from hyperimmunized BALBjC mice and myeloma cells were performed according to the procedure described by Kiihler and Milstein (1975). Only two experiments were successful. Twenty out of 100 wells (each containing lo6 cells) contained hybrid cells secreting antibodies which reacted with ‘H-labelled toxin y. The cells were cloned but only two clones developed suitably. They were grown i.p. in mice and approx. 2.5 ml of ascitic fluid per mouse were withdrawn. The two clones were designated as My, and My;_,, and correspond to mouse IgG, and IgG*b isotypes, respectively.

1332

JEAN-MARCGROGNETet al.

c3H j TOXIN added (nMi

0 0.1 Bound

(nM)

0.3

0.5 Bound (nM1

Fig. 2. Specificbinding of increasing amounts of labelled toxin to Mi, (upper left) and My,_,(upper right). The results are plotted according to Scatchard analysis (lower). Final antibody concns, expressed in terms of toxin binding sites, were equal to I and 0.5 n&f for MT, and Mj&, respectively. Binding of Mf, and II@_, to 3H-labelled toxin y

Neutralization of toxin y by monoclonal antibodies

Binding of 3H-labelled toxin to both monoclonal antibodies (My, and My,_,) is saturable (Fig. 2). There is little non-specific binding as revealed by the low level of bound radioactivity (= 10% of total radioactivity bound) when the binding experiments are performed in the presence of an excess of unlabelled toxin. Scatchard analysis of the data yielded straight line graphs, indicating the presence of a single class of binding sites (Fig. 2). Equilibrium dissociation constants derived from the slopes of these plots are equal to 0.4 and 0.2 nM for My, and MfZ_3, respectively. These values are comparable to those observed for specific binding between either a cobra neurotoxin (Boulain et al., 1982), myoglobin (Berzofsky et al., 1982) or a cobra cardiotoxin (Kfir et al., 1985) and specific monoclonal antibodies.

(1) In vivo. Thirty micrograms of toxin were injected into 20g mice in the absence or in the presence of a 3-fold molar excess (in terms of toxin binding sites) of either My, or My;_,. The results of this experiment (Table 1) indicate that neither of the two toxin-monocionai immunoglobulin complexes are toxic to mice. (2) In vitro. It has been well established that cardiotoxins from cobra venoms are capable of depolarizing the membranes of a variety of excitable cells (Harvey, 1985) including muscle fibres in culture (Harvey et al., 1983). This is also the case for Naja nigricollis toxin y (Fig. 3). This effect is clearly neutralized by each monocional antibody. The two types of experiments, made under in vivo and in vitro conditions, converge to indicate the

Two

neutralizing monoclonal antibodies for Naja nigricolliscardiotoxin

Table 1. Neutralization of the toxic action of toxin y by two monoclonal immunoglobulins Injected mixture

No. of dead mice

No. of injected mice

7 0 0

7 8 8

Toxin y (30 pg) Toxin y (30 pp) + I$, (1 mg) Toxin (3Opg) + My,, (I mg)

Injections were made i.v. using normal saline solutions (0.4ml). Animals were examined 30 min, 24 hr and I week after injections. Death occurred within 30 min, whereas survivors were all alive 1 week after injection.

potent neutralizing capacities of the antibodies. This is not, however, a property common to all cardiotoxin-specific monoclonal antibodies since it was recently reported that one of them, directed to iV. nivea cardiotoxin V”1, is not neutralizing (Kfir et al., 1985). Localization of epitopes

The epitopes have been localized on the basis of immunological crossreaction experiments using toxin y as the reference toxin, one chemically modified derivative of toxin y and a number of natural variants. (1) Preparation of Trp-1 1 modified toxin y. Toxin y possesses a single tryptophan residue located at position 11 (Fig. 1). Incorporation of 2-nitrophenylsulfenyl moiety at Tip-11 of toxin y was made according to Fontana and Scoffone (1972). The derivative displays a U.V. spectrum with two maxima centered at 365 and 282 nm (molar absorbancy = 16,000). The amino acid composition of the derivative was found to be similar to that of the parent compound (not shown). The U.V. circular dichroism spectra of native and derivatized toxins are similar, precluding any substantial conformational change subsequent to derivatization. LD5, values, determined by i.p. injections into 18-20 mg BALB/C mice,

01

6

1333

were 105 and 55 pg for modified and native toxin, respectively. Presumably, Trp-11 plays some role in the toxic action of toxin y. (2) Selection of natural variants. So far, more than 50 amino acid sequences of cardiotoxins have been elucidated (see review by Dufton and Hider, 1983). They all consist of a single polypeptide chain with 60 amino acids and four disulfide bonds. This chemical homology underlies a structural homogeneity which has been indeed pointed out by a variety of analyses including circular dichroism (Visser and Louw, 1978; Drake et al., 1980) and NMR (Lauterwein et al., 1977; Steinmetz et al., 1981). All these studies clearly indicate that the overall structure of cardiotoxins is similar to that of the homologous snake neurotoxins and consists of three main adjacent loops rich in p-sheet structure. Confi~ation of this high structural homogeneity between neuro- and cardiotoxins from snake venoms was definitely reported by Rees et al. (1983) who published the preliminary results of a crystallographic study of N. m. mossambica cardiotoxin V”4. This does not imply, obviously, that the detailed structure of any other cardiotoxin is strictly identical to that cardiotoxin. Rather, we recently observed some heterogeneity within the circular dichroism spectra of 17 cardiotoxins in which, however, the B-sheet structure clearly predominates (Grognet, 1984). Two major classes have been distinguished. Those belonging to one class are characterized by the presence of two extrema: an intense positive signal at 192 nm and a weaker negative signal at 214 nm. The spectra in the other class contain three extrema: a positive signal at 192 nm which is approx. three times less intense as that in the other group, a negative signal at approx. 210nm and a positive signal at 222 nm. Although it is not possible to assign precise differences in secondary structure to those two cardiotoxins classes, it is clear that some differences do

6

5

10

15

20

TIME Cminutrr) Fig. 3. Inhibition of the depolarizing effect of toxin y (0) on skeletal muscle fibers by My, (‘(I) and M& (m). The final concns of toxin y and monoclonal antibodies were 1 and 10 pA4, respectively. Fibers were sampled at random and values in each 2 min period in the presence of toxin y or toxin-antibody complexes were averaged, as previously described (Harvey et at., 1983).

1334

Table 2. Amino

N. N. N. N. N. N.

JEAN-MARC

et al.

acid sequence comparison of Naja nigricoliis toxin y with related cardiotoxins. toxin y(NPS) corresponds to a derivative chemically modified

toxin y m. mossambica toxin I m. mossambica toxin II m. mossambica toxin IV h. annulifera CM I I nigricdis toxin y(NPS) nigricollis

GROGNET

Only differences at Trp-1 I

IO 20 30 40 LKCNQLIPPFWKTCPKGKNLCYKMTMRAAPMVPVKRGCIDVCPKSSLLIKYMCCNTDKCN

are shown. N. nigricollis 50

60 N--

---U ---K

E

G-SK L-K Y-VS TLT----------

N-A-V-V---

N-N--

0

exist. In this study, we have selected cardiotoxins which display the same class of CD spectrum as that observed for Naja nigricollis toxin y (not shown). The sequences of the toxins selected for this study are shown in Table 2. (3) Competitive inhibition by cardiotoxin variants. Binding of tritium labelled toxin y to the monoclonal antibodies was examined in the presence of increasing amounts of chemically or naturally modified cardiotoxins. The competitive inhibition curves are shown in Fig. 4. The two panels of competitive inhibition curves are different from each other. The difference is particularly clear for some toxins like Trp-modified toxin and N. h. annulifera CM 11. Presumably the two antibodies bind at distinct epitopes and therefore the competition data have to be examined separately for each immunoglobulin. (3.1) Localization of the epitope recognized by My,. Naja mossambica mossambica toxins I and II are substituted at positions 57 and 28, 30 and 31, respectively. They behave like toxin y, indicating that these positions are not critically antigenic. Three of them, namely 28, 31 and 57, are also substituted in toxin IV, suggesting that one or both of the two remaining substitutions at positions 5 and 16 is associated with the slight decrease in affinity observed for this toxin. Presumably, therefore, the determinant specific for Mf, is on, or at proximity to loop I. Seven additional substitutions as afforded by Naja haje annulifera CM 11 (at positions 25, 27, 29, 30, 45, 47, 49 and 52), all located on loop II or loop III, do not induce further a decrease in affinity. The slight decrease in affinity observed for this toxin is probably due to the single substitution occurring in loop I, namely at position 5. The antigenic role of loop I is strongly confirmed by the behaviour of the Trp-1 1 modified derivative which has the lowest affinity for My,. Clearly, the determinant which is specific for this immunoglobulin is located on loop I and probably incorporates the indole ring at position 11. (3.2) Localization of the epitope recognized by My2_, . N. m. mossambica toxin I behaves like toxin y implying that its single substitution at position 57 is not involved in the epitope. In contrast to what has been observed in the case of My, (see above) modification of Trp-11 does not alter the affinity of toxin y for My,_,, implying that this residue is also excluded from the epitope. N. m. mossambica toxin IIy which is substituted at only three positions, all located at the tip of the central loop (28, 30 and 31), has a weaker affinity. The antibody binds presumably

at a site which involves, or is in proximity to, one or more of these three residues. The epitope, therefore, must be near the tip of the central loop. N. m. mossambica toxin IV which is also substituted in this part of the molecule, namely at positions 28 and 3 1, has a similarly weaker affinity than toxin II. The three other substitutions occurring in toxin IV, namely at positions 5, 16 and 57, are clearly remote from the tip of the central loop and do not induce a further decrease in affinity. Certainly, N. h. annufifera CM 11 bears one or more critical substitutions since this toxin has virtually no affinity for the antibody. Two of the 12 substitutions, at positions 5 and 57 cannot be responsible for this alteration. In contrast, one or more of the ten remaining substitutions which are all located in the same area, namely on the tip of the central loop and on the adjacent third loop, must be involved in, or at close proximity to the epitope. Taken together, these data indicate that the epitope which is recognized by MJ& is likely to involve certainly part of loop II and possibly part of loop III of toxin y. The localization of antigenic determinants as deduced from cross-reaction data are sometimes subject to criticisms (Atassi, 1984). Objections essentially arise from the fact that competition experiments between the reference antigen and variants are interpreted by assuming that: (1) all variants have similar structures; and (2) substitutions have no structural effects other than local. We have carefully examined both points. Concerning the first point, all variants have been selected for the high degree of similitude between their CD spectra and that of the reference toxin. Moreover, the Trp-11 modified derivative, which plays a critical role in the assessment that the two antibodies bind at different epitopes, is characterized by a far U.V. CD spectrum which is superimposable with that of native toxin y (not shown). We feel safe, therefore, in stating that all cardiotoxin homologs used in this study have very similar folding patterns. Our second remark addresses both assumptions. It is based on the fact that we used the same set of toxins to analyze two antibodies. A group of distinct toxins is well recognized by one antibody, suggesting that they do not differ markedly from the reference toxin. The same group of toxins binds poorly to the other antibody. Since we have two groups of toxins behaving in this respect in a symmetrical mode, most toxins can be shown to bind properly to one of the two antibodies. Each antibody acts as a global structural control for the experiments

Two neutralizing monocional antibodies for Nuja nigrieollis cardiotoxin

tC50

icc$(j (nM1

(nM)

6i2

5*2

CARDIOTOXIN I N. mossambica mosambica

3ooa

8112

10

NPS [Trp 111 CARDlOTOXlN N. nigricollis

6Oi

7t3

5 I

*’ CARDIOTOXIN II N. mossambica mossambica

40*5

60*5

*‘CARDIOTOXIN IZI N. mosssmbica moswnbica

40r5

>looO

TOXIN CM 11 N. haje annulifera

Fig. 4. Competition experiments between the ~diola~ll~ toxin y and a series of cardiotoxin homologues. The final concn of radiolabelled toxin y and the antibodies were equal to 3.5 and 1.7 n&4 (in terms of toxin binding sites), respectively. The left side of the Figure presents the competition data obtained with M& while the right side deals with Mf, . In all panels the continuous line represents the standard curve resulting from the inhibition of the binding of labelled toxin y by increasing amounts of unlabelled toxin y. The polypeptide chains of the five homologues used in this study have been folded schematically according to that of the homologous iV. m. mossambica toxin Vi4 (Rees et al., 1983 and personal communication), and are represented in the central part of the Figure. The amino acids which are modified, e.g. Trp-11, or substituted with respect to toxin y have been emphasized. The inhibition capacity of each homologue, toward Mfz_r and My,, is shown by data points on the left and right sides, respectively. IC& values of each analog for M&r (left) and Mf, (right) are also indicated. The IC,, values for unlabelled toxin y were 6 jI 2 nM and 5 f 2 nM, respectively.

JEAN-MARC GROGNET et af.

1336

carried out with the other one. More precisely, it was observed that: (i) suppression of a positive charge at position 57, in the C-terminal part of the molecule, does not alter the binding affinity of either antibody; (ii) incorporation of the bulky 2-nitrophenyisulfenyl group on residue 11 has no effect on the binding of My,_,; and (iii) substitutions in the tip of the central loop (Ala-28 Pro-30 and Met-31 in toxin y being replaced by Gly, Ser, and Lys in toxin N. m. massam bica II, respectively) do not modify the affinity of My,. This strongly indicates that these substituted positions do not belong to the respective epitopes and do not perturb the structure of the toxin, at least at these levels. Clearly, therefore, a number of regions of the toxin can be substituted or chemically modified without affecting the structure in remote parts of the molecule. GENERAL

DISCUSSION

Two monoclonal antibodies have been successfully prepared against N. nigricollis toxin y, a potent cardiotoxin, without preliminary modifications of the molecule to reduce its toxicity. This indicates that the toxin is a sufficiently powerful immunogen to stimulate the immune system at sublethal doses. This is true for a number of other toxins, including the potent neurotoxins (Boulain et al., 1982). Studies of the antigenic properties of “natiue” toxins can therefore be generalized. Unambiguously, the two immunoglobulins that have been prepared in this study bind at topographically different epitopes on the toxin surface. One of them includes part of loop II and possibly loop III, whereas the other is located on loop I, involving certainly Trp- 11, a residue which is on the edge of the latter loop (see Fig. I). Previously, we delineated the epitope recognized by another monoclonal antibody on the surface of the homologous toxin c( (Boulain et al., 1982), a neurotoxin present in the same Naja nigricollis venom (Fryklund and Eaker, 1975). It is striking that this epitope involves a number of residues that are aiso located on the edge of the first loop. These include in particular Lys-15, a residue which occupies in toxin tl, a nearly homologous position to that of Trp-1 1 in toxin y (see sequence lists of snake toxins in Dufton and Hider, 1983). It is possible therefore that the edge of the first loop constitutes an immunodominant area in both neuro- and cardiotoxins. Furthermore, it is of special interest that the monoclonal antibodies that bind at this level, either on toxin tl (Boulain et al., 1982) or on toxin y (this work) are potent at neutralizing the toxic actions of their antigens both under in uitro and in vivo conditions. These antigenic areas might prove important for the future developments of synthetic vaccines against both neuro- and cardiotoxins. We have also demonstrated that in spite of the topographical difference of their epitopes, the monoclonal antibodies that have been described in the

present study are both capable of neutralizing the lethal toxicity and depolarizing effects of toxin y. The question then arises as to the molecular mechanisms that are associated with their neutralizing properties. A number of non-mutually exclusive possibilities can be envisioned. The antibody may cover the “toxic” site, act by simple steric hindrance or perturb the conformation of the toxin molecule. Clearly, the answer requires a knowledge of the “toxic” site of the cardiotoxin. At present three hypotheses have been proposed which suggest that the “toxic” site is localized on one or more of the three main loops I, II or III (Lauterwein and Wiithrich, 1978; Hider and Khader, 1982; Dufourcq et al., 1982). There is some evidence based on chemical modifications of aromatic residues of two different cardiotoxins which supports the view that the first loop does indeed play some role in cardiotoxicity. Modification of Trp-I 1 of toxin y, as reported in this study, as well as formulation of the same residue or iodination of Tyr-11 of N. n. indian cytotoxin II all induce a slight but significant decrease in toxicity without producing detectable change in the confo~ation of the toxin architectures (to be published). Assuming then that the “toxic” site is localized in the vicinity of loop I, it is tempting to propose that My, neutralizes toxicity by masking the “toxic” site of toxin. Further experiments, however, are now required to confirm this proposal and also to clarify the neutralization mechanisms of My2_3. Acknowledgements-The

authors wish to thank very warmly Drs Rees, Moras and Thiery (Laboratoire de Biochimie et cristallographie biologique, Strasbourg, France) for having kindly communicated their X-ray data on the structure of a cardiotoxin prior to pub~jcation. They also thank Dr Boulain for his interest, Dr Joubert for his gift of toxins and Dr Bon for his help in toxicity measurements. REFERENCES

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