Molecular Immunolo~r, Vol. 27, No. Printed in Great Britain.

1,pp. 7-15, IWO

0161-5890/90 $3.00 + 0.00 0 1990 Pergamon Press plc





de Biologie, Batiment 142, Centre 91191 Gif sur Yvette, Cedex, France



de Saclay,

(Firsr received 4 April 1989; accepted in revised form 31 May 1989) Abstract-Notenin and nigexine are monomeric phospholipases A,(PLA,s) from the venoms of Nolechis scutatus scufatus and Nuju nigricollis, respectively. Polyclonal antibodies raised in mice against these antigenic proteins displayed non-reciprocal cross-reactivity; anti-notexin antibodies recognized notexin but not nigexine, whereas anti-nigexine antibodies recognized both antigens. Polyclonal antibodies raised by successive immunization with nigexine and notexin contained cross-reacting antibodies with affinities for both antigens that differed from those of antibodies present in anti-nigexine antiserum. A monoclonal antibody has been obtained from a mouse immunized with both PLA,s. This monoclonal antibody, called MN,, recognized notexin and nigexine with comparable high affinity (K,, = 10e9 M). It also recognized most purified PLA,s from elapid snake venoms and all PLA,-containing venoms from cobras and sea-snakes. This offers the first demonstration that most PLA,s from cobras and sea-snakes share a fine structure which is not restricted to the common catalytic site.

INTRODUCTION Phospholipases A,(PLA, s) are ubiquitous proteins present in a variety of tissues, including those of venom glands from poisonous animals. They hydrolyze the sn-2 acylester bond of phospholipids (E.C., and this conserved function is ensured by invariant residues forming the active site of the protein (Dijkstra et al., 1981). Experimental data suggest that overall folding of the polypeptide chains of PLA,s is similar, but that local fine structure may differ from one PLA, to another (Renetseder et al., 1985). Such topographical differences are clearly illustrated by the observation that polyclonal antibodies raised against one PLA, usually have a very limited range of cross-reactivity, as exemplified by snake venom PLA,s (Menez, 1985). Numerous immunological studies of PLA, s from snake venoms have revealed that cross-reaction occurs essentially between highly homologous isoenzyme antigens (Ramlau et aI., 1979; Gopalakrishnakone et al., 1981; Caratsch et al., 1982; Choumet et ul., 1989; Mollier et al., 1989). As PLA,s play a

*Present address: Unite d’Immunogenttique Cellulaire, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France. Abbreviations: PLA,, phospholipase A,; PEG, polyethylene glycol; TFA, trifluoroacetic acid; HPLC high performance liquid chromatography; RP, reverse phase; L.DSO, median lethal dose; RIA, radioimmunoassay; mAb, monoclonal antibody; Tris, Triss(hydroxymethy1) aminomethane-HCl; PS, physiological saline; FCA, Freund’s complete adjuvant; FIA, Freund’s incomplete adjuvant.

significant role in the lethal potency of venoms from animals, in particular snakes, the limited range of cross-reactivity of anti-PLA, antibodies constitutes a practical drawback in the field of serotherapy. However, as PLA,s have a number of common structural features, we anticipated that these molecules might share non-dominant epitopes which, as shown in the case of neurotoseins (Tremeau et al., 1986), could be identified by screening a large set of specific monoclonal antibodies. The aim of the present study was therefore to search for an antibody capable of recognizing a large set of PLA,s. Two monomeric PLA,s were chosen as antigens for the present study. One is notexin, a potent presynaptic PLA, from the Australian snake Notechis scutatus scutatus (Harris et al., 1973). The other is nigexine, a cytotoxic protein from the African cobra Nuju nigricollis (Chwetzoff et al., 1988), which also possesses some presynaptic and myotoxic activities (Harvey, personal communication). The purpose of the present paper was three-fold. First, we examined the reciprocal immunological properties of the two antigens. We observed that antibodies raised against notexin recognized notexin but not nigexine, whereas those raised against nigexine recognized both nigexine and notexin. Second, we investigated the properties of antibodies obtained after successive immunization by nigexine and notexin. We observed that the affinity of cross-reacting antibodies for notexin was substantially higher than that of antibodies present in anti-nigexine antiserum. Third, taking advantage of this observation we prepared hybridomas using spleen cells from mice immunized with both antigens, and screened the hybridomas



with the two antigens. A monoclonal antibody was thus obtained which recognized not only notexin and nigexine but also most PLA:s present in venoms from cobras and sea-snakes. The properties of this monoclonal antibody are presented.



Purification of‘ toxin antigens and iodination Notexin was purified from the venom of Notechis scutatus scutatus (Australian Reptile Park, Australia) according to Karlsson et al. (1972) using a Biorex-70 ion exchange column (Bio-Rad, Richmond, CA), to which a concave gradient of 0.09-1.4 M ammonium acetate was applied. The chromatographic fraction referred to as notexin (Karlsson et al., 1972) is composed of two main components, which can be separated by reversephase high-performance liquid chromatography (RPHPLC) (Waters, Milford, MA), using an acetronitrile gradient in 0.1% trifluoroacetic acid (TFA) as described elsewhere (Mollier et a/., 1990). The iodination of the main fraction (notechis Nr) of notexin was performed using Iodogen (Fraker and Speck, 1978). Experimental conditions were designed to favor mono-iodinated derivatives, using 3.5 nmol of a mixture of [‘251]Na (1.1 GBq, Oris Industries, Saclay, France) and INa for 7 nmol of notechis N, (100 pg). Mono-iodinated forms were separated from unreacted material and poly-iodinated forms using a Nucleosil butyl large-pore column (SFCC, NeuillyPlaisance, France) by HPLC. An acetonitrile gradient in 0.1% TFA was used: 2432% CH,CN for 120 min at a flow rate of 1 ml/min. The specific radioactivity of the mono-iodinated form obtained was 3.7 or 1.85 TBq/mmol. The iodinated product and native notexin had identical far-uv. circular dichroism spectra and in vivo toxicity (equal LD~” values in mice) (data not shown). Nigexine was purified from the venom of Naja nigricollis (Institut Pasteur, Paris) according to the procedure previously described (Chwetzoff et al., 1988). In brief, the venom was fractionated on a Biorex-70 column to which a linear gradient of 0.05-1.4 M ammonium acetate was applied. The most basic phospholipase A> was rechromatographed by HPLC on a butyl large-pore column using an acetonitrile gradient in 0.1% TFA. Radioiodination of nigexine with “‘1 was performed at room temperature using the chloramine-T method (Hunter and Greenwood, 1962), with 37 MBq of radioactive [12’I]Na for 75 nmol of nigexine in the presence of 75 nmol of chloramine-T. “‘I-Mono-labeled nigexine (3.7 TBq/mmol) was then separated from native nigexine and from other nigexine derivatives using RP-HPLC on a Nucleosil butyl large-pore column. This derivative had the same esterase activity and LD~~ in mice as native nigexine.


Other toxins and venoms Purified PLA, s from Bungarus multicinctus, Crotalus durissus durissus, Crotalus adamanteus, bee venoms, and pig pancreas were from Sigma Chemical Co. (St Louis, MO). Crotoxin subunits CA and CB from Crotalus durissus terrificus were kindly given to us by Dr G. Faure (Institut Pasteur, Paris). Naja mossambica mossambica CM II and CM III were kindly provided by Dr P. Marchot (U172 INSERM/ UAI 129 CNRS, Marseille). Guinea-pig alveolar macrophage and rat lymphocyte PLA,s were generous gifts from Dr J. Masliah (UA 524 CNRS, Paris). Aipysurus laevis venom was purchased during an expedition in Swain Reef. Naja melanoleuca, Naja haje, Naja oxiana and Naja kaouthia venoms were from Latoxan (Rosans, France). All others venoms were generously provided by Dr G. Mengden (Australia). Mouse immunization and monoclonal antibody (mAb) production BALB/c mice (IFFA CREDO, Lyon) were immunized with notexin alone, nigexine alone or both. The protocol described in Table 1 refers to the double immunization with nigexine and notexin. For immunization with a single PLA,, mice were injected according to the protocols for notexin alone or nigexine alone. Emulsions of Freund’s adjuvants (Difco Laboratories, Detroit, MI) were l/l and 3/5 (v/v) in physiological saline for nigexine and notexin, respectively. Preparation of’ antisera. Three days after the last booster injection, antiserum was withdrawn from the tail vein. Preparation of‘ monoclonal antibodies. All cell culture products were obtained from Seromed (Berlin). The fusion procedure was carried out according to Kiihler and Milstein (1975), 3 days after the last booster injection. Spleen cells (lo*) were mixed with 2 x lo7 X-63 NA-1 myeloma cells in the presence of polyethylene glycol (PEG) 4000 at a final concentration of 50% (w/v). After removal of PEG, cells were resuspended in hypoxanthinee aminopterin-thymidine selective medium and distributed in 24-well plates (Nunc, Roskilde, Denmark) containing BALB/c mice peritoneal macrophages. Cells were cultured with selective medium until disappearance of myeloma cells. Hybrid-containing wells were tested by radioimmunoassay (RIA) for anti-PLA? antibodies. Positive wells were cloned by limiting dilution in 96-well plates and tested by RIA. Monoclonal hybridoma cells producing anti-PLA, antibodies were injected intraperitoneally into BALBjc mice (5 x 10” cells per mouse). MAbs were purified by ammonium sulfate precipitation and ion-exchange HPLC on a polyvinylimidazole column (SFCC) using an Na,SO, gradient. The purity of the monoclonal antibody preparations was verified by SDS-PAGE (Phast system, Pharmacia).

A common epitope in elapid venom phospholipases A,



Two systems were routinely used. The PEG system was used with [1251]notexin,whereas double-antibody RIAs appeared to be more appropriate in the case of [“‘I]nigexine. PEG radioimmunoassay experiments with notexin.

RIAs were carried out using PEG 6000 at a final concentration of 10% (w/v) to precipitate the antigen-antibody complexes. All components were diluted in 0.05 M sodium phosphate buffer, 0.05% Tween-20 (pH 7.4). [1251]Notexin (100 ~1, 20,00Ocpm), 100~1 of antibody solution, 10~1 of normal horse serum (Nordic, Tilburg, The Netherlands) and 100 ~1 of buffer or dilutions of non-labeled competitors were mixed and incubated overnight at 4°C. PEG (300~1, 20%) was then added to the solution, which was stirred and centrifuged at 2000g and 4°C for 30min. Supernatants were discarded and radioactivity in the pellets was counted using a gamma counter (LKB, Bromma, Sweden). Double-antibody radioimmunoassay experiments with nigexine. The assay was performed in 0.1%

Tween-20, 0.1% bovine serum albumin, 0.02% 50 mM sodium phosphate (pH 7.4). NaN,, [‘251]Nigexine (100 ~1, 20,000 cpm), 100 ~1 of antibody solution and 100 ~1 of buffer or various dilutions of unlabeled competitor were mixed and incubated overnight at 4°C. The antibodies were then precipitated by adding rabbit antiserum to mouse immunoglobulins. Normal mouse serum (Nordic) (100 ~1, l/200) in an 8% PEG 6000 solution (Marsh et al., 1984) and 100 ~1 of rabbit anti-mouse immunoglobulins (Nordic) (dilution of l/7) in 0.2% Tween-20, 0.3 M NaCl, 0.01% NaN,, 0.1 M potassium phosphate (pH 7.4) were added. The mixture was further incubated for 60min, at room temperature, diluted with 2ml of phosphatebuffered saline containing 0.2% Tween-20 and centrifuged (2000g for 40min.). The supernatant was decanted and the radioactivity in the pellet was measured. In all experiments, the mean standard variation was +3% and the background varied from 5 to 10% of the total radioactivity with both radioactive antigens. In all competition experiments, antibodies were used at a constant concentration corresponding to their titer. The titer was previously determined as the dilution that binds half of the total available radioactivity (20,000 cpm/tube). Assays for toxin activity Toxicity in vivo. Toxins were injected into the tail vein of BALB/c mice (weight 20 f 2 g) in a final volume of 150 ~1 of physiological saline. Injected mice were examined 24 and 48 hr, and 1 week after injection. For neutralization experiments, antibodies were dialyzed against 20 mM Tris buffer (pH 7.7) and incubated with either nigexine or notexin overnight at 4°C before injection.






Io-8 [NI’i3EXlNEl








&++--.AT--r---y---r 10-e

10-4 ‘. 10-10






I 10-6




Fig. I. Recognition of notexin and nigexine by antisera raised against notexin, nigexine, or both. Competition experiments were performed between [‘251]notexin(A and B) or [‘251]nigexine(C and D) and unlabeled notexin and nigexine, for the binding to three antisera. A, Antiserum raised against notexin alone; +, antiserum raised against nigexine alone; n , antiserum raised against both nigexine and notexin. The relative antibody binding BIB, was determined as the ratio (%) of radioactivity bound by antibodies in the presence of competitor (B) to the radioactivity bound in control standard confining [“sI]antigen and no competitor (B,). The specific radioactivities of [‘2SI]notexin and [‘251]nigexine were 1.85 and 3.7 TBq/mmol, respectively. Antibody titers determined with [‘251]notexinwere t/200, \!I00 and l/l200 for anti-notexin, anti-nigex~ne, and anti-nigexine~notexin antisera, respectively. Antibody titers determined with [‘2SI]nigexine were l/2000 and l/1200 for anti-nigexine and anti-nigexine-notexin antisera, respectively.

&erase a&z&y in vitro. Enzymatic assays were performed using a titrimetric method according to Halpert and Karlsson (1976) with a Metrohm pH-stat apparatus (Her&au, Switzerland). Egg yolk phosphatidyi~hoIine {type IX-E, Sigma) was sonicated under nitrogen for 30min and then mixed with sodium deoxychoiate in a ~Phosphatid~ichoIine~~ [deoxycholate] ratio of 3.7. The phosphatidylcholine was diluted to 45 mM in assay mixtures containing 20 mM CaCl, and 120 mlM NaCl. The enzyme to be tested was then added and the enzymatic reaction was monitored by titration of the fatty acids released with 0.01 M NaOH, at 4O’C (pH = 8.0). For inhibition experiments, antibodies were dialyzed against 20 mM CaCI, and 120 mM NaCI, and enzymatic activities of notexin and nigexine were assessed in the presence or absence of antibodies.


Three antisera were obtained after immunization of mice against notexin, nigexine, or both. The immunization protocol with the highly toxic notexin consisted of injections of snblet~~I doses in physiological saline, followed by injections with mixtures containing a small proportion of Freund’s incomplete adjuvant. The double-immunization procedure first involved injection of the less toxic nigexine, and then injection of notexin (Table I). Titers (see Materials and Methods} of polyclonal antisera were measured using ‘151-labeled tracers of the same specific radioactivity (3.7 TBq/mmol). Titers were routinely l/2000 for anti-nigexine antisera, I!200 for anti-notexin antisera and 1,’1200 with both tracers for

A cornmen epitope in elapid venom phospholipases double-immunization antisera (data not shown). The higher immunization doses of nigexine could explain the higher titers obtained for anti-nigexine antisera. Recognition of notexin and nigexine by polyclonal antisera raised against notexin, nigexine, or both The ability of each antiserum to recognize notexin and nigexine was evaluated by homologous competition experiments with both tracers (Fig. 1A and 1C) and by heterologous competition experiments (i.e. [“‘I]notexin against unlabeled nigexine, and [‘251]nigexine against unlabeled notexin) (Fig. 1B and ID). As shown in Fig. lA, the binding of [‘2SI]notexin to anti-notexin antiserum was inhibited in a dosedependent manner by unlabeled notexin. In contrast, it was not inhibited by unlabeled nigexine (Fig. lB, curve a). The anti-notexin antiserum therefore contained specific anti-notexin antibodies, but no detectable nigexine cross-reacting antibodies. This was confirmed by the failure of anti-notexin antiserum to bind [‘2SI]nigexine (Fig. 1C and 1D). The binding of [r2’I]nigexine to anti-nigexine antiserum was specifically inhibited by unlabeled nigexine (Fig. 1C) but also, to a certain extent, by high concentrations of unlabeled notexin (Fig. lD, curve c). In contrast to anti-notexin antiserum, anti-nigexine antiserum therefore contained not only antibodies that specifically recognized nigexine, but also antibodies that cross-reacted with notexin. Direct binding of these cross-reacting antibodies to [‘251]notexin is shown in Fig. 1A and lB, curve c. It must be stressed that anti-nigexine antibody titer (see Materials and Methods) with [‘2SI]notexin fell to l/100, whereas it was equal to l/2000 with [rZ51]nigexine. “Double” antiserum raised against the two antigens contained specific anti-nigexine and anti-notexin antibodies (Fig. 1A and 1C). As shown in Fig. lB, curve b, the binding of [i2’I]notexin to the “double” antiserum was partially inhibited by unlabeled nigexine, and reciprocally, the binding of [‘251]nigexine to the “double” antiserum was partially inhibited by unlabeled notexin (Fig. lD, curve b). The “double” antiserum therefore contained antibodies that bound both notexin and nigexine. Of interest, however, is the observation that curves b and c in Fig. 1B and 1D are different. These differences suggest that the antibodies cross-reacting with nigexine and notexin in “double antiserum” had a lower affinity for nigexine (Fig. 1B) and a higher affinity for notexin (Fig. 1D) than those in the anti-nigexine antiserum. Preparation and reactivity of monoclonal antibodies As “double” antiserum contained cross-reacting antibodies, we attempted to obtain monoclonal crossreacting antibodies from double-immunized mice. After two fusions, two hybridomas were selected by RIA for secretion of mAbs which recognize both [“‘I]notexin and [“‘IJnigexine. As the two mAbs (MN, and MN,) had similar properties, only MN, is



described in this paper. MN, belongs to the IgG,K subclass isotype. The specific binding of [‘251]notexin or [‘251]nigexine to MN, was measured and the values were used to construct Scatchard plots (Fig. 2). Equilibrium dissociation constants for MN, derived from the slopes of these plots were 1.4 and 0.9 nM for [“‘I]notexin (Fig. 2A) and [i2’I]nigexine (Fig. 2B), respectively. In both cases, straight lines were obtained, which indicates that the antibody binds to a single class of sites. MN, recognizes several other PLA,s As shown in Table 2, we examined the capacity of MN, to recognize PLA,s other than the immunizing antigens and non-PLA, proteins, by competition

A c .-

Kk= 1.4nM


1 Bound










lOOO > 1000 > 1000 > 1000 > 1000 > 1000 >I000 > 1000 > 1000

Purified toxins Naja nigricollis r-neurotoxm Naja siamensis cobra toxin (toxin III)

> 1000 >I000

“Competition experiments were performed using [‘*‘I]notexin (3.7 TBq/mmol) and different PLA,s or non-PLA, toxins at concentrations varying between IO- ” and 10m4M. The mAb MN, titer was 1/50,000. *Median inhibition capacity of each competitor (I.C.,,) was defined as the molar concentration required to inhibit

50% of the binding

of [‘*SI]notexin to MN,.

experiments using [“‘I]notexin. MN, failed to recognize non-PLA, proteins, such as Naja nigricollis a-neurotoxin or Naja siamensis cobra toxin. All the PLA,s recognized (median inhibition capacity ranging between 6 and 500 nM) originated from snake venoms, and MN, did not bind to PLA, from other sources (bee venom, porcine pancreas, rat lymphocyte or guinea-pig alveolar macrophage). Moreover, we observed that the PLA,s recognized by MN, were all isolated from snakes of the same phylogenic family, namely Elapidae, according to Underwood’s classification (1979). To confirm this result, different crude venoms were also studied (Table 3). The Elapidae family is divided into three Elapinae, subfamilies: morphologically distinct Laticaudinae and Hydrophiinae (Underwood, 1979). Venom samples belonging to each subfamily were recognized by MN,. The overall results show that 25 different venoms from Elapidae from Africa, Asia, Australia, North and South America, and the Pacific seas were recognized by MN,. Venoms that do not contain PLA, were not recognized et al., by MN,, i.e. Dendroaspis genus (Karlsson 1985). /I-Bungarotoxin purified from the elapid Bungarus multicinctus was an exception and failed to bind MN, (Table 2). /I-Bungarotoxin contains a subunit that resembles PLA, but which is clearly atypical and differs both in sequence and disulfide bridges from other PLA,s (Kondo et al., 1982). However, crude Bungarus multicinctus venom was nevertheless recognized by MN,, which indicates that the venom contains at least one additional PLA, different from P-bungarotoxin. Indeed, the presence of a monomeric PLA, in the venom of Bungarus multicinctus has been reported (Kondo et a/., 1981).

Effects of MN, on notexin and nigexine activities EfSect on toxicity in mice. As shown in Table 4, when mice (20 + 2 g) were injected intravenously with 0.6 pg of notexin, they all died within 16 hr. Incubation of this dose of notexin before injection with MN,, even in a 700-fold molar excess, did not abolish the lethal effect. In contrast, anti-notexin polyclonal antibodies in 200-fold molar excess neutralized the same dose of notexin. The antibodies alone had no effect. In similar experiments (not shown), we observed that MN, was not able to neutralize 12 pg of nigexine/20 g mouse (LDIOO value of nigexine = 10.5 pgg/20 g mouse). Efict on esterase activity. Figure 3 shows the effect of MN, on the apparent maximal rate of phosphatidylcholine hydrolysis by either notexin (A) or nigexine (B). MN,, at a lo-500-fold excess, inhibited 50 and 54% of the esterase activity of notexin and nigexine, respectively. An unrelated monoclonal antibody of the same IgG, class was unable to inhibit the esterase activity of notexin. Anti-notexin polyclonal antibodies, on the other hand, inhibited 96% of the esterase activity of notexin.


Although notexin from Notechis scutatus scutatus and nigexine from Naja nigricollis are both monomeric PLA,s with 56% primary structure homology (Halpert and Eaker, 1975; Chwetzoff et al., 1989), we observed that anti-notexin antibodies were unable to recognize nigexine. This is in agreement with the findings that anti-notexin antibodies are highly specific for notexin and its closely related isoforms from the venoms of snakes belonging to the genus Notechis (Mollier et al., 1989). In sharp

A common epitope in elapid venom phospho~~~ses A,


Table 3. Comaetition between 1”‘Ilnotexin and different crude venoms for bindine to mAb MN,O Farnilv


Genera and saecies

Ic.” (en/ml)

Elapidae Elapinae Notechis scutatus scutatus Note&is ater kangaroo Note~his ater ater Norechis ater serventyi Notechis occident& Pseudonaja nuchalis Pseudon~jo butleri Pseudechis papuanus Hoplocephalus stephensii Acantophis antarricus Acontophis barkiey oxyrrranus s~ute~iat~s Micropeches Bungarusfmciatus Sungarus muiticincrus 5o~le~gerin~ ~~uiot~ Boulengerina christyi Naja melanoleuca melmtoleuca Naja meianoleuca subfuva Naja ~igricol~isnigricollis Naju haje (Mali) Naja oxiana Hemachatus haemachatus Wecrurus corallinus Dendroospis viridis Dendroaspis polylepis


0.1 0.1 0.4 50.0

1000 0.6 1.0 160 8.0


15.0 3.2 8.2 210 520 85.0 2.3 1.4 0.3 41.0 1.0 2500 0.6 > 20,000 > 20,000

Laticaudinae Laticauda Iaticaudata


Aipysutus iaeois


Hydrophiinae Viperidae Viperinae Vipera aspis Vipero russeifi Bitis arietans

s 20,000 > 20,000 > 20,000

Agkistrodon ~hodo~to~~

> 20,000

Crotalinae crotohts



“Competition experiments were performed as described in Table 2 using [“Qnotexin (3.7TBq/mmol) and different venoms at concentrations varying between 1ng/ml and 20 mg/ml.

contrast, antibodies raised against nigexine recognized both nigexine and notexin, with a lower affinity for notexin than for nigexine. These findings indicated, therefore, that both PLA,s have common antigenic surfaces which are immunogenic in nigexine only. A similar situation was previously described by Meijer et al. (197Q who studied the immunological properties of mamm~ian PLA,s. The presence or absence of PLA, cross-reacting antibodies in a serum

depends therefore on the immunogen used, a finding of relevance to the production of antivenom antisera of broader cross-reactivity. Following successive immunization with both PLA,s, the antiserum contained antibodies crossreacting with both antigens. Interestingly, however, the antibodies had different affinities for both antigens as compared with antibodies present in the serum obtained by immunization with nigexine alone. In particular, the affinity for notexin was higher. Table 4. Effects of monoclonal and poiyclonal antibodies on in D&O Definitive evidence in favor of the existence of notexin toxicity common antigenic sites in nigexine and notexin was Survival in mice provided by monoclonal antibody studies. The iv. injection” _~~. W) monoclonal antibody MN, recognized the two antiNotexin (44 pmol)* 0 gens with comparable high affinities, indicating Notexin (44 pmol) 0 + mAb MN, (30.8 nmol) greatly similar topographies for the antigens. MN, Notexin (44 pmol) 100 probably binds near the active site of PLA, as it + polyclonal antibodies (8.8 nmol) inhibited cu. 50% of the esterase activity of both mAb MN, (30.8 nmol) IO0 Polyclonal antibodies (8.8 nmol) 100 notexin and nigexine. However, we think it is unlikely Tris buffer 20 mM, pH 7.7 (150 ~1) 100 that the binding site overlaps the active site as (1) ‘The toxicity of notexin injected into mice was measured alone or in inhibition of esterase activity was only partial, (2) the presence of mAb MN, or anti-notexin polyclonal antibodies. No fewer than four mice were used per experiment, Death MN, did not recognize all PLA,s even though they occurred within 16 hr, whereas survivors were all alive I week share a highly conserved catalytic site, and (3) the after injection. catalytic pocket is unlikely to be an antigenic area ‘Forty-four pmoles of notexin correspond to 0.6pg. The LL),~ of notexin is 0.5 ng/20 g mouse. (Caratsch et al., 1982). MN, was unable to neutralize





1 NOTEXIN 1 (nM)









Fig. 3. Effects of monoclonal and polyclonal antibodies on the enzymatic activity of notexin and nigexine. Enzymatic activities of notexin (A) or nigexine (B) were measured in the absence [O), or presence (m) of MN,. In (A), the enzymatic activity of notexin was also measured in the presence of an unrelated mAb, (+), or anti-notexin polyclonal antibodies (A). All antibodies were used at 1.8 PM. The apparent maximal velocity (V,,,,,) was determined using a constant substrate concentration (45 mM phosphatidylcholine) and enzyme concentrations ranging between 0 and 10 nM, and is expressed in PM of NaOH per min. either notexin or nigexine in vivo, indicating that the in vivo toxic activity and the in vitro enzymatic activity of nigexine and notexin are not directly correlated. MN, is likely to be specific for the native conformation of the antigen, as is the case for most monoclonal antibodies raised against whole proteins (van Regenmortel, 1986), as preliminary experiments show that it no longer recognizes notexin after reduction and S-carboxymethylation (data not shown). Purified PLA,s recognized by MN, were from elapid snake venoms. It is of interest that all venoms from elapid and hydrophid snakes known to contain PLA,s (this is not the case for mamba, i.e. Dendroaspis genus venoms) were recognized by MN,, which indicates that they all contain antigens sharing a common antigenic surface, which is unrecognized by polyclonal antibodies (Mollier et al., 1989).

In contrast, MN, did not recognize PLA,s from Viperidae, bee venoms or mammals (intracellular or from pancreatic secretion). That the spectrum of MN, recognition coincides with a phylogenetic group of PLA,s is an unexpected observation. In fact, evolutionary trees derived from theoretical sequence comparisons indicate that the evolution of PLA,s does not parallel species evolution. It has recently been shown that an intracellular PLA, from rat platelets (Hayakama et al., 1988) is closer to Viperidae PLA,s in terms of primary structure than to mammalian pancreatic extracellular PLA,. Likewise, Dufton and Hider (1983) reported that PLA,s from the Elapidae family exhibit greater homology with mammalian pancreatic PLA,s than with the PLA,s from other snakes of the Viperidae family. In addition, comparison of PLA, cores shows that PLA,s

A common epitope in elapid venom phospholipases AZ from Elapidae subgroups (Naja, Hemachatus, Bungurus) present greater homoiogy with pancreatic PLA,S than with PLA,s from the other Elapidae subgroups (Notechis, Laticauda, Enhydrina, Oxyuranus) (Dufton and Hider, 1983). Our mAb MN, reveals structural homology in the PLA, of all Elapidae subgroups, but not in pancreatic PLA,s. On the basis of polypeptide chain length homologies and the position of disulfide bonds, Heinrikson er al. (1977) classified snake venom PLA,s into two classes, i.e. Viperidae and Elapidae enzymes. Mammalian pancreatic PLA,s were classified with the Elapidae PLA,s despite some differences in primary structure, such as the presence in pancreatic enzymes of an additional fragment numbered 5746 [for residue numbering, see Renetseder et al. (198511. As we can distinguish between pancreatic and Elapidae PLA,s on the basis of their recognition by MN,, we suggest that pancreatic enzymes fall into a different class from Elapidae enzymes. The use of mAbs as structural probes allows a new approach to the classification of water-soluble PLA,s, which, until now, has been based solely on comparison of primary structures. Acknowledgements-We are grateful to Anita Diu for helpful discussions and critical reading of this manuscript. We thank Mrs Francine Georges and Sophie Van Elstraete for expert secretarial assistance. We warmly thank Dr G. Mengden for donating venoms, REFERENCES

Caratsch B. M., Maranda B., Miledi R. F. R. S. and Strong P. N. (1982) Antibodies to ~-bun~arotoxin and its uhospholipase inactive derivative. Pro; R. Sot. Lond. B: 215, 365-373. Choumet V., Jiang M. S., Radvanyi F., Ownby C. and Bon C. (1989) Neutralization of lethal potency and inhibition of enzymatic activity of a phospholipase A, neurotoxin, crotoxin, by non-pr~ipitating antibodies (Fab). FEBS Lett. 244, 167-173. Chwetzoff S., Takeshi M., Fromageot P. and Mbnez A. (1988) La phospholipase basique du venin de Nqia nigricollis est cytotoxique. C.R. Acad. Sci. Ser. III-Vie 306, 31-33. Chwetzoff S.. Tsunasawa S., Sakiyama F. and Menez A. (1989) Nigexine, a phospholipase A, from cobra venom with cytotoxic properties not related to esterase activity; purification, amino acid sequence and biological properties. J. bial. Chem. 264, 13,289-l 3,297. Dijkstra B. W., Kalk K. H., HOI W. G. and Drenth _I.(1981 Structure of bovine pancreatic phospholipase A, at 1.7 d resolution. J. mofec. Biol. 147, 97-123. Dufton M. Y. and Hider R. C. (1983) Classification of phospholipases A, according to sequence. Eur. J. Biochem. 137, 545-551. Fraker P. J. and Speck J. C. (1978) Protein and cell membrane iodinations with a sparingly soluble chloramide 1,3,4,6-tetrachloro-3a,6a diphenylglycolu~l. Biochem. Biophys. Res. Commun. 80, 849-857. Gopalakrishnakone P., Hawgood B. J. and Theakston R. D. G. (1981) Specificity of antibodies to the reconstituted crotoxin complex, from the venom of South American rattlesnake (Crotalus durissus terrf$?cus),using enzyme-linked immunoabsorbent assay (ELISA) and double immunodiffusion. Toxicon 19, 13lll39. Halpert J. and Eaker D. (1975) Amino-acid sequence of a presynaptic neurotoxin from the venom of Note&is


scutatus scutatus (Australian tiger snake). J. biol. Chem. 250, 6990-6997. Halpert J. D. and Karlsson E. (1976) The role of phospholipase activity in the action of the presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake). FEBS Left. 61, 72-76. Harris J. B., Karlsson E. and Thesleff S. (1973) Effects of an isolated toxin from Australian tiger snake (Nofechis scutatus scufatus) venom at the neuromuscular junction. Er. J. Pharmac. 47, 141-146. Hayakama M., Kudo I., Tomita M., Nokima S. and Inoue K. (1988) The primary structure of rat platelet phospholipase A,. J. Biochem. 104, 7617772. Heinrikson R. L., Krueger E. T. and Keim P. S. (1977) Amino acid sequence of phospholipase AZ-~from the venom of Crotalus adamanteus. J. biol. Chem. 252,49134921. Hunter W. H. and Greenwood F. C. (1962) Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature, Lond. 194, 495-496. Karlsson E., Eaker D. and Ryden L. (1972) Purification of a presynaptic neurotoxin from the venom of the Australian tiger snake Notechis scutatus scutafus. Toxicon 10, 4055413. Karlsson E., Mbugua P. H. and Rodriguez-~thurraide D. (!985) Anticholinesterase toxins. Pharmac. Ther. 30, 2599276. Kohler G. and Milstein C. (1975) continuous cultures of fused cells secreting antibody of predehned specificity. Nature, Lond. 256, 495-497. Kondo K., Toda H. and Narita K. (1981) ~~fication and characterization of phospholipase A from Bungarus mulficinctus venom. J. Biochem. 89, 3147. Kondo K., Toda H., Narita K. and Lee C. Y. (1982) Amina acid sequences of three beta-bungarotoxins (beta 3-, beta 4- and beta 5-bungarotoxins) from Bungarus mulficinctus venom. Amino acid substitutions in the A chains. J. Biochem. 91, 1531-1548. Marsh D., Grassi J., Vigny M. and Massoulie J. (1984) An immunological study of rat a~tylcholinesterase: comparison with acetylcholinesterases from other vertebrates. J. Neurochem. 43, 204-213. Meijer H., Meddens M. J. M., Dijkman R., Sfotboom A. J. and de Haas G. H. (1978) Immunological studies on pancreatic ohosuholioases A,. J. biol. Chem. 253, 85648569. Menez A: (1985) Molecula> jmmunolo~ of snake toxins. Pharmac. Ther. 30, 9!- 113. Mollier P., Chwetzoff S., Bouet F., Harvey A. L. and Menez A. (1990) Tryptophan 110, a residue involved in the toxic and presynaptic activities but not in the enzymatic activity of notexin. Eur. J. Biochem. (in press). Mollier P., Chwetzoff S., Frachan P. and Minez A. (1989) Immunoiogica! properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Nofechis scufatus. FEBS Lett. 250, 479-482. Ramlau J., Bock E. and Fohlman J. (1979) Production of antivenom against detoxified taipoxin and immunochemical analysis of the alpha, beta and gamma subunits. Toxicon 17, 43-54. van Regenmortel M. H. V. (1986) Which structural features determine protein antigenicity? 7’IB.SII, 36-39. Renetseder RR., Brunie S., Dijkstra B. W., Drenth J. and Siegler P. B. (1985) A comparison of the crystal structures of phosphoiipases A, from bovine pancreas and Crotaius atrox venom. J. biol. Chem. 260, !!,627-11,634. Tremeau O., Boulain J. C., Couderc J., Fromageot P. and Mtnez A. (1986) A monoclonal antibody which recognized the functional site of snake neurotoxins and which neutralizes all short-chain variants. FEBS Left. 208, 236240. Underwood G. (1979) Classification and distribution of venomous snakes in the world. In Snake Venoms (Edited by Lee C. Y.), pp. 15-40. Springer-Verlag, Berlin.

A monoclonal antibody recognizing a conserved epitope in a group of phospholipases A2.

Notexin and nigexine are monomeric phospholipases A2(PLA2s) from the venoms of Notechis scutatus scutatus and Naja nigricollis, respectively. Polyclon...
973KB Sizes 0 Downloads 0 Views