Biochimica et Biophysica Acta 1339 Ž1997. 253–267
Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization a c Yveline Frobert a , Christophe Creminon , Xavier Cousin b, Marie-Helene , ´ ´ ` Remy ´ d e b a,) Jean-Marc Chatel , Suzanne Bon , Cassian Bon , Jacques Grassi a
CEA, SerÕice de Pharmacologie et d’Immunologie, DRM, batiment 136, Centre d’Etudes de Saclay, 91191 Gif sur YÕette Cedex, ˆ France b Unite´ des Venins, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France c Laboratoire de Pharmacologie et de Toxicologie Fondamentale, CNRS Unite´ 8221, Toulouse, France d INRA r CEA, SerÕice de Pharmacologie et d’Immunologie, DRM, CE r Saclay, 91191 Gif sur YÕette Cedex, France e Laboratoire de Neurobiologie, Ecole Normale Superieure, 75005 Paris, France ´ Received 23 October 1996; accepted 8 January 1997
Abstract We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes Ž Elapidae, Viperidae and Crotalidae.. We found high acetylcholinesterase ŽAChE. activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent ŽMPT. revealed that the AChE of all venoms have similar turnovers Ž6000 to 8000 sy1 . which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site Žedrophonium, tacrine. and at the peripheral site Žpropidium, fasciculin., as well as bis-quaternary ligands ŽBW284C51, decamethonium., were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition ŽIC 50 4 10y6 M., while Bungarus ŽIC 50 f 10y8 M. and especially Ophiophagus ŽIC 50 - 10y1 0 M. AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody ŽElec-410. previously described as a specific ligand for the
Abbreviations: AChE, acetylcholinesterase ŽEC 3.1.1.7.; BSA, bovine serum albumin; BuChE, butyrylcholinesterase ŽEC 3.1.1.8.; BW284C51, 1, 5-bisŽ4-allyldimethylammonium-phenyl.pentan-3-1-dibromide; EIA, enzyme immunoassay; MPT, O-ethyl S-w2-Ždiisopropylamino.ethylx methylphosphonothioate ) Corresponding author. Fax: q33 169 085907. E-mail:
[email protected] 0167-4838r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 0 0 9 - 5
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Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410. Keywords: Acetylcholinesterase; Snake venom; Butyrylcholinesterase
1. Introduction Acetylcholinesterase ŽAChE, EC.3.1.1.7. plays a critical role in cholinergic transmission by rapidly inactivating the neurotransmitter, acetylcholine. AChE belongs to the cholinesterase family which also includes butyrylcholinesterase ŽBuChE, EC.3.1.1.8.. Both enzymes hydrolyze choline esters faster than any other substrates and are inhibited by physostigmine Ž a natural carbamate alkaloid, also called eserine.. AChE differs from BuChE in that it is more active on acetylcholine than on propionyl or butyrylcholine. It is also characterized by excess substrate inhibition, which is not observed with BuChE. In addition, the two enzymes may be distinguished by their sensitivity to reversible inhibitors, specific either for AChE ŽBW284C51. or for BuChE Žethopropazine.. The catalytic center of AChE was traditionally considered as composed of an esterase subsite and an anionic subsite w1,2x. The esterase subsite contains the active serine residue which is the target of irreversible organophosphorous and carbamate inhibitors, leading to the formation of phosphorylated or carbamylated enzymes. The anionic subsite is responsible for the correct positioning of the substrate in the active site and constitutes the binding site of positively charged reversible inhibitors Žedrophonium, tacrine. . There is an allosteric regulatory site, called the ‘anionic peripheral site’, about 2 nm from the catalytic site w3,4x. Propidium, gallamine, Žq.-tubocurarine and fasciculin Ža snake venom toxin w5,6x. have been reported to bind specifically to this peripheral site. Strong reversible bis-quaternary inhibitors, such as BW284C51, decamethonium and ambenonium, can bind simultaneously to the active and peripheral sites w7,8x. Recently, both crystallographic and site-directed mutagenesis studies have shown that the active site is located at the bottom of a deep and narrow gorge
Žabout 2 nm long. lined predominantly with aromatic residues. It was hypothesized that this complex array of aromatic groups provides a guidance mechanism, facilitating the two-dimensional diffusion of acetylcholine from the surface of the protein to the catalytic site w9x. The peripheral site is located at the surface of the protein, close to the rim of the gorge, and the inhibitory effect of peripheral site ligand appears to result from an allosteric transition relayed by aromatic residues of the gorge from the peripheral site to the active site w10,11x. Excess substrate inhibition could be due to the same mechanism, as a consequence of the binding of acetylcholine to the peripheral site at high substrate concentration w12,13x. It has also been shown that the catalytic site does not contain an ‘anionic sub site’ sensu stricto and that the correct positioning of the substrate is due to the interaction of its quaternary ammonium group with aromatic residues Phe330 and Trp84 in Torpedo enzyme w7,9,14,15x. AChE presents a large variety of molecular forms, composed of homo- and hetero-oligomers containing catalytic subunits of similar catalytic activity. Globular forms Žnamed G1, G2 and G4. contain one, two or four catalytic subunits, while asymmetric forms Žnamed A4, A8 and A12. are characterized by the presence of a collagen-like tail which is associated with one, two or three tetramers. In addition, a distinction is currently made between amphiphilic and non-amphiphilic globular forms, depending on the presence of a hydrophobic domain responsible for anchoring the enzyme in membranes. The amphiphilic character is experimentally defined as the capacity of a given molecular form to bind detergent micelles under non-denaturing conditions, leading to significant modification of its hydrodynamic properties Žfor details see Massoulie´ et al. w16x.. In the case of vertebrates, it has been clearly demonstrated that AChE is encoded by a single gene and that the various molecular forms are generated by mRNA alternative splicing and post-translational modifica-
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tions. For instance, in Torpedo, alternative splicing produces two major types of coding sequences, encoding subunits which differ only in their C-terminal region, called H Žhydrophobic. and T Žtailed.. H subunits are processed into amphiphilic glycolipidanchored dimers while T subunits produce nonamphiphilic asymmetric and G4 forms, as well as amphiphilic G1, G2 and G4 forms. Studies on AChE have been considerably facilitated by the availability of fish electric organs, which are rich in cholinergic synapse components, including AChE. As a consequence, many of the decisive data concerning AChE, i.e., biochemical characterization w17,18x, molecular cloning w19,20x and crystallographic studies w9x, were obtained from Electrophorus electricus or Torpedo enzymes. The presence of AChE in snake venoms was first reported in 1938 w21x. AChE activity was found in Elapidae venoms but not in Viperidae and Crotalidae venoms w22x. Studies of AChE from the venoms of Bungarus fasciatus w23–25x and Naja naja oxiana w26–29x have demonstrated that the enzyme is particularly abundant in Bungarus venoms Ž 1–3 mgrg of dry venom. and is highly active Ž) 60 000 Ellman unitsrmg.. The Bungarus enzyme was first described as a non-amphiphilic dimer w23x, while more recent investigations indicated that it is a nonamphiphilic monomer w25x as the enzyme from Naja naja oxiana venom w26x. In the present study, we re-examined in detail the biochemical, immunological and enzymatic properties of AChE in various elapid venoms. In agreement with previous reports, we found that most elapid venoms contain high levels of a very active nonamphiphilic monomeric AChE. In general, the enzymes from different species have similar immunological and catalytic properties, but we found that they may differ very significantly in their sensitivity to peripheral site ligands, in particular fasciculin.
2. Materials and methods 2.1. Reagents and buffers Dry venoms were from the Unite´ des Venins, Institut Pasteur, France. Most were pooled venom samples from different snakes of the same species.
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They were reconstituted by adding 1 ml of 0.1 M phosphate buffer ŽpH 7.4. to 100 mg of dry venom with gentle stirring. O-Ethyl S-w2-Ž diisopropylamino . ethylx methylphosphonothioate ŽMPT. was kindly provided by the Centre d’Etudes du Bouchet Ž Vert-le-Petit, France. . Fasciculin 2, purified from Dendroaspis angusticeps venom, was a generous gift from Dr. ŽCEA, Departement Renee d’Ingenierie et ´ Menez ´ ´ ´ . d’Etudes des Proteines, C.E. Saclay, France . Unless ´ otherwise stated, all reagents were of analytical grade from Sigma ŽSt Louis, MO.. EAH-Sepharose 4B was from Pharmacia ŽSweden.. All reagents used for immunoassays were diluted in the following buffer ŽEIA buffer.: 0.1 M phosphate ŽpH 7.4., 0.15 M NaCl, 0.1% BSA, 0.01% sodium azide. The washing buffer used in enzyme immunoassays was 0.01 M phosphate buffer ŽpH 7.4. containing 0.05% Tween 20. 2.2. Purification and assay of AChE AChE from Elapidae venoms was purified by affinity chromatography on an m-carboxyphenyldimethylethylammonium-EAH Sepharose 4B column, as previously described w30,31x. For instance, Bungarus fasciatus AChE was purified as follows: 2 ml of reconstituted Bungarus fasciatus venom containing about 100 000 Ellman unitsrml was applied to an affinity column Ž2 ml. equilibrated with 0.1 M phosphate buffer pH 7.4. The gel was successively washed with 10 ml of this buffer and 10 ml of 0.1 M phosphate buffer Ž pH 7.4. containing 0.25 M NaCl. Retained AChE activity was eluted with 0.1 M phosphate buffer pH 7.4 containing 0.4 M NaCl and 0.02 M decamethonium bromide. Fractions of 1 ml were collected. Absorbance at 280 nm was determined and enzymatic activity was assayed as indicated below. Active fractions were pooled and extensively dialyzed Ž72 h. at 48C against 0.1 M phosphate buffer ŽpH 7.4.. The same procedure was used to purify AChE from 11 batches of venom: 3 venom batches from Bungarus fasciatus and 1 venom batch from each of the following species: Bungarus multicinctus, Bungarus caeruleus, Haemacatus haemacates, Naja haje, Naja kaouthia, Naja naja, Naja niÕea and Ophiophagus hannah.
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AChE activity was measured by means of the colorimetric method of Ellman w32x using 0.75 mM acetylthiocholine iodide as substrate, with 0.25 mM dithiobis nitrobenzoic acid ŽDTNB. in 0.01 M phosphate buffer ŽpH 7.4.. One Ellman unit was defined as the amount of enzyme producing an absorbance increase of 1 unit Ž412 nm. at 258C, in 1 min, in 1 ml of medium and for an optical path length of 1 cm. 2.3. Inhibition assays Inhibition tests were performed in 96-well microtiter plates. In most experiments, inhibitors and AChE Ž2.5 Ellman unitsrml. were diluted in 0.001 M phosphate buffer ŽpH 7.4. containing 0.1% BSA. In each well, 90 m l of purified AChE Žeither from snake venom or Electrophorus electric organ, control. were mixed with 90 m l of an inhibitor solution Ž from 2 10y4 to 2 10y10 M.. After 48-h reaction at q48C, the residual enzymatic activity was measured by adding 25 m l of a freshly prepared ten-fold concentrated Ellman medium Ž7.5 mM acetylthiocholine iodide with 2.5 mM dithiobis nitrobenzoic acid in 0.1 M phosphate buffer, pH 7.4.. Absorbance of individual wells was measured at 414 nm after 10-min enzyme reaction at 208C. Duplicates were used for each concentration of inhibitor. The control enzymatic activity Žmean of 8 values. was measured by mixing 90 m l of AChE solution with 90 m l of buffer. In some experiments, inhibition was determined in high ionic strength buffer: 0.1 M phosphate Ž pH 7.4. containing 0.4 M NaCl and 0.1% BSA. 2.4. Analysis of AChE actiÕity as a function of substrate concentration DTNB, acetylthiocholine, propionylthiocholine and butyrylthiocholine solutions were prepared in 0.001 M phosphate buffer ŽpH 7.4. containing 0.1% of BSA. Serial dilutions ranging from 400 mM to 10 m M were prepared for acetylthiocholine, propionylthiocholine and butyrylthiocholine. Aliquots of these dilutions were mixed with equal volumes of 0.5 mM DTNB. A 100-m l sample of purified snake venom AChE Ž2.5 Ellman unitsrml. was mixed with 1 ml of each of these substrate media. Hydrolysis of the substrate was then monitored at 412 nm, with a Gilford STASAR III apparatus. The spontaneous hy-
drolysis of the substrate was determined in the same way in the absence of enzyme and was routinely subtracted. 2.5. Immunization procedure and production of hybridoma Antibodies against AChE from Bungarus fasciatus were raised in mice Ž Biozzi strain. using the following procedure. On day zero, purified Bungarus AChE Ž20 m g. emulsified in Freund’s complete adjuvant was injected in the foot pad. The mice were bled two weeks later. Murine anti-AChE antibodies were detected by their capacity to bind Bungarus fasciatus venom AChE Žsee below.. Booster injections Ž20 m g. in the foot pad were given on days 21 and 35. Bleedings were done one week after each booster injection. Mice with the lowest EIA titer were selected for preparation of monoclonal antibodies. Three days before fusion they received a final booster injection Ž9 m g AChE, i.v. administration. . Spleen cells were collected and fused with NS1 myeloma cells as previously described w33x. Anti-Bungarus AChE antibodies in hybridoma culture supernatants were detected using an enzyme immunoassay ŽEIA, see below.. 2.6. Labeling of monoclonal antibodies with biotin Monoclonal antibodies ŽmAbs. were purified from ascitic fluids by successive precipitations with caprylic acid and ammonium sulfate w34x. The purity of the preparation was assessed by polyacrylamide gel electrophoresis under denaturing conditions. Biotin was covalently linked to the mAbs by reaction of an activated N-hydroxysuccinimide ester of biotin with amino groups of the purified mAbs. Briefly, to 1 mg of mAb dissolved in 1 ml of borate buffer ŽpH 9. were added 50 m l of 4 mgrml biotin-NHS in DMF. After 30-min reaction at room temperature, 4 ml of EIA buffer were added. Biotin-labeled mAbs were stored at y208C until used. 2.7. Enzyme immunoassays (EIA) In order to assay anti-AChE antibodies in antisera or in culture supernatants of hybridomas, 96-well microtiter plates were coated with specific second
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antibodies Žgoat anti-mouse IgG, w33x.. The test was performed in a total reaction volume of 100 m l:50 m l of each of two components, Bungarus AChE and antibodies. The concentration of Bungarus AChE was generally 2 Ellman unitsrml. After 18-h immunoreaction at q48C, the plates were washed and solid-phase bound AChE was measured using Ellman’s medium as described above. Immunoreactivity of AChE from other Elapidae venoms was determined by the same procedure using dilutions of crude venoms Ž5 Ellman unitsrml.. Non-specific binding Žnsb. was measured under the same conditions, in the absence of antibodies, and was - 0.02 absorbance units. We determined the capacity of pairs of mAbs to bind simultaneously to AChE by an immunometric assay, as previously described w34x. Bungarus AChE solution Ž100 m l of 10 Ellman unitsrml. was added to microtiter plate wells coated with one mAb. After 1-h reaction at room temperature with continuous agitation, 100 m l of a 10 m grml solution of another biotin-labeled mAb were added. After 18-h reaction at 48C, plates were washed before addition of 200 m l of an avidin-peroxidase conjugate solution ŽSigma, St Louis. . After a further 4-h incubation at room temperature, the plates were washed and peroxidase activity was measured using ortho-phenylene diamine as substrate. 2.8. Chromatographic characterization of Õenom AChE Molecular sieve chromatography was performed on a Superose 12 ŽHR 10r30. gel filtration column ŽPharmacia, Sweden. equilibrated in EIA buffer using FPLC equipment. AChE-containing samples Žeither purified or crude venom. Ž2 Ellman unitsr500 m l. were injected using a flow rate of 24 mlrh and 0.8 ml fractions were collected. The AChE activity of each fraction was measured by Ellman’s method. The column was calibrated with the following markers: Electrophorus AChE ŽG4 form. , 320 kDa; radiolabeled IgG, 150 kDa; interleukin 1 a ŽIL-1 a ., 20 kDa; potassium ferricyanide Žtotal volume marker.. 2.9. Titration of AChE actiÕe sites by MPT The turnovers of the different snake venom AChEs were determined by titrating active sites with the
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organophosphorous reagent O-ethyl S-w-Ž diisopropylamino.ethylx methylphosphothioate ŽMPT., as previously described w35x. Very similar results were obtained with affinity-purified enzyme and crude venom. Briefly, 50 m l of an MPT solution Žconcentrations ranging from 2.5 10y10 M to 10y9 M. were added to 450 m l of a 5 Ellman unitsrml solution of AChE. After 2 h of reaction at room temperature, the residual AChE activity was measured using Ellman’s method. All reagents were diluted in EIA buffer. Results were plotted as a function of MPT concentration and the turnovers were calculated from the slopes of the curves. Control experiments were performed with affinity-purified Electrophorus electricus enzyme ŽG4 form. in order to check the reactivity of MTP. 2.10. Sedimentation analysis in sucrose gradients AChE was analyzed by sedimentation in gradients containing 5% to 20% sucrose in 10 mM Tris-HCl ŽpH 7.0., 5 mM MgCl 2 , in the presence of 1% Brij-96. E. coli alkaline phosphatase Ž6.1 S. and beef liver catalase Ž11.3 S. were included with the sample, as internal standards of sedimentation coefficients. After centrifugation at 36 000 rpm for 18 h at q78C in a Beckman SW 41 rotor, about 45 fractions were collected from the bottom of the tubes and assayed for the different enzymatic activities. 2.11. SDS-PAGE and immunoblotting experiments SDS-PAGE was performed using a tricine buffer, as described previously by Schagger and von Jagow w36x. For immunoblot analysis, proteins were separated by 12% SDS-PAGE and electroblotted w37x onto PVDF membrane Ž Millipore.. After blotting, unspecific protein binding sites were blocked with 1% BSA in 50 mM Tris-HCl ŽpH 8., 150 mM NaCl, 0.5% Tween 20. The PVDF membranes were incubated overnight with a 1r500 dilution of the antiAChE BUNGA-46 or BUNGA-76 mAbs Žascitic fluids. in the same buffer. After washing, the membranes were incubated for 1 h with alkaline phosphatase-conjugated anti-mouse antibody Ž1r7000. ŽPromega.. Color development was obtained by adding BCIP and NBT, according the supplier’s instructions.
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3. Results 3.1. Studies of the enzymatic actiÕity of the Õenoms The AChE activities of venoms from snakes of the Elapidae, Viperidae and Crotalidae families are shown in Table 1. Venom samples were reconstituted under the same conditions Ž100 mg of dried venomrml of buffer. in order to allow comparison of their AChE activities. As previously reported by Zeller w22x, high AChE-like activity was observed in most of the elapid venoms, whereas viperid and crotalid samples exhibited no activity. In the cases of Bungarus and Naja genera, comparison of different species and sub-species, as well as different batches, showed that Bungarus venoms
contained at least twice as much AChE activity as Naja venoms. The mean activity Ž n s 8. for Bungarus was close to 654 000 " 120 000 Ellman unitsrg of dry venom; in Naja venoms mean activity was 150 000 " 108 000 Ellman unitsrg of dry venom Ž n s 8, two Naja nigricolis samples, which contained very low levels of AChE activity were not included in this calculation.. Bungarus venoms exhibited fairly uniform AChE activity, whereas Naja samples showed large variations. This could be related either to the heterogeneity of the pooled venoms or to differences between species and sub-species. Alternatively, it may reflect lower stability of the Naja enzymes, resulting in variable loss of enzyme activity during drying or storage of venom samples. The latter hypothesis was supported by our observation that
Table 1 Listing of the different snake venoms tested in this study, measurement of AChE activity and turn-over number ELAPIDAE
CROTALIDAE and VIPERIDAE
Venom
Activity ŽEll. unitsrg.
Turnover number Žs y 1.
Venom
Activity ŽEll. unitsrg.
Bungarus fasciatus Žbatch 5. Bungarus fasciatus Žbatch 6. Bungarus fasciatus Žbatch 8. Bungarus fasciatus ŽChina 1. Bungarus fasciatus ŽChina 2. Bungarus fasciatus ŽBombay. Bungarus multicinctus Bungarus caeruleus Dendroaspis Žbatch 5. Dendroaspis Žbatch 8. Haemacatus haemacates Naja haje Žbatch 16. Naja haje Žbatch 19. Naja kaouthia Žbatch 6. Naja kaouthia Žbatch 7. Naja nigricolis Žbatch 12. Naja nigricolis Žbatch 22. Naja nigricolis Žbatch 19. Naja naja naja Naja naja atra Naja niÕea Žbatch 1. Naja niÕea ŽMiami. Ophiophagus hannah Žbatch 1. Ophiophagus hannah Žbatch 2. Ophiophagus hannah Žbatch 3.
620000 630000 620000 890000 505000 557000 666000 747000 6 6 157000 331000 282000 79700 72900 880 380 22000 89200 82000 147000 238000 84800 65800 41800
6590 6920 6240 6130 6760 6470 7570 7340 ND ND 6430 7800 7470 7770 7520 ND ND 7550 7410 7690 7690 7600 6960 4350 6390
Bitis gabonica Žbatch 10. Bitis gabonica Žbatch 12. Bitis gabonica Žbatch 16. Bitis lachesis Žbatch 5. Bitis lachesis Žbatch 12. Bitis lachesis Žbatch 18. Bothrops atrox Žbatch 2. Bothrops atrox ŽMiami. Bothrops lanceolatus Echis carinatus Crotalus durissus terrificus Crotalus durissus terrificus Vipera aspis Žbatch 3. Vipera aspis Žbatch 5. Vipera berus Žbatch 17. Vipera russeli Žbatch 7. Vipera ammodytes Žbatch 3. Vipera ammodytes Žbatch 8. Trimereserus microsquamatus Trimereserus stejnegeri
0 0 0 146 6 0 0 0 0 0 0 6 6 0 0 6 0 0 0 0
AChE-like activity was measured on reconstituted venoms as described in Section 2. Results are expressed in terms of Ellman’s units per g of dry venom. Data in parenthesis refer to the classification of the venom batches as categorized in the ‘Unite´ des venins’ ŽInstitut Pasteur..
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affinity-purified AChE from Bungarus venoms is far more stable than Naja, Ophiophagus and Haemacatus enzymes Žresults not shown. . It is not surprising that the two samples from Dendroaspis presented no significant activity since it has been shown that venoms from this species contain high levels of fasciculin, a potent reversible inhibitor of AChE. The presence of fasciculin in Dendroaspis venoms was confirmed by the fact that mixing Dendroaspis venom with Bungarus venom Žvrv. resulted in a quite total inhibition of Bungarus AChE activity Žresults not shown. . Thus, the absence of AChE activity in Dendroaspis is venoms could be due either to enzyme inhibition by endogenous fasciculin or to the absence of AChE. These alternatives will be examined below. 3.2. Affinity-purification of Õenom AChEs and characterization of their quaternary molecular stucture AChEs from 11 venom batches, representative of the different elapid genera and species, were purified
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by affinity chromatography, as described in Section 2. As an illustration, the elution profile of Bungarus fasciatus AChE is presented in Fig. 1. AChE activity was almost entirely bound to the column, with less than 0.01% recovered in the eluate. Very little activity was desorbed from the column during the washing steps, and more than 85% was eluted in the presence of 0.02 M decamethonium. The capacity of the affinity gel was estimated to be about 80 000 to 90 000 Ellman units of Bungarus fasciatus enzymerml of packed gel. The affinity-purified AChE from Bungarus Õenom was analyzed by SDS-PAGE under reducing Žnot shown. and non-reducing conditions ŽFig. 1, inset. . In both cases, we observed a single protein band at 70 kDa, illustrating the efficacy of the purification procedure and indicating that the enzyme is monomeric. This was confirmed by hydrodynamic analysis performed on sucrose gradients with crude venom samples. AChEs from all analyzed venoms sedimented as a monodisperse peak of 4.5 S, the sedimentation of which was not influenced by addi-
Fig. 1. Affinity purification of Bungarus fasciatus AChE. AChEs from snake venoms were purified by affinity chromatography as described in Section 2. Characteristic chromatographic profiles obtained with a Bungarus fasciatus venom. `: Absorbance at 280 nm, v: AChE activity ŽEllman unitsrml= 10y3 .. Inset: SDS-PAGE analysis of affinity-purified AChE under non-reducing conditions. Lanes 1 and 3: molecular weight markers, lane 2: Bungarus fasciatus AChE.
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tion of the non-denaturating detergents Triton X-100 or Brij-96 in the gradients. This is illustrated in Fig. 2 in the case of Bungarus fasciatus AChE. In molecular sieve chromatography performed on a Superose 12 column Žsee Section 2., AChE was always eluted as a single peak with an apparent molecular weight of 70 kDa, compatible with a monomeric species Ž results not shown.. Taken together, these results unambiguously demonstrate that AChEs from Elapidae venoms comprise exclusively non-amphiphilic monomers in agreement with recent observations w25,38x. The monomeric structure of venom AChE was first reported by Raba et al. w26x for Naja naja oxiana, but Kumar and Elliott w23x described Bungarus fasciatus AChE as a dimer on the basis of sedimentation velocity, diffusion coefficient and gel filtration studies. The observation that Naja AChE dimerizes at concentrations above 0.2 mgrml w26x may account for this discrepancy, but we observed that this property is not general since Bungarus AChE sediments as a monomer in sucrose gradient at a concentration of 0.34 mgrml w38x.
Fig. 2. Sedimentation analysis in sucrose gradient. Bungarus fasciatus AChE was analyzed in sucrose gradient as described in Section 2. Sedimentation profile observed in the presence of 1% Brij-96. A sedimentation coefficient of 4.5 S was deduced from the position of the internal sedimentation standards, catalase Ž11.3 S. and alkaline phosphatase Ž6.1 S.. All venom AChEs analyzed Ž Naja haje, Haemacatus haemacates and Ophiophagus hannah. gave similar profiles with sedimentation coefficients ranging from 4.3 S to 4.6 S.
3.3. Production and characterization of monoclonal antibodies directed against affinity-purified Bungarus fasciatus AChE Three mice ŽBiozzi high responder strain. were immunized with affinity-purified AChE from Bungarus venom Ž see Section 2.. The presence of antiAChE antibodies in the antisera was checked by EIA. A marked immune response was observed in all immunized animals, from the first booster injection onwards. The two mice presenting the lowest titer Žabout 1r10 5 . were selected for fusion. The corresponding spleen cells were fused with NS1 myeloma as previously described w33x. One week after fusion, about 650 wells contained actively multiplying hybridomas. The presence of mouse anti-AChE antibodies was checked in all culture supernatants by testing their capacity to bind AChE from Bungarus venom using the same EIA. Eighty-three strongly positive culture supernatants were selected. Hybridomas were subcloned by limiting dilutions, leading to 27 stable secreting lines which were then expanded as ascitic tumours. The different monoclonal antibodies ŽmAbs. were purified using successively caprylic acid and ammonium sulfate precipitations ŽSection 2.. The Ouchterlony double diffusion technique was used to determine immunoglobulin isotypes Ž Table 2.. First, the binding compatibility of mAb pairs on a single monomeric AChE molecule was examined, using biotin-labeled antibody in EIA, as described in Section 2. These experiments indicated a fairly complicated pattern of compatibility which allowed classification of the mAbs into two main groups ŽA and B., which recognized two different regions of the AChE molecule ŽTable 2. . Second, we looked for possible cross-reactivity with AChEs from other elapid venoms in the same EIA, using dilutions of crude venom instead of affinity-purified AChE Ž Table 2.. Interestingly, each of the two compatibility groups appeared quite homogeneous in terms of cross-reactivity. Most mAbs of group A recognized AChEs from all Bungarus venoms equally well, showed partial cross-reactivity with some of the AChEs from Ophiophagus venoms, but did not recognize the AChEs from Haemacatus and Naja venoms. Most mAbs of group B very significantly cross-reacted with all the venoms tested, demonstrating that the different venom enzymes share
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Table 2 Cross-reactivity of AChE from the different venoms with mAbs directed against Bungarus fasciatus AChE
Empty square: bindingf nsb. Filled square: very strong binding ŽAbsorbance) 1.. Dark grey square: strong binding Ž0.1 - Absorbance - 1.. Light grey square: low binding Žnsb - Absorbance- 0.1.. nsb s non specific binding was currently - 0.02.
common epitopes. In these experiments, however, mAb Bunga-62 Ž group A. and mAbs Bunga-7 and Bunga-22 Žgroup B. appeared atypical since they behaved like members of the opposite group. We also examined whether anti-Bungarus mAbs would cross-react with AChE from electric fishes ŽA12 and G2 forms from Torpedo marmorata, G4 form from Electrophorus electricus . or mammals ŽG4 amphiphilic form from bovine brain. and with BuChE from human serum. We never observed any significant immunoreactivity. MAb Bunga-46 and Bunga-72, which strongly cross-reacted with all the venom AChEs tested Ž Table 2., were used in immunoblotting experiments, in order to check the possible presence of inactive AChE in Dendroaspis venoms. No immunoreactive material could be detected in Dendroaspis venoms diluted 1r10, while a 70 kDa band was clearly observed in Bungarus fasciatus venom even at 1r1000 dilution ŽFig. 3A.. This strongly suggests that the very low AChE-like activity measured in Dendroaspis ven-
oms Ž Table 1. was not due to inhibition of an endogenous enzyme by fasciculin, but rather reflects the absence of AChE in these venoms. Similar experiments were performed to compare the AChE immunoreactivity measured in two Naja nigricollis venom samples containing very different enzyme activities Žbatch 19, 22 000 Ellman unitsrg of dry venom; batch 22, 380 Ellman unitsrg of dry venom; see Table 1.. Samples were diluted so that the same AChE activity was loaded on the gel. In contrast with the Bungarus enzyme, most of the immunoreactivity observed in both Naja samples was distributed in several low molecular weight bands Žfrom 10 to 30 kDa, see Fig. 3B.. A faint 70 kDa band was visible only in the more active sample. Since AChE activity was equivalent in the two samples, this indicates that the more active venom also contained more immunoreactive material. Taken together, these observations suggest that the low AChE activity found in some Naja venom batches results in proteolytic degradation by venom proteases.
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Fig. 4. Substrate specificity. Hydrolysis of acetylthiocholine ŽAcSCh; v ., propionylthiocholine ŽPrSCh, '. and butyrykhiocholine ŽBuSCh, B. by affinity-purified Bungarus fasciatus venom AChE. For both AcSCh and PrSCh, excess substrate inhibition is clearly visible for substrate concentrations above 2–3 mM. Fig. 3. Immunoblotting characterization of snake venoms using monoclonal antibody Bunga-46. A: Comparison between Bungarus fasciatus and Dendroaspis venoms. a, b, c Bungarus and d, e, f Dendroaspis venoms diluted 1r10, 1r100 and 1r1000. All experiments were performed under non-reducing conditions. Similar results were obtained with mAb Bunga-72. B: Comparison between Bungarus fasciatus and Naja nigricolis venoms. a to c analysis under reducing conditions, d to f analysis under non-reducing conditions. a, d Bungarus venom diluted 1r100. b, e Naja nigricolis venom batch no. 19 Ž2200 Ellman unitsrml.. c and f Naja nigricolis venom batch no. 22 Ž38 Ellman unitsrnil.. All samples were diluted to the same AChE activity.
showed very little activity towards butyrylthiocholine Žsee example of Bungarus fasciatus AChE in Fig. 4.. Substrate inhibition was seen with all venom enzymes, at acetylthiocholine concentrations above 3 mM Ž see Figs. 4 and 5. . These properties identify the snake venom enzymes as true AChEs. This conclu-
Finally, we examined whether some of the antibodies might inhibit AChE. When purified Bungarus fasciatus AChE was reacted with an excess Ž at least 100-fold. concentration of mAbs and incubated for 48 h at room temperature, we observed no inhibition with any of the mAbs. 3.4. Venom AChEs correspond to true AChEs All venom AChEs tested, as affinity-purified enzyme or in crude venom, were efficiently inhibited ŽIC 50 f 2 10y9 M. by eserine Žphysostigmine., a characteristic feature of cholinesterases Ž results not shown.. The distinction with butyrylcholinesterase was made by examining substrate specificity. The 11 affinity-purified enzymes Žsee Section 2. hydrolyzed acetylthiocholine faster than propionylthiocholine and
Fig. 5. Excess substrate inhibition. The activities of affinity-purified AChEs from various snake venoms were measured as a function of substrate concentration Žacetylthiocholine. as described in Section 2. Electrophorus AChE was also tested as a reference. B: Bungarus fasciatus, v: Naja haje, ': Ophiophagus hannah, w: Haemacatus haemacates, I: Electrophorus electricus.
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sion was supported by their sensitivity to the specific inhibitor BW284C51 Žsee below.. 3.5. Measurement of the turnoÕer of AChE from different Õenoms Turnover was measured directly on crude venom samples, using serial dilutions of the organophosphorous compound MPT Žsee Section 2.. Affinity-purified AChE from Electrophorus electricus Ž G4 form. was used as a reference. The results of these experiments are presented in Table 1. The turnover of Electrophorus AChE was 14 711 " 500 sy1, in close agreement with previous measurements by Vigny et al. w35x. The venom enzymes exhibited turnovers between 6100 and 7800 sy1 Žexcept one of the Ophiophagus hannah samples: 4800 sy1 ., and thus appeared quite homogeneous in their catalytic activity. The values obtained with affinity-purified Bungarus fasciatus AChE were very similar to those obtained with crude venoms, indicating that measurements were not affected by the presence of other serine hydrolases in the venoms.
Fig. 6. Inhibition of Bungarus fasciatus AChE by various reversible inhibitors. The inhibition induced by different reversible inhibitors was measured after 48-h reaction at q48C as described in Section 2. B: fasciculin, v: BW 284C51, ': tacrine, w: edrophonium, `: decamethonium, I: propidium.
against the peripheral site Žpropidium, fasciculin., and with bis-quaternary ammonium ligands ŽBW284C51, decamethonium.. All measurements were performed at low ionic strength Ž 1 mM phosphate buffer, pH 7.4, containing 0.1% BSA. because inhibition was greatly reduced at high ionic strength, especially with peripheral site ligands. The results obtained with the 11 affinity-purified AChEs are listed in Table 3. As an illustration, inhibition curves obtained with Bungarus fasciatus are presented in Fig. 6. In general, the different AChEs were similarly sensitive to the different inhibitors, except fasciculin,
3.6. ReactiÕity of Õenom AChEs with reÕersible inhibitors In order to characterize further the catalytic properties of the different AChEs from elapid venoms, we examined their reactivity with reversible inhibitors directed against the active site Žtacrine, edrophonium.,
Table 3 Inhibition of snake venom AChEs by various reversible inhibitors Fasciculin Bungarus fasciatus Žlot 6. Bungarus fasciatus Žlot 8. Bungarus fasciatus ŽChina-1. Bungarus multicinctus Bungarus caeruleus Haemacatus haemacates Naja haje Naja kaoutia Naja naja Naja niÕea Ophiophagus hannah
y8
1.2 = 10 1.5 = 10y8 1.6 = 10y8 8.5 = 10y9 1.3 = 10y8 4 10y6 4 10y6 4 10y6 4 10y6 4 10y6 5 = 10y11
BW284C51 y9
4 = 10 3.6 = 10y9 3.2 = 10y9 4 = 10y9 5.2 = 10y9 7.3 = 10y9 7.5 = 10y9 6.4 = 10y9 9 = 10y9 6.7 = 10y9 4.5 = 10y9
Propidium y5
3.6 = 10 2.7 = 10y5 2.6 = 10y5 6.2 = 10y5 3 = 10y5 2.8 = 10y5 2.5 = 10y5 1.9 = 10y5 2 = 10y5 2.5 = 10y5 4.5 = 10y5
Tacrine
Edrophonium y8
4.5 = 10 5 = 10y8 5 = 10y8 5 = 10y8 4 = 10y8 4.6 = 10y8 7.3 = 10y8 7 = 10y8 8 = 10y8 8 = 10y8 4.2 = 10y8
y6
2.3 = 10 2.4 = 10y6 2.2 = 10y6 2.2 = 10y6 2.7 = 10y6 3.8 = 10y6 2 = 10y6 3 = 10y6 3.5 = 10y6 3.5 = 10y6 2.7 = 10y6
Decamethonium 7 = 10y6 7 = 10y6 12 = 10y6 13 = 10y6 7 = 10y6 18 = 10y6 15 = 10y6 13 = 10y6 15 = 10y6 13 = 10y6 11 = 10y6
Inhibition induced by various inhibitors was measured as described in Section 2, IC 50 ŽM. values were determined graphically from inhibition curves Žsee Fig. 6..
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One of these, Elec-410, which was unambiguously shown to be a peripheral site ligand, cross-reacted with some snake venom AChEs, significantly inhibiting catalytic activity. Fig. 8 shows that enzymes from Naja and Haemacatus were weakly inhibited by the mAb Elec-410 ŽIC 50 ) 10y6 M., while Ophiophagus ŽIC 50 f 5 10y7 M. and Bungarus enzymes ŽIC 50 f 10y9 M. were inhibited efficiently.
4. Discussion
Fig. 7. Inhibition of snake venom AChEs by fasciculin. Various concentrations of fasciculin were reacted for 48 h at q48C with different affinity-purified snake venom AChEs Žsee Section 2.. B: Bungarus fasciatus, v: Naja haje, ': Ophiophagus hannah, w: Haemacatus haemacates, I: Electrophorus electricus.
for which marked variations were observed. The sensitivity was low for Naja and Haemacatus ŽIC 50 ) 10y6 M., intermediate for Bungarus ŽIC 50 f 10y8 M. and high for Ophiophagus ŽIC 50 - 5 10y11 M, Fig. 7.. In previous work, we described a series of monoclonal antibodies directed against Electrophorus electricus AChE which strongly inhibit the enzyme w39x.
Fig. 8. Inhibition of snake venom AChEs by inhibitory mAb Elec-410. Various concentrations of mAb Elec-410 were reacted for 48 h at q48C with different affinity-purified snake venom AChEs Žsee Section 2.. B: Bungarus fasciatus, v: Naja haje, ': Ophiophagus hannah, w: Haemacatus haemacates, I: Electrophorus electricus.
By re-examining the AChE content of a series of 45 snake venoms, we have confirmed, on a larger scale, previous conclusions by Zeller w22x that most elapid venoms exhibit high AChE activity, while viperid and crotalid venoms do not contain this enzyme. Venoms from the Bungarus genus were particularly rich in AChE: close to 8 mgrg of dried venom Ž0.8% wrw. . To our knowledge, no other tissue or biological fluid contains comparable amounts of this enzyme, including electric organs from electric fishes Torpedo and Electrophorus, in which it does not exceed 0.05% wrw w18x. Snake venoms are thus, by far, the richest source of AChE known. As previously described w23x, Bungarus AChE can be efficiently purified by one-step affinity chromatography. Bungarus AChE is more stable than AChE from other elapid venoms and thus appears a very attractive source for biochemical characterization of snake AChE. A more detailed study of this enzyme is presented elsewhere w25x. Although lower than in Bungarus venom, AChE activity is still quite high in other elapid venoms, especially in Naja naja haje. However, Naja nigricolis samples contain low and variable AChE levels, in agreement with previous findings w22x. Since samples exhibiting low activity also show low immunoreactivity ŽFig. 3B., it is possible that the variation in AChE activity observed between Naja venoms is directly related to variations in their original AChE content. However, we observed considerable proteolytic degradation in these venoms, and the variable AChE activity may also reflect enzyme inactivation during lyophilization or storage of the pooled venoms Ž complete degradation leading to loss of immunoreactivity.. The presence of inactive AChE in Naja venom has been previously reported by Raba et al. w40x, who
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also observed intramolecular splitting of the enzyme and loss of activity correlated with aging. Proteolytic cleavage, however, may not be directly correlated with loss of activity. It has been observed, for instance, that Electrophorus AChE may be extensively cleaved while remaining active w17x. In contrast with Zeller w22x, we found essentially no activity in Dendroaspis venoms, which contain high amounts of fasciculin. Immunoblotting experiments performed with mAb Bunga-46 and Bunga-72, which cross-reacted with AChEs from all tested venoms, revealed no immunoreactivity. This strongly suggests that the absence of AChE activity reflects an absence of the protein rather than inhibition by fasciculin. Catalytic characterization of affinity-purified AChEs from the venoms of four elapid genera showed that snake venom AChEs share the features of other vertebrate AChEs: inhibition by eserine, preferential hydrolysis of acetyl rather than propionyl or butyryl esters, excess substrate inhibition and efficient inhibition by BW284C51 ŽIC50 F 9 10y9 M.. It is worth noting that we observed more pronounced substrate inhibition, even in the case of AChE from Naja venoms, than previous workers with AChE from Naja naja oxiana venom w29x. In addition, the catalytic activities of AChEs from the venoms of different snake species varied little, as demonstrated by their very similar turnovers Žsee Table 1. . Interestingly, snake venom AChEs are more active than Torpedo and mammalian AChEs w35x, although they are about two-fold less efficient than Electrophorus AChE. The close similarity between the different species is also illustrated by the fact that about half of the monoclonal antibodies directed against the Bungarus enzyme were found to recognize enzymes from other elapid genera and species. AChEs from the different venoms were also similarly sensitive to different inhibitors, directed either against the active site Žedrophonium, tacrine. or the peripheral site Žpropidium, fasciculin., and to bis-quaternary ammonium compounds. All snake venom AChEs were inhibited by propidium, indicating that they possess a peripheral site, in contradiction with previous reports w29x. This is confirmed by the fact that fasciculin and the inhibitory mAb Elec-410, which have both been characterized as peripheral site ligands, strongly inhibit AChEs from three of the elapid genera. The results
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obtained with fasciculin and the mAb Elec-410 are particularly interesting because inhibition varied widely between species. Naja and Haemacatus AChEs are resistant to fasciculin while Bungarus AChE ŽIC 50 f 10y8 M. was more sensitive and Ophiophagus AChE ŽIC 50 - 10y10 M. was inhibited as efficiently as Electrophorus or Torpedo enzymes. This species variability very likely explains differences between our results and those of Kreienkamp et al. w29x who reported that Naja naja oxiana AChE does not possess a peripheral site. Indeed, we found that AChEs from all Naja species are resistant to fasciculin inhibition. It is interesting to note that a similar order of immunoreactivity was observed with mAb Elec-410: Naja and Haemacatus venom AChEs being weakly inhibited while Ophiophagus and Bungarus enzymes are more sensitive. This confirms Elec-410 as a peripheral site ligand w39x. The binding of peripheral site ligands seems to be very sensitive to ionic strength, as previously shown in the case of propidium w4,41x. In the present study, we also observed that the effect of fasciculin and mAb Elec-410 was weakened by salt, the IC 50 being increased 100-fold in the presence of 0.4 M NaCl. Under the same conditions, inhibition by propidium was less affected Ž 4-fold increase in IC 50 . while inhibition by edrophonium remained essentially unmodified Žresults not shown. . It is therefore possible that such effects may be partly responsible for the differences observed between our results and those of Kreienkamp et al. w29x. It is worth noting that the same explanation could account for the very moderate substrate inhibition observed by these authors, since we also noted a significant reduction in substrate inhibition in high ionic strength buffer Ž results not shown.. Several recent studies w8,42–46x have examined the mechanism by which peripheral site ligands modulate AChE activity. Converging data obtained by kinetic analysis and site-directed mutagenesis strongly support the idea that inhibition is due to an allosteric transition coupling the peripheral site, at the rim of the gorge, to the choline-binding site, at the base of the gorge ŽTrp-84 and Phe-330 in Torpedo numbering.. The binding of ligands at the peripheral site would modify the orientation of Trp-84, which plays a critical role in the positioning of charged substrates Žor charged organophosphorus compounds. in the
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active site. This hypothesis, however, is in apparent contradiction with recent crystallographic data which show that fasciculin completely occludes the entrance of the AChE gorge w43,46x. The demonstration that snake venom AChEs possess a peripheral site and the large species differences in their sensitivity to fasciculin could help to define the mode of action of this inhibitor. The coding sequence of Bungarus fasciatus AChE has recently been determined w47x and allowed the identification of two of the residues that play a critical role in fasciculin and propidium inhibition. Valuable information should also be derived from the cloning of the Naja and Ophiophagus AChEs, which is in progress. In addition, the study of inhibitory antibodies directed to the peripheral site, such as Elec-410, would undoubtedly provide important information on the mechanism of inhibition, since they bind to slightly different sites. We have shown that AChE in snake venoms exists exclusively as a non-amphiphilic monomer. To our knowledge, this is a unique situation, since the monomeric forms which have been identified in other tissues have been characterized as amphiphilic molecules of class II w48,49x, consisting of T-subunits w50,51x. In conclusion, snake venom constitutes a valuable source of AChE, providing large amounts of a highly active non-amphiphilic monomeric enzyme, which can be easily purified by affinity chromatography. Analysis of venom AChE should improve understanding of the biochemical and catalytic properties of this enzyme.
Etudes Techniques and from the Institut National de la Recherche Agronomique. References w1x w2x w3x w4x w5x w6x w7x
w8x
w9x w10x
w11x w12x w13x
w14x w15x
Acknowledgements The authors are indebted to Dr. Jean Massoulie´ for helpful discussions and critical reading of the manuscript. They thank Patricia Lamourette and Marc Plaisance for expert technical assistance and Dr. Renee for providing purified fasciculin. This ´ Menez ´ research was supported in part by grants from the Direction des Recherches et Etudes Techniques, the Human Capital and Mobility program of the European community and the Association Franc¸aise contre les Myopathies. X.C. was the recipient of the fellowships from the Direction des Recherches et
w16x w17x w18x w19x
w20x w21x
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