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Binding of radiolabeled taipoxin to PC-12 cells
290
Ninth European Symposium
A simple method to evaluate the digestive effect of snake venoms. J. Merna, CH . Aot.vrt and G. SuxeEtt-Cbexi
(Pentapharm Ltd, 4002 Basel, Switzerland) .
Ttu: Mosr prominent and primary biological function of snake venoms is to weaken and immobilize large prey organisms before being swallowed. Secondly, they play an important role in defence. Since there is evidence that We different venom components evolved from ancestral digestive enzymes it is generally accepted that snake venoms also have some digestive functions. However, most of the attempts to show such digestive effects failed . A simple method is presented which allows to investigate the digestive function of snake venoms . For this purpose, a newborn white mouse of about 3 g body weight is killed by breaking its neck. Then, 0.5 ml of snake venom dilution or physiological saline respectively is i.p. injected through the thorax . The mouse is then put into a glass funnel closed with parafihn . After some days the liquified digest may be collected in a calibration cylinder. With this method it has been easily shown that liquid digests are found e.g. already 6 days after injection of the venom from the common lance head Bothrops atrox moojeni (lOmg/ml), whereas digestive juices appear only after 12 days in the control animals. The method has been shown to be useful in investigating the influence of proteinase inhibitors and divalent rations on the digestive activity of Viperidae snake venoms . Protection against snake toxins using genetically and chemically engineered immunogens . Axnxi Mixez, M~ct~L
Lsoxern, L,~utt~vce Pu,t~r, Fxfofattc Ducexcet. and J~x-CutroE Houtrttx (Service de Biochimie des Protéines, Laboratoire d'Ingéniérie des protéines, CEN Saclay, 91191, Gif- sur- Yvette, France). Cbxexiaur~rtc toxins bind to the nicotinic acetylcholine receptor (AcChoR) with selectivity and high affinity. They block neuromuscular transmission and death occurs by respiratory failure. The toxins are folded into three adjacent loops (I, II and III) rich in ß-sheet structure. Residues located on loop II (residues 27, 29, 31-34, 36, 38) and loop III (residues 46, 47, 49, 52) together with residue 8 have been previously proposed to constitute the AcChoR binding site of curaremimetic toxins (Mfavez et al., 1984) . In agreement with this proposal, chemical modifications at positions 27, 29 and 47 as well as recent genetic substitutions at positions 8, 31 and 34, induced substantial decreases in affinity of the toxin for the receptor . In contrast, changes at positions outside the putative AcChor binding site, i .e . chemical modifications at N-terminal, Lys-15 and Lys-51 and genetic deletion of residue 18, did not really affect toxin affinity . We used two different approaches to elicit antibodies capable of neutralizing a curaremimetic toxin. In the first approach we identified the region wmprised between residues 24 and 41 as the dominant T-cell epitope of toxin a from Naja nigricollis in BALB/c mice (Lnoxern et al., 1990). Only this segment, which corresponds to the second loop of the toxin, elicited antibodies without any coupling to a carrier protein. However, the antibodies were directed against the cognate sequence of the unfolded toxin. Conformational restriction was then brought to the peptide by cyclizing it. The cyclic peptide not only remained capable of stimulating T cells but also elicited antibodies that recognized the native toxin as well as a set of toxin variants . Though of relatively low titers these antibodies did neutralize binding of the toxin to the receptor . Therefore, neutralization of neurotoxicity can be achieved by antibodies selectively elicited by a free peptide which mimics part of the functional site of the toxin. The principle of this approach could be more generally applied to other toxic proteins . With the view to improve titers of anti-toxin antisera we also constructed a plasmid encoding a hybrid protein composed of the sequences of protein A from S. aureus and erabutoxin a from L. semifasciata (Ducnxcer. et al., 1989). The fused toxin was markedly less toxic than the native toxin. Presumably, this was due to the bulkiness of protein A which, though coupled to a residue apart from the AcChoR binding site, hindered the binding of the toxin to the receptor. Interestingly enough, immunogenicity of the toxin dramatically increased upon fusion with protein A, since 1 nmole of hybrid and 14 nmoles of native toxin gave rise, in rabbits, to comparable titers of antitoxin antibodies . Moreover, these antibodies were equally potent at neutralizing neurotoxicity . Fused toxins may be of great value for future development of potent antisera against small toxic proteins .
REFERENCES Ducwvcet., F., Hout.etrt, J.-C., TxHa~wu, O. and MfrrFa, A. (1989) Protein Engineering 3, 139-143. Leoxsrn, M., Pit,tsr, L., L.~t~tx, H., Ftucxox, P., Counsxc, J. and MÉxez, A. (1990) J. lmmwral. (in press) . Mi~z et al . (1984) J. Physiol. (Paris) 79, 19t~206. Binding ojradiolabeled taipoxin to PC-12 cells. Jot~n~r L. MIDDLEBROOK (Dept of Toxinology, Pathology Division,
U.S . Army Medical Research Institute of Infectious Diseases, Frederick, MD 21701, U.S .A.).
PHOSPHOLIPASE A, (PLAN neurotoxins are found in venoms of snakes around the world. These toxins are
Ninth European Symposium
29 1
implicated as major factors in deaths due to the bites of several species. PLAZ toxins arc known to inhibit the release of neurotransmitters from the presynaptic nerve terminus, thus producing paralysis . However, details concerning the mechanism by which this inhibition is induced arc not available. In order to gain some insights into the molecular mechanism of action of the PLAZ toxins, we studied the binding of taipoxin to a cultured cell line (PC-12) employed by many laboratories as a nerve cell model. Taipoxin was labeled with 'ZSI using chloramine T. The toxin retained its biological activity as judged by the mouse lethality assay. Binding of 'ZSI-taipoxin to PC-12 cells was saturable, of high affinity, reversible and specific, properties suggestive of a receptor type of interaction. The dissociation constant was approximately 10 s M. The half-life for dissociation at 37°C was about 1 hr . Binding of 'ZSI-taipoxin was blocked by an excess of unlabelled taipoxin, but not by similar excesses of other PLA Z toxins . These and other data suggest that the PC-12 cell line is a good model with which to delineate the molecular events involved in the mechanism of action of taipoxin .
Factors in snake venoms that increase capillary permeability. R. A. Mt~t-en and A . T . Tu (Department of Biochemistry, Colorado State University, Fort Collies, CO 80523, U.S .A .). CAPILLARY permeability increasing (CPI) activity is a phenomenon of the microvasculaturc caused by many agents including snake venoms. A CPi factor from Naja naja afro (Taiwan cobra) venom was isolated using i.v . injections of Evan's blue dye as the indicator of increased permeability and the factor's properties were extensively studied. Cardiotoxin from Naja raja kaouthia (Thailand cobra) and Mojave toxin from Crotales scutulatus scutulatus (Mojave rattlesnake) venoms demonstrated CPI activity . Post-synaptic neurotoxins from an elapid and a hydrophilid and myotoxin a from Crotaless viridis viridis (Prairie rattlesnake) showed no CPI activity at dose studied. The purified CPI active component from Naja raja afro venom was found to have cardiotoxic activity . Sequence analysis verified this Elapidae CPI factor is cardiotoxin . However, CPI activity is not due to cardiotoxins alone as the pre-synaptic neurotoxin, Mojave toxin, also showed CPI activity . The histamine H, receptor blockers: diphenylhydramine, promethazine and cyproheptadine were effective against Naja naja afro venom and Crotales scutulatus scutulates venom in preventing increased capillary permeability. These results suggested that histamine release activity is the most likely mechanism resulting in CPI activity from these venoms .
lnjleence ofprotein C activator from Agkistrodon halys halys venom on blood coagulation. K. MBI,LER, A. Tzttw, A. Mvucsn~tt, A. E. Koc~N' and S. M. $TRUKOVA' (Institute of Chemical Physics and Biophysics of the Estonian Academy of Sciences, Tallinn, and Moscow State University, Moscow, U.S .S .R .) . AN ANTICOAGULANT glycoprotein of mol. wt 40,000-50,000 with protein C activating properties has bcen found in Agkistrodon italys venom (MSL~-RA et al., 1990). The protein has been purified 60-fold for bovine zymogen protein C activation in amidolytic test ; it was inactive on prothrombin, factor X, plasminogen, fibrin and did not clot fibrinogen . The protein C activator exerted species specific prolongation of the partial thromboplastin time (PTT) in bovine, rat, sheep, human, rabbit and pig plasmas and it did not change the PTT in horse plasma . Remarkable prolongation of the activated partial thromboplastin dme (APTT) in rat plasma enabled us to develop a method for protein C determination in rat blood making use of the activator from Agkistrodon halys halys venom. For comparison, the venom of Agkistrodon contortrix contortrix has been fractionated by the same method as the venom of Agkistrodon halys halys using gel filtration on Sephadex G-100 sf followed by ion-exchange chromatography on DEAE cellulose. Both venoms showed similar distribution of anticoagulant activity in the gel filtration fractions . The reactions of the purified activators from Agkistrodon halys halt's and Agkistrodon contortrix contortrix venoms with typical low-molecular ester substrates for arginine~irected proteases have been studied. The activator from Agkistrodon halt's halt's venom can be used in the functional protein C assay in human plasma by MARTINOLI and STOCKER (1986).
REFERENCES MARTINOLI, J. L. and $TOCKER, K. (1986) Fast functional protein C assay using Protac, a novel protein C activator. Thromb . Res. 43, 253-264. MSLt-ER, K., T~Ra, A., Anvltcs~wR, A., KOGAN, A. E. and Srxvtcov~, S. M. (1990) Protein C activator from the venom of Agkistrodon halt's halt's, Abstr. 20th FEBS Meeting, Budapest, p. 198, P-Tu 453.
Ninth European Symposium
A simple method to evaluate the digestive effect of snake venoms. J. Merna, CH . Aot.vrt and G. SuxeEtt-Cbexi
(Pentapharm Ltd, 4002 Basel, Switzerland) .
Ttu: Mosr prominent and primary biological function of snake venoms is to weaken and immobilize large prey organisms before being swallowed. Secondly, they play an important role in defence. Since there is evidence that We different venom components evolved from ancestral digestive enzymes it is generally accepted that snake venoms also have some digestive functions. However, most of the attempts to show such digestive effects failed . A simple method is presented which allows to investigate the digestive function of snake venoms . For this purpose, a newborn white mouse of about 3 g body weight is killed by breaking its neck. Then, 0.5 ml of snake venom dilution or physiological saline respectively is i.p. injected through the thorax . The mouse is then put into a glass funnel closed with parafihn . After some days the liquified digest may be collected in a calibration cylinder. With this method it has been easily shown that liquid digests are found e.g. already 6 days after injection of the venom from the common lance head Bothrops atrox moojeni (lOmg/ml), whereas digestive juices appear only after 12 days in the control animals. The method has been shown to be useful in investigating the influence of proteinase inhibitors and divalent rations on the digestive activity of Viperidae snake venoms . Protection against snake toxins using genetically and chemically engineered immunogens . Axnxi Mixez, M~ct~L
Lsoxern, L,~utt~vce Pu,t~r, Fxfofattc Ducexcet. and J~x-CutroE Houtrttx (Service de Biochimie des Protéines, Laboratoire d'Ingéniérie des protéines, CEN Saclay, 91191, Gif- sur- Yvette, France). Cbxexiaur~rtc toxins bind to the nicotinic acetylcholine receptor (AcChoR) with selectivity and high affinity. They block neuromuscular transmission and death occurs by respiratory failure. The toxins are folded into three adjacent loops (I, II and III) rich in ß-sheet structure. Residues located on loop II (residues 27, 29, 31-34, 36, 38) and loop III (residues 46, 47, 49, 52) together with residue 8 have been previously proposed to constitute the AcChoR binding site of curaremimetic toxins (Mfavez et al., 1984) . In agreement with this proposal, chemical modifications at positions 27, 29 and 47 as well as recent genetic substitutions at positions 8, 31 and 34, induced substantial decreases in affinity of the toxin for the receptor . In contrast, changes at positions outside the putative AcChor binding site, i .e . chemical modifications at N-terminal, Lys-15 and Lys-51 and genetic deletion of residue 18, did not really affect toxin affinity . We used two different approaches to elicit antibodies capable of neutralizing a curaremimetic toxin. In the first approach we identified the region wmprised between residues 24 and 41 as the dominant T-cell epitope of toxin a from Naja nigricollis in BALB/c mice (Lnoxern et al., 1990). Only this segment, which corresponds to the second loop of the toxin, elicited antibodies without any coupling to a carrier protein. However, the antibodies were directed against the cognate sequence of the unfolded toxin. Conformational restriction was then brought to the peptide by cyclizing it. The cyclic peptide not only remained capable of stimulating T cells but also elicited antibodies that recognized the native toxin as well as a set of toxin variants . Though of relatively low titers these antibodies did neutralize binding of the toxin to the receptor . Therefore, neutralization of neurotoxicity can be achieved by antibodies selectively elicited by a free peptide which mimics part of the functional site of the toxin. The principle of this approach could be more generally applied to other toxic proteins . With the view to improve titers of anti-toxin antisera we also constructed a plasmid encoding a hybrid protein composed of the sequences of protein A from S. aureus and erabutoxin a from L. semifasciata (Ducnxcer. et al., 1989). The fused toxin was markedly less toxic than the native toxin. Presumably, this was due to the bulkiness of protein A which, though coupled to a residue apart from the AcChoR binding site, hindered the binding of the toxin to the receptor. Interestingly enough, immunogenicity of the toxin dramatically increased upon fusion with protein A, since 1 nmole of hybrid and 14 nmoles of native toxin gave rise, in rabbits, to comparable titers of antitoxin antibodies . Moreover, these antibodies were equally potent at neutralizing neurotoxicity . Fused toxins may be of great value for future development of potent antisera against small toxic proteins .
REFERENCES Ducwvcet., F., Hout.etrt, J.-C., TxHa~wu, O. and MfrrFa, A. (1989) Protein Engineering 3, 139-143. Leoxsrn, M., Pit,tsr, L., L.~t~tx, H., Ftucxox, P., Counsxc, J. and MÉxez, A. (1990) J. lmmwral. (in press) . Mi~z et al . (1984) J. Physiol. (Paris) 79, 19t~206. Binding ojradiolabeled taipoxin to PC-12 cells. Jot~n~r L. MIDDLEBROOK (Dept of Toxinology, Pathology Division,
U.S . Army Medical Research Institute of Infectious Diseases, Frederick, MD 21701, U.S .A.).
PHOSPHOLIPASE A, (PLAN neurotoxins are found in venoms of snakes around the world. These toxins are
Ninth European Symposium
29 1
implicated as major factors in deaths due to the bites of several species. PLAZ toxins arc known to inhibit the release of neurotransmitters from the presynaptic nerve terminus, thus producing paralysis . However, details concerning the mechanism by which this inhibition is induced arc not available. In order to gain some insights into the molecular mechanism of action of the PLAZ toxins, we studied the binding of taipoxin to a cultured cell line (PC-12) employed by many laboratories as a nerve cell model. Taipoxin was labeled with 'ZSI using chloramine T. The toxin retained its biological activity as judged by the mouse lethality assay. Binding of 'ZSI-taipoxin to PC-12 cells was saturable, of high affinity, reversible and specific, properties suggestive of a receptor type of interaction. The dissociation constant was approximately 10 s M. The half-life for dissociation at 37°C was about 1 hr . Binding of 'ZSI-taipoxin was blocked by an excess of unlabelled taipoxin, but not by similar excesses of other PLA Z toxins . These and other data suggest that the PC-12 cell line is a good model with which to delineate the molecular events involved in the mechanism of action of taipoxin .
Factors in snake venoms that increase capillary permeability. R. A. Mt~t-en and A . T . Tu (Department of Biochemistry, Colorado State University, Fort Collies, CO 80523, U.S .A .). CAPILLARY permeability increasing (CPI) activity is a phenomenon of the microvasculaturc caused by many agents including snake venoms. A CPi factor from Naja naja afro (Taiwan cobra) venom was isolated using i.v . injections of Evan's blue dye as the indicator of increased permeability and the factor's properties were extensively studied. Cardiotoxin from Naja raja kaouthia (Thailand cobra) and Mojave toxin from Crotales scutulatus scutulatus (Mojave rattlesnake) venoms demonstrated CPI activity . Post-synaptic neurotoxins from an elapid and a hydrophilid and myotoxin a from Crotaless viridis viridis (Prairie rattlesnake) showed no CPI activity at dose studied. The purified CPI active component from Naja raja afro venom was found to have cardiotoxic activity . Sequence analysis verified this Elapidae CPI factor is cardiotoxin . However, CPI activity is not due to cardiotoxins alone as the pre-synaptic neurotoxin, Mojave toxin, also showed CPI activity . The histamine H, receptor blockers: diphenylhydramine, promethazine and cyproheptadine were effective against Naja naja afro venom and Crotales scutulatus scutulates venom in preventing increased capillary permeability. These results suggested that histamine release activity is the most likely mechanism resulting in CPI activity from these venoms .
lnjleence ofprotein C activator from Agkistrodon halys halys venom on blood coagulation. K. MBI,LER, A. Tzttw, A. Mvucsn~tt, A. E. Koc~N' and S. M. $TRUKOVA' (Institute of Chemical Physics and Biophysics of the Estonian Academy of Sciences, Tallinn, and Moscow State University, Moscow, U.S .S .R .) . AN ANTICOAGULANT glycoprotein of mol. wt 40,000-50,000 with protein C activating properties has bcen found in Agkistrodon italys venom (MSL~-RA et al., 1990). The protein has been purified 60-fold for bovine zymogen protein C activation in amidolytic test ; it was inactive on prothrombin, factor X, plasminogen, fibrin and did not clot fibrinogen . The protein C activator exerted species specific prolongation of the partial thromboplastin time (PTT) in bovine, rat, sheep, human, rabbit and pig plasmas and it did not change the PTT in horse plasma . Remarkable prolongation of the activated partial thromboplastin dme (APTT) in rat plasma enabled us to develop a method for protein C determination in rat blood making use of the activator from Agkistrodon halys halys venom. For comparison, the venom of Agkistrodon contortrix contortrix has been fractionated by the same method as the venom of Agkistrodon halys halys using gel filtration on Sephadex G-100 sf followed by ion-exchange chromatography on DEAE cellulose. Both venoms showed similar distribution of anticoagulant activity in the gel filtration fractions . The reactions of the purified activators from Agkistrodon halys halt's and Agkistrodon contortrix contortrix venoms with typical low-molecular ester substrates for arginine~irected proteases have been studied. The activator from Agkistrodon halt's halt's venom can be used in the functional protein C assay in human plasma by MARTINOLI and STOCKER (1986).
REFERENCES MARTINOLI, J. L. and $TOCKER, K. (1986) Fast functional protein C assay using Protac, a novel protein C activator. Thromb . Res. 43, 253-264. MSLt-ER, K., T~Ra, A., Anvltcs~wR, A., KOGAN, A. E. and Srxvtcov~, S. M. (1990) Protein C activator from the venom of Agkistrodon halt's halt's, Abstr. 20th FEBS Meeting, Budapest, p. 198, P-Tu 453.