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NMR three-dimensional structure determination of a novel crustacean toxin from the venom of the scorpion Centruroides limpidus limpidus Karsch
Report and Abstracts
1127
N M R three-dimensional structure determination of a novel crustacean toxin from the venom of the scorpion Centruroides limpidus limpidus Karsch. F. Lebreton, ~ M. Delepierre, ~ A. N. Ramirez, 2 C. Balderas 2 and L. D. Possani 2 (~Institut Pasteur, 25 Rue du Dr Roux, 75724, Paris Cedex 15, France; and 2Department of Biochemistry, Biotechnology Institute, National A u t o n o m o u s University of Mexico, Av. Universidad, 2001 Cuernavaca, Mexico 62271). A crustacean-specific toxin from the Mexican scorpion Centruroides limpidus limpidus was purified and its primary sequence determined, including disulfide bonds. This toxin has 66 amino acid residues and is stabilized by four disulfide bridges (Cys12-Cys65, Cysl6-Cys41, Cys25-Cys46 and Cys29-Cys48). A detailed nuclear magnetic resonance structure of this protein was obtained using a combination of two-dimensional proton N M R experiments. The N M R parameters that gave 69 dihedral restraints and 418 distances constraints were used in molecular dynamic calculations (SA) to determine the solution conformation of the toxin. It is composed of a short or-helix and a three-stranded antiparallel r-sheet. Although the regular secondary structure of this crustacean toxin is c o m m o n to the structural motif of other scorpion toxins, detailed conformational analysis was performed to highlight structural features that might be responsible for the differential modulation of the toxin on sodium channels o f distinct tissues: m a m m a l i a n s versus crustaceans.
Biochemistry (1994) 33, 11,135.
Unexplained toxicity in molluscs found in the course of phytoplankton monitoring. C. Marcaillou-LeBaut and Z. Amzil (Laboratoire I F R E M E R , Phycotoxines et Nuisances). A m o n g the toxic species of phytoplankton occurring in French coastal waters, two genera are liable to pose problems, Dinophysis and Alexandrium. The French Phytoplankton Monitoring Network, R E P H Y , watches for developments of these algae. W h e n concentrations cause concern, it carries out toxicological tests on mice, using bivalves (generally mussels) that have been exposed to the algae. So far this has led to the detection of two classes of toxin: diarrhoeic (or 'diarrhoetic') and paralytic. Only when there is any doubt are physico-chemical analyses performed to confirm toxin identities. During the last 2 years, episodes of toxicity to mice have occurred during R E P H Y monitoring, in which the cause has remained unexplained, particularly since no toxigenic phytoplankton was observed at the sites concerned. Such episodes have occurred at different Atlantic and Mediterranean zones, but have been different in their progress, extent and duration, and there is no proof that the same toxin was involved in each incident. In each case, however, the usual mouse tests indicated intense toxicity, but the physico-chemical tests indicated either no toxicity at all or only traces which could not have accounted for the short survival times found. The m a n y non-toxic controls carried out have eliminated the idea of a possible artefact, and the positive tests found by various coastal laboratories during R E P H Y work have been confirmed by the research teams at Nantes. Several hundred kilograms of toxic mussels are now stored frozen, and research is under way to identify the toxic component(s). In particular, at the Mediterranean site of Toulon, preliminary work has shown that the active principle is heat stable and water soluble, acts as a neurotoxin and passes through the intestinal barrier in mice. Its action on h u m a n s remains u n k n o w n since the sale of the affected mussels has been banned since the toxicity was discovered.
Purification and characterization of two serine-proteases from the venom of Cerastes cerastes, cerastocytin and cerastotin. N. Marrakchi, ~.2,3 R. Barbouche, ~ R. Zingali, 2 C. Bon, 2 S. Guermazi, 4 H. Karoui, ~ and M. El Ayeb ~ (~ Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Tunis, Tunisia; 2Unit6 des Venins, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris, France; 3Facult6 de M6decine de Tunis, Tunis, Tunisia; and 4Laboratoire d'H6mostase, Institut Pasteur de Tunis, Tunis, Tunisia). The venom of the horned viper Cerastes cerastes is rich in proteases, nucleotidases, and phospholipases A2, and contains proteins that interfere with blood coagulation (e.g. protein C activators and fibrinogenases). A m o n g them, two proaggregant proteins, cerastocytin and cerastotin, which share functional properties with thrombin, have been purified to homogeneity and their mechanism of action has been characterized in detail. Cerastocytin induces aggregation of washed platelets at nanomolar concentrations, while the proaggregant activity of cerastotin is observed only in the presence of fibrinogen. Both proteins are made of a single polypeptide chain of 38,000 tool. wt characterized by very similar N-terminal sequence, although their isoelectric points are quite different, 9 and 6.8, respectively. Cerastocytin and cerastotin are serine proteases that are irreversibly inhibited by P M S F and P P A C K , and reversibly by benzamidine, but are insensitive to hirudin and antithrombin III/heparin. They catalyse the hydrolysis of various synthetic substrates ( T A M E and S-2238), as well as that of blood coagulation factors (e.g. fibrinogen, factor X, factor XIII). The proaggregant activity of cerastocytin is inhibited by mepacrin, chlorpromazin and theophyUin, as that of thrombin, but it is insensitive to PAF-acether antagonists and cyclooxygenase inhibitors. Furthermore, washed platelets desensibilized to thrombin do not aggregate in the presence of cerastocytin, while they remain active in the presence of other agonists (e.g. PAF-acether and collagen), suggesting that cerastocytin and thrombin recognize the same platelet receptors. TOX33/~-B
1127
N M R three-dimensional structure determination of a novel crustacean toxin from the venom of the scorpion Centruroides limpidus limpidus Karsch. F. Lebreton, ~ M. Delepierre, ~ A. N. Ramirez, 2 C. Balderas 2 and L. D. Possani 2 (~Institut Pasteur, 25 Rue du Dr Roux, 75724, Paris Cedex 15, France; and 2Department of Biochemistry, Biotechnology Institute, National A u t o n o m o u s University of Mexico, Av. Universidad, 2001 Cuernavaca, Mexico 62271). A crustacean-specific toxin from the Mexican scorpion Centruroides limpidus limpidus was purified and its primary sequence determined, including disulfide bonds. This toxin has 66 amino acid residues and is stabilized by four disulfide bridges (Cys12-Cys65, Cysl6-Cys41, Cys25-Cys46 and Cys29-Cys48). A detailed nuclear magnetic resonance structure of this protein was obtained using a combination of two-dimensional proton N M R experiments. The N M R parameters that gave 69 dihedral restraints and 418 distances constraints were used in molecular dynamic calculations (SA) to determine the solution conformation of the toxin. It is composed of a short or-helix and a three-stranded antiparallel r-sheet. Although the regular secondary structure of this crustacean toxin is c o m m o n to the structural motif of other scorpion toxins, detailed conformational analysis was performed to highlight structural features that might be responsible for the differential modulation of the toxin on sodium channels o f distinct tissues: m a m m a l i a n s versus crustaceans.
Biochemistry (1994) 33, 11,135.
Unexplained toxicity in molluscs found in the course of phytoplankton monitoring. C. Marcaillou-LeBaut and Z. Amzil (Laboratoire I F R E M E R , Phycotoxines et Nuisances). A m o n g the toxic species of phytoplankton occurring in French coastal waters, two genera are liable to pose problems, Dinophysis and Alexandrium. The French Phytoplankton Monitoring Network, R E P H Y , watches for developments of these algae. W h e n concentrations cause concern, it carries out toxicological tests on mice, using bivalves (generally mussels) that have been exposed to the algae. So far this has led to the detection of two classes of toxin: diarrhoeic (or 'diarrhoetic') and paralytic. Only when there is any doubt are physico-chemical analyses performed to confirm toxin identities. During the last 2 years, episodes of toxicity to mice have occurred during R E P H Y monitoring, in which the cause has remained unexplained, particularly since no toxigenic phytoplankton was observed at the sites concerned. Such episodes have occurred at different Atlantic and Mediterranean zones, but have been different in their progress, extent and duration, and there is no proof that the same toxin was involved in each incident. In each case, however, the usual mouse tests indicated intense toxicity, but the physico-chemical tests indicated either no toxicity at all or only traces which could not have accounted for the short survival times found. The m a n y non-toxic controls carried out have eliminated the idea of a possible artefact, and the positive tests found by various coastal laboratories during R E P H Y work have been confirmed by the research teams at Nantes. Several hundred kilograms of toxic mussels are now stored frozen, and research is under way to identify the toxic component(s). In particular, at the Mediterranean site of Toulon, preliminary work has shown that the active principle is heat stable and water soluble, acts as a neurotoxin and passes through the intestinal barrier in mice. Its action on h u m a n s remains u n k n o w n since the sale of the affected mussels has been banned since the toxicity was discovered.
Purification and characterization of two serine-proteases from the venom of Cerastes cerastes, cerastocytin and cerastotin. N. Marrakchi, ~.2,3 R. Barbouche, ~ R. Zingali, 2 C. Bon, 2 S. Guermazi, 4 H. Karoui, ~ and M. El Ayeb ~ (~ Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, Tunis, Tunisia; 2Unit6 des Venins, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris, France; 3Facult6 de M6decine de Tunis, Tunis, Tunisia; and 4Laboratoire d'H6mostase, Institut Pasteur de Tunis, Tunis, Tunisia). The venom of the horned viper Cerastes cerastes is rich in proteases, nucleotidases, and phospholipases A2, and contains proteins that interfere with blood coagulation (e.g. protein C activators and fibrinogenases). A m o n g them, two proaggregant proteins, cerastocytin and cerastotin, which share functional properties with thrombin, have been purified to homogeneity and their mechanism of action has been characterized in detail. Cerastocytin induces aggregation of washed platelets at nanomolar concentrations, while the proaggregant activity of cerastotin is observed only in the presence of fibrinogen. Both proteins are made of a single polypeptide chain of 38,000 tool. wt characterized by very similar N-terminal sequence, although their isoelectric points are quite different, 9 and 6.8, respectively. Cerastocytin and cerastotin are serine proteases that are irreversibly inhibited by P M S F and P P A C K , and reversibly by benzamidine, but are insensitive to hirudin and antithrombin III/heparin. They catalyse the hydrolysis of various synthetic substrates ( T A M E and S-2238), as well as that of blood coagulation factors (e.g. fibrinogen, factor X, factor XIII). The proaggregant activity of cerastocytin is inhibited by mepacrin, chlorpromazin and theophyUin, as that of thrombin, but it is insensitive to PAF-acether antagonists and cyclooxygenase inhibitors. Furthermore, washed platelets desensibilized to thrombin do not aggregate in the presence of cerastocytin, while they remain active in the presence of other agonists (e.g. PAF-acether and collagen), suggesting that cerastocytin and thrombin recognize the same platelet receptors. TOX33/~-B