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Different cytoskeletal targets for clostridium difficile toxins
156
8th European Symposium-Abstracts
Preliminary crystallographic studies offasciculin 2 from green mamba venom . M. H. LE Du,' P. MARCHOT,~ P. E.
Bouclez and J. C. FotvrECll.l.w-Cweo?s' ('Lab. de . Cristallographie et Cristallisation des Macromolécules Biologiques, URA 232 CNRS, Fac. de Médecine, Sector Nord, Bd Pierre Dramard, 13326 Marseille, Cedex 15, France, and z Lab. de Biochimie CNRS UA 1179 - INSERM U172, Fac. de Médecine, Sector Nord, Bd Pierre Dramard, 13326 Marseille, Cedex 15, France). Ttn: vExor~t of the green mamba Dendroaspis angusticeps contains at least two toxic proteins that can induce severe muscular fasciculations when injected in the mouse. This effect is provoked by a very strong inhibition of acetylcholinesterase . These toxins which have been called fasciculin 1 and 2 are constituted of 61 amino acid residues and are crosslinked by 4 disulfide bridges. Amino acid sequence comparisons show that fasciculins are structurally related to snake neurotoxins and cardiotozins . We report here the crystallization of fasciculin 2 and a preliminary computer model of this molecule to be used in the resolution of the three-dimensional structure by the molecular replacement method. Fasciwlin 2 crystals belong to the tetragonal space group P4,2,2 or its enantionmorph. The cell parameters are a = 48,96 A and c = 82,03 A . Density measurements using organic solvents indicate that there are 2 molecules in the asymmetric unit. Examination of precession photographs of the h k o plane shows that the crystals are pseudo centered with apparent I4,22 symmetry . This wnfu-ms the existence of 2 molecules in the asymmetric unit and imposes restrictions in the way the two crystallographically independent molecules are oriented relative to each other. Computing modelling of fasciculin 2 was carried out using the known three-dimensional structures of erabutoxin and cardiotoxin . Subsequently, the model was subjected to an energy minimization procedure to remove all the bad contacts resulting from the modelling process. Interestingly, 2 of the arginine side chains of fasciculin 2 are found at the tip of the central loop . In erabutoxin this region is thought to be partially responsible for the toxic action. By analogy it is possible, then, to speculate that these positively charged arginine residues may play a role in the inhibition of acetylcholinesterase.
Molecular recognition : from snake venom newotoxin directly to complementary receptor binding site . BARBARA W. Low (Columbia University, Department of Biochemistry and Molecular Biophysics).
DelEltumvwnorr of the 3-dimensional molecular structure of one prototype a-toxin at high resolution provided a detailed view of the toxin reactive site with both stable and highly mobile regions, well~haracterized . Consideration of the probable binding mode of this amphipathic toxin domain (Low and CORFIELD, 1986) led us directly to identification of the complementary principal ligand-binding domain on the a-subunit of the acetylcholine receptor (Low and CoRl~l .n, 1988). This unique highly conserved a-subunit domain 177-193, studied as synthetic peptides, has been shown to bind a-toxins extremely tightly (RADDING et al., 1988). The process of molecular recognition we employed in using the 3-dimensional structure of an a-toxin as stereochemical probe of receptor binding domain will be described. The fit of the a-peptide within the reactive site concavity leaves certain short and long chain series~onserved residues in other toxin regions free to engage in further binding interactions. Changes in prototype binding model, responsive to the anomalous features of one receptor a-subunit domain (human) are required as are some modifications in the sequential binding mode of some long and short chain a-toxins . The significance of relatively minor differences between different a-toxins can now be studied. Specific residue-residue interactions at the complementary toxin and peptide binding surfaces have been monitored in collaborative NMR studies (Bo~n~tlat-Bv et al., 1989). Two of the methods used and to be employed in these studies were designed to provide information about the roles in binding of both a-peptide and a-toxin Trp residues . REFERENCES BOTHNER-BY, A. A., Mlswtw, P. K. and Low B. W. (1989) Proceedings of the U.C.L .A . Symposium, January 1989 . To be published in Frontiers ojNMR in Molecular Biology (Live, D., ARMITAGE, I. and Pw~tEt., D. eds). Alan R. Lies. Low, H. W. and CORFIELD, P. W. R. (1986) Eur. J. Biochem . 161, 579-586. Low, B. W. and Coxr+>Lt.n P. W. R. (19$7) Asia Pac. J. Pharm . 2, 115-127. RADDING, W., CoRl~l.n, P. W. R., LEVnvsox, L. S., Hwsln~t, G. A. and Low, B. W. (1988) FEES Lett . 231, 212-216.
Different cytoskeletal targets for clostridium difficile toxins. W . MALORTiI, C. FIORErmrrl, S. PARwnlsl and G.
DorIEt.l .l (Department of Ultrastructures, istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy) . TIIE ANAEROBIC bacterium Clostridium difficile has been recognized as the major setiological agent responsible for the antibiotic-associated pseudomembranous colitis in humans. It produces at least two different protein toxins,
8th European Symposium-Abstracts
157
named toxins A and B, which are implicated in the pathogenesis of the disease (Bwrrtao et al ., 1984). Previous experimental studies have demonstrated that toxin A is an enterotoxin which elicits severe epithelial damages when injected into rabbit deal loops, while toxin B appears to be devoid of such an activity . Both toxins induce cytopathogenic effects in tisauei;ultured cells and belong to the group of intracellularly acting proteins which have to be internalized and processed in order to exert their cytotoxic activity (FLOxnv and Tr~t.ESrw~, 1983). Our in vitro studies are focused on the mechanisms underlying C. di,~tcile toxins cytotoxicity (F~ORENTIHI et al., 1989). Results obtained can be summarized as follows: (a) toxin A treatment (4 pg/ml) of cultured epithelial cells induced a characteristic series of morphological changes mainly represented by cell rounding and subsequent nuclear displacement. Studies performed by using cytoskeleton perturbating agents seem to indicate miaotubule system as responsible for such a nuclear polarization; (b) the exposure of different epithelial cell lines to 0 .15 pg/ml toxin B induced cell retraction, cell rounding and the formation, on the cell surface, of bulb-like structures filled with ribosomes and devoid of other cell organelles . Immunocytochemical studies performed on the cytoskeletal elements seem to indicate that microfiLiment system can play a role in the appearance of such a blebbing phenomenon . Thus, both toxins caused cell retraction and rounding in all cell types considered and seemed to exert their effect on cell morphology via a rearrangement of some cytoskeletal components. However, the toxin B-induced blebbing and the toxin A-induced nuclear polarization seem to be dependent on different cytoskeletal elements . REFERENCES BANNO, Y., et al. (1984) Rev. Infect. Dis. 6, I1-20. F~oxerrrna~, C. et al., (1989) Toxicon 27, 1209-1218 . FLORIN, I. and Tt-n?LESrw~t, M. (1983) Biochim. Biophys. Acta 763, 383-392.
Yenomorcr snakes and snake venom poisoning in Ewope. Z. Mwxar[~ and F. E. RUa9ELL (Medicinski Centar, 52000 Pula, Yugoslavia). N~nE st~ECn s and 18 subspecies of Vipers are generally recognized in Europe . One Crotalidae, Agkistrodon halys, an Asiatic species, is found as far west as the Caspian Sea. Two rear-fanged snakes, Malpolon monspessulatws and Telescopes jallax are also said to be "mildly venomous". Although reliable statistics on the number of bites and envenomations by venomous snakes in Europe are not available, those gathered from individual countries over the past two decades would indicate that the total number would not exceed 8000, and although the number of deaths per year may be as high as 50, the probable number is closer to 20. The present report treats the general biology of the venomous snakes in Europe, the chemistry and pharmacology of their venons, and the clinical problem of venom poisoning.
Re-examination ojthe protease inhibitor activities ojthe dendrotoxins from mamba venons . D. L. MwRacrwr L and A. L. HARVEY (Department of Physiology and Pharmacology, University of Strathclyde, Glasgow GI iXW, U.K .) . Try nENnßo~roz~rrs are small proteins isolated from mamba venons (HARVEY and At~rnEasort, 1985). They are highly homologous to Kursitz serine protease inhibitors, such as bovine pancreatic trypsin inhibitor (BPTI) . Although the dendrotoxins are known for their ability to block neuronal potassium ion channels and to facilitate the release of neurotransmitters (e.g . ANDERSON and HARVEY, 1988), less attention has been given to their potential anti-protease activity. Initial studies revealed little, if any, ability to block trypsin or chymotrypsin, possibly because very short incubation times were used (S~rnYnota, 1976 ; JouRERr and TwLtwwRD, 1980). We have tested the effects of three dendrotozins (toxins I and K from black mamba Derrdroaspis polylepis and dentrotoxin from Eastern green mamba Dendroaspis angesticeps) on trypsin, chymotrypsin and kallikrein . All assays were performed at 25°C in buffer containing 5 mM CaCl 2. Appropriate concentrations of specific substrates were used for each enzyme . The three toxins produced a concentration~ependent inhibition of trypsin. When measured after maximum inhibition had been reached after 60 min incubation, the rc,~ values were 0.9, 0.5 and 6.0 pM for toxin I, toxin K and dendrotoxin, respectively . The corresponding value for BPTI was 0.02 pM . The toxins acted as competitive inhibitors of kallikrein from pig pancreas. Maximum inhibition was reached by 10 min incubation . The K, values were 1.3, 0.07 and 1 .4 uM for toxin I, toxin K and dendrotoxin, respectively . None of the toxins inhibited the activity of chymotrypsin . Toxin 1 was tested at 6 KM for 75 min; toxin K at 30 pM for 60 min; and dendrotoxin at 0.7 pM for 150 min.
8th European Symposium-Abstracts
Preliminary crystallographic studies offasciculin 2 from green mamba venom . M. H. LE Du,' P. MARCHOT,~ P. E.
Bouclez and J. C. FotvrECll.l.w-Cweo?s' ('Lab. de . Cristallographie et Cristallisation des Macromolécules Biologiques, URA 232 CNRS, Fac. de Médecine, Sector Nord, Bd Pierre Dramard, 13326 Marseille, Cedex 15, France, and z Lab. de Biochimie CNRS UA 1179 - INSERM U172, Fac. de Médecine, Sector Nord, Bd Pierre Dramard, 13326 Marseille, Cedex 15, France). Ttn: vExor~t of the green mamba Dendroaspis angusticeps contains at least two toxic proteins that can induce severe muscular fasciculations when injected in the mouse. This effect is provoked by a very strong inhibition of acetylcholinesterase . These toxins which have been called fasciculin 1 and 2 are constituted of 61 amino acid residues and are crosslinked by 4 disulfide bridges. Amino acid sequence comparisons show that fasciculins are structurally related to snake neurotoxins and cardiotozins . We report here the crystallization of fasciculin 2 and a preliminary computer model of this molecule to be used in the resolution of the three-dimensional structure by the molecular replacement method. Fasciwlin 2 crystals belong to the tetragonal space group P4,2,2 or its enantionmorph. The cell parameters are a = 48,96 A and c = 82,03 A . Density measurements using organic solvents indicate that there are 2 molecules in the asymmetric unit. Examination of precession photographs of the h k o plane shows that the crystals are pseudo centered with apparent I4,22 symmetry . This wnfu-ms the existence of 2 molecules in the asymmetric unit and imposes restrictions in the way the two crystallographically independent molecules are oriented relative to each other. Computing modelling of fasciculin 2 was carried out using the known three-dimensional structures of erabutoxin and cardiotoxin . Subsequently, the model was subjected to an energy minimization procedure to remove all the bad contacts resulting from the modelling process. Interestingly, 2 of the arginine side chains of fasciculin 2 are found at the tip of the central loop . In erabutoxin this region is thought to be partially responsible for the toxic action. By analogy it is possible, then, to speculate that these positively charged arginine residues may play a role in the inhibition of acetylcholinesterase.
Molecular recognition : from snake venom newotoxin directly to complementary receptor binding site . BARBARA W. Low (Columbia University, Department of Biochemistry and Molecular Biophysics).
DelEltumvwnorr of the 3-dimensional molecular structure of one prototype a-toxin at high resolution provided a detailed view of the toxin reactive site with both stable and highly mobile regions, well~haracterized . Consideration of the probable binding mode of this amphipathic toxin domain (Low and CORFIELD, 1986) led us directly to identification of the complementary principal ligand-binding domain on the a-subunit of the acetylcholine receptor (Low and CoRl~l .n, 1988). This unique highly conserved a-subunit domain 177-193, studied as synthetic peptides, has been shown to bind a-toxins extremely tightly (RADDING et al., 1988). The process of molecular recognition we employed in using the 3-dimensional structure of an a-toxin as stereochemical probe of receptor binding domain will be described. The fit of the a-peptide within the reactive site concavity leaves certain short and long chain series~onserved residues in other toxin regions free to engage in further binding interactions. Changes in prototype binding model, responsive to the anomalous features of one receptor a-subunit domain (human) are required as are some modifications in the sequential binding mode of some long and short chain a-toxins . The significance of relatively minor differences between different a-toxins can now be studied. Specific residue-residue interactions at the complementary toxin and peptide binding surfaces have been monitored in collaborative NMR studies (Bo~n~tlat-Bv et al., 1989). Two of the methods used and to be employed in these studies were designed to provide information about the roles in binding of both a-peptide and a-toxin Trp residues . REFERENCES BOTHNER-BY, A. A., Mlswtw, P. K. and Low B. W. (1989) Proceedings of the U.C.L .A . Symposium, January 1989 . To be published in Frontiers ojNMR in Molecular Biology (Live, D., ARMITAGE, I. and Pw~tEt., D. eds). Alan R. Lies. Low, H. W. and CORFIELD, P. W. R. (1986) Eur. J. Biochem . 161, 579-586. Low, B. W. and Coxr+>Lt.n P. W. R. (19$7) Asia Pac. J. Pharm . 2, 115-127. RADDING, W., CoRl~l.n, P. W. R., LEVnvsox, L. S., Hwsln~t, G. A. and Low, B. W. (1988) FEES Lett . 231, 212-216.
Different cytoskeletal targets for clostridium difficile toxins. W . MALORTiI, C. FIORErmrrl, S. PARwnlsl and G.
DorIEt.l .l (Department of Ultrastructures, istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy) . TIIE ANAEROBIC bacterium Clostridium difficile has been recognized as the major setiological agent responsible for the antibiotic-associated pseudomembranous colitis in humans. It produces at least two different protein toxins,
8th European Symposium-Abstracts
157
named toxins A and B, which are implicated in the pathogenesis of the disease (Bwrrtao et al ., 1984). Previous experimental studies have demonstrated that toxin A is an enterotoxin which elicits severe epithelial damages when injected into rabbit deal loops, while toxin B appears to be devoid of such an activity . Both toxins induce cytopathogenic effects in tisauei;ultured cells and belong to the group of intracellularly acting proteins which have to be internalized and processed in order to exert their cytotoxic activity (FLOxnv and Tr~t.ESrw~, 1983). Our in vitro studies are focused on the mechanisms underlying C. di,~tcile toxins cytotoxicity (F~ORENTIHI et al., 1989). Results obtained can be summarized as follows: (a) toxin A treatment (4 pg/ml) of cultured epithelial cells induced a characteristic series of morphological changes mainly represented by cell rounding and subsequent nuclear displacement. Studies performed by using cytoskeleton perturbating agents seem to indicate miaotubule system as responsible for such a nuclear polarization; (b) the exposure of different epithelial cell lines to 0 .15 pg/ml toxin B induced cell retraction, cell rounding and the formation, on the cell surface, of bulb-like structures filled with ribosomes and devoid of other cell organelles . Immunocytochemical studies performed on the cytoskeletal elements seem to indicate that microfiLiment system can play a role in the appearance of such a blebbing phenomenon . Thus, both toxins caused cell retraction and rounding in all cell types considered and seemed to exert their effect on cell morphology via a rearrangement of some cytoskeletal components. However, the toxin B-induced blebbing and the toxin A-induced nuclear polarization seem to be dependent on different cytoskeletal elements . REFERENCES BANNO, Y., et al. (1984) Rev. Infect. Dis. 6, I1-20. F~oxerrrna~, C. et al., (1989) Toxicon 27, 1209-1218 . FLORIN, I. and Tt-n?LESrw~t, M. (1983) Biochim. Biophys. Acta 763, 383-392.
Yenomorcr snakes and snake venom poisoning in Ewope. Z. Mwxar[~ and F. E. RUa9ELL (Medicinski Centar, 52000 Pula, Yugoslavia). N~nE st~ECn s and 18 subspecies of Vipers are generally recognized in Europe . One Crotalidae, Agkistrodon halys, an Asiatic species, is found as far west as the Caspian Sea. Two rear-fanged snakes, Malpolon monspessulatws and Telescopes jallax are also said to be "mildly venomous". Although reliable statistics on the number of bites and envenomations by venomous snakes in Europe are not available, those gathered from individual countries over the past two decades would indicate that the total number would not exceed 8000, and although the number of deaths per year may be as high as 50, the probable number is closer to 20. The present report treats the general biology of the venomous snakes in Europe, the chemistry and pharmacology of their venons, and the clinical problem of venom poisoning.
Re-examination ojthe protease inhibitor activities ojthe dendrotoxins from mamba venons . D. L. MwRacrwr L and A. L. HARVEY (Department of Physiology and Pharmacology, University of Strathclyde, Glasgow GI iXW, U.K .) . Try nENnßo~roz~rrs are small proteins isolated from mamba venons (HARVEY and At~rnEasort, 1985). They are highly homologous to Kursitz serine protease inhibitors, such as bovine pancreatic trypsin inhibitor (BPTI) . Although the dendrotoxins are known for their ability to block neuronal potassium ion channels and to facilitate the release of neurotransmitters (e.g . ANDERSON and HARVEY, 1988), less attention has been given to their potential anti-protease activity. Initial studies revealed little, if any, ability to block trypsin or chymotrypsin, possibly because very short incubation times were used (S~rnYnota, 1976 ; JouRERr and TwLtwwRD, 1980). We have tested the effects of three dendrotozins (toxins I and K from black mamba Derrdroaspis polylepis and dentrotoxin from Eastern green mamba Dendroaspis angesticeps) on trypsin, chymotrypsin and kallikrein . All assays were performed at 25°C in buffer containing 5 mM CaCl 2. Appropriate concentrations of specific substrates were used for each enzyme . The three toxins produced a concentration~ependent inhibition of trypsin. When measured after maximum inhibition had been reached after 60 min incubation, the rc,~ values were 0.9, 0.5 and 6.0 pM for toxin I, toxin K and dendrotoxin, respectively . The corresponding value for BPTI was 0.02 pM . The toxins acted as competitive inhibitors of kallikrein from pig pancreas. Maximum inhibition was reached by 10 min incubation . The K, values were 1.3, 0.07 and 1 .4 uM for toxin I, toxin K and dendrotoxin, respectively . None of the toxins inhibited the activity of chymotrypsin . Toxin 1 was tested at 6 KM for 75 min; toxin K at 30 pM for 60 min; and dendrotoxin at 0.7 pM for 150 min.