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Chemical and genetic engineering of snake toxins
8th European Symposium-Abstracts
159
It has bcen shown that none of the drying procedures has a general advantage over the others, since they are dependent on the substrate used and the snake venom investigated differences in enzyme activities found were common. There was a general decrease in ~-amino acid oxidase activity after drying in all venoms investigated . Most surprisingly, however, a tremendous increase in enzyme activities on several substrates was observed with Echis carinates sochureki venom after drying with some .differences according to the drying procedure used. To date, it remains open, whether (an) extremely labile inhibitors) is (are) present in this venom or whether (an) activation peptides) is (are) split off during the drying procedures . Me~rt et al. (1985) Toxicon 23, 393-397.
REFERENCE
Biochemical andpharmacological investigation ojEchis carinates venons jrom three dtfferertt geographical regions .
Jüxo M>~re' and Gb~rz Nowwrc~ ('Pentapharm Ltd, CH-4002 Basle, Switzerland, and ZInstitute of Pharmacology and Toxicology, Med. Academy, DDR-50 Erfurt, G.D.R .). Trn: Sww-scwr Fn or Carpet viper (Echis carinates) is extensively distributed from Central and North Africa over Near and Middle East to the Indian subcontinent . It has been shown that E. carirtatus in most of these regions is also the principal cause of snake bite morbidity. In this study E. carinates venoms from three geographical regions (Mali, WHO reference venom; Iran, WHO reference venom; Pakistan, from snakes kept in our own serpentarium) were investigated by biochemical and pharmacological methods. Differences in SDS-PAGE and iscelectric focusing patterns were obtained . Determination of enzymatic activities using chromogenic proteinase substrates revealed that the venoms from Iran and Pakistan exerted high proteinase activities whereas no such activities were detected in Mali venom. The prothrombin activating potential ("Ecarin Activity") was high in the venoms from Iran and Pakistan, but very low in venom from Mali . The development of lung microthrombosis induced by the venoms was investigated in rats using "'In-platelets and' 2'I-fibrinogen. In the case of E. carirtatus venoms from Pakistan and Iran platelet- as well as fibrinogen-derived radioactivity was strongly increased in the lung, whereas with E. carinates Mali venom, no platelet~erived radioactivity was observed, whereas its effect on fibrinogen was in the same range as with E. carirtatus Pakistan venom. Differences were also obtained in inhibition experiments with a-Hirudin. The latest results of this ongoing study will be discussed. Chemical and genetic engineering of snake toxins. ANnxi Mfr~z, Fxkofserc DUCANCEI, and JEwN-L`twunE
Hovr.wrx (Service de Biochimie, CEN Saclay, 91191, Gif- sur-Yvette, France).
SxwrcE venoms contain a wide variety of pharmacologically different toxins which, remarkably, only belong to two structural families . These are (i) toxins with a polypeptide chain folded into three main adjacent loops (I, II and III) rich in ß-sheets and (ü) toxins with a phospholipase A=-like structure. We cloned and elucidated the cDNA sequences of several toxins from each group. Postsynaptically acting neurotoxins belong to the first structural group. They bind to the nicotinic aatylchofine receptor (AcChoR) with high affinity . Previous work based on sequence comparisons suggested that the AcChoR binding site involves several conserved residues (at positions 8, 27, 29, 31, 32, 33, 34, 38, 46, 47, 49 and 52) mostly located on loops II and III. In agreement with this view, chemical modifications of Lys-27, Trp-29 and Lys-47 resulted in a decrease in toxicity and in affinity of the toxin for AcChoR. At present, however, no clear data are available to establish the actual participation of the other putative residues in the formation of the toxin-AcChoR complex. We constructed a recombinant expression plasmid encoding a protein A-neurotoxin fusion protein. The protein was directly expressed in the periplasmic space of E. coli and was purified in the milligramme range in a single immuno-afiinity step . The fused protein was toxic in vivo, specifically bound to AcChoR and triggered high antitoxin antibody titers in mice . By site-directed mutagenesis, we further investigated the function of loop II using fused toxin variants mutated on the tip of the loop at positions 33, 34 and across 31-34, and on a more central location at position 38 . We report on the binding properties of these mutants toward both AcChoR and a toxin-specific monoclonal antibody. Protection by silymarirt ojmicrocystirt-LR-induced acute hepatotoxicity: biochemistry, histopathology and lethality .
K. A. Meansx, D. R. Rwar .wrvn and D. A. CxFasu (U .S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21701-SOI1, U.S .A.).
MrcxocsrsrrN-LR produces acute hemorrhaggc necrosis of the liver in mice and rats. Histopathological examination of liver tissues of mice and rats that received microcystin-LR showed diffuse, severe antrilobular hepatocellular necrosis and hemorrhage . These pathological changes were correlated with increased serum activities of sorbitol dehydrogenase, alanine aminotransferase, and lactate dehydrogenase. Pretreatment of either rats or mice with a single dose of silymarin, a flavonolignane isolated from the Wild Artichoke (Silybum mariartum L. Gaertn) completely abolished the lethal effects, pathological changes, and significantly decreased the activities of serum enzymes induced by microcystin-LR .
159
It has bcen shown that none of the drying procedures has a general advantage over the others, since they are dependent on the substrate used and the snake venom investigated differences in enzyme activities found were common. There was a general decrease in ~-amino acid oxidase activity after drying in all venoms investigated . Most surprisingly, however, a tremendous increase in enzyme activities on several substrates was observed with Echis carinates sochureki venom after drying with some .differences according to the drying procedure used. To date, it remains open, whether (an) extremely labile inhibitors) is (are) present in this venom or whether (an) activation peptides) is (are) split off during the drying procedures . Me~rt et al. (1985) Toxicon 23, 393-397.
REFERENCE
Biochemical andpharmacological investigation ojEchis carinates venons jrom three dtfferertt geographical regions .
Jüxo M>~re' and Gb~rz Nowwrc~ ('Pentapharm Ltd, CH-4002 Basle, Switzerland, and ZInstitute of Pharmacology and Toxicology, Med. Academy, DDR-50 Erfurt, G.D.R .). Trn: Sww-scwr Fn or Carpet viper (Echis carinates) is extensively distributed from Central and North Africa over Near and Middle East to the Indian subcontinent . It has been shown that E. carirtatus in most of these regions is also the principal cause of snake bite morbidity. In this study E. carinates venoms from three geographical regions (Mali, WHO reference venom; Iran, WHO reference venom; Pakistan, from snakes kept in our own serpentarium) were investigated by biochemical and pharmacological methods. Differences in SDS-PAGE and iscelectric focusing patterns were obtained . Determination of enzymatic activities using chromogenic proteinase substrates revealed that the venoms from Iran and Pakistan exerted high proteinase activities whereas no such activities were detected in Mali venom. The prothrombin activating potential ("Ecarin Activity") was high in the venoms from Iran and Pakistan, but very low in venom from Mali . The development of lung microthrombosis induced by the venoms was investigated in rats using "'In-platelets and' 2'I-fibrinogen. In the case of E. carirtatus venoms from Pakistan and Iran platelet- as well as fibrinogen-derived radioactivity was strongly increased in the lung, whereas with E. carinates Mali venom, no platelet~erived radioactivity was observed, whereas its effect on fibrinogen was in the same range as with E. carirtatus Pakistan venom. Differences were also obtained in inhibition experiments with a-Hirudin. The latest results of this ongoing study will be discussed. Chemical and genetic engineering of snake toxins. ANnxi Mfr~z, Fxkofserc DUCANCEI, and JEwN-L`twunE
Hovr.wrx (Service de Biochimie, CEN Saclay, 91191, Gif- sur-Yvette, France).
SxwrcE venoms contain a wide variety of pharmacologically different toxins which, remarkably, only belong to two structural families . These are (i) toxins with a polypeptide chain folded into three main adjacent loops (I, II and III) rich in ß-sheets and (ü) toxins with a phospholipase A=-like structure. We cloned and elucidated the cDNA sequences of several toxins from each group. Postsynaptically acting neurotoxins belong to the first structural group. They bind to the nicotinic aatylchofine receptor (AcChoR) with high affinity . Previous work based on sequence comparisons suggested that the AcChoR binding site involves several conserved residues (at positions 8, 27, 29, 31, 32, 33, 34, 38, 46, 47, 49 and 52) mostly located on loops II and III. In agreement with this view, chemical modifications of Lys-27, Trp-29 and Lys-47 resulted in a decrease in toxicity and in affinity of the toxin for AcChoR. At present, however, no clear data are available to establish the actual participation of the other putative residues in the formation of the toxin-AcChoR complex. We constructed a recombinant expression plasmid encoding a protein A-neurotoxin fusion protein. The protein was directly expressed in the periplasmic space of E. coli and was purified in the milligramme range in a single immuno-afiinity step . The fused protein was toxic in vivo, specifically bound to AcChoR and triggered high antitoxin antibody titers in mice . By site-directed mutagenesis, we further investigated the function of loop II using fused toxin variants mutated on the tip of the loop at positions 33, 34 and across 31-34, and on a more central location at position 38 . We report on the binding properties of these mutants toward both AcChoR and a toxin-specific monoclonal antibody. Protection by silymarirt ojmicrocystirt-LR-induced acute hepatotoxicity: biochemistry, histopathology and lethality .
K. A. Meansx, D. R. Rwar .wrvn and D. A. CxFasu (U .S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21701-SOI1, U.S .A.).
MrcxocsrsrrN-LR produces acute hemorrhaggc necrosis of the liver in mice and rats. Histopathological examination of liver tissues of mice and rats that received microcystin-LR showed diffuse, severe antrilobular hepatocellular necrosis and hemorrhage . These pathological changes were correlated with increased serum activities of sorbitol dehydrogenase, alanine aminotransferase, and lactate dehydrogenase. Pretreatment of either rats or mice with a single dose of silymarin, a flavonolignane isolated from the Wild Artichoke (Silybum mariartum L. Gaertn) completely abolished the lethal effects, pathological changes, and significantly decreased the activities of serum enzymes induced by microcystin-LR .