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Gene structure of Vipera ammodytes phospholipases A2
Tenth European Symposium
52S
10 20 30 40 50 60 1/VRDAYIAKNYNCVYEC-FRDSYCNDLCTKNGASSGYCQWAGKYGNACWCYALPDNVPIRVPGK-CH 2/VRDAYIAKNYNCVYEC-FRDAYCNELCTKNGASSGYCQWAGKYGNACWCYALPDNVPIRVPGK-CR 3/--DGYIRKRDGCKLSCLFGNEGCNKECKSYGGSYGYC-WTWGLA--CWCEGLPDEKTWKSETNTCG 4/--DGYIRRRDGCKVSCLFGNEGCDKECKAYGGSYGYC-WTWGLA--CWCEGLPEEKTWKDETNTCG 5/VKDGYIVDDVNCTYFCGR-NAYCNEECTKLKGESGYCQWASPYGNACYCYKLPDHVRTKGPGR-CH (1) LgqIIi-(2) LghaIT-(3) LgqITZ-(4) HaIT2-(5) AaH II . Fta . 1 .
CraAtv®t, T. Hturroo, H . Rocxer and F . MtaArroA (CNRS, URA 1455, Laboratoire de Hiachimie, Faculté de Médocine secteur Nord, bd. P. Dramard, 13326 Marseille Cedex 15, France). Taomv III of LeGaua qulrtquestr7atus gidnquestriatus Mq III), active both on mammals and insects, was purified according to MiAANUA et d. We report here its primary structure obtained by the sequencing of the reduced and S~arboxymethylated protein and derived tryptic peptides . It is a 64 amino-acid polypeptide reticulated by four disulfide bridges. This toxin of the a-type, as attested by pharmacological tests, is highly potent on mice (~z -I pg/2t1 g mouse) aced insects (rbw - 3 ag/50 mg Blatella geimanica). We found that Lqq III sequence differs from the sequence of LghaIT an anti-insect toxin from the scorpion Lairaus grdAqurstriotus hebraerrs (ErrAN, et d., 1990), Only m poaUons 20, 24 and 64 (Ser- ~ Ala, Asp- -. alu, Hnr ~ Arg). However, LghaIT u clarmed to be practically non-toxic to mice and potent at the dose of 140 ng/g of body weight on insects and isopode. Thus, three amino acid changes could modify the epecifi~ty of the toxins. Another toxin, LgqIT~ (Kori?rwiv et d., 1990), Purified from Lahore q.q. and which inducts a flaccid paralysis strictly on insects, was injected into rabbits to raise antibodies . These antibodies were used to set up an ELISA test based on competition of LgqIT~ with toxins of the same type. Using this technique, it was possible to screen rapidly the `fiaocid' anti-insect activity in chromatographic fiBCtioas obtained in the Brat step of the puriûcation (gel filtration) of Buthacus arYnicola. BaIT= was Wus detected, purified in one run of HPLC and its sequence established by Edman degradation of protein aced peptides obtained by action of lysine C protease. BaIT i and other anti-iasxt toxins of this type are single polypeptide chains of 61 amino acids, pr+esentiag, surprisiaglY, strong homologies with the most active anti-mammal a-toxins, such as AaH II (toxin II of A+rd}octtnne erustrdis Hector) . These molecule may prove to be useful for the studies on etructute-epe~ity developed in our laboratory . See Fig. 1 . ErrAx, M ., Fowt.sa, E., Ht~rAtvx, R ., Dwer., A., Per~twre, M . and Zr .o~xnv, E. (1990) Biocleenrirtry 29, 5941-594?. Kort=.rAx, C.,1Ner~ tß r~, P., SA~~u, F ., HaArroo, T ., BAHOtAOUi, E . M ., Rocxwr, H. and GxAN~, C. (1990) FEBS Last. 261, 42326 . MtxANnA, F., Kot~etrAx, C ., RocHAr, H ., RocrrAr, C. and Lrssrrznr, S. (1970) Eru . !. Bioclrcm . 16, 514-523 .
tîrxe atructtor of Vipers ammodytes phospJwlipaus A : D. Kortnt3 and F. Cruam~ (Department of Biochemistry, Jobef Stefan Institute, Jamova 39, Ljubljana, Slovenia) . Ttm vt~rn of the bag-nosed viper (Ytpera annrrodytas) is a complex mixture of biologically active compounds with lethal, hemorrhagic, proteolytic and arterial blood depressing effects. Important constituents of the venom are phospholipase Az (PL .A~ isozymes. Some of them possess high preaynaptic neurotoxicity such as ammadytoxins A, H sad C while some are strongly myotoxic such as ammodytin L and some are nontoxic such as ammodytin I=. Ln contrast to mammalian PLAre several atnecturally similar PLA= enzymes or their homologues were isolated from a single snake species. Southern blot analyses of human and rat chromosomal DNA indicate that very likely a single gene exists for mammalian group II PLA1s . At present the gcae structure of the snake group II PLA NS is still unknown. However, we can show that snake PLA~s are encoded by multiple gone families . A genomic library has ban constructed in Lambda C3EM-12 phage using high molecular mass DNA isolated from the V. amtrrodytas liver. Complete cDNA Bona coding for ammodytoxins B aced C were used as hybridization probe . Five positive clones were found, two strongly (ammodytoxin gene) and three weakly hybridizing (ammodytin geaa) after restriction digestion and Southern blot hybridization with an ammodytoxin C cDNA probe. Hybridizing restriction fragments were isolated, subcloned and sequenced. The ammodytoxin and ammodytin geaa spanned about 3.5 kb pairs . Comparison of the genomic aced cDNA eequenees reveals that the ammodytoxin end ammodytin genes have 5 axons and 4 introas. Exon 1 eaoodea the 5' nontraaslated region, exoa 2 encodes the signal peptide, aroas 3-5 epode regions of the protein of residua -2 to 43 (axon 3), 43 to 77 (axon 4~ 77 to 121 plw 3' nontranslated region (axon 5). Southern blot analysis of V. mnmodytes genomic DNA
526
Tenth European Symposium
indicates that the ammodytoxim and ammodydas are encoded by multiple genes within the Y. amnrodyur genorae and therdore mpraent a multigene family. The intron/exon structure of the V anottodyua PLA= genes is similar to that of the mammalian group I sad II PLArs. Is~olattar and partfa! eharaeterlsation of lumrorrlutgic tax6t jrom the lorg-rto~sr~d vlpn (Vipaa ammodytas ammodytes) voran. I. Kartar and F. Ciu~ (Department of Biochemistry, Josef Stefan Institute, Jartrova 39, Ljubljana, Slovenia). Oro; oe the most striking manifestations of Yfperidoe and Crotalidbe poisoning is extensive hemorrhage which may spread in severe cases m that bleeding in organs such as brain, heart, lung, intestine and kidney is eaoountered . In contrast to neurotoxic erects, induced by snake envenomstion, the local etfacts, such as hemorrhage, necaosis and edema, ate poorly understood at the molecular level. In general, hemorrhagins are proteins which usually have proteolytic activity. They vary widely is structure, but appear to be timt7ar in their ability to induce hemorrhage by dirxt actions on the blood vessel wall. In order to atndy this interosting group of toxins a hemorrhgic toxin from the venom of European viper (Ytpera anurrodytea mnmtodyus) was isohrted in a five step isolation procedure. Attar initial CM-cellulose ohromatogtsphY, Sephadex G100, DEAFrSapMod, lentil-écrin Sepharose and hydrophobic interaction chromatographies were need . Isolated hemorrhagin (HRI) is a 70,000 mol. wt ttumotneric glycoprotein with a multiple pl about S. It possesses a very potent hemorrhagic activity. HRl is a metalbpmteinase. The aseinolytic activity was inhibited by EDTA, which destroyed also hemorrhagic activity . It cleaved the inadin H-chain prderentially at the position AIaN-Leuu whit cleavages at Tyru_Leu" and His'°-Leuu oocurrod much moro slowly. The molecub is N-terrrrinally bloclced. CNBr fragmentation of the protein was made and some internal parts of ib segoenoe were established. Comparing the sequenced part of HRl to the corresponding parts in the other atrt>cturally known hemoahagios, the levcl of identity is the highest with lachsla mots mtua hemorrhagic fs~ctor LHF II (62.5X° of identity) and 7Ylamercawacr Jlarnviridit high molecular mass hemorrhagic protein HR-1B (5g.3°iG of identity). Synthrsis of an crabs of sarajotaxbr-b (?fb2) .uaafotazfn-b . H. Last'rxet~t,' C. CrtmmVOt+,' J. daasar,= Z wor ila~~: A. Bnotaa,' S x«~rv~3 andA. Mimez' ('Département a'Etuaea et d'Ingénierie sea Protbines, Bat. 152, C. E. Saclay, 91191 C3if%Yvette Cedex, France; =Département de Recherche en Imagerie, Pharmacologie et Physiobgle (DRIPP), C.E. Saclsy, 91 l91 (3if/Yvette Cedex, France; and 3Departmeat of Zoology, ßeorge S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69 978 Tel Aviv, Israel). Seaer+ol+ormva consritute a family of isopeptidea of 21 amino acids with two disulfide bonds that are found in the venom of the burrowing asp Atroctaspla engaddensLr. The SRTXs are lethal, catdiotoxic and itdnce oonatricrion of blood vessels and contraction of other smooth muscles. SRTX~ aad SRTX-d ate less toxic and weaker pharmacologically thaw the other SRTXs (a end b). These diB'erences were proposed to be associated with the presence of a threoniae residue at position 2 itutead of a serine in the other iaopeptides. we have now syatherized the ('Thr~-3RTX-b analog by solid phase synthesis and studied its biological and ®muaological properties. Preliminary data suggest that the synthetic analog is about as toxic as SRTX-b, has a lower vasoconstriction et&acy in the rabbit aorta and is about as potent as SRTX-b in influencing contraction in rat uterw. Tetuaeu et al. (1991) have shown that the same analog syatheaiaed by them poaesses a contida~ably lower vasownstriction efl>cacy in the denuded aorta and a diminished constriction potency in the mesenteric arteries of the rat. It may thw be concluded that lethality and uteri contraction, on the one hand, and vasooonattidion, on the other, can be discriminated by the single Thr to Sec mutation at position 2. Ftirrthermore, the synthetic peptide cross-reacted with both the monoclonal antibodies anti-N-tar-side of human endothelia-1 and with the monoclonal antibodies anti-C-tar-aide of hutoan er~dothelin-1 as displayed by the binding assays . Secondaryatructwe andproteinjo(d ojblaat mamba dsndrotaxbr I m determined by ~X-NMR coward amokcrdar bastr jor the .nkctlro~r blockage of voltgge nenstttve potasrhem chwureir. M:F. Faster,' J:M. L~uvc~.nv,' M. Hor.rBCS®t~ and D. Meartnv' ('Institut de Hiologle Structurale, 41, Avenue des Martyrs, 38027 C3reaoble, France; and =Centr+e de Biophysique Moléculaire, lA, Avenue de la Recherche Scientifique, 43071 Orléans, France). Trm aacormertY structure of dendrotoxin I, an important conadtuent of the venom of the African bladt mamba snake Dendrompta polylepirpalyJepta, was determined in agtxous solution by two-dimendonal nuclear magnetic resonance rathods. Complete sequence-specific proton NMR assignment wen obtained at the exception of the
52S
10 20 30 40 50 60 1/VRDAYIAKNYNCVYEC-FRDSYCNDLCTKNGASSGYCQWAGKYGNACWCYALPDNVPIRVPGK-CH 2/VRDAYIAKNYNCVYEC-FRDAYCNELCTKNGASSGYCQWAGKYGNACWCYALPDNVPIRVPGK-CR 3/--DGYIRKRDGCKLSCLFGNEGCNKECKSYGGSYGYC-WTWGLA--CWCEGLPDEKTWKSETNTCG 4/--DGYIRRRDGCKVSCLFGNEGCDKECKAYGGSYGYC-WTWGLA--CWCEGLPEEKTWKDETNTCG 5/VKDGYIVDDVNCTYFCGR-NAYCNEECTKLKGESGYCQWASPYGNACYCYKLPDHVRTKGPGR-CH (1) LgqIIi-(2) LghaIT-(3) LgqITZ-(4) HaIT2-(5) AaH II . Fta . 1 .
CraAtv®t, T. Hturroo, H . Rocxer and F . MtaArroA (CNRS, URA 1455, Laboratoire de Hiachimie, Faculté de Médocine secteur Nord, bd. P. Dramard, 13326 Marseille Cedex 15, France). Taomv III of LeGaua qulrtquestr7atus gidnquestriatus Mq III), active both on mammals and insects, was purified according to MiAANUA et d. We report here its primary structure obtained by the sequencing of the reduced and S~arboxymethylated protein and derived tryptic peptides . It is a 64 amino-acid polypeptide reticulated by four disulfide bridges. This toxin of the a-type, as attested by pharmacological tests, is highly potent on mice (~z -I pg/2t1 g mouse) aced insects (rbw - 3 ag/50 mg Blatella geimanica). We found that Lqq III sequence differs from the sequence of LghaIT an anti-insect toxin from the scorpion Lairaus grdAqurstriotus hebraerrs (ErrAN, et d., 1990), Only m poaUons 20, 24 and 64 (Ser- ~ Ala, Asp- -. alu, Hnr ~ Arg). However, LghaIT u clarmed to be practically non-toxic to mice and potent at the dose of 140 ng/g of body weight on insects and isopode. Thus, three amino acid changes could modify the epecifi~ty of the toxins. Another toxin, LgqIT~ (Kori?rwiv et d., 1990), Purified from Lahore q.q. and which inducts a flaccid paralysis strictly on insects, was injected into rabbits to raise antibodies . These antibodies were used to set up an ELISA test based on competition of LgqIT~ with toxins of the same type. Using this technique, it was possible to screen rapidly the `fiaocid' anti-insect activity in chromatographic fiBCtioas obtained in the Brat step of the puriûcation (gel filtration) of Buthacus arYnicola. BaIT= was Wus detected, purified in one run of HPLC and its sequence established by Edman degradation of protein aced peptides obtained by action of lysine C protease. BaIT i and other anti-iasxt toxins of this type are single polypeptide chains of 61 amino acids, pr+esentiag, surprisiaglY, strong homologies with the most active anti-mammal a-toxins, such as AaH II (toxin II of A+rd}octtnne erustrdis Hector) . These molecule may prove to be useful for the studies on etructute-epe~ity developed in our laboratory . See Fig. 1 . ErrAx, M ., Fowt.sa, E., Ht~rAtvx, R ., Dwer., A., Per~twre, M . and Zr .o~xnv, E. (1990) Biocleenrirtry 29, 5941-594?. Kort=.rAx, C.,1Ner~ tß r~, P., SA~~u, F ., HaArroo, T ., BAHOtAOUi, E . M ., Rocxwr, H. and GxAN~, C. (1990) FEBS Last. 261, 42326 . MtxANnA, F., Kot~etrAx, C ., RocHAr, H ., RocrrAr, C. and Lrssrrznr, S. (1970) Eru . !. Bioclrcm . 16, 514-523 .
tîrxe atructtor of Vipers ammodytes phospJwlipaus A : D. Kortnt3 and F. Cruam~ (Department of Biochemistry, Jobef Stefan Institute, Jamova 39, Ljubljana, Slovenia) . Ttm vt~rn of the bag-nosed viper (Ytpera annrrodytas) is a complex mixture of biologically active compounds with lethal, hemorrhagic, proteolytic and arterial blood depressing effects. Important constituents of the venom are phospholipase Az (PL .A~ isozymes. Some of them possess high preaynaptic neurotoxicity such as ammadytoxins A, H sad C while some are strongly myotoxic such as ammodytin L and some are nontoxic such as ammodytin I=. Ln contrast to mammalian PLAre several atnecturally similar PLA= enzymes or their homologues were isolated from a single snake species. Southern blot analyses of human and rat chromosomal DNA indicate that very likely a single gene exists for mammalian group II PLA1s . At present the gcae structure of the snake group II PLA NS is still unknown. However, we can show that snake PLA~s are encoded by multiple gone families . A genomic library has ban constructed in Lambda C3EM-12 phage using high molecular mass DNA isolated from the V. amtrrodytas liver. Complete cDNA Bona coding for ammodytoxins B aced C were used as hybridization probe . Five positive clones were found, two strongly (ammodytoxin gene) and three weakly hybridizing (ammodytin geaa) after restriction digestion and Southern blot hybridization with an ammodytoxin C cDNA probe. Hybridizing restriction fragments were isolated, subcloned and sequenced. The ammodytoxin and ammodytin geaa spanned about 3.5 kb pairs . Comparison of the genomic aced cDNA eequenees reveals that the ammodytoxin end ammodytin genes have 5 axons and 4 introas. Exon 1 eaoodea the 5' nontraaslated region, exoa 2 encodes the signal peptide, aroas 3-5 epode regions of the protein of residua -2 to 43 (axon 3), 43 to 77 (axon 4~ 77 to 121 plw 3' nontranslated region (axon 5). Southern blot analysis of V. mnmodytes genomic DNA
526
Tenth European Symposium
indicates that the ammodytoxim and ammodydas are encoded by multiple genes within the Y. amnrodyur genorae and therdore mpraent a multigene family. The intron/exon structure of the V anottodyua PLA= genes is similar to that of the mammalian group I sad II PLArs. Is~olattar and partfa! eharaeterlsation of lumrorrlutgic tax6t jrom the lorg-rto~sr~d vlpn (Vipaa ammodytas ammodytes) voran. I. Kartar and F. Ciu~ (Department of Biochemistry, Josef Stefan Institute, Jartrova 39, Ljubljana, Slovenia). Oro; oe the most striking manifestations of Yfperidoe and Crotalidbe poisoning is extensive hemorrhage which may spread in severe cases m that bleeding in organs such as brain, heart, lung, intestine and kidney is eaoountered . In contrast to neurotoxic erects, induced by snake envenomstion, the local etfacts, such as hemorrhage, necaosis and edema, ate poorly understood at the molecular level. In general, hemorrhagins are proteins which usually have proteolytic activity. They vary widely is structure, but appear to be timt7ar in their ability to induce hemorrhage by dirxt actions on the blood vessel wall. In order to atndy this interosting group of toxins a hemorrhgic toxin from the venom of European viper (Ytpera anurrodytea mnmtodyus) was isohrted in a five step isolation procedure. Attar initial CM-cellulose ohromatogtsphY, Sephadex G100, DEAFrSapMod, lentil-écrin Sepharose and hydrophobic interaction chromatographies were need . Isolated hemorrhagin (HRI) is a 70,000 mol. wt ttumotneric glycoprotein with a multiple pl about S. It possesses a very potent hemorrhagic activity. HRl is a metalbpmteinase. The aseinolytic activity was inhibited by EDTA, which destroyed also hemorrhagic activity . It cleaved the inadin H-chain prderentially at the position AIaN-Leuu whit cleavages at Tyru_Leu" and His'°-Leuu oocurrod much moro slowly. The molecub is N-terrrrinally bloclced. CNBr fragmentation of the protein was made and some internal parts of ib segoenoe were established. Comparing the sequenced part of HRl to the corresponding parts in the other atrt>cturally known hemoahagios, the levcl of identity is the highest with lachsla mots mtua hemorrhagic fs~ctor LHF II (62.5X° of identity) and 7Ylamercawacr Jlarnviridit high molecular mass hemorrhagic protein HR-1B (5g.3°iG of identity). Synthrsis of an crabs of sarajotaxbr-b (?fb2) .uaafotazfn-b . H. Last'rxet~t,' C. CrtmmVOt+,' J. daasar,= Z wor ila~~: A. Bnotaa,' S x«~rv~3 andA. Mimez' ('Département a'Etuaea et d'Ingénierie sea Protbines, Bat. 152, C. E. Saclay, 91191 C3if%Yvette Cedex, France; =Département de Recherche en Imagerie, Pharmacologie et Physiobgle (DRIPP), C.E. Saclsy, 91 l91 (3if/Yvette Cedex, France; and 3Departmeat of Zoology, ßeorge S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69 978 Tel Aviv, Israel). Seaer+ol+ormva consritute a family of isopeptidea of 21 amino acids with two disulfide bonds that are found in the venom of the burrowing asp Atroctaspla engaddensLr. The SRTXs are lethal, catdiotoxic and itdnce oonatricrion of blood vessels and contraction of other smooth muscles. SRTX~ aad SRTX-d ate less toxic and weaker pharmacologically thaw the other SRTXs (a end b). These diB'erences were proposed to be associated with the presence of a threoniae residue at position 2 itutead of a serine in the other iaopeptides. we have now syatherized the ('Thr~-3RTX-b analog by solid phase synthesis and studied its biological and ®muaological properties. Preliminary data suggest that the synthetic analog is about as toxic as SRTX-b, has a lower vasoconstriction et&acy in the rabbit aorta and is about as potent as SRTX-b in influencing contraction in rat uterw. Tetuaeu et al. (1991) have shown that the same analog syatheaiaed by them poaesses a contida~ably lower vasownstriction efl>cacy in the denuded aorta and a diminished constriction potency in the mesenteric arteries of the rat. It may thw be concluded that lethality and uteri contraction, on the one hand, and vasooonattidion, on the other, can be discriminated by the single Thr to Sec mutation at position 2. Ftirrthermore, the synthetic peptide cross-reacted with both the monoclonal antibodies anti-N-tar-side of human endothelia-1 and with the monoclonal antibodies anti-C-tar-aide of hutoan er~dothelin-1 as displayed by the binding assays . Secondaryatructwe andproteinjo(d ojblaat mamba dsndrotaxbr I m determined by ~X-NMR coward amokcrdar bastr jor the .nkctlro~r blockage of voltgge nenstttve potasrhem chwureir. M:F. Faster,' J:M. L~uvc~.nv,' M. Hor.rBCS®t~ and D. Meartnv' ('Institut de Hiologle Structurale, 41, Avenue des Martyrs, 38027 C3reaoble, France; and =Centr+e de Biophysique Moléculaire, lA, Avenue de la Recherche Scientifique, 43071 Orléans, France). Trm aacormertY structure of dendrotoxin I, an important conadtuent of the venom of the African bladt mamba snake Dendrompta polylepirpalyJepta, was determined in agtxous solution by two-dimendonal nuclear magnetic resonance rathods. Complete sequence-specific proton NMR assignment wen obtained at the exception of the