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Ichthyotoxic proteins of marine origin
Tenth European Symposium
SOS
71k ant~esicity oj the earbohydrate mokty oj honeybee (Apir mdltfera) phospholipare Ai, an alhTgentc gtycoproteie: strrrctrant and fierctianal studies. L. Mxrtz (Institut fùr Chemie, Universitilt fur Hadenkultur Gregr-Mendelstraeae 33, A-1180 Vieans, Austrie). Pt ot m~ A1 (PLA; E.C . 3.1 .1 .4) from hoaeybx (Apia nsrltjjera) venom l a glycoprotein with a carbohydrate unit linked to aspangine-13 of the polypeptide. Isolation and analysis of this N-glycan revealed a high degree of chemical heterogeneity, u it contains l4 variants. While four of them repr+eaeat extended, complex, structures with novel structural f~atures, the other ten are of the oligomannoeidic structural type with a1,3-and/or al,frfucosyl exte~ona at the asn-bound N-saetylgluoosamine. In earlier studies we had shown that sera of beo-atingallergic patients contain IgE antibodies, which apecißcally react with the oGgosaccharide unit of bee venom PLA. This led us to believe that a peculiar structural feature, not shared by glycoproteina of vertebrate origin, may be responsible for this interaction. A rabbit polycloncl serum, raised against PLA, was subsequently shown to contain antibodies, which react with the enzyme's carbohydrate, and to cross-react with plant glyeoproteins. ELISA binding and inhibition studies, in combination with selective deglyeosylation experiments, identified a1,3-fuooaylation of the ass-bound N-acetyl-gluoosamine as the antigenic determinant. This part structure is also rxognized by carbohhydrate-directed IgE antibodies of patient sera. Mammalian glyooproteins Lick this particrilar modification . This work l supported by the Bundesministerium tür Wissenschaft and Forschung and the Fonda zur Forschung FBrdenmg der ' . Ichtlryotoxicproteins ojmarine origin. D. Mns (Zentrum der Rechbmedizin, University of Frankfurt, Frankfurt, F.R.G .) . Prtm~a isolated from sea anemones and sponges from the Partific and Red Sea wen found to exhibit considerable toxicity when tested on fish (added to the water). Thex eomponenta are also potent haemolysins. Homogenates of the samples (sea anemones or sponges) were fractionated by gel filtration and the active (haemolytic and ichthyotoxic) oomponenta were further purified by ion~xchange chromatography . Icthyotoxina (haemolysins) from sea anemones (Hetaactir nragn~rca, Gyrwtonra hdimethre) possess mol. wta of about 20,000 and are structurally homologue to similar proteins such as tenebrosin or equiaatoxin . Hiatological studies indicate that they affect the giW of fish causing severe membrane disruptions and impairing the respiratory functions when added to the water even in very low eonaatrationa. Exprcssian to E. cob of a recombinant colortmetrtc antibody that recognizes craannrLnetic toxins and mbnica nicotint aottyicholine receptors. F. Ducniv®,, D. Gal$r, O. Tciirm~u, J.-C. Hour.~av and A. Mffiv~ (Département d'Etudes et d'Ingénierie des Protéines, Laboratoire d'Ingénierie Géntique des Protéines, Bat. 152, C.E .N. de Saclay, 91191 Gif-sur-Yvette, France). Wa n~at>ee a genetic construction that allows the direct expression in E. colt of an IgG-like molecule which r+ecogoizea curaremimetic toxins from snake veaoma aced which possess a oolorimetric activity . The Fab parts of this hybrid are similar to those found is Mat-3, a monoclonal antibody that binds to all ahort~hsin curaremimetic toxins, wher+eaa the Fc part consists of alkaline phoaphatase . The preeeatatioa will be divided into two parts. First, it will be shown that Mat-3 mimics the nicotinic aoetyleholi~ receptor (AcChoR). This is mainly based on the observation that (1) Mat-3 and Ad hoR recognize identical residues at the surface of curar+emimetic toxins; (2) when Mat-3 and AcChoR are independently injected in animals they both trigger immu~ responses that induce signs of Myasthenia gravir and the production of anti-AcChoR antibodies; (3) parts of the a-subunit of AcChoR and some CDRa of Mat-3 share clear amino acid sequence analogies. The second part will be devoted to the description of the genetic oonstnrction of the oolorimetric Mat-3 and to the characterization of its biological properties. The potential offered by this new type of colorimetric antibody will be discussed. Tetamrs tox6r it a stuc protein and its inhibition ojnaaotransnritter rehears Lr z6rc dependent. C. MOtrrBr,-uooo,' G. Scrawo,' O. Ito~n+o' and B. Pout.~nv~ ('Dipertimento di Scieaze Hiomediche, University di Padoue, Italy; and'C .N.R .S. Neurobiologie Molxulaire et Cellulaire, Gif-cur-Yvette, Frana) . Tsrexus (TeTx) sad botuliaum neurotoxins are the most potent toxins known. They are di~hein proteins (H, 100,000 and L, 50,000 mol. wt) which bind to nerve ceW, penetrate the cytosol and block neurotraaamitter release . Comparison of their amino acid sequenoea evideatiatea in the middle of the L~haia the presence of a
SOS
71k ant~esicity oj the earbohydrate mokty oj honeybee (Apir mdltfera) phospholipare Ai, an alhTgentc gtycoproteie: strrrctrant and fierctianal studies. L. Mxrtz (Institut fùr Chemie, Universitilt fur Hadenkultur Gregr-Mendelstraeae 33, A-1180 Vieans, Austrie). Pt ot m~ A1 (PLA; E.C . 3.1 .1 .4) from hoaeybx (Apia nsrltjjera) venom l a glycoprotein with a carbohydrate unit linked to aspangine-13 of the polypeptide. Isolation and analysis of this N-glycan revealed a high degree of chemical heterogeneity, u it contains l4 variants. While four of them repr+eaeat extended, complex, structures with novel structural f~atures, the other ten are of the oligomannoeidic structural type with a1,3-and/or al,frfucosyl exte~ona at the asn-bound N-saetylgluoosamine. In earlier studies we had shown that sera of beo-atingallergic patients contain IgE antibodies, which apecißcally react with the oGgosaccharide unit of bee venom PLA. This led us to believe that a peculiar structural feature, not shared by glycoproteina of vertebrate origin, may be responsible for this interaction. A rabbit polycloncl serum, raised against PLA, was subsequently shown to contain antibodies, which react with the enzyme's carbohydrate, and to cross-react with plant glyeoproteins. ELISA binding and inhibition studies, in combination with selective deglyeosylation experiments, identified a1,3-fuooaylation of the ass-bound N-acetyl-gluoosamine as the antigenic determinant. This part structure is also rxognized by carbohhydrate-directed IgE antibodies of patient sera. Mammalian glyooproteins Lick this particrilar modification . This work l supported by the Bundesministerium tür Wissenschaft and Forschung and the Fonda zur Forschung FBrdenmg der ' . Ichtlryotoxicproteins ojmarine origin. D. Mns (Zentrum der Rechbmedizin, University of Frankfurt, Frankfurt, F.R.G .) . Prtm~a isolated from sea anemones and sponges from the Partific and Red Sea wen found to exhibit considerable toxicity when tested on fish (added to the water). Thex eomponenta are also potent haemolysins. Homogenates of the samples (sea anemones or sponges) were fractionated by gel filtration and the active (haemolytic and ichthyotoxic) oomponenta were further purified by ion~xchange chromatography . Icthyotoxina (haemolysins) from sea anemones (Hetaactir nragn~rca, Gyrwtonra hdimethre) possess mol. wta of about 20,000 and are structurally homologue to similar proteins such as tenebrosin or equiaatoxin . Hiatological studies indicate that they affect the giW of fish causing severe membrane disruptions and impairing the respiratory functions when added to the water even in very low eonaatrationa. Exprcssian to E. cob of a recombinant colortmetrtc antibody that recognizes craannrLnetic toxins and mbnica nicotint aottyicholine receptors. F. Ducniv®,, D. Gal$r, O. Tciirm~u, J.-C. Hour.~av and A. Mffiv~ (Département d'Etudes et d'Ingénierie des Protéines, Laboratoire d'Ingénierie Géntique des Protéines, Bat. 152, C.E .N. de Saclay, 91191 Gif-sur-Yvette, France). Wa n~at>ee a genetic construction that allows the direct expression in E. colt of an IgG-like molecule which r+ecogoizea curaremimetic toxins from snake veaoma aced which possess a oolorimetric activity . The Fab parts of this hybrid are similar to those found is Mat-3, a monoclonal antibody that binds to all ahort~hsin curaremimetic toxins, wher+eaa the Fc part consists of alkaline phoaphatase . The preeeatatioa will be divided into two parts. First, it will be shown that Mat-3 mimics the nicotinic aoetyleholi~ receptor (AcChoR). This is mainly based on the observation that (1) Mat-3 and Ad hoR recognize identical residues at the surface of curar+emimetic toxins; (2) when Mat-3 and AcChoR are independently injected in animals they both trigger immu~ responses that induce signs of Myasthenia gravir and the production of anti-AcChoR antibodies; (3) parts of the a-subunit of AcChoR and some CDRa of Mat-3 share clear amino acid sequence analogies. The second part will be devoted to the description of the genetic oonstnrction of the oolorimetric Mat-3 and to the characterization of its biological properties. The potential offered by this new type of colorimetric antibody will be discussed. Tetamrs tox6r it a stuc protein and its inhibition ojnaaotransnritter rehears Lr z6rc dependent. C. MOtrrBr,-uooo,' G. Scrawo,' O. Ito~n+o' and B. Pout.~nv~ ('Dipertimento di Scieaze Hiomediche, University di Padoue, Italy; and'C .N.R .S. Neurobiologie Molxulaire et Cellulaire, Gif-cur-Yvette, Frana) . Tsrexus (TeTx) sad botuliaum neurotoxins are the most potent toxins known. They are di~hein proteins (H, 100,000 and L, 50,000 mol. wt) which bind to nerve ceW, penetrate the cytosol and block neurotraaamitter release . Comparison of their amino acid sequenoea evideatiatea in the middle of the L~haia the presence of a