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Some structural characteristics of Sticholysins I and II, Cytolysins from the sea anemone Stichodactyla helianthus and their implications for the interaction with membranes
Abstracts/Toxlcon 38 (2000) 487-595
491
antithrombin as well as platelet aggregation inhibitor. Many substances have been only partially characterized while others have been cloned and prepared in recombinant form. The compounds identified can be classified in five groups according to their function: Group I: Activities interfering with the thrombinfibrinogen reaction, most of them are antithrombins. Examples are found in Rhodnius prolixus, Triatoma pallidipennis, Eutriatoma maculatus, Ixodes ricinus, Ornithodorous moubata, Aedes and Anophelins. Group II: Compounds acting on the platelet function: inducing aggregation, as in Apis mellifera and Loxoseles reclusa or having inhibitory effect on adhesion or aggregation as in Glossina morsitans, Triatoma pallidipennis, Ornithodorus moubata and Anophelins. Group III: Compounds acting on the prothrombinase complex; the majority of them function as inhibitors of FXa as in Rhodnius prolixus and Ornithodorus moubata. In Lonomia achelous a prothrombin activator and a FXa-like activity have been detected while in Lonomia obliqua activators of prothrombin and FX have been reported. Group IV: Compounds acting on the fibrinolytic system as in, Glossina austeni, Rhodnius prolixus, Lonomia achelous and Scolopendra subepinipes mutilans. Group V: An heterogeneous and poorly characterized group of compounds, such as inhibitor from Haementeria ghillianis; a FXIII proteolytic activity from Lonomia achelous, an c~V-B/3 inhibitor from Dermacentor variabilis and a LA-like protein from Loxosceles reclusa. In this Group are also included activities that have not been studied in depth. It is important to mention that apart from scorpions and spiders which induced DIC, but whose activities have not been well characterized, caterpillars of the Lonomia genus are the only arthropods which really affects clinically the hemostatic mechanism, inducing a severe bleeding syndrome which can cause death. In conclusion: Every day new compounds with activities interfering with the hemostatic mechanism are identified in arthropods. These compounds are of interest as usefull tools for the understanding of the hemostatic mechanisms and as potential therapeutic drugs.
Some structural characteristics of Sticholysins I and II, CytoIysins from the sea anemone StichodactyIa helianthus and their implications for the interaction with membranes. M.E. Lanio a , C. Alvarez a, V. Morerab," F. Pazos a, M. Tejuca a, D. Martinez a, G. Menestrina c, A.M. Campos d, E. Lissi d (~University of Havana, Cuba; bCIGB, Cuba; CCeFSA, [taly; dUniversity of Santiago de, Chile). Sticholysin I (St I) and II (St II) are two basic cytolysins (MW 20 kDa) purified from the sea anemone Stichodactyla helianthus. They exhibit potent hemolytic activities (HA) and increase the membrane permeability by forming oligomeric pores. In the present work, the comparison of some structural characteristics of St I and St II and their structural modifications was carried out to deepen into the mechanism of interaction with membranes. Primary structure analysis of St I and St II (Edman's degradation and Mass Spectrometry) revealed a high homology between them suggesting they are isoforms of the same cytolysin. Similarly to other sea anemone cytolysins, St I and St II also contain a 22 amino acid insertion fragment absent in C III, the major hemolysin reported in S. helianthus.
492
Abstracts/Toxtcon 38 (2000) 487-595
The high homology between St II and C III and the revaluation of C III MW suggest that C III and St II are the same protein. St I and C I (Kern and Dunn, 1988) showed complete homology in the small C I fragment analyzed. Structural predictions of St I & II indicated they are hydrophylic polypeptides with a single highly hydrophobic fragment corresponding to the N-terminal probably involved as a leader peptide in the interaction with membranes. CNBr treatment of St II yielded three main peptides. One of them contained the N-terminal of the protein and was able to inhibit St II activity probably due to their competition for the membrane. The rest of the peptides did not elicit any effect on St lI HA, suggesting the significance of its N-terminal fragment for the interaction with membranes. Pre-incubation of St II with 2,2-azo-bis(2amidinopropane), a source of peroxyl radicals reduced its HA and significantly modified only Trp residues. The loss of HA correlates with the decrease of the protein fluorescence due the radicals. St II association with liposomes increased protein fluorescence indicating a more hydrophobic environment of St II Trp groups. St II bound to liposomes reduced the rate of fluorescence loss during its modification by free radicals. The results suggest the existence of two populations of Trp quenched by acrylamide and inactivated by free radicals at different rates. Modification of one of the critical Trp leads to cytolysin inactivation pointing its relevant role to the function of the protein. St I modified by AAPH gave similar results to those above described for St II.
Elucidation and exploitation of the functional architectures of animal toxins. Andr+ M6nez (D~partement d'Ing6nierie et d']~tudes des Prot6ines, CEA Saclay, 91191, Gif-sur-Yvette, France). This paper will describe some rules associated with the major functional and structural properties of animal toxic proteins and will show how these rules may be exploited in protein engineering. Though being often small (less than 120 amino acids), animal toxins exert a wide diversity of biological functions. Extensive studies by N M R spectroscopy and X-ray crystallography, have revealed that functionally unrelated toxins produced by a phylogenetically related group of venomous animals (a family, an order, etc.) adopt the same structural fold whereas functionally related toxins produced by phylogenetically distinct venomous animals adopt distinct folds. The topographies by which a toxin fold exerts diverse functions have also been subjected to extensive studies. This is the case, for example, of various functionally different toxins which adopt the so called 'three-fingered fold' as well as various structurally different toxins which competitively block voltage-gated potassium channels. These studies revealed that (i) a functional topography consists of a homogeneous surface of 5-10 residues among which 2-4 play a most critical role: (ii) distinct regions of a fold can accommodate unrelated functional sites; (iii) individual members of a family of functionally related toxins can display different though overlapping functional topographies. The functional site of each member of a toxin family comprises at least two components. First, it includes a small functional core composed of few
491
antithrombin as well as platelet aggregation inhibitor. Many substances have been only partially characterized while others have been cloned and prepared in recombinant form. The compounds identified can be classified in five groups according to their function: Group I: Activities interfering with the thrombinfibrinogen reaction, most of them are antithrombins. Examples are found in Rhodnius prolixus, Triatoma pallidipennis, Eutriatoma maculatus, Ixodes ricinus, Ornithodorous moubata, Aedes and Anophelins. Group II: Compounds acting on the platelet function: inducing aggregation, as in Apis mellifera and Loxoseles reclusa or having inhibitory effect on adhesion or aggregation as in Glossina morsitans, Triatoma pallidipennis, Ornithodorus moubata and Anophelins. Group III: Compounds acting on the prothrombinase complex; the majority of them function as inhibitors of FXa as in Rhodnius prolixus and Ornithodorus moubata. In Lonomia achelous a prothrombin activator and a FXa-like activity have been detected while in Lonomia obliqua activators of prothrombin and FX have been reported. Group IV: Compounds acting on the fibrinolytic system as in, Glossina austeni, Rhodnius prolixus, Lonomia achelous and Scolopendra subepinipes mutilans. Group V: An heterogeneous and poorly characterized group of compounds, such as inhibitor from Haementeria ghillianis; a FXIII proteolytic activity from Lonomia achelous, an c~V-B/3 inhibitor from Dermacentor variabilis and a LA-like protein from Loxosceles reclusa. In this Group are also included activities that have not been studied in depth. It is important to mention that apart from scorpions and spiders which induced DIC, but whose activities have not been well characterized, caterpillars of the Lonomia genus are the only arthropods which really affects clinically the hemostatic mechanism, inducing a severe bleeding syndrome which can cause death. In conclusion: Every day new compounds with activities interfering with the hemostatic mechanism are identified in arthropods. These compounds are of interest as usefull tools for the understanding of the hemostatic mechanisms and as potential therapeutic drugs.
Some structural characteristics of Sticholysins I and II, CytoIysins from the sea anemone StichodactyIa helianthus and their implications for the interaction with membranes. M.E. Lanio a , C. Alvarez a, V. Morerab," F. Pazos a, M. Tejuca a, D. Martinez a, G. Menestrina c, A.M. Campos d, E. Lissi d (~University of Havana, Cuba; bCIGB, Cuba; CCeFSA, [taly; dUniversity of Santiago de, Chile). Sticholysin I (St I) and II (St II) are two basic cytolysins (MW 20 kDa) purified from the sea anemone Stichodactyla helianthus. They exhibit potent hemolytic activities (HA) and increase the membrane permeability by forming oligomeric pores. In the present work, the comparison of some structural characteristics of St I and St II and their structural modifications was carried out to deepen into the mechanism of interaction with membranes. Primary structure analysis of St I and St II (Edman's degradation and Mass Spectrometry) revealed a high homology between them suggesting they are isoforms of the same cytolysin. Similarly to other sea anemone cytolysins, St I and St II also contain a 22 amino acid insertion fragment absent in C III, the major hemolysin reported in S. helianthus.
492
Abstracts/Toxtcon 38 (2000) 487-595
The high homology between St II and C III and the revaluation of C III MW suggest that C III and St II are the same protein. St I and C I (Kern and Dunn, 1988) showed complete homology in the small C I fragment analyzed. Structural predictions of St I & II indicated they are hydrophylic polypeptides with a single highly hydrophobic fragment corresponding to the N-terminal probably involved as a leader peptide in the interaction with membranes. CNBr treatment of St II yielded three main peptides. One of them contained the N-terminal of the protein and was able to inhibit St II activity probably due to their competition for the membrane. The rest of the peptides did not elicit any effect on St lI HA, suggesting the significance of its N-terminal fragment for the interaction with membranes. Pre-incubation of St II with 2,2-azo-bis(2amidinopropane), a source of peroxyl radicals reduced its HA and significantly modified only Trp residues. The loss of HA correlates with the decrease of the protein fluorescence due the radicals. St II association with liposomes increased protein fluorescence indicating a more hydrophobic environment of St II Trp groups. St II bound to liposomes reduced the rate of fluorescence loss during its modification by free radicals. The results suggest the existence of two populations of Trp quenched by acrylamide and inactivated by free radicals at different rates. Modification of one of the critical Trp leads to cytolysin inactivation pointing its relevant role to the function of the protein. St I modified by AAPH gave similar results to those above described for St II.
Elucidation and exploitation of the functional architectures of animal toxins. Andr+ M6nez (D~partement d'Ing6nierie et d']~tudes des Prot6ines, CEA Saclay, 91191, Gif-sur-Yvette, France). This paper will describe some rules associated with the major functional and structural properties of animal toxic proteins and will show how these rules may be exploited in protein engineering. Though being often small (less than 120 amino acids), animal toxins exert a wide diversity of biological functions. Extensive studies by N M R spectroscopy and X-ray crystallography, have revealed that functionally unrelated toxins produced by a phylogenetically related group of venomous animals (a family, an order, etc.) adopt the same structural fold whereas functionally related toxins produced by phylogenetically distinct venomous animals adopt distinct folds. The topographies by which a toxin fold exerts diverse functions have also been subjected to extensive studies. This is the case, for example, of various functionally different toxins which adopt the so called 'three-fingered fold' as well as various structurally different toxins which competitively block voltage-gated potassium channels. These studies revealed that (i) a functional topography consists of a homogeneous surface of 5-10 residues among which 2-4 play a most critical role: (ii) distinct regions of a fold can accommodate unrelated functional sites; (iii) individual members of a family of functionally related toxins can display different though overlapping functional topographies. The functional site of each member of a toxin family comprises at least two components. First, it includes a small functional core composed of few