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Bacterial ADP-ribosylating toxins
390
Eleventh European Meeting
regions of toxin VII. The PCR amplified genomic DNA was cloned in the plasmid pKS, and was shown to hybridise with oligos specific for this toxin, containing approximately 800 base pair, compatible with the idea of the presence of an intron plus the corresponding structural region, which mrpressa toxin VII, or toxin gamma (Passwxr et aJ., 1992). Supported in part by Howard Hughes Medical Institute grant No . 75191-527104, and grants from the National University of Mexico (DC3APA No . IN205893) and Mexican National Council for Science and Txhnology (CONACyT No., 0018-N9105) . Bee~en., $. et al. (1993) Gene 128, 165-171. M~rtrnv-Frtucuma, M:F. ct al. (1992) FEBS Lctt. 302, 220-222. Pas~tvt ct al. (1992) !. bioJ. Chcm. 266, 3178-3185. Bacteria! ADP-rJbosylating toxins. R. Rerruou (IRIS, Immunobiological Research Institute Siena, Via
Fioreatina 1, 53100 Siena, Italy) . BACr~tL\L ADP-ribosylating toxins are proteins, usually secreted in the extraallular medium by pathogenic bacteria, that cause disease by killing or altering the metabolism of eukaryotic cells. Cassically, bacterial toxins are considered to be composed of two functionally different domains: the enzymatically active domain which has ADP-ribosylating activity and is responsible for the toxicity (A aubunit) and the binding domain which interacts with the receptors on the sodas of eukaryotic cells (B subunit) . The A aubunits have a similar enzymatic activity that is reflected by a similarity in size, Primary and tertiary structure, while the B subunits bind different receptors on eukaryotic sells and have developed different strategies to translocate the A subunit into the all. These differ+enoes are reflected by the variety of structures and composition of the B aubunits. Most of the bacterial ADP-ribosylating toxins can be divided into three major families . The first family comprises diphtheria toxin (DT) and Pseudonronas exotoxin A (PASTA). These are composed by a single polypeptide chain containing both the A and H subunits that in the case of diphtheria toxin are also linked by a disulfide bridge . The second family of toxins comprises cholera toxin (CT), heat labile toxins 1 and 2 (LTl and LT2) and pertussia toxin (PT) . These proteins have a general structure that can be described as A, Bs, where one molecule of the A aubunit is linked to a pentameric B aubunit (which is composed of five monomers). The third family of toxins comprises the remaining toxins like ClattrJdfum bondinum C2 and C3 toxins, CJostridhon perfringens iota toxin and related proteins, Pseudomonas exoenryme S and the mosquidocidal toxin (MTJC) from BaciJJus sphaericus. During the last 10 years, the primary and tertiary structure of many of the toxins has become available, and thin has allowed identification of the amino acid residues that are important for the activity of these toxins . These studies have indicated that in spite of the different primary structure, all ADP-ribosylating toxins have a common structure of the active site. On the structure-actFotty relatiorrsldps ojscorpfon toxin blockers ojpotassiwn chma~els. H. Rocxwr (CNRS URA
1455, Faculté de Médxine Nord, Biochimie, Bd. P. Dramard 13916 Marseille Cedex 20, France). Ix ascExr years more and more peptidic toxins have been purified from scorpion venoms as they have proved to be useful tools for studying ionic channels. The first toxins which have been characterized are those active on sodium channels and responsible for the high toxicity of scorpion venoma . More recently, shorter toxins (31 to 39 residues, thra disulfide bridges) have been isolated according to their blocking activity on potassium selective channels . These toxins are generally present in very small amount in scorpion venoms but they may be chemically synthesised. Using natural and synthetic toxins, synthetic analogsof the toxins and shorter peptides derived from there, a structure-activity relationship study was made (Caasr er aJ., 1992; S~~mrr et a1., 1993x, b ; Rasst et aJ., 1994; Mstnvmr et aJ., 1994) that leads to a better understanding of the structural features that may be involved in recognition by leiurotoxin I-like toxins of the apamin-sensitive Ca=+-activated K+ channel and by kaliotoxin of the high conductance BK-type neuronal channel. Carssr, M. tt aJ. (1992) !, bioJ. Chem. 267, 1640-1647. IN$ux~e, S. et aJ. (1994) Biochemistry (in press) . Rosa, R. et aJ . (1994) !. bioJ. Chcm. (in pry) . S~rn:a, J. M. et d. (1993x) J. Biochcm. 32, 2763-2770. S~~r, J. M. et aJ. (19936) Int. !. Peptide Protein Res. (in press) . The intraceJJuiar actJUtty of tetamrs and botuliruan mrurotoxins . (i . SCraAVO,' O. Rosser.I'o,' B. Pouurx ; F.
Bewservr+rr,' C. C. SrsoriE,' B. R. D~sGtrnrt.' and C.MorrtECUOCO' ('Centra CNR Biomembrane and Dip. Seisms Hiomediche; =CNRS Neurobiologie Moleculaire, Gyf-sur-Yvette, France; 3Iatituto di Fisiologia Umana, Università di Modem, Italy; 'PHLS, Porton Down, Salisbury, U.K .; and °Food Research Institute, University of Wisconsin, Madison, U.S .A .) . T>rrexus (TeTx) and botulinum neurotoxina (BoNT, seven different serotypea A-G) are responsible for tetanus and botulism, respectively. In their active form, they are composed of two fragments held together by a disulfide bridge and noa~ovalent forces. The heavy chain (H, 100 kDa) is involved in the neuroapecific binding and the all penetration of the &ght chain (L, 50 kDa) which is responsible for the blockade of neurotransmitter release .
Eleventh European Meeting
regions of toxin VII. The PCR amplified genomic DNA was cloned in the plasmid pKS, and was shown to hybridise with oligos specific for this toxin, containing approximately 800 base pair, compatible with the idea of the presence of an intron plus the corresponding structural region, which mrpressa toxin VII, or toxin gamma (Passwxr et aJ., 1992). Supported in part by Howard Hughes Medical Institute grant No . 75191-527104, and grants from the National University of Mexico (DC3APA No . IN205893) and Mexican National Council for Science and Txhnology (CONACyT No., 0018-N9105) . Bee~en., $. et al. (1993) Gene 128, 165-171. M~rtrnv-Frtucuma, M:F. ct al. (1992) FEBS Lctt. 302, 220-222. Pas~tvt ct al. (1992) !. bioJ. Chcm. 266, 3178-3185. Bacteria! ADP-rJbosylating toxins. R. Rerruou (IRIS, Immunobiological Research Institute Siena, Via
Fioreatina 1, 53100 Siena, Italy) . BACr~tL\L ADP-ribosylating toxins are proteins, usually secreted in the extraallular medium by pathogenic bacteria, that cause disease by killing or altering the metabolism of eukaryotic cells. Cassically, bacterial toxins are considered to be composed of two functionally different domains: the enzymatically active domain which has ADP-ribosylating activity and is responsible for the toxicity (A aubunit) and the binding domain which interacts with the receptors on the sodas of eukaryotic cells (B subunit) . The A aubunits have a similar enzymatic activity that is reflected by a similarity in size, Primary and tertiary structure, while the B subunits bind different receptors on eukaryotic sells and have developed different strategies to translocate the A subunit into the all. These differ+enoes are reflected by the variety of structures and composition of the B aubunits. Most of the bacterial ADP-ribosylating toxins can be divided into three major families . The first family comprises diphtheria toxin (DT) and Pseudonronas exotoxin A (PASTA). These are composed by a single polypeptide chain containing both the A and H subunits that in the case of diphtheria toxin are also linked by a disulfide bridge . The second family of toxins comprises cholera toxin (CT), heat labile toxins 1 and 2 (LTl and LT2) and pertussia toxin (PT) . These proteins have a general structure that can be described as A, Bs, where one molecule of the A aubunit is linked to a pentameric B aubunit (which is composed of five monomers). The third family of toxins comprises the remaining toxins like ClattrJdfum bondinum C2 and C3 toxins, CJostridhon perfringens iota toxin and related proteins, Pseudomonas exoenryme S and the mosquidocidal toxin (MTJC) from BaciJJus sphaericus. During the last 10 years, the primary and tertiary structure of many of the toxins has become available, and thin has allowed identification of the amino acid residues that are important for the activity of these toxins . These studies have indicated that in spite of the different primary structure, all ADP-ribosylating toxins have a common structure of the active site. On the structure-actFotty relatiorrsldps ojscorpfon toxin blockers ojpotassiwn chma~els. H. Rocxwr (CNRS URA
1455, Faculté de Médxine Nord, Biochimie, Bd. P. Dramard 13916 Marseille Cedex 20, France). Ix ascExr years more and more peptidic toxins have been purified from scorpion venoms as they have proved to be useful tools for studying ionic channels. The first toxins which have been characterized are those active on sodium channels and responsible for the high toxicity of scorpion venoma . More recently, shorter toxins (31 to 39 residues, thra disulfide bridges) have been isolated according to their blocking activity on potassium selective channels . These toxins are generally present in very small amount in scorpion venoms but they may be chemically synthesised. Using natural and synthetic toxins, synthetic analogsof the toxins and shorter peptides derived from there, a structure-activity relationship study was made (Caasr er aJ., 1992; S~~mrr et a1., 1993x, b ; Rasst et aJ., 1994; Mstnvmr et aJ., 1994) that leads to a better understanding of the structural features that may be involved in recognition by leiurotoxin I-like toxins of the apamin-sensitive Ca=+-activated K+ channel and by kaliotoxin of the high conductance BK-type neuronal channel. Carssr, M. tt aJ. (1992) !, bioJ. Chem. 267, 1640-1647. IN$ux~e, S. et aJ. (1994) Biochemistry (in press) . Rosa, R. et aJ . (1994) !. bioJ. Chcm. (in pry) . S~rn:a, J. M. et d. (1993x) J. Biochcm. 32, 2763-2770. S~~r, J. M. et aJ. (19936) Int. !. Peptide Protein Res. (in press) . The intraceJJuiar actJUtty of tetamrs and botuliruan mrurotoxins . (i . SCraAVO,' O. Rosser.I'o,' B. Pouurx ; F.
Bewservr+rr,' C. C. SrsoriE,' B. R. D~sGtrnrt.' and C.MorrtECUOCO' ('Centra CNR Biomembrane and Dip. Seisms Hiomediche; =CNRS Neurobiologie Moleculaire, Gyf-sur-Yvette, France; 3Iatituto di Fisiologia Umana, Università di Modem, Italy; 'PHLS, Porton Down, Salisbury, U.K .; and °Food Research Institute, University of Wisconsin, Madison, U.S .A .) . T>rrexus (TeTx) and botulinum neurotoxina (BoNT, seven different serotypea A-G) are responsible for tetanus and botulism, respectively. In their active form, they are composed of two fragments held together by a disulfide bridge and noa~ovalent forces. The heavy chain (H, 100 kDa) is involved in the neuroapecific binding and the all penetration of the &ght chain (L, 50 kDa) which is responsible for the blockade of neurotransmitter release .