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Novel polypeptide toxins with crab lethality in the sea anemone Anemonia erythraea
488
Abstracts
to determine the lytic activity of a large group of cytolysins. According to the amino acid sequence information obtained by molecular cloning, SNTX has been predicted to contain several cc-helices. The axial projection of these he&s indicates that the hydrophobic and hydrophilic residues in some of the helices segregate to form two different surfaces. Interestingly, the hydrophilic surface in amphiphilic a-helices such as G(Sand px are rich in cationic residues such as lysine. To test the role of the cationic site in the haemolytic activity of SNTX, the positive charges of lysine and arginyl residues were modified in the present study. SNTX lost its haemolytic activity when the positively charged side-chains of lysine residues were neutralized or changed to negatively charged side-chains upon carbamylation or succinylation, respectively. SNTX was also sensitive to the modification of positively charged arginyl residues using butanedione. These results were corroborated by competitive inhibition of haemolysis using negatively charged lipids such as phosphatidylserine, cardiolipin and monosialogangliosides. The present studies thus support the prediction that cationic residues are essential for the haemolytic activity of SNTX. Srructure-activity relationship of mastoparan B isolatedfrom the hornet (Vespa basalis) venom. C. L. Ho, W. C. Chen and Y. L. Lin (Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, R.O.C.). Mastoparan B (MP-B) is a cationic tetradecapeptide (LKLKSIVSWAKKVL-CONH2) isolated from the venom of Vespa basalis, which is the most dangerous species of hornet found in Taiwan. The peptide has a primary structure distinct from other vespine mastoparans. Unlike other mastoparans, MP-B was shown to be active both in vivo and in vitro. It caused a dose-dependent swelling in rat hindpaw and showed potent haemolytic activity in guinea-pig red cells. Studies on the structure-activity relationship of the peptide showed that replacing Lys’ by Asn in MP-B sequence caused a remarkable decrease in its oedema-inducing and haemolytic activities, while the same substitution at Lys4 caused no significant change in either activity. Replacing either Lys” or Lys” by Leu caused a slight or no decrease in the oedema-inducing and haemolytic activities. However, decreases in both activities were observed when both Lys” and Lys’? were replaced by Leu. Replacing Trp’ by Tyr or Phe in MP-B sequence almost abolished its haemolytic activity, but the oedema-inducing activity was only partially inhibited. Circular dichroism spectra of the peptides measured in 20% trifluoroethanol revealed that substitution of Lys and Trp did not cause a significant change in the conformation of MP-B. It appears that Lys* is crucial for both haemolytic and oedema-inducing activities, while Trp9 is of special importance to the haemolytic activity of MP-B. Lys” and Lys12 in MP-B probably play a smaller role in both activities. Effects of rick on the ability of the rabbit aorta to relax IO relaxant agents. C. Robinson, L. Zhang Hsu (College of Pharmacy, University of Oklahoma, HSC, Oklahoma City, OK 73190, U.S.A.).
and C.-H.
Ricin, a toxic lectin from the castor bean, causes many changes in the mammalian cardiovascular system, including decreasing blood pressure and altering blood flow and distribution. In order to investigate further the mechanism of its toxicity, the current experiment was carried out to determine whether the beta-adrenergic receptor system, the adenosine receptor system or the potassium channel coupled relaxing system was altered by ricin administration. Rabbits were given 0.44 pg/kg of ricin i.v. and 48 hr later the aortas were removed and their abilities to relax to isoproterenol, adenosine and pinacidil were measured. The maximal relaxations to the three agents were not changed by ricin administration. However, their relaxations to pinacidil were increased after ricin administration by 12% and 23% at concentrations of 3 x l0-b M and lO-o M. These differences were significant at the level of P I 0.05. Although the relaxation to adenosine showed a tendency to increase in the aortas from the rabbits receiving ricin, differences were not statistically significant. Thus, the increased sensitivity of the rabbit aorta to potassium channel openers will decrease vascular tension and reduce blood pressure. Supported
by DOD
contract
DAMD
l7-90-C-Dl09.
Novelpolypeptide toxins with crab lethality in the sea anemone Anemonia erythraea. K. Shiomi, W.-H. Qian, X.-Y. Lin, K. Shimakura, Y. Nagashima and M. Ishida (Department of Food Science and Technology, Tokyo University of Fisheries, Konan-4, Minato-ku, Tokyo 108, Japan). The aqueous extract of the sea anemone Anemonia eryrhraea was found to be potently lethal to crabs. Three polypeptide toxins (named AETX I, II and III) were isolated from specimens collected at Banda, Chiba Prefecture, by gel filtration on Sephadex G-SO and reverse-phase high-performance liquid chromatography (HPLC) on TSKgel ODS-120Ts. Specimens collected at two other places lacked AETX I or II, suggesting a regional variation in toxin composition. The LDso against crabs of AETX I, II and III were estimated to be 2.2, 0.52 and 0.28 pg/kg, respectively, but none of the toxins showed lethality in mice. The complete amino acid sequences of the three toxins were determined and their accuracy was supported by the results in both amino acid analyses and mol. wt determinations by MALDI/TOFMS. AETX I comprises 47 amino acid residues including six half-Cys residues, being an analogue of type I toxins compared to the known sea anemone polypeptide toxins. In contrast, AETX II and III, which are highly homologous with each other, are quite different from the known sea anemone polypeptide toxins in that they are composed of 59 residues including ten half-Cys residues. Interestingly, both toxins have a 36% amino acid homology with the neurotoxin (TX]) isolated from the Brazilian ‘armed’ spider Phoneutria nigriventer.
Abstracts
to determine the lytic activity of a large group of cytolysins. According to the amino acid sequence information obtained by molecular cloning, SNTX has been predicted to contain several cc-helices. The axial projection of these he&s indicates that the hydrophobic and hydrophilic residues in some of the helices segregate to form two different surfaces. Interestingly, the hydrophilic surface in amphiphilic a-helices such as G(Sand px are rich in cationic residues such as lysine. To test the role of the cationic site in the haemolytic activity of SNTX, the positive charges of lysine and arginyl residues were modified in the present study. SNTX lost its haemolytic activity when the positively charged side-chains of lysine residues were neutralized or changed to negatively charged side-chains upon carbamylation or succinylation, respectively. SNTX was also sensitive to the modification of positively charged arginyl residues using butanedione. These results were corroborated by competitive inhibition of haemolysis using negatively charged lipids such as phosphatidylserine, cardiolipin and monosialogangliosides. The present studies thus support the prediction that cationic residues are essential for the haemolytic activity of SNTX. Srructure-activity relationship of mastoparan B isolatedfrom the hornet (Vespa basalis) venom. C. L. Ho, W. C. Chen and Y. L. Lin (Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, R.O.C.). Mastoparan B (MP-B) is a cationic tetradecapeptide (LKLKSIVSWAKKVL-CONH2) isolated from the venom of Vespa basalis, which is the most dangerous species of hornet found in Taiwan. The peptide has a primary structure distinct from other vespine mastoparans. Unlike other mastoparans, MP-B was shown to be active both in vivo and in vitro. It caused a dose-dependent swelling in rat hindpaw and showed potent haemolytic activity in guinea-pig red cells. Studies on the structure-activity relationship of the peptide showed that replacing Lys’ by Asn in MP-B sequence caused a remarkable decrease in its oedema-inducing and haemolytic activities, while the same substitution at Lys4 caused no significant change in either activity. Replacing either Lys” or Lys” by Leu caused a slight or no decrease in the oedema-inducing and haemolytic activities. However, decreases in both activities were observed when both Lys” and Lys’? were replaced by Leu. Replacing Trp’ by Tyr or Phe in MP-B sequence almost abolished its haemolytic activity, but the oedema-inducing activity was only partially inhibited. Circular dichroism spectra of the peptides measured in 20% trifluoroethanol revealed that substitution of Lys and Trp did not cause a significant change in the conformation of MP-B. It appears that Lys* is crucial for both haemolytic and oedema-inducing activities, while Trp9 is of special importance to the haemolytic activity of MP-B. Lys” and Lys12 in MP-B probably play a smaller role in both activities. Effects of rick on the ability of the rabbit aorta to relax IO relaxant agents. C. Robinson, L. Zhang Hsu (College of Pharmacy, University of Oklahoma, HSC, Oklahoma City, OK 73190, U.S.A.).
and C.-H.
Ricin, a toxic lectin from the castor bean, causes many changes in the mammalian cardiovascular system, including decreasing blood pressure and altering blood flow and distribution. In order to investigate further the mechanism of its toxicity, the current experiment was carried out to determine whether the beta-adrenergic receptor system, the adenosine receptor system or the potassium channel coupled relaxing system was altered by ricin administration. Rabbits were given 0.44 pg/kg of ricin i.v. and 48 hr later the aortas were removed and their abilities to relax to isoproterenol, adenosine and pinacidil were measured. The maximal relaxations to the three agents were not changed by ricin administration. However, their relaxations to pinacidil were increased after ricin administration by 12% and 23% at concentrations of 3 x l0-b M and lO-o M. These differences were significant at the level of P I 0.05. Although the relaxation to adenosine showed a tendency to increase in the aortas from the rabbits receiving ricin, differences were not statistically significant. Thus, the increased sensitivity of the rabbit aorta to potassium channel openers will decrease vascular tension and reduce blood pressure. Supported
by DOD
contract
DAMD
l7-90-C-Dl09.
Novelpolypeptide toxins with crab lethality in the sea anemone Anemonia erythraea. K. Shiomi, W.-H. Qian, X.-Y. Lin, K. Shimakura, Y. Nagashima and M. Ishida (Department of Food Science and Technology, Tokyo University of Fisheries, Konan-4, Minato-ku, Tokyo 108, Japan). The aqueous extract of the sea anemone Anemonia eryrhraea was found to be potently lethal to crabs. Three polypeptide toxins (named AETX I, II and III) were isolated from specimens collected at Banda, Chiba Prefecture, by gel filtration on Sephadex G-SO and reverse-phase high-performance liquid chromatography (HPLC) on TSKgel ODS-120Ts. Specimens collected at two other places lacked AETX I or II, suggesting a regional variation in toxin composition. The LDso against crabs of AETX I, II and III were estimated to be 2.2, 0.52 and 0.28 pg/kg, respectively, but none of the toxins showed lethality in mice. The complete amino acid sequences of the three toxins were determined and their accuracy was supported by the results in both amino acid analyses and mol. wt determinations by MALDI/TOFMS. AETX I comprises 47 amino acid residues including six half-Cys residues, being an analogue of type I toxins compared to the known sea anemone polypeptide toxins. In contrast, AETX II and III, which are highly homologous with each other, are quite different from the known sea anemone polypeptide toxins in that they are composed of 59 residues including ten half-Cys residues. Interestingly, both toxins have a 36% amino acid homology with the neurotoxin (TX]) isolated from the Brazilian ‘armed’ spider Phoneutria nigriventer.