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Gene cloning and expression of two mammalian neurotoxins from scorpion (Buthus martensii Karsch)
482
Abstracts
Molecular and.fimctional characters qf neurotoric peptidesfrom Chinese scorpion Buthus Ji (Shanghai Institute of Physiology, Shanghai 200031, P. R. China).
martensi
Karsch. Y.-H.
Chinese scorpion venom a rich source of polypeptide ligands of ion channels, Two anti-mammal neurotoxins (BmK I and II), an excitatory neurotoxin (BmK IT), four depressant neurotoxins (BmK ITI-IT5) and an r-type (BmK GRIT)anti-insect neurotoxin have been purified from the venom of the Chinese scorpion, Buthus nzartensi Karsch (BmK) (Ji et al., 1988, 1993, 1994a-c). These toxins are composed of 61-70 amino acids selectively targeted against voltage-dependent Na+ channels on excitable membranes. Another new peptide designated as BmK AS, composed of 63 amino acids, was found recently to be a potent activator of ryanodine receptors on rabbit skeletal muscle, and also enhances remarkably [‘Hlnoradrenaline release on rat hippocampal slices and increases Na+ permeability on voltage-gated Na+ channels of NGl,,x.15 cells (Ji 6’1a/., 199613; Huang et rd., 1995). In addition, a reversible blocking effect of the venom and a minipeptide fraction on transient outward K’ channels in neonatal rat ventricular cells and K,’ channels in NG,,,x.,s cells were recorded, respectively (Liu et nl., 1996). Here we will briefly describe the molecular and functional characteristics of various polypeptide ligands of ion channels from the venom of the Chinese scorpion, as well as the molecular genesis and evolutionary connotation of each type of scorpion toxin on the basis of their structural resemblance and functional divergence. 1st Biennial Meeting Huang, H. Y. et al. (1995) Chinese Society for Neuroscience, Ji, Y. H. et al. (1988) Comp. Biochem. Physiol. 9OC, 237-240. Ji, Y. H. et a/. (1993) Chin. Sci. Bull. 38, I21 I-1215. Ji, Y. H. et al. (1994) Sci. China 37, 42-49. Ji, Y. H. et al. (1994b) Sci. China 37, 9.54-963. Ji, Y. H. ef al. (1994~) Chin. Sci. Bull. 39, 945-949. Ji, Y. H. et a/. (1996a) Toxicon, forthcoming. Ji, Y. H. et nl. (l996b) Chin. Sci. Bull., forthcoming. Liu, Y. et al. (1996) Annual Physiolog!> Symposium. Hong Kong, 2627 April.
(Abstract),
p. 49.
Gene cloning and expression qf’tlvo mammalian neuroto.uin.s.fiom scorpion (Buthus martensii Karsch). Y. Xiong.’ M. Ling,’ T. Zhao,’ D. Wang’ and C. Chi2 (‘Beijing Institute of Biophysics, Academia Sinica, Beijing 100101, P.R. China; and ?Shanghai Institute of Biochemistry, Academia Sinica. Shanghai 200031, P.R. China). The cDNA library of the BmK (Buthus martensii Karsch) scorpion venom gland was constructed, and two cDNAs encoding the mammalian neurotoxins were screened and sequenced. The deduced amino acid sequence from one cDNA corresponded to that of the known Bmk ml toxin, while another deduced sequence has not been reported before, but was similar to Bmk md with only two replacement residues. The deduced amino acid sequences were composed of three parts: a 19 amino acid signal peptide, the mature toxin with 64 amino acid which was removed during the protein residues and an additional Arg tail at the carboxyl-terminus, postprocessing. The elucidated Bmk cDNA sequences showed 87.2% homology with those of African scorpion LqH. The Bmk ml cDNA was cloned into plasmid pVT102U/sc and has been successfully expressed in Saccharomyces ceret:isiae with a yield of around 2 mg per litre. The gene product was purified and found to be identical to the natural one, except for two additional residues at the amino-terminus of the toxin which were derived from the linker between the a-factor leader peptide and the toxin downstream, postprocessed by KEX2 during the yeast secretion system. Function qf the 28,000 and 66,000 mol. bvt protein torins of’ Bacillus thuringiensis. Y. Nitzan, Pechatnikov (Department of Life Sciences, Bar-Ilan University, Ramat Can 52900, Israel).
R. Cahan
and I.
A 28,000 mol. wt protein toxin was purified to homogeneity from the crystal toxin of the bacteria Bacillus thuringiensis israelensis. This protein contained haemolytic and cytolytic activity. The mechanism by which toxin exerts its effect on mature muscle cultures shows that the toxin inhibited Na+/K+-ATPase activity, as revealed by XLRb influx. A 50% inhibition of Na+jK+ATPase activity was obtained with 0.2 leg/ml of the toxin. The inhibition was time and dose dependent, and it was reversible with low doses of the toxin (up to 0.2 fig/ml). A considerable release of “ORb was obtained by doses greater than 0.2 /cg/ml. The XhRb release was also time and dose dependent. This effect is probably non-specific, since “Ca influx is also accelerated by toxin-treated cultures. Preincubation of the toxin with phosphotidylserine (PS) antagonized the toxin. It is concluded that the toxin is a hydrophobic protein which interacts with the membrane. In low doses this interaction reduces the activity of the sodium pump and in high doses it causes non-specific permeability of the sarcolemma. Another protein toxin was also purified from the crystal toxin of the bacteria. The second toxin had a mol. wt of 66,000 and had a mosquitocidal activity on the larvae of Aedes aegypti (LDn, 12-14 pg/ml). A combination (1: 1) of the 66,000 and 29,000 mol. wt proteins resulted in a 5-fold increase in the toxin’s effect on the larvae (LD~,,46 pg/ml). Our results indicate synergism between these two proteins: the first is a pore-forming toxin and the other is an intracellular active protein which penetrates through these pores and inactivates the cells. ELISA anal?& Pharmaceutical
of‘microcystins, algal hepatotosins, in cwrironmental water. Y. Ueno and S. Nagata (Faculty Sciences, Science University of Tokyo, Ichigdya, Shinjuku-Ku, Tokyo 162, Japan).
of
Several species of cyanobacteria (blue-green algae) such as Microcj,.stis aeruginosa often result in toxic water blooms in eutrophic lakes, reservoirs and rivers in many regions of the world. This blooming is closely associated
Abstracts
Molecular and.fimctional characters qf neurotoric peptidesfrom Chinese scorpion Buthus Ji (Shanghai Institute of Physiology, Shanghai 200031, P. R. China).
martensi
Karsch. Y.-H.
Chinese scorpion venom a rich source of polypeptide ligands of ion channels, Two anti-mammal neurotoxins (BmK I and II), an excitatory neurotoxin (BmK IT), four depressant neurotoxins (BmK ITI-IT5) and an r-type (BmK GRIT)anti-insect neurotoxin have been purified from the venom of the Chinese scorpion, Buthus nzartensi Karsch (BmK) (Ji et al., 1988, 1993, 1994a-c). These toxins are composed of 61-70 amino acids selectively targeted against voltage-dependent Na+ channels on excitable membranes. Another new peptide designated as BmK AS, composed of 63 amino acids, was found recently to be a potent activator of ryanodine receptors on rabbit skeletal muscle, and also enhances remarkably [‘Hlnoradrenaline release on rat hippocampal slices and increases Na+ permeability on voltage-gated Na+ channels of NGl,,x.15 cells (Ji 6’1a/., 199613; Huang et rd., 1995). In addition, a reversible blocking effect of the venom and a minipeptide fraction on transient outward K’ channels in neonatal rat ventricular cells and K,’ channels in NG,,,x.,s cells were recorded, respectively (Liu et nl., 1996). Here we will briefly describe the molecular and functional characteristics of various polypeptide ligands of ion channels from the venom of the Chinese scorpion, as well as the molecular genesis and evolutionary connotation of each type of scorpion toxin on the basis of their structural resemblance and functional divergence. 1st Biennial Meeting Huang, H. Y. et al. (1995) Chinese Society for Neuroscience, Ji, Y. H. et al. (1988) Comp. Biochem. Physiol. 9OC, 237-240. Ji, Y. H. et a/. (1993) Chin. Sci. Bull. 38, I21 I-1215. Ji, Y. H. et al. (1994) Sci. China 37, 42-49. Ji, Y. H. et al. (1994b) Sci. China 37, 9.54-963. Ji, Y. H. ef al. (1994~) Chin. Sci. Bull. 39, 945-949. Ji, Y. H. et a/. (1996a) Toxicon, forthcoming. Ji, Y. H. et nl. (l996b) Chin. Sci. Bull., forthcoming. Liu, Y. et al. (1996) Annual Physiolog!> Symposium. Hong Kong, 2627 April.
(Abstract),
p. 49.
Gene cloning and expression qf’tlvo mammalian neuroto.uin.s.fiom scorpion (Buthus martensii Karsch). Y. Xiong.’ M. Ling,’ T. Zhao,’ D. Wang’ and C. Chi2 (‘Beijing Institute of Biophysics, Academia Sinica, Beijing 100101, P.R. China; and ?Shanghai Institute of Biochemistry, Academia Sinica. Shanghai 200031, P.R. China). The cDNA library of the BmK (Buthus martensii Karsch) scorpion venom gland was constructed, and two cDNAs encoding the mammalian neurotoxins were screened and sequenced. The deduced amino acid sequence from one cDNA corresponded to that of the known Bmk ml toxin, while another deduced sequence has not been reported before, but was similar to Bmk md with only two replacement residues. The deduced amino acid sequences were composed of three parts: a 19 amino acid signal peptide, the mature toxin with 64 amino acid which was removed during the protein residues and an additional Arg tail at the carboxyl-terminus, postprocessing. The elucidated Bmk cDNA sequences showed 87.2% homology with those of African scorpion LqH. The Bmk ml cDNA was cloned into plasmid pVT102U/sc and has been successfully expressed in Saccharomyces ceret:isiae with a yield of around 2 mg per litre. The gene product was purified and found to be identical to the natural one, except for two additional residues at the amino-terminus of the toxin which were derived from the linker between the a-factor leader peptide and the toxin downstream, postprocessed by KEX2 during the yeast secretion system. Function qf the 28,000 and 66,000 mol. bvt protein torins of’ Bacillus thuringiensis. Y. Nitzan, Pechatnikov (Department of Life Sciences, Bar-Ilan University, Ramat Can 52900, Israel).
R. Cahan
and I.
A 28,000 mol. wt protein toxin was purified to homogeneity from the crystal toxin of the bacteria Bacillus thuringiensis israelensis. This protein contained haemolytic and cytolytic activity. The mechanism by which toxin exerts its effect on mature muscle cultures shows that the toxin inhibited Na+/K+-ATPase activity, as revealed by XLRb influx. A 50% inhibition of Na+jK+ATPase activity was obtained with 0.2 leg/ml of the toxin. The inhibition was time and dose dependent, and it was reversible with low doses of the toxin (up to 0.2 fig/ml). A considerable release of “ORb was obtained by doses greater than 0.2 /cg/ml. The XhRb release was also time and dose dependent. This effect is probably non-specific, since “Ca influx is also accelerated by toxin-treated cultures. Preincubation of the toxin with phosphotidylserine (PS) antagonized the toxin. It is concluded that the toxin is a hydrophobic protein which interacts with the membrane. In low doses this interaction reduces the activity of the sodium pump and in high doses it causes non-specific permeability of the sarcolemma. Another protein toxin was also purified from the crystal toxin of the bacteria. The second toxin had a mol. wt of 66,000 and had a mosquitocidal activity on the larvae of Aedes aegypti (LDn, 12-14 pg/ml). A combination (1: 1) of the 66,000 and 29,000 mol. wt proteins resulted in a 5-fold increase in the toxin’s effect on the larvae (LD~,,46 pg/ml). Our results indicate synergism between these two proteins: the first is a pore-forming toxin and the other is an intracellular active protein which penetrates through these pores and inactivates the cells. ELISA anal?& Pharmaceutical
of‘microcystins, algal hepatotosins, in cwrironmental water. Y. Ueno and S. Nagata (Faculty Sciences, Science University of Tokyo, Ichigdya, Shinjuku-Ku, Tokyo 162, Japan).
of
Several species of cyanobacteria (blue-green algae) such as Microcj,.stis aeruginosa often result in toxic water blooms in eutrophic lakes, reservoirs and rivers in many regions of the world. This blooming is closely associated