Postsynaptic short-chain neurotoxins from Pseudonaja textilis. cDNA cloning, expression and protein characterization.

Two lethal proteins, which specifically bind to the nAChR from Torpedo californica, were isolated from the venom of Pseudonaja textilis, the common brown snake from Australia. The isolated proteins have masses of 6236 and 6345 Da and are structurally related to short-chain neurotoxins from other elapids. Six cDNAs encoding isoforms of related neurotoxins were cloned using the RT-PCR of the venom gland mRNAs. The sequences of the corresponding proteins consist of 57-58 amino acid residues and display several unique features when compared with all known short-chain neurotoxins. Accordingly, they grouped separately in phylogenetic analysis. The six cDNAs were expressed in Escherichia coli and the recombinant proteins were characterized. They have similar masses and display similar toxicities and binding constants to the nAChR as the native toxins isolated from the venom. Thus, a new group of short-chain postsynaptic neurotoxins from the venom of an Australian elapid has been characterized.

[1]  D. Barnett,et al.  Pseudonajatoxin b: unusual amino acid sequence of a lethal neurotoxin from the venom of the Australian common brown snake, Pseudonaja textilis. , 1987, European journal of biochemistry.

[2]  D. Mebs Progress in the characterization of venoms and standardization of antivenoms: 44 pp. Geneva: WHO Offset Publication No. 58 (1981) , 1981 .

[3]  H. Boedtker,et al.  RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. , 1977, Biochemistry.

[4]  D. Botes,et al.  Snake venom toxins. The amino acid sequences of toxins b and d from Naja melanoleuca venom. , 1972, The Journal of biological chemistry.

[5]  S. Zinn-Justin,et al.  Genetic engineering of snake toxins. Role of invariant residues in the structural and functional properties of a curaremimetic toxin, as probed by site-directed mutagenesis. , 1993, The Journal of biological chemistry.

[6]  A. Ménez,et al.  Refolding of reduced short neurotoxins: circular dichroism analysis. , 1980, Biochemistry.

[7]  D. J. Strydom,et al.  Snake venom toxins. Purification and properties of three toxins from Naja nivea (Linnaeus) (Cape cobra) venom and the amino acid sequence of toxin delta. , 1971, The Journal of biological chemistry.

[8]  B. Dobberstein,et al.  Transfer to proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components , 1975, The Journal of cell biology.

[9]  F. Joubert,et al.  Purification, some properties and the primary structures of three reduced and S-carboxymethylated toxins (CM-5, CM-6 and CM-10a) from Naje haje haje (Egyptian cobra) venom. , 1978, Biochimica et biophysica acta.

[10]  D. J. Strydom,et al.  A Neurotoxin, Toxin α, from Egyptian Cobra (Naja haje haje) Venom I. PURIFICATION, PROPERTIES, AND COMPLETE AMINO ACID SEQUENCE , 1969 .

[11]  P. Gopalakrishnakone,et al.  Postsynaptic alpha-neurotoxin gene of the spitting cobra, Naja naja sputatrix: structure, organization, and phylogenetic analysis. , 1999, Genome research.

[12]  H. Rochat,et al.  Amino Acid Sequences of Neurotoxins I and III of the Elapidae Snake Naja mossambica mossambica , 1977 .

[13]  B. W. Low The Three-Dimensional Structure of Postsynaptic Snake Neurotoxins: Consideration of Structure and Function , 1979 .

[14]  J. Porath,et al.  The Amino Acid Sequence of a Neurotoxin from Naja nigricollis Venom , 1967 .

[15]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[16]  C. Diniz,et al.  Purification and amino acid sequences of six Tx3 type neurotoxins from the venom of the Brazilian 'armed' spider Phoneutria nigriventer (Keys). , 1993, Toxicon : official journal of the International Society on Toxinology.

[17]  G. Kreil Transfer of proteins across membranes. , 1981, Annual review of biochemistry.

[18]  C. Takasaki Amino acid sequence of a long-chain neurotoxin homologue, Pa ID, from the venom of an Australian elapid snake, Pseudechis australis. , 1989, Journal of biochemistry.

[19]  J. Mallet,et al.  Cloning and sequence analysis of the cDNA encoding a snake neurotoxin precursor. , 1985, Biochimie.

[20]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[21]  S. Sutherland Progress in the characterization of venoms and standardization of antivenoms. , 1981, WHO offset publication.

[22]  J. Hauert,et al.  The major lethal neurotoxin of the venom of Naja naja philippinensis. Purification, physical and chemical properties, partial amino acid sequence. , 2009, International journal of peptide and protein research.

[23]  N. Maeda,et al.  Isolation, properties and amino acid sequences of three neurotoxins from the venom of a sea snake, Aipysurus laevis. , 1976, The Biochemical journal.

[24]  A. Tu,et al.  Isolation and primary structure of the major toxin from sea snake, Acalyptophis peronii, venom. , 1988, Archives of biochemistry and biophysics.

[25]  P. Gopalakrishnakone,et al.  Cloning and characterization of cDNAs encoding three isoforms of phospholipase A2 in Malayan spitting cobra (Naja naja sputatrix) venom. , 1997, Toxicon : official journal of the International Society on Toxinology.

[26]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[27]  D Bertrand,et al.  Only Snake Curaremimetic Toxins with a Fifth Disulfide Bond Have High Affinity for the Neuronal α7 Nicotinic Receptor* , 1997, The Journal of Biological Chemistry.

[28]  T. Tsuchiya,et al.  Structure of the snake short-chain neurotoxin, erabutoxin c, precursor gene. , 1990, European journal of biochemistry.

[29]  A. Ménez,et al.  Genetic Engineering of Snake Toxins , 1995, The Journal of Biological Chemistry.

[30]  A. Ménez,et al.  Structure of snake toxins and their affinity to the acetylcholine receptor of fish electric organ. , 1977, Toxicon : official journal of the International Society on Toxinology.

[31]  N. Tamiya,et al.  Isolation and amino acid sequence of a short-chain neurotoxin from an Australian elapid snake, Pseudechis australis. , 1985, The Biochemical journal.

[32]  N. Maeda,et al.  Three neurotoxins from the venom of a sea snake Astrotia stokesii, including two long-chain neurotoxic proteins with amidated C-termini. , 1978, The Biochemical journal.

[33]  A. Armugam,et al.  Structure and organization of the cardiotoxin genes in Naja naja sputatrix 1 , 1998, FEBS letters.

[34]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[35]  M. Sasa Diet and snake venom evolution: can local selection alone explain intraspecific venom variation? , 1999, Toxicon : official journal of the International Society on Toxinology.

[36]  J. Halpert,et al.  Amino acid sequence of a postsynaptic neurotoxin from the venom of the Australian tiger snake Notechis scutatus scutatus. , 1979, Biochimie.

[37]  P. Gopalakrishnakone,et al.  Molecular cloning of a cardiotoxin structural gene from Malayan spitting cobra (Naja naja sputatrix). , 1993, Toxicon : official journal of the International Society on Toxinology.

[38]  J. Fox,et al.  Amino acid sequence of a snake neurotoxin from the venom of Lapemis hardwickii and the detection of a sulfhydryl group by laser Raman spectroscopy , 1977, FEBS letters.

[39]  P. Hains,et al.  Acanthoxin, a toxic phospholipase A2 from the venom of the common death adder (Acanthophis antarcticus). , 1997, Toxicon : official journal of the International Society on Toxinology.

[40]  J. Fontecilla-Camps,et al.  The Structures and Evolution of Snake Toxins of the Three-Finger Folding Type , 1996 .

[41]  P Gopalakrishnakone,et al.  Four new postsynaptic neurotoxins from Naja naja sputatrix venom: cDNA cloning, protein expression, and phylogenetic analysis. , 1998, Toxicon : official journal of the International Society on Toxinology.

[42]  S. Chiou,et al.  Sequence characterization of venom toxins from Thailand cobra. , 2009, International journal of peptide and protein research.

[43]  N. Tamiya,et al.  Isolation, properties and amino acid sequence of a long-chain neurotoxin, Acanthophis antarcticus b, from the venom of an Australian snake (the common death adder, Acanthophis antarcticus). , 1981, The Biochemical journal.

[44]  M. Hattori,et al.  Unusually high conservation of untranslated sequences in cDNAs for Trimeresurus flavoviridis phospholipase A2 isozymes. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[45]  S. Sato,et al.  The amino acid sequences of erabutoxins, neurotoxic proteins of sea-snake (Laticauda semifasciata) venom. , 1971, The Biochemical journal.

[46]  T. Endo,et al.  Current view on the structure-function relationship of postsynaptic neurotoxins from snake venoms. , 1987, Pharmacology & therapeutics.

[47]  J. Daltry,et al.  Diet and snake venom evolution , 1996, Nature.

[48]  Y. Ovchinnikov,et al.  Amino acid sequence of neurotoxin II from Naja naja oxiana venom , 1973, FEBS letters.

[49]  T. Tsuchiya,et al.  Sequence analysis of a cDNA encoding a erabutoxin b from the sea-snake Laticauda semifasciata. , 1989, Nucleic acids research.

[50]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[51]  N. Tamiya,et al.  The amino acid sequence and position of the free thiol group of a short-chain neurotoxin from common-death-adder (Acanthophis antarcticus) venom. , 1981, The Biochemical journal.

[52]  M. Howden,et al.  Studies on the subunit structure of textilotoxin, a potent presynaptic neurotoxin from the venom of the Australian common brown snake (Pseudonaja textilis). 3. The complete amino-acid sequences of all the subunits. , 1993, Biochimica et biophysica acta.

[53]  B. Martin,et al.  Two novel alpha-neurotoxins isolated from the taipan snake, Oxyuranus scutellatus, exhibit reduced affinity for nicotinic acetylcholine receptors in brain and skeletal muscle. , 1996, Biochemistry.

[54]  P. Gopalakrishnakone,et al.  Postsynaptic a -Neurotoxin Gene of the Spitting Cobra, Naja naja sputatrix : Structure, Organization, and Phylogenetic Analysis , 1999 .

[55]  R. Kini Venom phospholipase A[2] enzymes : structure, function, and mechanism , 1997 .

[56]  M. Howden,et al.  The complete amino acid sequence of a post-synaptic neurotoxin isolated from the venom of the Australian death adder snake Acanthophis antarcticus. , 1990, Comparative biochemistry and physiology. B, Comparative biochemistry.

[57]  R. Stöcklin,et al.  Isolation and amino acid sequence of a new long-chain neurotoxin with two chromatographic isoforms (Aa el and Ae e2) from the venom of the Australian death adder (Acanthophis antarcticus). , 1997, Toxicon : official journal of the International Society on Toxinology.

[58]  D. J. Strydom Snake venom toxins. Purification and properties of low-molecular-weight polypeptides of Dendroaspis polylepis polylepis (black mamba) venom. , 1976, European journal of biochemistry.