The amino acid sequence of protein CM-3 from Dendroaspis polylepis polylepis (black mamba) venom

Protein CM-3 from Dendroaspis polylepis polylepis venom was purified by gel filtration and ion exchange chromatography. It comprises 65 amino acids including eight half-cystines. The complete amino acid sequence of protein CM-3 has been elucidated. The sequence (residues 1-50) resembles that of the N-terminal sequence of the subunits of a synergistic type protein and residues 51-65 that of the C-terminal sequence of an angusticeps type protein. Mixtures of protein CM-3 and angusticeps type proteins showed no apparent synergistic effect, in that their toxicity in combination was no greater than the sum of their individual toxicities.     

Physicochemical characterization and functional analysis of some snake venom toxin proteins and related non-toxin proteins of other chordates

Snake venom contains a diverse array of proteins and polypeptides. Cytotoxins and short neurotoxins are non-enzymatic polypeptide components of snake venom. The three-dimensional structure of cytotoxin and short neurotoxin resembles a three finger appearance of three-finger protein super family. Different family members of three-finger protein super family are employed in diverse biological functions. In this work we analyzed the cytotoxin, short neurotoxin and related non-toxin proteins of other chordates in terms of functional analysis, amino acid compositional (%) profile, number of amino acids, molecular weight, theoretical isoelectric point (pI), number of positively charged and negatively charged amino acid residues, instability index and grand average of hydropathy with the help of different bioinformatical tools. Among all interesting results, profile of amino acid composition (%) depicts that all sequences contain a conserved cysteine amount but differential amount of different amino acid residues which have a family specific pattern. Involvement in different biological functions is one of the driving forces which contribute the vivid amino acid composition profile of these proteins. Different biological system dependent adaptation gives the birth of enriched bio-molecules. Understanding of physicochemical properties of these proteins will help to generate medicinally important therapeutic molecules for betterment of human lives.     

Snake venom toxins. The amino-acid sequences of three toxins (9B, 11 and 12A) from Hemachatus haemachatus (Ringhals) venom.

Three toxins (9B, 11 and 12A) were purified from the venom of Hemachtus haemachatus as described previously. Whereas toxin 11 and 12A comprise 61 amino acid residues, toxin 9B contains 63 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have elucidated. The properties of the toxins were compared with those of the cytotoxin group. The toxicities, the sequences and some of the invariant residues of toxin 11 and 12A resemble the corresponding properties of the cytotoxin group. However their immunochemical properties indicate that they are distinct from both the cytotoxin and neurotoxin groups. The sequence of toxin 9B shows that it is related to the cytotoxins, but its toxicity is much lower than those encountered among members of this group.     

Some properties and the complete primary structures of two reduced and S-carboxymethylated polypeptides (S5C1 and S5C10) from Dendroaspis jamesoni kaimosae (Jameson's mamba) venom.

Two polypeptides (protein S5C1 and toxin S5C10) were purified from Dendroaspis jamesoni kaimosae venom. Whereas protein S5C1 comprises 61 amino acid residues, toxin S5C10 contains 58 and they each comprise four disulphide bridges. The complete primary structures of the two polypeptides have been elucidated. The sequences of protein S5C1 and toxin S5C10 are structurally homologous to the short neurotoxins Type I, but they are much less toxic. In toxin S5C10 one of the functionally invariant amino acid residues, lysine 26, of the Type I neurotoxin has been replaced by a serine. In contrast protein S5C1 has the feature that it contains ten or eleven structurally invariant amino acids and apparently only one of the five functionally invariant residues.     

Snake venom toxins. The primary structure of protein S4C11. A neurotoxin homologue from the venom of forest cobra (Naja melanoleuca).

Six minor protein constituents (S4C10-S4C15) have been isolated from the venom of Naja melanoleuca. The complete amino acid sequence of S4C11 has been established and indicates that it is a homologue of the neurotoxins which are found in elapid venoms. The other proteins appear from the amino acid compositions to be homologues of the cyto- or cardiotoxins found in cobra venoms. Protein S4C11 has a low toxicity, failing to kill mice at an intravenous dose of 20 mug/g body weight. The sequence of the first 25 residues out of the total of 65, was determined using the automatic sequenator. The remainder of the sequence was derived with the aid of tryptic and chymotryptic peptides. The sequence showed the unusual feature of having 65 amino acid residues including 10 half-cystine residues.     

The primary structure of the cytotoxin restrictocin

The complete amino acid sequence of the single polypeptide chain of cytotoxin restrictocin has been determined. Its structure was established by automated Edman degradation of the intact molecule reduced and [14C]carboxymethylated and of fragments obtained by chemical cleavage of the protein with cyanogen bromide and BNPS-skatole and by enzymatic cleavage of the polypeptide chain with trypsin. The molecule consists of 149 amino acid residues with a calculated relative molecular mass of 16836. The protein presents two disulfide bridges, one between cysteine residues at positions 5 and 147 and the other one formed by cysteine residues at positions 75 and 131. The amino acid sequence of restrictocin shows a high degree of homology (86%) with that of the cytotoxin named alpha-sarcin.     

Amino acid sequence of cytotoxin IIa isolated from the venom of the Indian cobra (Naja naja)

A cytotoxic basic polypeptide, designated as cytotoxin IIa, was purified to homogeneous state from the venom of the Indian cobra (Naja naja) by a combination of gel filtration on Sephadex G-50, CM-cellulose chromatography, and fast protein liquid chromatography. Cytotoxin IIa is a single polypeptide consisting of 60 amino acid residues with four intramolecular disulfide linkages. The toxin showed high cytotoxicity toward Yoshida sarcoma and ascites hepatoma cells as did cytotoxins I and II isolated from the same venom. Analysis of the amino acid sequence revealed that cytotoxin I, IIa, and II are highly homologous in their primary structures and that cytotoxin IIa differs from cytotoxin I only in having Phe 25 and Val 52 in place of Tyr 25 and Glu 52 residues.     

Partial amino acid sequence of the heavy and light chains of botulinum neurotoxin type A

The dichain (nicked) type A botulinum neurotoxin is a protein (mol. wt. 145,000) composed of a heavy and a light chain (mol. wt. 97,000 and 53,000, respectively) that are held together by disulfide bond(s). We report here the sequence of the first 17 amino acid residues of the light chain, and the first 10 residues of the heavy chain. The heavy chain was isolated from the neurotoxin by two different methods, while the light chain was isolated by the only available method. The identical amino acid sequence was found in both preparations of heavy chain. Two samples of the light chain isolated from two separately prepared batches of the neurotoxin also had identical sequences.     

Pseudomonas aeruginosa cytotoxin: the nucleotide sequence of the gene and the mechanism of activation of the protoxin

The gene encoding cytotoxin (ctx) was cloned from Pseudomonas aeruginosa 158 and the nucleotide sequence was determined. The structural gene of ctx encodes the procytotoxin of 286 amino acid residues with a molecular mass of 31,681 Daltons. Procytotoxin was activated by removal of 20 amino acid residues from the C terminus with trypsin. The cloned ctx gene was not expressed in either an Escherichia coli strain or a cytotoxin non-producing strain of P. aeruginosa. An expression system for the ctx gene was constructed by placing the structural gene of ctx downstream of tac promoter on a broad host-range vector plasmid.     

Sequence analysis and phylogenetic study of some toxin proteins of snakes and related non-toxin proteins of chordates

Snakes are equipped with their venomic armory to tackle different prey and predators in adverse natural world. The venomic composition of snakes is a mix of biologically active proteins and polypeptides. Among different components snake venom cytotoxins and short neurotoxin are non-enzymatic polypeptide candidates with in the venom. These two components structurally resembled to three-finger protein superfamily specific scaffold. Different non-toxin family members of three-finger protein superfamily are involved in different biological roles. In the present study we analyzed the snake venom cytotoxins, short neurotoxins and related non-toxin proteins of different chordates in terms of amino acid sequence level diversification profile, polarity profile of amino acid sequences, conserved pattern of amino acids and phylogenetic relationship of these toxin and nontoxin protein sequences. Sequence alignment analysis demonstrates the polarity specific molecular enrichment strategy for better system adaptivity. Occurrence of amino acid substitution is high in number in toxin sequences. In non-toxin body proteins there are less amino acid substitutions. With the help of conserved residues these proteins maintain the three-finger protein scaffold. Due to system specific adaptation toxin and non-toxin proteins exhibit a varied type of amino acid residue distribution in sequence stretch. Understanding of Natural invention scheme (recruitment of venom proteins from normal body proteins) may help us to develop futuristic engineered bio-molecules with remedial properties.     

Nucleotide sequence of the gene coding for non-proteolyticClostridium botulinum type B neurotoxin: Comparison with other clostridial neurotoxins

The neurotoxin gene of non-proteolyticClostridium botulinum type B (strain Eklund 17B) was cloned as a series of overlapping polymerase chain reaction (PCR) fragments generated with primers designed to conserved regions of published botulinal toxin (BoNT) sequences. The 3 end of the gene was obtained by using primers designed to the determined sequence of non-proteolytic BoNT/B and a published downstream region of BoNT/B gene from a proteolytic strain. Translation of the nucleotide sequence derived from cloned PCR fragments demonstrated the toxin gene encodes a protein of 1291 amino acid residues. Comparative alignment of the derived BoNT/B sequence with those of other published botulinal neurotoxins revealed highest sequence relatedness with BoNT/B of proteolyticC. botulinum. The sequence identity between non-proteolytic and proteolytic BoNT/B was 97.7% for the light chain (corresponding to 10 amino acid changes) and 90.2% for the heavy chain (corresponding to 81 amino acid changes), with most differences occurring at the C-terminal end. A genealogical tree constructed from all known botulinal neurotoxin sequences revealed marked topological differences with a phylogenetic tree ofC. botulinum types based upon small-subunit (16S) ribosomal RNA sequences.     

Primary structure of protein S13 from the small subunit of escherichia coli ribosomes.

The experimental details which led to the determination of the complete primary structure of protein S13 from the small subunit of Escherichia coli ribosomes are presented. S13 consists of 117 amino acid residues and has the following composition: Asp6, Asn2, Thr6, Ser6, Glu6, Gln2, Pro4, Gly11, Ala11, Cys1, Val7, Met2, Ile12, Leu9, Tyr2, Phe1, His3, Lys11 and Arg15. Tryptophan was not found. The molecular weight of protein S13 as derived from the sequence shown in Fig. 1 is 12970. The amino acid sequence of the protein was determined by combining the results obtained from liquid phase Edman degradation of the intact protein with those from the peptides isolated after enzymatic digestions with trypsin, Staphylococcus aureus protease and thermolysin. Additional information about the primary structure was derived from analysis of the chymotryptic peptides of protein S13 and from its digestion with carboxypeptidase C. The amino acid sequence of protein S13 was compared with the published sequences of the other ribosomal proteins of E. coli and predictions for the secondary structure of this protein were made.     

Amino acid sequence of a short chain neurotoxin from the venom of banded krait (Bungarus fasciatus)

The amino acid sequence of a short chain neurotoxin obtained from Bungarus fasciatus venom consists of 64 amino acid residues: Arg-Ile-Cys-Leu-Asn-Gln-Gln-Gln-Ser- Thr-Pro-Glu-Asp-Gln-Pro-Thr-Asn-Gly-Gln-Cys-Tyr-Ile-Lys-Thr-Asp-Cys-Gln- Asn-Lys - Thr-Trp-Asn-Thr-His-Arg-Gly-Ser-Arg-Thr-Asp-Arg-Gly-Cys-Gly-Cys-Pro-Lys- Val-Lys - Pro-Gly-Ile-Asn-Leu-Arg-Cys-Cys-Lys-Thr-Asp-Lys-Cys-Asn-Glu. The above result was obtained primarily from the amino acid analyses and sequencing of tryptic peptides accompanied with the necessary analyses and sequencing of the chymotryptic and lysyl endopeptidic peptides for alignment.     

Snake venom toxins--II. The primary structures of cytotoxin homologues S3C2 and S4C8 from Aspidelaps scutatus (shield or shield-nose snake) venom

1. Cytotoxin homologues S3C2 and S4C8 from Aspidelaps scutatus were purified by gel filtration and ion exchange chromatography. 2. They consist of 63 amino acids including eight half-cystines. The toxicities of S3C2 and S4C8 were determined and LD50 values of 6.6 and 9.4 micrograms/g mouse were, respectively, found. 3. The complete primary structures of toxins S3C2 and S4C8 have been determined. The two toxins resemble the cytotoxin type toxins and in the cytotoxin homologues the ten structurally invariant amino acids of the neurotoxins and the cytotoxins are conserved.     

Molecular composition of progenitor toxin produced by Clostridium botulinum type C strain 6813

The molecular composition of the purified progenitor toxin produced by a Clostridium botulinum type C strain 6813 (C-6813) was analyzed. The strain produced two types of progenitor toxins (M and L). Purified L toxin is formed by conjugation of the M toxin (composed of a neurotoxin and a non-toxic nonhemagglutinin) with additional hemagglutinin (HA) components. The dual cleavage sites at loop region of the dichain structure neurotoxin were identified between Arg444-Ser445 and Lys449-Thr450 by the analyses of C-terminal of the light chain and N-terminal of the heavy chain. Analysis of partial amino acid sequences of fragments generated by limited proteolysis of the neurotoxin has shown to that the neurotoxin protein produced by C-6813 was a hybrid molecule composed of type C and D neurotoxins as previously reported. HA components consist of a mixture of several subcomponents with molecular weights of 70-, 55-, 33-, 26~21- and 17-kDa. The N-terminal amino acid sequences of 70-, 55-, and 26~21-kDa proteins indicated that the 70-kDa protein was intact HA-70 gene product, and other 55- and 26~21-kDa proteins were derived from the 70-kDa protein by modification with proteolysis after translation of HA-70 gene. Furthermore, several amino acid differences were exhibited in the amino acid sequence as compared with the deduced sequence from the nucleotide sequence of the HA-70 gene which was common among type C (strains C-St and C-468) and D progenitor toxins (strains D-CB16 and D-1873).     

Amino acid sequence of neurotoxin III of the scorpion Androctonus austrialis Hector.

The amino acid sequence of neurotoxin III, purified from the venom of the North African scorpion Androctonus australis Hector, has been determined by Edman degradation using a liquid-phase sequencer. Carboxypeptidase A hydrolyses confirmed not only the sequence of the five last residues but also the presence of a free alpha-carboxylic group at the C-terminus. Edman degradation was conducted on one hand with the Quadrol [N,N,N',N'-tetrakis(2-hydroxypropyl)ethylene diamine] program and S-alkylated protein before or after coupling with sulfophenylisothiocynate (the first 34 residues were thus identified), on the other hand on tryptic and chymotryptic peptides with a dimethylbenzylamine program (residues 1--23 and 31--34 were confirmed, the positions of residues 35-64 were established). Neurotoxin III was found to belong to the same group of scorpion toxins active on mammals as neurotoxin I purified from the same venom (50 homologous positions exist in the two proteins).     

Complete amino acid sequence of the Aspergillus cytotoxin mitogillin

The complete amino acid sequence of the cytotoxin mitogillin has been determined by sequencing the intact chain and peptide fragments produced by cleavage at methionyl, arginyl, lysyl, and tryptophanyl residues and at one aspartic acid-proline bond. The protein consists of 149 amino acid residues with alanine at the NH2 terminus and histidine at the COOH terminus. The calculated Mr of the native mitogillin was 16 867. The native molecule presents two disulfide bridges, one between cysteine residues at positions 5 and 147 and another one between cysteine residues at positions 75 and 131. The amino acid sequence of mitogillin shows 86% homology with another cytotoxic protein called alpha-sarcin.     

The purification and amino acid sequence of the lethal neurotoxin Tx1 from the venom of the Brazilian 'armed' spider Phoneutria nigriventer

A lethal neurotoxic polypeptide of Mr 8 kDa was purified from the venom of the South American 'armed' or wandering spider Phoneutria nigriventer by centrifugation, gel filtration on Superose 12, and reverse phase FPLC on columns of Pharmacia PepRPC and ProRPC. The purified neurotoxin Tx1 had an LD50 of 0.05 mg/kg in mice following intracerebroventricular injection. The complete amino acid sequence of the neurotoxin was determined by automated Edman degradation of the native and S-carboxymethylated protein in pulsed liquid and dual phase sequencers, and by the manual DABITC/PITC double coupling method applied to fragments obtained after digestions with the S. aureus V8 protease and trypsin. The neurotoxin Tx1 consists of a single chain of 77 amino acid residues, which contains a high proportion of cysteine. The primary structure showed no homology to other identified spider toxins.