Snake venoms. The amino-acid sequence of protein S2C4 from Dendroaspis jamesoni kaimosae (Jameson's mamba) venom.

A major component (S2C4) was purified from Jameson's mamba by gel filtration on Sephadex G-50 and ion-exchange chromatography on CM-cellulose. Protein S2C4 comprises 62 amino acid residues including 8 half-cystine residues. The complete amino acid sequence of the protein has been established. The sequence and the invariant amino acid residues of protein S2C4 resemble a short neurotoxin, a long neurotoxin, a cytotoxin and an angusticeps type protein. However, the position of its four disulphide bridges differs from those encountered in a short neurotoxin or a cytotoxin. Mixtures of protein S2C4 and angusticeps type proteins revealed a marked synergistic effect, in that their toxicity in combination was greater than the sum of their individual toxicities.     

A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.     

Orpotrin: a novel vasoconstrictor peptide from the venom of the Brazilian stingray Potamotrygon gr. orbignyi

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of crude samples, techniques such as chromatography and mass spectrometry have been employed. The present study describes the isolation, biochemical characterization, and sequence determination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The natural peptide was shown to be effective in microcirculatory environment causing a strong vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as the novel peptide. Its amino acid sequence, HGGYKPTDK, aligns only with creatine kinase residues 97–105, but has no similarity to any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by limited proteolysis is discussed. Taken together, the results indicate the usefulness of this single-step approach for low molecular mass compounds in complex samples such as venoms.     

The purification and amino acid sequence of toxin CM-13b from Naja haje annulifera (Egyptian cobra) venom.

Toxin CM-13b was purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. The toxin comprises 65 amino acid residues and is cross-linked by five disulphide bridges. The complete amino acid sequence of toxin CM-13b was elucidated. The reduced and S-carboxymethylated toxin was digested with trypsin and chymotrypsin and the peptides purified by DEAE-cellulose chromatography and chromatography or electrophoresis on paper. The amino acid sequences of the intact toxin and its constituent peptides were determined by the Edman-Begg procedure, either through the use of the automatic sequenator or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The primary structure of toxin CM-13b shows a high degree of homology with that of protein S4C11 from Naja melanoleuca venom[1], but their toxicities are very different.     

Three neurotoxins from the venom of a sea snake Astrotia stokesii, including two long-chain neurotoxic proteins with amidated C-termini.

From the venom of a sea snake Astrotia stokesii three neurotoxic components, toxins Astrotia stokesii a, b and c were isolated in 40, 15 and 5% yield by weight respectively of the whole venom. Their LD50 values for 20g mice were 0.13, 0.096 and 0.098 microgram/g body wt. respectively and accounted for almost all the lethal activity of the venom. Their amino acid sequences were determined. Astrotia stokesii a was composed of 60 amino acid residues with nine half-cystine residues and was quite homologous to other sea-snake short-chain neurotoxins in its amino acid sequence. Toxins Astrotia stokesii b and c were composed of 70 and 72 amino acid residues respectively with 10 half-cystine residues. They are the first long-chain neurotoxins with high activity isolated from sea-snake venoms. The C-terminal carboxy groups of toxins b and c were found to be amidated; the amidation is known for some polypeptides, but is novel for a protein. The amide group may make a hydrogen-bond with glutamic acid-39, which replaces a lysine that has so far been found invariably in long-chain neutrotoxins. Astrotia stokesii b and c are also novel in having phenylalanine-25 and isoleucine- or valine-42. The ordinary Tyr-Glu pair, which is observed in X-ray structure [Low, Preston, Sato, Rosen, Searl, Rudko & Richardson (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2991-2994] and n.m.r.study [Inagaki, Tatsumi, Miyazawa, Hori & Tamiya (1977) Abstr. Int. Congr. Pure Appl. Chem. 26th, p. 336] on erabutoxins may be replaced by a hydrophobic pair. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 5009o (30 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7B1, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Molecular cloning and sequence analysis of cDNAs coding for a serine proteinase and a Kunitz-type inhibitor in the venom gland of the Vipera nikolskii viper

Serine proteinases and Kunitz-type inhibitors are widely represented in the venoms of snakes belonging to different genera. During the studies of the venoms of snakes inhabiting Russia, we have cloned cDNAs coding for novel proteins of these families. A novel serine proteinase that we named nikobin was identified in the venom gland of the Nikolsky viper. The amino acid sequence of nikobin deduced from the cDNA sequence slightly differs from those of the serine proteinases found in other snakes, displaying 15 unique amino acid substitutions. This is the first serine proteinase from a viper of the Vipera genus for which the complete amino acid sequence has been determined. A cDNA coding for a Kunitz-type inhibitor has also been cloned. The deduced amino acid sequence of the inhibitor displays overall homology to the already known sequences of analogous proteins from vipers of the Vipera genus. However, several unusual amino acid substitutions that can cause a change of the inhibitor activity have been detected.     

Molecular cloning and nucleotide sequence analysis of a cDNA encoding the main beta-neurotoxin from the venom of the South American scorpion Tityus serrulatus.

A cDNA encoding the main Tityus serrulatus beta-neurotoxin was isolated from a venom gland cDNA library by using an oligonucleotide probe. The amino acid sequence deduced from the cDNA nucleotide sequence indicated that the toxin is the processed product of a precursor containing: (i) a signal peptide of 20 residues; (ii) the amino acid sequence of the mature toxin; and (iii) an extra Gly-Lys-Lys tail at the C-terminal end before the termination codon. Thus, in addition to the removal of the signal peptide by a signal peptidase, the generation of the mature toxin requires both a post-translational cleavage by a carboxypeptidase specific for basic residues and the action of an alpha-amidating enzyme. These results also show that the biosynthetic pathway for beta-toxins of 'New World' scorpion venoms is similar to that already described for alpha-toxins of 'Old World' scorpion venoms.     

Amino acid sequence and characterization of C-type lectin purified from the snake venom of Crotalus ruber

Galactoside-binding lectin was purified from the snake venom of Crotalus ruber by affinity chromatography on a lactose-agarose column, and the complete amino acid sequence was determined. The C. ruber venom lectin (CRL) showed a single band of 28 kDa by SDS-polyacrylamide electrophoresis under non-reducing conditions, but it showed a single band of 15 kDa under reducing conditions, indicating that CRL is a disulfide-linked homodimer of 15 kDa subunit. CRL specifically recognized beta-galactosides such as thiodigalactoside followed by N-acetylgalactosamine when examined with their inhibitory effects on CRL-induced hemagglutination. A CRL subunit was composed of 135 residues containing nine Cys residues and showed a high similarity to other C-type galactoside-binding lectins from snake venoms. C. atrox lectin (CAL) showed almost the same sequence except for eight amino acid residues. Neither CRL nor CAL induced platelet aggregation by itself or inhibited platelet aggregation mediated by von Willebrand factor or fibrinogen with agonists. CRL showed a similar oligomeric form and the sugar specificity as CAL, but it showed different divalent cation sensitivity such as Mn(2+) and Ni(2+). Homology modeling suggested that the amino acid substitution found in CRL does not affect sugar recognition of the lectin but might alter the conformation and influence the sugar binding pocket induced by the metal-ion binding.     

Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum

A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.     

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.     

A novel peptide from the ACEI/BPP-CNP precursor in the venom of Crotalus durissus collilineatus

In crotaline venoms, angiotensin-converting enzyme inhibitors [ACEIs, also known as bradykinin potentiating peptides (BPPs)], are products of a gene coding for an ACEI/BPP-C-type natriuretic peptide (CNP) precursor. In the genes from Bothrops jararaca and Gloydius blomhoffii, ACEI/BPP sequences are repeated. Sequencing of a cDNA clone from venom glands of Crotalus durissus collilineatus showed that two ACEIs/BPPs are located together at the N-terminus, but without repeats. An additional sequence for CNP was unexpectedly found at the C-terminus. Homologous genes for the ACEI/BPP-CNP precursor suggest that most crotaline venoms contain both ACEIs/BPPs and CNP. The sequence of ACEIs/BPPs is separated from the CNP sequence by a long spacer sequence. Previously, there was no evidence that this spacer actually coded any expressed peptides. Aird and Kaiser (1986, unpublished) previously isolated and sequenced a peptide of 11 residues (TPPAGPDVGPR) from Crotalus viridis viridis venom. In the present study, analysis of the cDNA clone from C. d. collilineatus revealed a nearly identical sequence in the ACEI/BPP-CNP spacer. Fractionation of the crude venom by reverse phase HPLC (C(18)), and analysis of the fractions by mass spectrometry (MS) indicated a component of 1020.5 Da. Amino acid sequencing by MS/MS confirmed that C. d. collilineatus venom contains the peptide TPPAGPDGGPR. Its high proline content and paired proline residues are typical of venom hypotensive peptides, although it lacks the usual N-terminal pyroglutamate. It has no demonstrable hypotensive activity when injected intravenously in rats; however, its occurrence in the venoms of dissimilar species suggests that its presence is not accidental. Evidence suggests that these novel toxins probably activate anaphylatoxin C3a receptors.     

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.     

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.     

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.     

Characterization of cerastobin, a thrombin-like enzyme from the venom of Cerastes vipera (Sahara sand viper)

Cerastobin, a thrombin-like enzyme, was isolated from the venom of Cerastes vipera (Sahara sand viper) in homogeneous form. Cerastobin had a molecular weight of 38,000 with 348 amino acid residues. It had an isoelectric point of 7.7 (a pH optimum of 7.9 and a temperature optimum of 45 degrees C). Cerastobin hydrolyzed arginine-containing synthetic substrates such as TAME, BAME, and BAEE, but BAPNA was not hydrolyzed. Cerastobin had thrombin-like activity, producing fibrin from fibrinogen and also hydrolyzing chromogenic substrates for thrombin such as 2AcOH.H-D-CHG-But-Arg-pNA (CBS 34.47) and H-D-Phe-Pip-Arg-pNA (S-2238). It showed kallikrein-like activity and hydrolyzed kallikrein substrates 2AcOH.H-D-Phe-Gly-Arg-pNA (CBS 33.27) and H-D-Pro-Phe-Arg-pNA (S-2302). It produced bradykinin from bradykininogen, as uterus contraction was observed. A serine inhibitor, DFP, exerted a pronounced inhibitory effect, suggesting that cerastobin is a serine-type protease. The sequence of 37 residues from the amino-terminal end was investigated. The amino-terminal amino acid was valine as it is in most other thrombin-like enzymes. The amino acid sequence of cerastobin was similar to that of thrombin in some residues and had some homology with that of kallikrein. However, cerastobin showed a high degree of homology to thrombin-like enzymes isolated from various snake venoms. Factor X was partially degraded by cerastobin. It was also found that antithrombin III was degraded by the enzyme. The alpha and beta chains of fibrin monomer were preferentially hydrolyzed by cerastobin, but the gamma chain was quite resistant.     

Snake venom toxins--I. The primary structure of a long neurotoxin S4C6 from Aspidelaps scutatus (shield or shield-nose snake) venom

1. Long neurotoxin S4C6 from Aspidelaps scutatus venom was purified by gel filtration and ion exchange chromatography (Joubert, 1987). 2. It contains 68 amino acids including 10 half-cystines. The toxicity of toxin S4C6 was determined and a LD50 of 0.13 +/- 0.04 micrograms/g mouse was found. 3. The complete primary structure of long neurotoxin S4C6 has been elucidated. In the toxin the 10 structurally invariant amino acids of the neurotoxins and cytoxins and the five functionally invariant amino acids of the neurotoxins are conserved.     

The complete amino acid sequence of the haemorrhagic factor LHFII, a metalloproteinase isolated from the venom of the bushmaster snake (Lachesis muta muta)

The complete amino acid sequence the haemorrhagic agent LHFII, a Zn and Ca containing metalloproteinase isolated from the venom of the Bushmaster snake Lachesis muta muta was determined by automated and DABITC/PITC microsequencing of the intact protein, fragments derived by cleavage with cyanogen bromide, and peptides resulting from enzymatic digestions with trypsin and the protease from S. aureus V8. The protein is composed of 200 residues and exhibits considerable sequence homology with the haemorrhagic toxins from a number of other snake venoms, and some metalloproteinases in the region of the putative Zn-binding sites.     

Molecular dissection of venom from Chinese scorpion Mesobuthus martensii: identification and characterization of four novel disulfide-bridged venom peptides

Scorpion venom is composed of a large repertoire of biologically active polypeptides. However, most of these peptides remain to be identified and characterized. In this paper, we report the identification and characterization of four novel disulfide-bridged venom peptides (named BmKBTx, BmKITx, BmKKx1 and BmKKx2, respectively) from the Chinese scorpion, Mesobuthus martensii (also named Buthus martensii Karsch). BmKBTx is composed of 58 amino acid residues and cross-linked by three disulfide bridges. The sequence of BmKBTx shows some similarities to that of the toxin, birtoxin, and its analogs. It is likely that BmKBTx is a beta-toxin active on Na+ channels, which is toxic to either insects or mammals. BmKITx is composed of 71 amino acid residues with four disulfide bridges. It is the longest venom peptide identified from M. martensii so far. BmKITx shows little sequence identity with scorpion alpha-toxins toxic to insects. It is likely that BmKITx is a new type of Na+ -channel specific toxin active on both insects and mammals. BmKKx1 contains 38 amino acid residues cross-linked by three disulfide bridges and shows 84% sequence identity with BmTx3, an inhibitor of A-type K+ channel and HERG currents. BmKKx1 has been classified as alpha-KTx-15.8. BmKKx2 is composed of 36 residues and stabilized by three disulfide bridges. BmKKx2 is a new member of the gamma-K+ -channel toxin subfamily (classified as gamma-KTx 2.2). The venoms of scorpions thus continue to provide novel toxins with potential novel actions on targets.     

Amino acid sequence homology of mammalian type C RNA virus major internal proteins.

The NH2-terminal amino acid sequence of the major group-specific antigen, the major internal virion protein (p30; approximate molecular weight 30,000) of several mammalian type C RNA viruses was determined by the Edman degradation procedure using an automated protein sequenator. All of the proteins analyzed show a high degree of over-all sequence homology and also contain specific regions or single residues. All p30s begin with the sequence prolyl-leucylarginyl (Pro-Leu-Arg) and have an invariant, conserved region from residues 11 to 24. In this region only a single amino acid difference appears between the cat and mouse p30s. At position 17 alanine is found in the cat, and serine in all the mouse proteins. This homologous region starts at position 10 for RD-114 and baboon virus p30s, and at position 18 in the protein of the virus isolated from gibbon ape. The region extending from residue 4 to 10 shows considerable variability between p30s isolated from different mammalian species. Out of 24 residues compared, only a single amino acid difference was found between six different mouse p30s. At position 4, three have leucine, two have alanine, and one has serine. The comparative sequence data demonstrate that the viral p30s are products of related genes in the viruses from various mammalian species.     

Primary structures of cytotoxic factors isolated from habu (Trimeresurus flavoviridis) venom

The amino acid sequence of a cytotoxic factor, CTF-I, isolated from the venom of the Japanese habu snake (Trimeresurus flavoviridis) has been determined through automatic phenylisothiocyanate degradation of the PE-protein and derived proteolytic peptides. CTF-I consists of 72 amino acids and contains an Arg-Gly-Asp sequence present in trigramin-like peptides isolated from other snake venoms. The primary structure of another cytotoxic factor, CTF-II, consisting of 75 amino acids, was deduced to comprise that of CTF-1 with an additional Glu-Leu-Leu-sequence at its N-terminal.