Characterization and isolation of <Emphasis Type="SmallCaps">L</Emphasis>-to-<Emphasis Type="SmallCaps">D</Emphasis>-amino-acid-residue isomerase from platypus venom

Summary. Platypus venom contains an isomerase that reversibly interconverts the second amino-acid residue in some peptides between the L-form and the D-form. The enzyme acts on the natriuretic peptides OvCNPa and OvCNPb, and on the defensin-like peptides DLP-2 and DLP-4, but it does not act on DLP-1. While the isomerization of DLP-2 to DLP-4 is inhibited by the amino-peptidase inhibitor amastatin, it is not affected by the leucine amino-peptidase inhibitor bestatin. The enzyme, that is only present in minute quantities in an extract of the venom gland, is thermally stable up to 55 °C, and it was found by anion-exchange chromatography to be acidic. Isolation of the isomerase was carried out by combined ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC).     

Substrate specificity of platypus venom L-to-D-peptide isomerase

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.     

Illicit secretion of a cytoplasmic protein into the periplasm of Escherichia coli requires a signal peptide plus a portion of the cognate secreted protein. Demarcation of the critical region of the mature protein

The beta-lactamase signal peptide alone is not sufficient to direct secretion of chicken muscle triosephosphate isomerase, a normally cytoplasmic protein, into the periplasm of Escherichia coli. The signal peptide and at least the first 3 residues of the mature beta-lactamase are required before any secretion of the isomerase can be observed. At this point the level of secretion is very low, but the addition of further residues of the mature beta-lactamase enhances the secretion of the hybrid protein. The maximum level of secretion is achieved when 12 or more residues of the mature beta-lactamase intervene between the signal peptide and the isomerase. It is the proximity of an arginine residue at position 3 of the isomerase that is responsible for the blockade to secretion of these hybrid proteins (see Summers, R.G., Harris, C.R., and Knowles, J.R. (1989) J. Biol. Chem. 264, 20082-20088). With 12 residues of the mature beta-lactamase between the signal peptide and the isomerase, the offending arginine now lies at position 15 of the hybrid. The 14 residues that immediately follow the signal peptide therefore define a region of constrained properties that is critical to the secretability of proteins from E. coli.     

Inhibition of ClC-2 Chloride Channels by a Peptide Component or Components of Scorpion Venom

ClC chloride channels play essential roles in membrane excitability and maintenance of osmotic balance. Despite the recent crystallization of two bacterial ClC-like proteins, the gating mechanism for these channels remains unclear. In this study we tested scorpion venom for the presence of novel peptide inhibitors of ClC channels, which might be useful tools for dissecting the mechanisms underlying ClC channel gating. Recently, it has been shown that a peptide component of venom from the scorpion L. quinquestriatus hebraeus inhibits the CFTR chloride channel from the intracellular side. Using two-electrode voltage clamp we studied the effect of scorpion venom on ClC-0, -1, and -2, and found both dose- and voltage-dependent inhibition only of ClC-2. Comparison of voltage-dependence of inhibition by venom to that of known pore blockers revealed opposite voltage dependencies, suggesting different mechanisms of inhibition. Kinetic data show that venom induced slower activation kinetics compared to pre-venom records, suggesting that the active component(s) of venom may function as a gating modifier at ClC-2. Trypsinization abolished the inhibitory activity of venom, suggesting that the component(s) of scorpion venom that inhibits ClC-2 is a peptide.     

Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus

We have shown that the venom of the South American rattlesnake Crotalus durissus terrificus induces a long-lasting antinociceptive effect mediated by activation of kappa- and delta-opioid receptors. Despite being mediated by opioid receptors, prolonged treatment with the crotalid venom does not cause the development of peripheral tolerance or abstinence symptoms upon withdrawal. In the present study, we have isolated and chemically characterized a novel and potent antinociceptive peptide responsible for the oral opioid activity of this crotalid venom. The amino acid sequence of this peptide, designated crotalphine, was determined by mass spectrometry and corroborated by solid-phase synthesis to be 相似文献   

D-Amino acid residue in the C-type natriuretic peptide from the venom of the mammal,Ornithorhynchus anatinus,the Australian platypus

The C-type natriuretic peptide from the platypus venom (OvCNP) exists in two forms, OvCNPa and OvCNPb, whose amino acid sequences are identical. Through the use of nuclear magnetic resonance, mass spectrometry, and peptidase digestion studies, we discovered that OvCNPb incorporates a D-amino acid at position 2 in the primary structure. Peptides containing a D-amino acid have been found in lower forms of organism, but this report is the first for a D-amino acid in a biologically active peptide from a mammal. The result implies the existence of a specific isomerase in the platypus that converts an L-amino acid residue in the protein to the D-configuration.     

Specialisation of the venom gland proteome in predatory cone snails reveals functional diversification of the conotoxin biosynthetic pathway

Conotoxins, venom peptides from marine cone snails, diversify rapidly as speciation occurs. It has been suggested that each species can synthesize between 1000 and 1900 different toxins with little to no interspecies overlap. Conotoxins exhibit an unprecedented degree of post-translational modifications, the most common one being the formation of disulfide bonds. Despite the great diversity of structurally complex peptides, little is known about the glandular proteins responsible for their biosynthesis and maturation. Here, proteomic interrogations on the Conus venom gland led to the identification of novel glandular proteins of potential importance for toxin synthesis and secretion. A total of 161 and 157 proteins and protein isoforms were identified in the venom glands of Conus novaehollandiae and Conus victoriae, respectively. Interspecies differences in the venom gland proteomes were apparent. A large proportion of the proteins identified function in protein/peptide translation, folding, and protection events. Most intriguingly, however, we demonstrate the presence of a multitude of isoforms of protein disulfide isomerase (PDI), the enzyme catalyzing the formation and isomerization of the native disulfide bond. Investigating whether different PDI isoforms interact with distinct toxin families will greatly advance our knowledge on the generation of cone snail toxins and disulfide-rich peptides in general.     

Proexosite-1-dependent recognition and activation of prothrombin by taipan venom

An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate prothrombin to thrombin. To determine whether, similar to prothrombinase, taipan venom utilizes proexosite-1 on prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg(35), Lys(36), Arg(67), Lys(70), Arg(73), Arg(75), and Arg(77)) are also crucial for recognition and rapid activation of the substrate by taipan venom. This was evidenced by the observation that the K(m) and k(cat) values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) were markedly impaired. Competitive kinetic studies with the Tyr(63)-sulfated hirudin(54-65) peptide revealed that although the peptide inhibits the activation of the wild type zymogen by taipan venom with a K(D) of approximately 2 microm, it is ineffective in inhibiting the activation of mutant zymogens (K(D) > 4-30 microm). Interestingly, an approximately 50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom.     

灭鼠安、灭鼠优现场灭效试验、解毒及毒理探讨

灭鼠安、灭鼠优是70年代出现的新单剂量灭鼠剂。国外经试验对多种鼠有杀灭效果,适口性较好,对非杀灭对象比较安全,对抗凝血剂有耐药性的鼠能够杀灭等优点。国内军事医学科学院等单位也有合成,我站在1978-1979年对这两类药进行了现场灭效和解毒试验,并对毒理机制进行了探讨,今报告如下。     

Identification of sites in the cysteine-rich domain of the class P-III snake venom metalloproteinases responsible for inhibition of platelet function

Atrolysin A and jararhagin are class P-III snake venom metalloproteinases (SVMPs) with three distinct domains: a metalloproteinase, a disintegrin-like and a cysteine-rich. The metalloproteinase and the disintegrin-like domains of atrolysin A and jararhagin contain peptide sequences that interact with alpha2beta1 integrin and inhibit the platelet responses to collagen. Recently, the recombinant cysteine-rich domain of atrolysin A was shown to have similar effects, but the sequence(s) responsible for this is unknown. In this report, we demonstrate two complete peptide sequences from the homologous cysteine-rich domains of atrolysin A and jararhagin that inhibit both platelet aggregation by collagen and adhesion of alpha2-expressing K562 cells to this protein. In addition, the peptide effects on platelets do not seem to involve an inhibition of GPVI. These results identify, for the first time, sites in the cysteine-rich domain of SVMPs that inhibit cell responses to collagen and reveal the complexity of the potential biological effects of these enzymes with multifunctional domains.     

Function and solution structure of hainantoxin-I, a novel insect sodium channel inhibitor from the Chinese bird spider Selenocosmia hainana

Hainantoxin-I is a novel peptide toxin, purified from the venom of the Chinese bird spider Selenocosmia hainana (=Ornithoctonus hainana). It includes 33 amino acid residues with a disulfide linkage of I-IV, II-V and III-VI, assigned by partial reduction and sequence analysis. Under two-electrode voltage-clamp conditions, hainantoxin-I can block rNa(v)1.2/beta(1) and the insect sodium channel para/tipE expressed in Xenopus laevis oocytes with IC(50) values of 68+/-6 microM and 4.3+/-0.3 microM respectively. The three-dimensional solution structure of hainantoxin-I belongs to the inhibitor cystine knot structural family determined by two-dimensional (1)H nuclear magnetic resonance techniques. Structural comparison of hainantoxin-I with those of other toxins suggests that the combination of the charged residues and a vicinal hydrophobic patch should be responsible for ligand binding. This is the first report of an insect sodium channel blocker from spider venom and it provides useful information for the structure-function relationship studies of insect sodium channels.     

Alpha-neurotoxin gene expression in Naja sputatrix: identification of a silencer element in the promoter region

Alpha-neurotoxin (alpha-NTX) from the venom of cobra, Naja sputatrix, is a highly lethal post-synaptic toxin that is responsible for the lethality caused by the venom. However, this toxin is found at low levels (3%) in the crude venom. The expression of its gene is determined by a promoter which is 90% similar to the promoter of another three-fingered toxin, cardiotoxin (CTX), which is produced in large amounts (60%) in the same venom. Functional analysis of the NTX-2 gene promoter demonstrated the presence of a silencer element of 24 nucleotides (nt -678 to -655) at its 5(') flanking region. This element has been found to play a major role in the down-regulation of NTX-2 gene expression. A point mutation on this silencer appears to attenuate its repressive property in CTX-2 gene.     

Identification of Conus amadis disulfide isomerase: minimum sequence length of peptide fragments necessary for protein annotation

Protein disulfide isomerase (PDI) has been identified in a protein extract from the venom duct of the marine snail C. amadis. In-gel tryptic digestion of a thick protein band at approximately 55 kDa yields a mixture of peptides. Analysis of tryptic fragments by MALDI-MS/MS and LC-ESI-MS/MS methods permits sequence assignment. Three tryptic fragments yield two nine residue sequences (FVQDFLDGK and EPQLGDRVR ) and an eleven residue sequence (DQESTGALAFK ). Database analysis using peptides and were consistent with the sequence of PDI and peptide appears to be derived from a co-migrating protein. In identifying proteins based on the characterization of short peptide sequences the question arises about the reliability of identification using peptide fragments. Here we have also demonstrated the minimum length of peptide fragment necessary for unambiguous protein identification using fragments obtained from the experimentally derived sequences. Sequences of length > or =7 residues provide unambiguous identification in conjunction with protein molecular mass as a filter. The length of sequence necessary for unambiguous protein identification is also established using randomly chosen tryptic fragments from a standard dataset of proteins. The results are of significance in the identification of proteins from organisms with unsequenced genomes.     

Snake venomics of two poorly known Hydrophiinae: Comparative proteomics of the venoms of terrestrial Toxicocalamus longissimus and marine Hydrophis cyanocinctus

The venom proteomes of Toxicocalamus longissimus and Hydrophis cyanocinctus, a fossorial and a marine species, respectively, of the Hydrophiinae genus of Elapidae, were investigated by Edman degradation of RP-HPLC isolated proteins, and de novo MS/MS sequencing of in-gel derived tryptic peptide ions. The toxin arsenal of T. longissimus is made up of 1-2 type-I PLA(2) molecules, which account for 6.5% of the venom proteins, a minor PIII-SVMP (1.4% of the venom toxins), and ~20 members of the 3FTx family comprising 92% of the venom proteome. Seventeen proteins (5 type-I PLA(2)s and 12 3FTxs) were found in the venom of H. cyanocinctus. Three-finger toxins and type-I PLA(2) proteins comprise, respectively, 81% and 19% of its venom proteome. The simplicity of the H. cyanocinctus venom proteome is highlighted by the fact that only 6 venom components (3 short-chain neurotoxins, two long-chain neurotoxins, and one PLA(2) molecule) exhibit relative abundances >5%. As expected from its high neurotoxin abundance, the LD(50) for mice of H. cyanocinctus venom was fairly low, 0.132μg/g (intravenous) and 0.172μg/g (intraperitoneal). Our data indicate that specialization towards a lethal cocktail of 3FTx and type-I PLA(2) molecules may represent a widely adopted trophic solution throughout the evolution of Elapidae. Our results also points to a minimization of the molecular diversity of the toxin arsenal of the marine snake Hydrophis cyanocinctus in comparison to the venom proteome of its terrestrial relatives, and highlight that the same evolutionary solution, economy of the toxin arsenal, has been convergently adopted by different taxa in response to opposite selective pressures, loss and gain of neurotoxicity.     

蝎毒液肽基因内含子剪接信号分析

从中国蝎Buthus martensii Karsch基因组DNA中分离到两个毒液基因的内含子。在此基础上,通过编辑和分析目前已出版的蝎毒液肽基因内含子序列,确定了这类基因内含子剪接信号的共有序列,并将其与其它其它物种进行了比较。本文的结果对于研究蝎毒液肽前体mRNA的剪接机制以及比较不同物种之间内含子进化和功能的关系具有参考价值。     

Contryphan-Vn: a novel peptide from the venom of the Mediterranean snail Conus ventricosus.

The isolation, purification, and biochemical characterization of the novel peptide Contryphan-Vn, extracted from the venom of the Mediterranean marine snail Conus ventricosus, is reported. Contryphan-Vn is the first Conus peptide described from a vermivorous species and the first purified from the venom of the single Mediterranean Conus species. The amino acid sequence of Contryphan-Vn is As with other contryphans, Contryphan-Vn contains a d-tryptophan residue, is amidated at the C-terminus, and maintains the five-residue intercystine loop size. However, Contryphan-Vn differs from the known contryphans by the insertion of the Asp residue at position 2, by the lack of hydroxylation of Pro(4), and, remarkably, by the presence of the basic residue Lys(6) within the intercystine loop. Although the biological function(s) of contryphans is still unknown, these characteristics suggest distinct molecular target(s) and/or function(s) for Contryphan-Vn.     

Activation of the ryanodine receptor Ca2+ release channel of sarcoplasmic reticulum by a novel scorpion venom.

We identified a peptide fraction from the venom of the scorpion Buthotus hottentota that stimulated binding of [3H]ryanodine to ryanodine receptors of skeletal and cardiac sarcoplasmic reticulum and brain microsomes in a highly specific manner. Activity was concentrated in a peptide fraction of Mr 5,000-8,000. Assuming a single active peptide in this fraction, we estimated a dissociation constant of 20-30 nM for the interaction of the peptide with the ryanodine receptor. The whole venom and the purified fraction activated skeletal ryanodine receptor Ca2+ release channels incorporated into planar lipid bilayers. The venom produced a 10-fold increase in the mean open time and induced the appearance of a long lasting subconductance state not seen in controls. Changes were reversible and could be induced by the partially purified venom fraction. This novel scorpion venom should be helpful in establishing the role of ryanodine receptors in the initiation of intracellular Ca2+ release in striated muscle and in nonmuscle cells containing functional ryanodine receptors such as neurons and secretory cells.     

Venomic and antivenomic analyses of the Central American coral snake, Micrurus nigrocinctus (Elapidae)

The proteome of the venom of Micrurus nigrocinctus (Central American coral snake) was analyzed by a "venomics" approach. Nearly 50 venom peaks were resolved by RP-HPLC, revealing a complex protein composition. Comparative analyses of venoms from individual specimens revealed that such complexity is an intrinsic feature of this species, rather than the sum of variable individual patterns of simpler composition. Proteins related to eight distinct families were identified by MS/MS de novo peptide sequencing or N-terminal sequencing: phospholipase A(2) (PLA(2)), three-finger toxin (3FTx), l-amino acid oxidase, C-type lectin/lectin-like, metalloproteinase, serine proteinase, ohanin, and nucleotidase. PLA(2)s and 3FTxs are predominant, representing 48 and 38% of the venom proteins, respectively. Within 3FTxs, several isoforms of short-chain α-neurotoxins as well as muscarinic-like toxins and proteins with similarity to long-chain κ-2 bungarotoxin were identified. PLA(2)s are also highly diverse, and a toxicity screening showed that they mainly exert myotoxicity, although some are lethal and may contribute to the known presynaptic neurotoxicity of this venom. An antivenomic characterization of a therapeutic monospecific M. nigrocinctus equine antivenom revealed differences in immunorecognition of venom proteins that correlate with their molecular mass, with the weakest recognition observed toward 3FTxs.     

Modulation of Conotoxin Structure and Function Is Achieved through a Multienzyme Complex in the Venom Glands of Cone Snails

The oxidative folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a “non-native” peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the oxidative folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.     

Interactions between discrete neuronal membrane binding sites for the putative K+-channel ligands beta-bungarotoxin, dendrotoxin and mast-cell-degranulating peptide

1. beta-Bungarotoxin, a presynaptically active neurotoxin from the venom of Bungarus multicinctus, was radiolabelled with 125I and its binding to synaptic membranes from rat brain was analyzed. The interaction of these binding sites with those for dendrotoxin (a convulsant polypeptide from mamba venom) and mast-cell-degranulating peptide (from bee venom) was examined in the light of the known effects of all three toxins on voltage-dependent K+ currents. 2. When measured in Krebs/phosphate buffer, the binding appeared monotonic at low concentrations of radioiodinated beta-bungarotoxin (Kd 0.4 nM; Bmax 0.42 pmol/mg protein); higher concentrations of labelled toxin revealed an additional binding component of lower affinity, but computer analysis of the data failed to provide well-defined estimates of its Kd and Bmax values. 3. Equilibrium binding experiments conducted in imidazole-based buffers yielded distinctly biphasic Scatchard plots; computer analysis of the data revealed two populations of sites [Kd 0.26 (+/- 0.30) nM and 6.14 (+/- 5.68) nM; Bmax 0.16 (+/- 0.20) and 2.65 (+/- 1.21) pmol/mg protein]. 4. In Krebs medium, beta-bungarotoxin was a very weak antagonist of the binding of 125I-labelled dendrotoxin. In imidazole medium, however, the efficacy of the inhibition was markedly increased; analysis of this inhibition showed it to be non-competitive. 5. Dendrotoxin inhibited the binding of radioiodinated beta-bungarotoxin in Krebs medium with high potency, although the interaction was by a complex, non-competitive mechanism. 6. Mast-cell-degranulating peptide inhibited non-competitively the binding of both radiolabelled dendrotoxin and beta-bungarotoxin but with relatively low potency. 7. A speculative schematic model of the dendrotoxin/beta-bungarotoxin/mast-cell-degranulating peptide binding component(s) is proposed. Findings are discussed in terms of the likely involvement of these sites with voltage-dependent K+-channel proteins.