Structural and functional analogs of the novel mammalian neuropeptide, neuromedin S (NmS), in the dermal venoms of Eurasian bombinid toads.

We report the isolation and structural characterization of two neuromedin S (NmS) analogs, (NmS-17 and NmS-33), from the dermal venoms of Eurasian bombinid toads. NmS is a novel neuromedin U (NmU)-related peptide with potent anorexigenic and circadian rhythm-modulating properties recently discovered in mammals. Cloning of NmS precursor-encoding cDNAs from skin venom-derived libraries revealed the presence of a high degree of transcript splice variation comparable to that found previously for NmU in both amphibian skin and mammalian brain. Synthetic replicates of both amphibian NmS peptides evoked robust and dose-dependent transient increases in intracellular calcium ion concentrations in CHO cells that had been stably transfected with either FM-3/GPR66 or FM-4/TGR-1 human NmU receptors. The potency and efficacy of these amphibian skin peptides at such receptors were comparable to those observed with human NmS and rat NmS. These data show that NmS and NmU genes had already diverged at the level of the Amphibia and that differential splicing of their transcribed mRNAs has been highly conserved throughout tetrapod vertebrate evolution indicative of fundamental biological function. NmS is additionally a novel neuropeptide homolog that can be added to the biologically active peptide arsenal of amphibian venom/defensive skin secretions.

Unmasking venom gland transcriptomes in reptile venoms

While structural studies of reptile venom toxins can be achieved using lyophilized venom samples, until now the cloning of precursor cDNAs required sacrifice of the specimen for dissection of the venom glands. Here we describe a simple and rapid technique that unmasks venom protein mRNAs present in lyophilized venom samples. To illustrate the technique we have RT-PCR-amplified a range of venom protein transcripts from cDNA libraries derived from the venoms of a hemotoxic snake, the Chinese copperhead (Deinagkistrodon acutus), a neurotoxic snake, the black mamba (Dendroaspis polylepis), and a venomous lizard, the Gila monster (Heloderma suspectum). These include a metalloproteinase and phospholipase A2 from D. acutus, a potassium channel blocker, dendrotoxin K, from D. polylepis, and exendin-4 from H. suspectum. These findings imply that the apparent absence and/or lability of mRNA in complex biological matrices is not always real and paves the way for accelerated acquisition of molecular genetic data on venom toxins for scientific and potential therapeutic purposes without sacrifice of endangered herpetofauna.

Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers.

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.

Helokinestatin-7 peptides from the venoms of Heloderma lizards

Helokinestatins 1–6 constitute a family of bradykinin antagonist peptides originally isolated from the venoms of the Gila Monster, Heloderma suspectum and the Mexican beaded lizard, Heloderma horridum. Here we report the identification, isolation and preliminary pharmacological characterization of two novel tridecapeptides, named helokinestatin-7S (FDDDSTELILEPR – 1550 Da) and helokinestatin-7H (FDDDSRKLILEPR – 1604 Da), whose primary structures were predicted from cDNAs cloned from venom libraries of respective Heloderma lizards. Computed molecular masses of putative helokinestatin-7 peptides were used as tools to locate these peptides in archived LC/MS fractions from respective venoms and sequences were confirmed by MS/MS fragmentation. A synthetic replicate of helokinestatin-7H was found to antagonize the relaxation effect of bradykinin on rat arterial smooth muscle but to have no measurable effects alone. In contrast, synthetic helokinestatin-7S was found to directly contract this preparation. Studies on related natural peptides with subtle differences in primary structure can provide the tools for structure/activity studies in pharmacological investigations directed toward unraveling the molecular basis of venom toxicity and for the evaluation of potential therapeutic leads.

Cloning of cDNAs and molecular characterisation of C-type lectin-like proteins from snake venoms

C-type lectin-like proteins (CTLPs) isolated from snake venoms are the largest and most complex non-mammalian vertebrate C-type lectin-like domain family. In the present study, we simultaneously amplified four cDNAs encoding different types of CTLP subunits from the venoms of two different species of snakes by RT-PCR with a single sense primer and a nested universal primer - two CTLP subunit-encoding cDNAs were cloned from Deinagkistrodon acutus venom and two from Agkistrodon halys Pallas venom. All four cloned CTLP subunits exhibited typical motifs in their corresponding domain regions but with relatively-low sequence similarities to each other. Compared with previously-published CTLPs, the four cloned CTLPs subunits showed slight variations in the calcium-binding sites and the disulphide bonding patterns. To our knowledge, these data constitute the first example of co-expression of CTLP platelet glycoprotein Ib-binding subunits and coagulation factors in Agkistrodon halys Pallas venom.

Cloning and characterisation of three novel disintegrin precursors from the venoms of three Atheris species: Atheris chlorechis, Atheris nitschei and Atheris squamigera

Snake venom constitutes one of the most complex mixtures of naturally-occurring toxic proteins/polypeptides and a large number of these possess very profound biological activities. Disintegrins, that are commonly found in viper venoms, are low molecular weight proteins that usually contain an -Arg-Gly-Asp- (-RGD-) motif that is known to be involved in cell adhesion ligand recognition, binding specifically to cell surface integrin receptors and also exhibiting platelet anti-aggregation activity.

Here, we report for the first time, the successful cloning of three cDNAs encoding disintegrin precursors from lyophilised venom-derived libraries of Atheris chlorechis, Atheris nitschei and Atheris squamigera, respectively. All of these disintegrins belong to the short-coding class and all exhibit high degrees of structural identity, both in their amino acid sequences and in the arrangement of their functional domains. Mass spectrometric analyses of the HPLC-separated/in-gel digested venom proteins was performed to characterise the mature disintegrins as expressed in the venom proteome. Studies on both the structures and conserved sites within these disintegrins are of considerable theoretical interest in the field of biological evolution and in the development of new research tools or novel templates for drug design.

Isolation and cloning of exendin precursor cDNAs from single samples of venom from the Mexican beaded lizard (Heloderma horridum) and the Gila Monster (Heloderma suspectum)

Reptile venoms are complex cocktails of bioactive molecules, including peptides. While the drug discovery potential of most species remains unrealized, many are endangered and afforded protection under international treaties. In this study, we describe how potential clinically important bioactive peptides and their corresponding mRNAs can be structurally characterized from single, small samples of reptile venom. The potential type-2 diabetes therapeutics, exendin-3 and exendin-4, from the Mexican beaded lizard (Heloderma horridum) and the Gila monster (Heloderma suspectum), respectively, have been characterized at both protein and nucleic acid levels to illustrate the efficacy of the technique and its contribution to biodiversity conservation.

Molecular cloning of a novel putative potassium channel-blocking neurotoxin from the venom of the North African scorpion, Androctonus amoreuxi

Scorpion venoms are a particularly rich source of neurotoxic proteins/peptides that interact in a highly specific fashion with discrete subtypes of ion channels in excitable and non-excitable cells. Here we have employed a recently developed technique to effect molecular cloning and structural characterization of a novel putative potassium channel-blocking toxin from the same sample of venom from the North African scorpion, Androctonus amoreuxi. The deduced precursor open-reading frame is composed of 59 amino acid residues that consists of a signal peptide of approximately 22 amino acid residues followed by a mature toxin of 37 amino acid residues. The mature toxin contains two functionally important residues (Lys27 and Tyr36), constituting a functional dyad motif that may be critical for potassium channel-blocking activity that can be affirmed from structural homologs as occurring in the venoms from other species of Androctonus scorpions. Parallel proteomic/transcriptomic studies can thus be performed on the same scorpion venom sample without sacrifice of the donor animal.

Isolation of scorpion (Androctonus amoreuxi) alpha-neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60–70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.

Helokinestatin: a new bradykinin B2 receptor antagonist decapeptide from lizard venom.

Synthetic bradykinin antagonist peptides/peptoids have been powerful tools for delineating the roles of kinins in both normal physiology and in pathological states. Here, we report the identification of a novel, naturally occurring bradykinin B2 receptor antagonist peptide, helokinestatin, isolated and structurally characterized from the venoms of helodermatid lizards—the Gila monster (Heloderma suspectum) and the Mexican beaded lizard (Heloderma horridum). The primary structure of the peptide was established by a combination of microsequencing and mass spectroscopy as Gly-Pro-Pro-Tyr-Gln-Pro-Leu-Val-Pro-Arg (Mr 1122.62). A synthetic replicate of helokinestatin was found to inhibit bradykinin-induced vasorelaxation of phenylephrine pre-constricted rat tail artery smooth muscle, mediated by the B2 receptor sub-type, in a dose-dependent manner. Natural selection, that generates functional optimization of predatory reptile venom peptides, can potentially provide new insights for drug lead design or for normal physiological or pathophysiological processes.

Antimicrobial peptides in the venom of the European hornet, Vespa crabro, identified as mastoparan C and crabrolin

Amphibian skin secretions are rich sources of cationic amphipathic peptides which often possess potent and broad-spectrum antimicrobial activity. However, the venoms of other animals such as hymenopteran insects, also contain peptides with these characteristics and the literature is unclear as to their antimicrobial potential. Here we subjected the venom of the European hornet, Vespa crabro, to reverse phase HPLC fractionation followed by screening of aliquots of individual fractions in bacterial zonal inhibition assays. Two major peptides possessing activity in these assays were further purified by HPLC and subjected to MALDI-TOF MS analysis and MS/MS fragmentation using an ESI mass spectrometer. The peptides were identified as mastoparan C (LNLKALLAVAKKILamide) and crabrolin (FLPLILRKIVTALamide). Replicates of both peptides were synthesised by solid-phase methodology and mean inhibitory concentrations (MICs) established against Staphylococcus aureus and Escherichia coli. Mastoparan C was found to be a potent antimicrobial with MIC values of 2 µM and 4 µM against S. aureus and E. coli, respectively. Crabrolin was found to be less potent with MIC values of > 160 µM and 40 µM for S. aureus and E. coli. Hornet venom thus contains a potent antimicrobial peptide that has been unambiguously identified as mastoparan C, a peptide that is known to affect profound histamine release from mast cells and to generally activate membrane G protein-linked receptors. It is thus highly probable that its antimicrobial effects, like those previously documented, are a result of a generalized membrane interactive and disruptive function — perhaps reflective of the authentic role of amphibian skin antimicrobials.

Molecular cloning and deduced organization of the pHpG/CNP precursor from the venom of the African forest viper, Atheris squamigera

The discovery that the hypotensive sequela of envenomation by the South American viper, Bothrops jararaca, was mediated by peptides, represented a milestone in drug discovery research that led to the introduction of ACE inhibitors. These bradykinin-potentiating peptides (BPPs) have been found in the venoms of many species of viper and molecular cloning of biosynthetic precursors has revealed that each encodes several different BPPs in tandem with a single copy of a C-type natriuretic peptide (CNP) located at the C-terminus. Venoms of the African forest vipers (Atheris) have been poorly studied possibly because they do not represent a major danger to humans. However, initial studies have indicated that they contain some of the “classical” protein toxins of viper venoms and a novel class of peptide, the polyglycine/polyhistidine (pGpH) peptides. These peptides occur in several molecular forms with different numbers of repetitive glycine and histidine repeats. We have cloned the biosynthetic precursor of A. squamigera pGpH peptides from a venom-derived cDNA library and have confirmed that a single copy of CNP is located at the C-terminus and additionally that, like BPPs in other vipers, pGpH peptides are encoded in tandem within this single precursor. Solid phase peptide synthesis of pGpH peptides has proven to be extremely difficult but is progressing and acquisition of synthetic replicates of each peptide is a necessary prerequisite for systematic pharmacological characterisation as establishment of a biological function for these peptides remains elusive. pGpH peptides may prove to play a role as fundamental as that of the BPPs.

A novel and potent trypsin inhibitor peptide, AC-17, from the skin secretion of the edible frog, Rana esculenta

Protease inhibitors are found in many venoms and evidence suggests that they occur widely in amphibian skin secretions. Kunitz inhibitors have been found in the skin secretions of bombinid toads and ranid frogs, Kazal inhibitors in phyllomedusine frogs and Bowman–Birk inhibitors in ranid frogs. Selective protease inhibitors could have important applications as therapeutics in the treatment of diseases in which discrete proteases play an aetiologcal role. Here we have examined the skin secretion of the edible frog, Rana esculenta, for protease inhibitors using trypsin as a model. HPLC fractions of secretions were screened for inhibitory activity using a chromogenic substrate as reporter. Three major peptides were resolved with trypsin inhibitory activity in HPLC fractions — one was a Kunitz-type inhibitor, a second was a Bowman–Birk inhibitor but the third represented a novel class of trypsin inhibitor in European frog skin. Analysis of the peptide established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17. Peptide AC-17 resembled a typical “Rana box” antimicrobial peptide but while it was active against Escherichia coli (MIC 30 µM) it was devoid of activity against Staphylococcus aureus and of haemolytic activity. In contrast, the peptide was a potent inhibitor of trypsin with a Ki of 5.56 µM. AC-17 represents the prototype of a novel trypsin inhibitor from the skin secretion of a European ranid frog that may target a trypsin-like protease present on the surface of Gram-negative bacteria.