The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard (Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)⁵-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides.     

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.     

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.     

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10,000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (S[combining low line]QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.     

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.     

Chemical, immunological and biological properties of peptides like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide extracted from the venom of two lizards (Heloderma horridum and Heloderma suspectum)

Having previously isolated helodermin, the major peptide like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide, from the venom of the lizard Heloderma suspectum, we decided on a systematic exploration of all (VIP-PHI)-like peptides present in the venom of another lizard of the Helodermatidae family: Heloderma horridum. Six (VIP-PHI)-like peptides (PHH1 to 6) were purified to homogeneity from the venom of the lizard H. horridum with PHH3 and PHH4 representing two minor forms. All peptides cross-reacted in radioimmunoassays for helodermin and PHI but not for VIP. They yielded four fragments (T1 to T4) after trypsin digestion. T1, T2 and T3 showed the same retention time by reverse-phase HPLC and the same amino acid composition; the differences were confined to T4, the C-terminal sequence. PHH5 and PHH6 were found to be identical to synthetic helospectins I and II respectively. PHH1 and PHH3 probably resulted from a secondary modification of PHH5, while PHH2 and PHH4 derived from PHH6. Thus, the VIP-like peptides, previously called helospectins, are in fact typical of H. horridum venom. We confirmed that helodermin is the major (VIP-PHI)-like peptide of the venom of H. suspectum and observed its absence in H. horridum venom. Also, we found that positions 8 and 9 of helodermin are occupied by two Glu residues instead of two Gln as previously published. Helospectin-like material was also present in H. suspectum venom but in very small amount. In both venoms all VIP-like peptides were equally potent and efficient when tested for (a) their ability to occupy VIP as well as secretin receptors in rat pancreatic membranes and VIP receptors in rat liver membranes, and (b) the ensuing activation of adenylate cyclase in both membrane preparations.     

Structure and pharmacology of spider venom neurotoxins

Spider venoms are complex mixtures of neurotoxic peptides, proteins and low molecular mass organic molecules. Their neurotoxic activity is due to the interaction of the venom components with cellular receptors, in particular ion channels. Spider venoms have proven to be a rich source of highly specific peptide ligands for selected subtypes of potassium, sodium and calcium channels, and these toxins have been used to elucidate the structure and physiological roles of the channels in excitable and non-excitable cells. Spider peptides show great variability in their pharmacological activity and primary structure but relative homogeneity in their secondary structure. Following diverse molecular evolution mechanisms, and in particular selective hypermutation, short spider peptides appear to have functionally diversified while retaining a conserved molecular scaffold. This paper reviews the composition and pharmacology of spider venoms with emphasis on polypeptide toxin structure, mode of action and molecular evolution.     

Primary structure and biological activity of bradykinin potentiating peptides fromBothrops insularis snake venom

Several bradykinin potentiating peptides (BPPs) were isolated from the venom of the Brazilian arboricole snake Bothrops insularis by gel filtration on Sephadex G-150-120, followed by sequencial high-voltage paper electrophoreses atpH 3.5, 6.5, and 2.1. The BPPs were assayed by their ability to potentiate the contractile activity, on the isolated guinea pig ileum, and the hypotensive activity, on anesthetized rats, of bradykinin. Eight BPPs, containing 3–13 amino acid residues, were sequenced and their primary structures were shown to have a marked degree of homology with those of several BPPs from other venoms.     

Biochemical characterization of the lizard toxin gilatoxin

The Gila monster (genus Heloderma) is the only known lizard to produce and inject a venomous secretion. Little is known about the venom from these lizards, and none of the toxins have been isolated until this time. This paper reports the isolation and characterization of a major lethal toxin (gilatoxin) from the venoms of Heloderma suspectum and Heloderma horridum. Gilatoxins from both species were similar in amino acid composition, electrophoretic mobility, pI, and immunological reactivity. They are acidic proteins possessing molecular weights of 35 000-37 500 and isoelectric points of 4.25 and consist of a single polypeptide chain. Neither is antigenically related to the venoms of snakes. The toxins are devoid of phospholipase A2 activity and proteolytic, hemorrhagic, and hemolytic activities, with lethality being the only biological activity detectably expressed. The toxins appear to be unique and distinct from those of other venomous animals.     

Characterisation of neurotoxic polypeptides from <Emphasis Type="Italic">Cyanea capillata</Emphasis> medusae (Scyphozoa)

Cnidarian venoms include neurotoxins, which are able to paralyse prey organisms immediately. Important targets for neurotoxins are voltage-gated ion channels in membranes of excitable cells. By blocking specific receptor sites, neurotoxic components disturb the physiological ion channel functions. Here, we describe the isolation and characterisation of potential neurotoxic polypeptides from the crude tentacle venom of the boreal scyphomedusan Cyanea capillata. Partially purified venom fractions were obtained by size-exclusion and subsequent reversed-phase chromatography. To assess the blocking activity of the venom on voltage-gated sodium channels, we modified a mouse neuroblastoma (MNB) cell assay. Venom fractions containing channel-blocking activity were analysed by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The resulting mass spectra revealed a cluster of singly charged peptides within a mass range from 3,900 to 7,000 Da. A group of three potentially neurotoxic peptides with molecular masses of 3983.4, 5795.4 and 6962.1 Da could be tracked throughout the purification process. This investigation of the crude venom is part of a multidimensional assay-guided approach for the isolation and structural characterisation of toxic polypeptides in northern Scyphozoa.     

Identification of second lipolysis activating protein from scorpion Buthus occitanus tunetanus

Besides the previously described LVP1, a second protein, LVP2, inducing a lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 2% of crude venom proteins, with pHi = 6 and molecular mass of 16889 Da. The reduction and the alkylation of LVP2 revealed an heterodimeric structure. Isolated alpha and beta chains of LVP2 have a molecular weight (MW) of 8822 Da and 8902, respectively. This protein was not toxic to mice and stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 = 2 +/- 0.75 microg/ml. LVP2 subunits did not display any lipolytic activity. As previously described for venom and LVP1, beta adrenergic receptor (beta AR) antagonists interfere with LVP2 activity. Furthermore, it is shown that LVP2 competes with [3H] CGP 12177 (beta1/beta2 AR antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10(-7)M. Thus, these results bring original information on the existence of proteins that are present in scorpion venoms and can exert a distinct biological activity on adipocyte lipolysis through a beta-type adreno-receptor pathway.     

Characterization of the venom of the vermivorous cone snail Conus fulgetrum

Over 200 components with molecular mass ranging mainly from 400 to 4000 Da were characterized from the venom of the vermivorous cone snail Conus fulgetrum that inhabit Egyptian Red Sea. One major component having a molecular mass of 2946 Da was purified by HPLC, and its primary structure was determined by a combination of Edman degradation and MS/MS analysis.     

Primary structure and biological activity of bradykinin potentiating peptides fromBothrops insularis snake venom

Several bradykinin potentiating peptides (BPPs) were isolated from the venom of the Brazilian arboricole snake Bothrops insularis by gel filtration on Sephadex G-150-120, followed by sequencial high-voltage paper electrophoreses atpH 3.5, 6.5, and 2.1. The BPPs were assayed by their ability to potentiate the contractile activity, on the isolated guinea pig ileum, and the hypotensive activity, on anesthetized rats, of bradykinin. Eight BPPs, containing 3–13 amino acid residues, were sequenced and their primary structures were shown to have a marked degree of homology with those of several BPPs from other venoms.     

Purification and structure of exendin-3, a new pancreatic secretagogue isolated from Heloderma horridum venom.

An amino-terminal histidyl structure (His1) is characteristic of most peptides in the glucagon superfamily. An assay for His1 peptides performed by amino-terminal amino acid sequencing was used to screen venom from the Gila monster lizard, Heloderma horridum. Two His1 peptides were identified: helospectin and a new His1 peptide that has been named exendin-3 to indicate that it is the third peptide to be found in an exocrine secretion of Heloderma lizards which has endocrine activity, the first two being helospectin (exendin-1) and helodermin (exendin-2). In the lot of H. horridum venom tested, exendin-3 was 5-10-fold more abundant in molar concentration than helospectin. The structure of exendin-3 was analyzed by amino acid sequencing and mass spectrometry. Exendin-3 is a 39-amino acid peptide with a mass of 4200. It contains a carboxyl-terminal amide and has a strong homology with secretin at its amino-terminal 12 amino acids. The complete structure of exendin-3 is His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala- Val-Arg - Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser- amide. It is 32 and 26% homologous with helospectin and helodermin, respectively. It has greatest homology with glucagon (48%) and human glucagon-like peptide-1 (50%). Exendin-3 (3 microM) stimulated increases in cellular cAMP and amylase release from dispersed guinea pig pancreatic acini.     

Myotoxic phospholipases A(2) isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: cytotoxic effect on microorganism and tumor cells

This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A(2) (PLA(2)s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M(r) approximately 14,000 for the monomer and 28,000Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA(2)s from snake venoms, MTX-I belonging to Asp49 PLA(2) class, enzymatically active, and MTX-II to Lys49 PLA(2)s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA(2) and anticoagulant activities, corroborating the importance of residue His48 and Ca(2+) ions for the enzymatic catalysis. Both PLA(2)s induced myotoxic activity and dose-time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115-129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA(2) proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer.     

Peptide diversity in the venom of the social wasp Polybia paulista (Hymenoptera): A comparison of the intra- and inter-colony compositions

The venoms of the social wasps evolved to be used as defensive tools to protect the colonies of these insects against the attacks of predators. Previous studies estimated the presence of a dozen peptide components in the venoms of each species of these insects, which altogether comprise up to 70% of the weight of freeze-dried venoms. In the present study, an optimized experimental protocol is reported that utilizes liquid chromatography coupled to electrospray ionization mass spectrometry for the detection of peptides in the venom of the social wasp Polybia paulista; peptide profiles for both intra- and inter-colonial comparisons were obtained using this protocol. The results of our study revealed a surprisingly high level of intra- and inter-colonial variability for the same wasp species. We detected 78–108 different peptides in the venom of different colonies of P. paulista in the molar mass range from 400 to 3000 Da; among those, only 36 and 44 common peptides were observed in the inter- and intra-colony comparisons, respectively.     

Isolation and characterization of exendin-4, an exendin-3 analogue, from Heloderma suspectum venom. Further evidence for an exendin receptor on dispersed acini from guinea pig pancreas.

The recent identification in Heloderma horridum venom of exendin-3, a new member of the glucagon superfamily that acts as a pancreatic secretagogue, prompted a search for a similar peptide in Heloderma suspectum venom. An amino acid sequencing assay for peptides containing an amino-terminal histidine residue (His1) was used to isolate a 39-amino acid peptide, exendin-4, from H. suspectum venom. Exendin-4 differs from exendin-3 by two amino acid substitutions, Gly2-Glu3 in place of Ser2-Asp3, but is otherwise identical. The structural differences make exendin-4 distinct from exendin-3 in its bioactivity. In dispersed acini from guinea pig pancreas, natural and synthetic exendin-4 stimulate a monophasic increase in cAMP beginning at 100 pM that plateaus at 10 nM. The exendin-4-induced increase in cAMP is inhibited progressively by increasing concentrations of the exendin receptor antagonist, exendin-(9-39) amide. Unlike exendin-3, exendin-4 does not stimulate a second rise in acinar cAMP at concentrations greater than 100 nM, does not stimulate amylase release, and does not inhibit the binding of radiolabeled vasoactive intestinal peptide to acini. This indicates that in dispersed pancreatic acini, exendin-4 interacts only with the recently described exendin receptor.     

Leftward Shift in the Voltage-Dependence for Ca2+ Currents Activation Induced by a New Toxin from Phoneutria reidyi (Aranae, Ctenidae) Venom

Summary Various neurotoxins have been described from the venom of the Brazilian spider Phoneutria nigriventer, but little is known about the venoms of the other species of this genus. In the present work, we describe the purification and some structural and pharmacological features of a new toxin (PRTx3-7) from Phoneutria reidyi that causes flaccid paralysis in mice. The observed molecular mass (4627.26 Da) was in accordance with the calculated mass for the amidated form of the amino acid sequence (4627.08 Da). The presence of an α-amidated C-terminus was confirmed by MS/MS analysis of the C-terminal peptide, isolated after enzymatic digestion of the native protein with Glu-C endoproteinase. The purified protein was injected (intracerebro-ventricular) into mice at dose levels of 5 μg/mouse causing immediate agitation and clockwise gyration, followed by the gradual development of general flaccid paralysis. PRTx3-7 at 1 μM inhibited by 20% the KCl-induced increase on [Ca²⁺]_i in rat brain synaptosomes. The HEK cells permanently expressing L, N, P/Q and R HVA Ca²⁺ channels were also used to better characterize the pharmacological features of PRTx3-7. To our surprise, PRTx3-7 shifted the voltage-dependence for activation towards hyperpolarized membrane potentials for L (−4 mV), P/Q (−8 mV) and R (−5 mV) type Ca²⁺ currents. In addition, the new toxin also affected the steady state of inactivation of L-, N- and P/Q-type Ca²⁺ currents. L. B. Vieira and A. M. C. Pimenta contributed equally to this work.