Search

The platypus is a venomous monotreme. Male platypuses possess a spur on their hind legs that is connected to glands in the pelvic region. They produce venom only during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in- and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of the breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland, and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified included antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines, and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine, and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution.

Gene duplication followed by adaptive selection is believed to be the primary driver of venom evolution. However, to date, no studies have evaluated the importance of gene duplications for venom evolution using a genomic approach. The availability of a sequenced genome and a venom gland transcriptome for the enigmatic platypus provides a unique opportunity to explore the role that gene duplication plays in venom evolution. Here, we identify gene duplication events and correlate them with expressed transcripts in an in-season venom gland. Gene duplicates (1,508) were identified. These duplicated pairs (421), including genes that have undergone multiple rounds of gene duplications, were expressed in the venom gland. The majority of these genes are involved in metabolism and protein synthesis not toxin functions. Twelve secretory genes including serine proteases, metalloproteinases, and protease inhibitors likely to produce symptoms of envenomation such as vasodilation and pain were detected. Only 16 of 107 platypus genes with high similarity to known toxins evolved through gene duplication. Platypus venom C-type natriuretic peptides and nerve growth factor do not possess lineage-specific gene duplicates. Extensive duplications, believed to increase the potency of toxic content and promote toxin diversification, were not found. This is the first study to take a genome-wide approach in order to examine the impact of gene duplication on venom evolution. Our findings support the idea that adaptive selection acts on gene duplicates to drive the independent evolution and functional diversification of similar venom genes in venomous species. However, gene duplications alone do not explain the “venome” of the platypus. Other mechanisms, such as alternative splicing and mutation, may be important in venom innovation.

Male platypus (Ornithorhynchus anatinus) venom has a peptidyl aminoacyl L/D-isomerase (hereafter called peptide isomerase) that converts the second amino acid residue in from the N-terminus from the L- to the D-form, and vice versa. A reversed-phase high-performance liquid chromatography (RP-HPLC) assay has been developed to monitor the interconversion using synthetic hexapeptides derived from defensin-like peptide-2 (DLP-2) and DLP-4 as substrates. It was hypothesised that animals other than the platypus would have peptide isomerase with the same substrate specificity. Accordingly, eight mouse tissues were tested and heart was shown to have the activity. This is notable for being the first evidence of a peptide isomerase being present in a higher mammal and heralds finding the activity in man.

Only five mammalian species are known to be venomous, and while a large amount of research has been carried out on reptile venom, mammalian venom has been poorly studied to date. Here we describe the status of current research into the venom of the platypus, a semi-aquatic egg-laying Australian mammal, and discuss our approach to platypus venom transcriptomics. We propose that such construction and analysis of mammalian venom transcriptomes from small samples of venom gland, in tandem with proteomics studies, will allow the identification of the full range of mammalian venom components. Functional studies and pharmacological evaluation of the identified toxins will then lay the foundations for the future development of novel biomedical substances. A large range of useful molecules have already been identified in snake venom, and many of these are currently in use in human medicine. It is therefore hoped that this basic research to identify the constituents of platypus venom will eventually yield novel drugs and new targets for painkillers.

The platypus, as an egg-laying mammal, displays an unusual mixture of reptilian and mammalian characteristics. It is also venomous, and further investigations into its little-studied venom may lead to the development of novel pharmaceuticals and drug targets and provide insights into the origins of mammalian venom. Here we investigate the expression patterns of antimicrobial genes called defensins, and also the venom peptides called defensin-like peptides (OvDLPs). We show, in the first expression study on any platypus venom gene, that the OvDLPs are expressed in a greater range of tissues than would be expected for genes with specific venom function, and thus that they may have a wider role than previously suspected.

When the platypus (Ornithorhynchus anatinus) was first discovered, it was thought to be a taxidermist’s hoax, as it has a blend of mammalian and reptilian features. It is a most remarkable mammal, not only because it lays eggs but also because it is venomous. Rather than delivering venom through a bite, as do snakes and shrews, male platypuses have venomous spurs on each hind leg. The platypus genome sequence provides a unique opportunity to unravel the evolutionary history of many of these interesting features. While searching the platypus genome for the sequences of antimicrobial defensin genes, we identified three Ornithorhynchus venom defensin-like peptide (OvDLP) genes, which produce the major components of platypus venom. We show that gene duplication and subsequent functional diversification of beta-defensins gave rise to these platypus OvDLPs. The OvDLP genes are located adjacent to the beta-defensins and share similar gene organization and peptide structures. Intriguingly, some species of snakes and lizards also produce venoms containing similar molecules called crotamines and crotamine-like peptides. This led us to trace the evolutionary origins of other components of platypus and reptile venom. Here we show that several venom components have evolved separately in the platypus and reptiles. Convergent evolution has repeatedly selected genes coding for proteins containing specific structural motifs as templates for venom molecules.

Nuclear magnetic resonance spectroscopy was used to investigate the conformations of the platypus venom C-type natriuretic peptide A (OvCNPa) in aqueous solutions and in solutions containing sodium dodecyl sulfate (SDS) micelles. The chemically synthesized OvCNPa showed a substantial decrease in flexibility in aqueous solution at 10 degreesC, allowing the observation of medium- and long-range nuclear Overhauser enhancement (NOE) connectivities. Three-dimensional structures calculated using these data showed flexible and reasonably well-defined regions, the locations of which were similar in the two solvents. In aqueous solution, the linear part that spans residues 3-14 was basically an extended conformation while the cyclic portion, defined by residues 23-39, contained a series of beta-turns. The overall shape of the cyclic portion was similar to that observed for an atrial natriuretic peptide (ANP) variant in aqueous solution. OvCNPa adopted a different conformation in SDS micelles wherein the N-terminal region, defined by residues 2-10, was more compact, characterised by turns and a helix, while the cyclic region had turns and an overall shape that was fundamentally different from those structures observed in aqueous solution. The hydrophobic cluster, situated at the centre of the ring of the structure in aqueous solution, was absent in the structure in the presence of SDS micelles. Thus, OvCNPa interacts with SDS micelles and can possibly form ion-channels in cell membranes. (C) 2002 Elsevier Science Ltd. All rights reserved.

Envenomation by platypus (Ornithorhynchus anatinus) venom (OaV) causes severe local effects, including intense pain, hyperalgesia and plasma extravasation (oedema) (de Plater et al. 1995), as well as hypotension and peripheral vasodilation in systemically administered experimental animals (Fenner et al. 1992). The molecular mechanisms underlying these OaV-induced effects are not known. Using the lipid bilayer technique it has been shown that platypus venom (OaV), which contains 19 peptide-protein components (de Plater et al. 1995; de Plater, 1998), formed inward anion and outward slow and fast cation-selective channels (J. I. Kourie unpublished data). The OaV-formed anion channel has a maximum conductance of 857 ± 23 pS in 250/50 mM KCl cis/trans. The current-voltage relationship of this channel shows strong inward rectification. The channel activity undergoes time-dependent inactivation that can be removed by depolarizing voltage steps more positive than +40 mV, the equilibrium potential for chloride (ECl). The conductance values for the slow and fast channels are 22·5 ± 2·6 and 38·8 ± 4·6 pS in 250/50 mM KCl cis/trans and 41·38 ± 4·2 and 60·7 ± 7·1 pS in 750/50 mM KCl cis/trans, respectively. The kinetics of the slow ion channels are voltage dependent. The channel open probability (Po) is between 0·1 and 0·8 at potentials between 0 and +140 mV. The channel frequency (Fo) increases with depolarizing potentials between 0 and +140 mV, whereas the mean open time (To) and mean closed time (Tc) decrease. The channel has conductance values of 21·47 ± 2·3 and 0·53 ± 0·1 pS in 250 mM KCl and choline chloride, respectively. The amplitude of the single channel current is dependent on cytoplasmic [K+] ([K+]cis) and the reversal potential (Erev) value responds to increases in [K+]cis by shifting to more negative voltages. The increase in current amplitude as a function of increasing [K+]cis is described by the Michaelis-Menten equation. At +140 mV, γmax and KS (maximal single channel conductance and concentration for half-maximal γ, respectively), have values of 38·6 pS and 380 mM and decline at 0 mV to 15·76 pS and 250 mM, respectively. The permeability values for PK:PNa:PCs:Pcholine are 1:1:0·63:0·089, respectively. The fast cation channel has not yet been studied in detail. The aims of this study are: (a) to characterize the biophysical properties of the OaV-formed fast cation-selective channel; (b) to compare these properties with those of a fast cation-selective channel formed by a synthetic C-type natriuretic peptide (OaCNP-39), which has been identified as a major component of Ornithorhynchus anatinus venom (de Plater, 1998); and (c) to determine the role of the 17 residue N-terminal fragment, i.e. OaCNP-39(1-17) of OaCNP-39 in the channel activity.

A peptide which relaxes rat uterine smooth muscle and exhibits homology with the mammalian C-type natriuretic peptide (CNP) has previously been identified in platypus (Ornithorhynchus anatinus) venom from its partial N-terminal amino acid sequence. In this study we describe the purification, detailed structure, synthesis and pharmacological characteristics of this peptide, which has been designated ovCNP-39 (Ornithorhynchus venom C-type natriuretic peptide). Elucidation of the 39-residue amino acid sequence confirms the homology with mammalian CNPs. These peptides produce hypotension in vivo and relax smooth muscle in vitro, but are poorly characterised in terms of physiological function. ovCNP-39 is equipotent with human/rat/porcine CNP-22 in eliciting cyclic guanosine 5'-monophosphate (cGMP) elevation in cultured vascular smooth muscle cells, suggesting that, like CNP, it acts through the ANPB natriuretic peptide receptor subtype. The direct elevation of cGMP in vascular smooth muscle by ovCNP-39 may underlie the vasodilatory effects of platypus envenomation.

In this study several activities of the venom of Ornithorhynchus anatinus have been investigated. Whole venom induced local oedema after subplantar injection and produced relaxation of the rat uterus in vitro. The relaxant activity was partially purified by gel permeation HPLC and subsequent analyses by SDS-PAGE revealed that this activity was associated with a 4200 mol. wt peptide. The N-terminal partial sequence of this peptide exhibited substantial identity with human and porcine C-type natriuretic peptide (CNP). Three other major proteins isolated from the venom had mol. wts of 140,000, 55,000 and 16,000. None was found to have any sequence homology with proteins listed in the SwissProt database. The 140,000 mol. wt protein exhibited hyaluronidase activity but the nature of the 55,000 and 16,000 mol. wt proteins remains to be determined. Platypus venom also exhibits protease activity, although the concentration of proteolytic enzymes was too low to be visualised by SDS-PAGE using Coomassie staining.

International audience; We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co- opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non- protein- coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation.

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 the process of delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used substrate segments from the N-terminus of the natriuretic peptide (OvCNP) and defensin-like peptides-2 and -4 (DLP-2 and DLP-4) from the venom. Synthetic hexapeptides containing the first three amino acid residues (IMF) of DLP-4 and DLP-2 were linked to the tripeptide srs (lower case letters denote the corresponding D-amino acid), to increase peptide stability and water solubility. The DLP analogues IMFsrs and ImFsrs were found to be effective substrates for the isomerase. Mutants of these hexapeptides were synthesized with the second amino acid replaced, respectively, by the L- and D-forms of Abu, Ala, His, Ile, Leu, Lys, Nle, Phe, Trp, Tyr, and Val. The relative rates of peptide isomerization were measured by using partially purified isomerase extracted from venom glands and separating the reactants with high performance liquid chromatography (HPLC). The isomerase was active with the mutants that contained L- or D-forms of Phe or Nle residues at the second position; but the amino acids that contained shorter, bta-branched- or long side-chains with terminal polar groups, viz., Ala, Abu, Ile, Leu, Val, Lys and Tyr respectively, were not substrates. For each of these non-substrates, newly formed peptides that eluted from HPLC earlier than the substrate were isolated and were deduced to have been formed through the loss of N-terminal Ile in each case. None of the hexapeptides based on LLH, the first three amino acid residues of OvCNPalpha were substrates, but when LLH was attached to full-length OvCNP it became a substrate for the isomerase; thus modulation of the substrate specificity is brought about by other sections of the longer peptide. Based on the action of the isomerase with the various substrates a model of the ‘van der Waals outline’ of the active site is proposed.

Cone snail venom is a rich source of bioactives, in particular small disulfide rich peptides that disrupt synaptic transmission. Here, we report the discovery of conomap-Vt (Conp-Vt), an unusual linear tetradecapeptide isolated from Conus vitulinus venom. The sequence displays no homology to known conopeptides, but displays significant homology to peptides of the MATP (myoactive tetradecapeptide) family, which are important endogenous neuromodulators in molluscs, annelids and insects. Conp-Vt showed potent excitatory activity in several snail isolated tissue preparations. Similar to ACh, repeated doses of Conp-Vt were tachyphylactic. Since nicotinic and muscarinic antagonists failed to block its effect and Conp-Vt desensitised tissue remained responsive to ACh, it appears that Conp-Vt contractions were non-cholinergic in origin. Finally, biochemical studies revealed that Conp-Vt is the first member of the MATP family with a D-amino acid. Interestingly, the isomerization of L-Phe to D-Phe enhanced biological activity, suggesting that this post-translational modified conopeptide may have evolved for prey capture. (c) 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

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 degrees 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).

The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met2. The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution.

In this study we characterise the ability of a C-type natriuretic peptide from platypus (Ornithorhynchus anatinus) venom (ovCNP-39) to relax the rat uterus in vitro and we investigate the possibility that ovCNP-39 contributes to the acute effects of envenomation, which include oedema, pain and erythema. We have found that both ovCNP-39 and the endogenous C-type natriuretic peptide, CNP-22, produce oedema in the rat paw and release histamine from rat peritoneal mast cells. Two synthetic peptides, ovCNP-39(1–17) and ovCNP-39(18–39), corresponding to the N- and C-termini, respectively, are equipotent histamine releasers, suggesting that ovCNP-39 and other natriuretic peptides do not act through conventional natriuretic peptide receptors on mast cells.

Background: To date, few peptides in the complex mixture of platypus venom have been identified and sequenced, in part due to the limited amounts of platypus venom available to study. We have constructed and sequenced a cDNA library from an active platypus venom gland to identify the remaining components. Results: We identified 83 novel putative platypus venom genes from 13 toxin families, which are homologous to known toxins from a wide range of vertebrates (fish, reptiles, insectivores) and invertebrates (spiders, sea anemones, starfish). A number of these are expressed in tissues other than the venom gland, and at least three of these families (those with homology to toxins from distant invertebrates) may play non-toxin roles. Thus, further functional testing is required to confirm venom activity. However, thepresence of similar putative toxins in such widely divergent species provides further evidence for the hypothesis that there are certain protein families that are selected preferentially during evolution to become venom peptides. We have also used homology with known proteins to speculate on the contributions of each venom component to the symptoms of platypus envenomation. Conclusions: This study represents a step towards fully characterizing the first mammal venom transcriptome. We have found similarities between putative platypus toxins and those of a number of unrelated species, providing insight into the evolution of mammalian venom.

Objective To describe in detail for the first time, the clinical course and medical management of a significant human envenomation by the Australian platypus (Ornithorhynchus anatinus). Clinical features A 57-year-old man was envenomated via two spur wounds to the right hand from each hind leg of a male platypus. Pain was immediate, sustained, and devastating; traditional first aid analgesic methods were ineffective. Intervention and outcome On admission to hospital, narcotics administered intravenously, both intermittently and by infusion, provided inadequate analgesia. A right wrist block was dramatically effective. After the blockade narcotic analgesic support was required for several days. The patient spent six days in hospital, and the envenomated area remained painful, swollen and with little movement for three weeks. Significant functional impairment of the hand persisted for three months, the cause of which is uncertain. Conclusions Male platypus venom remains largely unstudied. It produces savage local pain and marked local swelling, but no apparent tissue ischaemia. No antivenom is available; in its absence the only effective analgesia appears to be regional nerve blockade, when the envenomation site and available skills permit. Immobilisation assists.

The presence of d-amino-acid-containing polypeptides, defensin-like peptide (DLP)-2 and Ornithorhyncus venom C-type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian d-amino-acid-residue isomerase(s) responsible for the modification of the all-l-amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP-2 from DLP-4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is approximately 50-60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known l-to-d-amino-acid-residue isomerase in a mammal.

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.