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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.

Venomous mammals are rare, and only a few species in the orders Insectivora and Monotremata produce toxic venom. Among them, the duckbill platypus (Ornithorhynchus anatinus) is one of the two venomous Australian mammals. The adult male platypus carries a spur on each hind leg, which it uses to inject competitors with poison. However, the structure and function of the poison’s active compounds are still imcompletely characterized. We found that crude platypus venom produced potent Ca2+ influx in human neuroblastoma IMR‑32 cells. Guided by this assay, we identified 11 unique peptides, including peptide H–His–Asp–His–Pro–Asn–Pro–Arg–OH, which coincided with the N-terminal domain residues of Ornithorhynchus venom C-type natriuretic peptide (OvCNP). This heptapeptide induced a significant increase in [Ca2+]i in IMR-32 cells at 75 μM; had relatively specific affinities for glutamate, histamine, and GABAA receptors; and facilitated neurogenic twitching in guinea pig ileum specimens at 30 μM. We also established that its proteinous venom fraction strongly hydrolyzed Pro–Phe–Arg–MCA and cleaved a human low-molecular-weight kininogen (LK), similar to porcine pancreas kallikrein. These results strongly indicated that platypus venom contains tissue kallikrein-like protease(s), and its proteolytic activity might synergistically contribute to toxicity through the specific cleavage of other venom constituents., Conference：27th International Symposium on the Chemistry of Natural Products and the 7th International Conference on Biodiversity (ISCNP-27 & ICOB-7), Brisbane, Australia, 10–15 July 2011

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.

The discovery of new ecologically active compounds often triggers the development of basic scientific concepts in the field of biological sciences, since such compounds have direct physiological and behavioral effects on other living organisms. We have focused on the identification of natural key compounds that control biologically and physiologically intriguing phenomena. We describe three recent aspects of chemical ecology that we have investigated: natural toxins, coral communities, and symbiotic relationships. Blarina toxin (BLTX) is a lethal mammalian venom that was isolated from the short-tailed shrew. Duck-billed platypus venom shows potent Ca2+ influx in neuroblastoma cells. The venom of the solitary wasp contains arginine kinase-like protein and is used to paralyze its prey to feed its larva. The ecological behaviors of corals are controlled by combinations of small molecules. The polyol compound symbiodinolide may serve as a defense substance for symbiotic dinoflagellates to prevent digestion of their host animals. These compounds reveal the wonder of nature, in both terrestrial and marine ecological systems.

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.

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.

The venoms of predatory marine snails (Conus spp.) contain diverse mixtures of peptide toxins with high potency and selectivity for a variety of voltage-gated and ligand-gated ion channels. Here we describe the chemical and functional characterization of three novel conotoxins, αD-VxXIIA, αD-VxXIIB, and αD-VxXIIC, purified from the venom of Conus vexillum. Each toxin was observed as an ∼11-kDa protein by LC/MS, size exclusion chromatography, and SDS-PAGE. After reduction, the peptide sequences were determined by Edman degradation chemistry and tandem MS. Combining the sequence data together with LC/MS and NMR data revealed that in solution these toxins are pseudo-homodimers of paired 47-50-residue peptides. The toxin subunits exhibited a novel arrangement of 10 conserved cystine residues, and additional post-translational modifications contributed heterogeneity to the proteins. Binding assays and two-electrode voltage clamp analyses showed that αD-VxXIIA, αD-VxXIIB, and αD-VxXIIC are potent inhibitors of nicotinic acetylcholine receptors (nAChRs) with selectivity for α7 and β2 containing neuronal nAChR subtypes. These dimeric conotoxins represent a fifth and highly divergent structural class of conotoxins targeting nAChRs.

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.

Three defensin-like peptides (DLPs) were isolated from platypus venom and sequenced. One of these peptides, DLP-1, was synthesized chemically and its three-dimensional structure was determined using NMR spectroscopy. The main structural elements of this 42-residue peptide were an anti-parallel β-sheet comprising residues 15-18 and 37-40 and a small 310 helix spanning residues 10-12. The overall three-dimensional fold is similar to that of β-defensin-12, and similar to the sodium-channel neurotoxin ShI (Stichodactyla helianthusneurotoxin I). However, the side chains known to be functionally important in β-defensin-12 and ShI are not conserved in DLP-1, suggesting that it has a different biological function. Consistent with this contention, we showed that DLP-1 possesses no anti-microbial properties and has no observable activity on rat dorsal-root-ganglion sodium-channel currents.

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.

The monotremes (the echidnas and the platypus) display both mammalian and reptilian features. Male monotremes have a bilateral crural gland that is connected via a duct to a spur on each hind limb. Male echidnas appear not to use their spurs as weapons in aggressive acts, but the crural system may have a role in reproductive behaviour because it appears only to be active during the breeding season. The secretions produced by the echidna’s crural gland have not hitherto been biochemically or pharmacologically characterised. We used reverse-phase high-performance liquid chromatography (RP-HPLC) to separate the components of echidna venom and compared the chromatograms with those from platypus venom. The echidna venom appears to contain fewer proteins and peptides than platypus venom; however, it appears to have defensin-like peptides that behave similarly on RP-HPLC to those in platypus venom. Like platypus venom, echidna venom has peptidyl aminoacyl l/d-peptide isomerase activity. An RP-HPLC-based assay showed that the second amino acid residue, of a probe synthetic hexapeptide, was converted into the d-form, when incubated with echidna venom.

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 venom of the platypus (Ornithorhynchus anatinus) has been poorly studied to date. The recent publication of the platypus genome heralds a new era for mammalian venom research and is a useful starting tool for functional studies of venom components. We report here the patterns of tissue expression of two venom genes, OvNGF and OvCNP, in order to provide some insight into the functions of the proteins they produce and to pave the way for further functional and pharmacological studies, which may lead to the development of novel therapeutic agents.

An anatomical feature of the platypus (Ornithorhynchus anatinus) that is seen in only one other mammal, the echidna, is that the male has a crural glandular system that produces venom that is used for defence and territorial–breeding functions; whether the echidna is similarly venomous is not yet established. Platypus venom contains many novel proteins and peptides that are different from those in reptilian venom. It also causes pain and symptoms that are not experienced by any other kind of envenomation. Five types of proteins and peptides have been isolated and identified from platypus venom, namely: defensin-like peptides (DLPs); Ornithorhynchus venom C-type natriuretic peptides (OvCNPs); Ornithorhynchus nerve growth factor; hyaluronidase; and l-to-d-peptide isomerase. The structures of DLPs and OvCNPs have already been studied and they are very similar to β-defensin-12 and mammalian C-type natriuretic peptides, respectively. A special mammalian l-to-d-peptide isomerase that is responsible for interconverting the l- and d-peptide isomers is also found in platypus venom. Isomerase activity has recently been discovered in platypus tissues other than the venom gland. It is possible that similar kinds of enzymes might exist in other mammals and play important, as yet unknown, biological roles. Considering the fact that some animal venoms have already been widely used in pharmaceutical applications, research into platypus venom may lead to the discovery of new molecules and potent drugs that are useful biomedical tools.

Platypuses (Ornithorhynchus anatinus) and other monotremes occupy an important position as an early offshoot from the evolutionary lineage leading from reptiles to mammals. One of the most intriguing characteristics of these mammals is that the males produce venom during the mating season. O. anatinus venom contains defensin-like peptide, C-type natriuretic peptide and nerve growth factor, as well as other unidentified fractions. These components of O. anatinus venom display similarity to components of the venom of other species such as sea anemones, snakes and shrews. Here we review available literature on O. anatinus venom and venom in other species. Further research into O. anatinus venom will offer some insight into the evolution and functions of venom components.

Curiosity and controversy have surrounded the function of the crural system of the platypus since its discovery in the late 18th century. Early work on the venom confused rather than clarified the biological significance of the crural system. Many experiments gave conflicting results, especially concerning the coagulation effects of intravenous injections of venom extracts, although consistent observations were made of general vasodilation following intravenous injection into rabbits. More recent studies have shown that crural (venom) gland activity is seasonal and in synchrony with the breeding season. Secretion from the crural glands shows proteolytic activity and contains at least three major proteins, one of which has hyaluronidase activity. Subcutaneous injection of venom produced mild toxic effects whereas intravenous doses (75-90mg protein/kg) in mice were lethal. Whole venom induced local oedema after subplantar injection in rats and a 4.2kD peptide isolated from the venom caused relaxation of rat uterus in vitro. At least 16 incidents of envenomation by the platypus have been recorded in humans but no fatalities have been reported. In most human cases, envenomation resulted in immediate and severe local pain and oedema, sometimes associated with nausea, cold sweats, dull gastric pain and vomiting, hyperaesthesia and swelling of the axillary lymph nodes. Significant functional impairment of the upper limb for some weeks or months has been observed. Various treatments have been used to alleviate the symptoms of envenomation with differing successes. Envenomation has also been recorded in platypuses and dogs. The effects of these envenomations will be discussed.

In this study we investigated the possibility that the C-type natriuretic peptide from platypus venom (ONP-39) contributes to the acute effects of envenomation, which include oedema, pain and erythema. These effects may result from the release of auto pharmacological mediators from mast cells. Using an in vitro assay we have demonstrated that both ONP-39 and the endogenous C-type natriuretic peptide (CNP-22) release histamine from rat peritoneal mast cells, an effect similar to the structurally homologous atrial natriuretic peptide (ANP) (Opgenorth et al., 1990, Peptides 11(5):1003-7). Two synthetic peptides, ONP-39(1-17) and ONP-39(18-39), corresponding to the N- and C- termini respectively, are equipotent, suggesting that ONP-39 and other natriuretic peptides do not act through conventional ANP receptors on mast cells. The ability of ONP-39 to promote histamine release suggests that it may contribute to the acute symptoms of envenomation.

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.