Platypus Venom Research Articles

Research roundup: January 2017

In brief About a quarter of nursing home residents experience a medication error, while three-quarters receive a potentially inappropriate drug, a new systematic review shows About two-thirds of falls among older people occur in those who took at least one high-risk medication during the preceding 24 hours Individuals with albuminuria are at increased risk of cognitive impairment and dementia Duck-billed platypus venom could lead to a new drug for type 2 diabetes

Interaction of hibernation and male reproductive function in wild Tasmanian echidnasTachyglossus aculeatus setosus

The short-beaked echidna Tachyglossus aculeatus is a seasonally breeding mammal with a near ubiquitous distribution throughout Australia. In Tasmania, breeding follows a period of deep hibernation, and males begin mating approximately 30 days after the termination of hibernation. The echidna has exceptionally large testes, which may reach a maximum of 1% of body mass during the mating period. As involution of gonads is considered a prerequisite for entering hibernation and hibernation typically suppresses all reproductive function, this raises questions about the timing of testes recrudescence in the Tasmanian echidna. We measured plasma testosterone concentrations and used ultrasonography to measure testicular and crural gland volume through the annual cycle in wild Tasmanian echidnas. Testes were at their minimum size (0.06% of body mass) in December (early summer); testes recrudescence occurred prior to entry into hibernation when plasma testosterone concentrations were low; and testes were maintained at 75% of their maximum volume throughout the hibernation period. The crural glands, which are secondary reproductive structures in the echidna, also exhibited an annual pattern of recrudescence and involution, with recrudescence occurring after males emerged from hibernation, when plasma testosterone was rising. We suggest that the unusual strategy of testes recrudescence occurring prior to hibernation in the Tasmanian echidna is a consequence of extremely high competition between males.

Echidna Venom Gland Transcriptome Provides Insights into the Evolution of Monotreme Venom

Monotremes (echidna and platypus) are egg-laying mammals. One of their most unique characteristic is that males have venom/crural glands that are seasonally active. Male platypuses produce venom during the breeding season, delivered via spurs, to aid in competition against other males. Echidnas are not able to erect their spurs, but a milky secretion is produced by the gland during the breeding season. The function and molecular composition of echidna venom is as yet unknown. Hence, we compared the deeply sequenced transcriptome of an in-season echidna crural gland to that of a platypus and searched for putative venom genes to provide clues into the function of echidna venom and the evolutionary history of monotreme venom. We found that the echidna venom gland transcriptome was markedly different from the platypus with no correlation between the top 50 most highly expressed genes. Four peptides found in the venom of the platypus were detected in the echidna transcriptome. However, these genes were not highly expressed in echidna, suggesting that they are the remnants of the evolutionary history of the ancestral venom gland. Gene ontology terms associated with the top 100 most highly expressed genes in echidna, showed functional terms associated with steroidal and fatty acid production, suggesting that echidna “venom” may play a role in scent communication during the breeding season. The loss of the ability to erect the spur and other unknown evolutionary forces acting in the echidna lineage resulted in the gradual decay of venom components and the evolution of a new role for the crural gland.

Proteomics and Deep Sequencing Comparison of Seasonally Active Venom Glands in the Platypus Reveals Novel Venom Peptides and Distinct Expression Profiles

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.

Bioorganic studies on the venom from duckbill platypus

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 ofOrnithorhynchusvenom C-type natriuretic peptide (OvCNP). This heptapeptide induced a significant increase in [Ca2+]iin IMR-32 cells at 75 μM; had relatively specific affinities for glutamate, histamine, and GABAAreceptors; 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.

A Limited Role for Gene Duplications in the Evolution of Platypus Venom

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.

Mammalian Peptide Isomerase: Platypus‐Type Activity Is Present in Mouse Heart

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.

L-to-D-peptide isomerase in male echidna venom

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.

Novel venom gene discovery in the platypus

BackgroundTo 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.ResultsWe 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, the presence 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.ConclusionsThis 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.

Morphological and Histochemical Observations on the Crural Gland-Spur Apparatus of the Echidna (Tachyglossus aculeatus) together with Comparative Observations on the Femoral Gland-Spur Apparatus of the Duckbilled Platypus (Ornithorhyncus anatinus)

The echidna and platypus have a crural/femoral gland that is linked by a large duct to a canalized, keratinous spur located on the medial side of the ankle. The echidna crural gland, like the femoral gland of the platypus, exhibits cyclic activity, being prominent in both monotremes when they are sexually active. In the present study, we compared the structure and histochemistry of these glands. During the active phase, the secretory epithelium forming the respective glands of both species increased in height and became packed with secretory granules that differed markedly in structure. Secretory granules of the echidna crural gland were electron dense and characterized by cores or areas of increased electron density. Those of the platypus were initially electron dense, but then became less dense and coalesced into irregular complexes of secretory material. Large cytoplasmic blebs extended from epithelial cell apices and appeared to be shed into the lumen, resulting in an apocrine mode of secretion. Exocytosis was also observed. A similar form of release of secretory product was not observed in the echidna. Secretory granules of both species were periodic acid-Schiff positive and stained for protein, suggesting that much of the secretory product was glycoprotein. Myoepithelial cells enveloped the secretory tubules of the platypus femoral gland, whereas they were not observed surrounding tubules comprising the echidna crural gland. During the quiescent phase, the epithelial cells of both species lost their secretory granules and decreased in height. As a result, the secretory tubules became smaller, intralobular connective tissue increased and the glands decreased in overall size.

Recent aspects of chemical ecology: Natural toxins, coral communities, and symbiotic relationships

Abstract 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.

Platypus venom: source of novel compounds

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.

Understanding and utilising mammalian venom via a platypus venom transcriptome

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.

Expression patterns of platypus defensin and related venom genes across a range of tissue types reveal the possibility of broader functions for OvDLPs than previously suspected

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.

Defensins and the convergent evolution of platypus and reptile venom genes

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.

Characterization and isolation of L-to-D-amino-acid-residue isomerase from platypus venom

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).

Mammalian l-to- d-amino-acid-residue isomerase from platypus venom

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 ∼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.

D-Amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties

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.

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

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

Conformations of platypus venom C-type natriuretic peptide in aqueous solution and sodium dodecyl sulfate micelles

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 °C, 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 β-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.