Feature: Platypus venom spurs drug discovery

This feature appeared in the May/June 2011 issue of Australian Life Scientist. To subscribe to the magazine, go here.

The duck-billed platypus is the strangest of beasts, down to its deepest evolutionary roots. Ornithorhynchus anatinus males pack a venomous punch – or puncture – in two sharp spurs that project from the rear legs. If not picked up safely by the tail, a male platypus can suddenly wrap its rear legs around an arm or a leg and thrust its spurs downward into the flesh of its incautious handler.

Nobody has died from a platypus sting, but in lucid moments during a three-day delirium of excruciating agony, rare victims of platypus envenomtion have probably wished they had. For a 57-year old angler stung when he handled a platypus in the Broken River near Mackay in 1991, only a nerve-blocking agent directly injected in the elbow’s 'funny bone' nerve ganglion dulled his agony.

The Medical Journal of Australia describes the patient’s suffering: "Pain was immediate, sustained, and devastating; traditional first aid analgesic methods were ineffective… Significant functional impairment of the hand persisted for three months, the cause of which is uncertain… [The venom] produces savage local pain… No antivenom is available."

What kind of brew of proteins and non-peptide agents causes systemic pain that almost nothing from the medicine cabinet of modern analgesics, nor even morphine, can assuage?

The persistence of severe pain in the presence of analgesics and hypnotics with well-characterised actions upon specific nerve-cell receptors implied that components of platypus venom acted upon unidentified pain-sensing systems against novel receptors.

That was the pharmacologically intriguing question that a joint US-Australian project set out to answer early in 2010, using DNA sequence data from the recently completed Platypus Genome Project.

The project involved Professor Wes Warren’s team at the University of Washington Genome Centre in Missouri, which coordinated the Platypus Genome Project, Associate Professor Kathy Belov’s molecular genetics group at the University of Sydney’s Faculty of Veterinary Sciences at Camperdown, in NSW, and Dr Anthony Papenfuss of the Walter and Eliza Hall Medical Research Institute’s Bioinformatics Division.

Mysterious monotreme

The platypus, a monotreme, is one of only a few venomous mammals in the world, and the first to have its genome sequenced. Only a few peptide toxins from platypus venom have been identified and sequenced previously because venom is rarely available for study.

Most of the venom’s complex mix of components remained unidentified, but an Illumina next-generation sequencer made short working of finding all the venom genes.

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Belov’s PhD student Camilla Whittington was lead author on the paper, ‘Novel venom gene discovery in the platypus’, published in the open-access journal Genome Biology last November.

Papenfuss developed algorithms that allowed Emily Wong, a postdoctoral researcher in Belov’s laboratory, to compare the transcriptomes – the full complement of cDNA transcripts – of the venom-gland with other specialised platypus tissues. After eliminating shared transcripts, Wong identified 83 genes that are expressed only in the venom gland; their encoded peptides represented 13 different classes of toxins.

Whittington then searched international databases for homologues of the platypus venom genes in other venomous creatures to develop evolutionary trees for the different venom components.

Juvenile females possess spurs that regress in adulthood,and lack venom glands. The platypus’ distant cousin, the echidna, Tachyglossus aculeatus, has a relict, non-functional venom gland inherited from a monotreme ancestor shared with the platypus, but all other venomous mammals are eutherians that deliver their venoms by biting.

With the exception of the primitive Asian primate genus Nycticebus - the slow lorises - all are soricomorph (“shrew-form”) mammals like North America’s Blarina short-tailed shrews, the Eurasian water shrew Neomys fodiens, Old World Talpus moles, and two Solenodon species from Cuba and Hispaniola.

Wong says most of the toxic components of platypus venom are peptides. and most are homologs of previously characterised peptide toxins from the venoms of species such as sea anemones, starfish, arachnids, fish, venomous lizards and snakes.

At the peptide level, the degree of homology ranges from as little as 25 per cent for some alpha-latrotoxins, to 68 per cent for helothermines (Gila monsters) and cysteine-rich venom proteins (snakes).

Whittington’s efforts to reconstruct phylogenetic trees assumed these homologous venom peptides evolved from an archaic set of genes whose proteins contained peptides with structures and functions that saw unrelated species independently co-opt and adapt them as components of their venoms.

In certain taxa, like the venomous Soricomorpha and widow spiders, the toxins are more likely conserved, being relatively recent hand-me-downs from a venomous ancestor. But in the case of the platypus, the lorises and the Soricomorpha, the venom components have almost certainly evolved independently at different times.

A 2005 comparison of the mitochondrial DNAs of monotremes and eutherians (placental mammals) and marsupials placed the monotreme-eutherian divergence in the early to mid-Jurassic, between 231 and 217 million years ago – much too early for their venom components to be the legacy of a common ancestor.

“Basically, we tried to figure out the evolutionary paths that the venom components have taken,” says Wong. “We used a phylogenetic program to compare peptide sequences from putative venom peptides in the platypus with homologs from other venomous species. The program performs a clustering analysis that allows us to retrace the changes, and reconstruct the ancestral genes.”

Whittington et al found that the dominant toxin genes in platypus venom code for serine proteases - 23 in all - including kallikreins, a class of enzymes with roles in inflammation, lowering blood pressure through vasodilation, blood coagulation and pain. Related peptide toxins are found in the venoms of Blarina shrews and North America’s Heloderma lizards, the Gila Monster and the Mexican Beaded Lizard.

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Eighteen of the venom genes code for stonustoxin-like peptides, related to those in stone fish venom, and ohanin, from the venom of the king cobra. These peptides destroy red blood cells and cause oedema and intense pain.

Platypus venom contains 10 Kunitz-type protease inhibitors, similar to beta-bungatoxin, from the venom of the Sydney region’s Broad-Headed Snake, Hopocephalus bungaroides, which has caused several human deaths.

The platypus’ arsenal of toxins includes seven zinc metalloproteinases, similar to those found in the venom of many snakes. Zinc metalloproteinases cause inflammation and destroy muscle cells.

Redback-like toxins

Interestingly, it also includes seven toxins related to the alpha-latrotoxins from the venom of widow spiders like the Australian redback, Latrodectus hasseltii. Alpha-latrotoxins cause intense pain by inducing nerve cells to fire uncontrollably, releasing neurotransmitters like acetylcholine.

The other major venom components are six cysteine-rich secretory proteins (CRISPs), similar to those found in the venoms of Gila Monsters and Mexican Button Lizards. CRISPs cause muscle wasting and also relax smooth muscle in artery walls, causing loss of blood pressure.

Minor components of platypus venom variously share structure and activity with actinoporins from sea anemone venom (haemolysis, pain and pore-formation in cells), Mamba intestinal toxin-like proteins (activation of calcium, potassium and sodium channels in nerve cells), snake sarafotoxins, vascular-endothelial growth factor (oedema and leakage from blood vessels) and DNAse II, similar to plancitoxin from the Crown-of-Thorns Starfish (apoptosis and DNA degradation).

Wong says next-gen sequencing allowed them to complete and publish the study in little more than 12 months, where previously it would have taken years. “To discover new cDNAs before next-gen sequencing we would have to design primers for conserved regions in peptide toxins in other venoms and then try to clone and sequence the platypus counterparts,” she says.

“That would mean looking for each gene individually, which would have taken a very long time. Now that we have the platypus genome sequence, and next-gen sequencing, we are able to map all the transcribed reads from the venom-gland transcriptome back to the annotated genome, and quickly get an idea of which expressed genes contribute to the full venom transcriptome.”

Wong says the novel peptides identified in platypus venom offer potential leads to new drug targets once more is known about what they actually do. “Certainly, down the track we would like to do some functional studies to determine the actual function of some of the platypus venom peptides for which we only have inferred functions from their putative homologs,” says Wong.

These studies might hopefully lead to new drug candidates for severe pain, low blood pressure and other conditions related to the medical problems caused by envenomation by these mysterious, cute and highly venomous monotreme.

This feature appeared in the May/June 2011 issue of Australian Life Scientist. To subscribe to the magazine, go here.