Feature: Plumbing the peptides of spider toxins

This feature appeared in the September/October 2009 issue of Australian Life Scientist. To subscribe to the magazine, go here.

Some 40,000 spider species have colonised almost every environmental niche on six continents, preying on a vast smorgasbord of insects and other invertebrates – even the odd, small vertebrate.

French toxinologist Dr Pierre Escoubas, an invited speaker at next month’s annual conference of the Australian Peptide Association, has spent much of his research career analysing spider venoms and assessing their peptide toxins for pharmacological activity. An assistant professor in the Institute of Molecular and Cellular Pharmacology at the University of Nice-Sophia Antipolis, in Valbonne, Escoubas has set up a biotechnology company, VenomeTech, to develop and commercialise his group’s discoveries.

Only a few years ago, guesstimates of the number of peptide toxins in a typical spider venom ranged from a dozen to one hundred. Using high-end mass spectrometry, Escoubas and his colleagues have come up with a figure at least 10 times higher. Some species cover all contingencies by subduing their prey du jour with as many as 1,000 peptide toxins. Multiply that total by 40,000 species, and spider venoms become an immense resource for drug-lead discovery; up to 40 million biologically active peptides await analysis.

According to Escoubas, a mere 760 spider venom peptides have been sequenced, and most have not been assayed thoroughly for pharmacological activity – less than one spider’s worth. He visits Australia at least once a year to work with Australian collaborators Dr Graham Nicholson at the University of Technology, Sydney, and Dr Glenn King of the University of Queensland.

Arachnophobic?

Only four spider families have species with venoms that can kill or severely harm humans: Australian funnel-webs (Atrax and Hadronyche spp, Hexathelidae); widow spiders (Latrodectus spp, Theridiidae); recluse or fiddle-back spiders (Loxosceles spp, Sicariidae); and Brazilian wandering spiders (Phoeneutria spp, Ctenidae).

King is interested in venom evolution, including the role of gene-duplication events in creating the proteins from which the peptide toxins are cleaved. They complement each other, having expertise in complementary methods of venom analysis. “Glenn is doing the cDNA work and I’ve been doing the mass spectrometry,” says Escoubas. Together they are analysing and comparing peptide toxins in the venoms of Atrax and Hadronyche funnel-webs, specifically the Fraser island form of the Toowoomba funnelweb, Hadronyche infensa.

Forms of H. infensa range from south-west Queensland to the Blue Mountains, west of Sydney. Escoubas says the Fraser Island form is genetically homogeneous and has a distinctly different peptide profile to mainland H. infensa populations, a consequence of long genetic isolation on the world’s largest sand island.

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The collaborative research program has two aims: “The first is academic – we wanted to develop techniques to mine the full depth of the venom proteomes. Spider venoms are comparable in complexity to cone shell venoms. The diversity, structure and range of pharmacological activities is absolutely enormous.”

Most spider peptide toxins fall within the size range between 30 and 40 amino acids. They are somewhat longer than the conotoxins of Conus marine snails, but have similar effects on the nervous systems of prey. “Like conotoxins, many appear to target potassium, sodium and calcium channels,” says Escoubas. “We’ve found some very interesting activity, including peptides that are selective for particular ion-channel subtypes. Some target acid-sensing ion channels – they respond to pH variation – which makes them candidates for treating pain.”

Escoubas and his colleagues use a high-resolution orbitrap mass spectrometer from Thermo Scientific to mine spider venoms to the last peptide. Venom proteins are separated by nano chromatography then fragmented in the orbitrap to generate partial peptide sequences called ‘tags’. In parallel, venom-gland messenger RNAs are converted into cDNAs and sequenced, and correlated with the peptide toxin sequence tags, allowing the genes coding for the original venom proteins to be identified, cloned and fully sequenced.

According to Escoubas, the traditional approach of starting with whole-cell messenger RNAs extracts and random cloning can miss low-abundance mRNAs. The dual approach using the orbitrap and mass spectrometry-based peptide sequencing misses almost nothing. It is sensitive enough to detect more than 1,000 peptides in a single venom extract, irrespective of size or abundance.

Size matters

Escoubas spent 15 years studying tarantulas. Despite their impressive size, tarantulas do not have particularly toxic venom. Thinking laterally, Escoubas reasoned that the venoms of small spiders are likely to contain peptides at least as potent as those of notoriously venomous large spiders. His team has developed techniques for extracting venom from the tiny venom glands of very small spiders, as small as 4mm, which constitute the majority of the world’s species.

Escoubas says small spiders face similar problems to cone shells that need to rapidly paralyse fish. “If you’re a jumping spider and want to subdue your prey rapidly, you need enormous punching power in your venom.” He said the camouflaged crab spiders that lurk in flowers can catch bees and bumblebees much larger than themselves, so their peptide toxins are likely to be especially potent, and potentially specific for particular ion channel sub-types. “Our challenge is to access the full range of spider diversity – the large, dangerous species are really just the tip of the iceberg,” he says.