January 2011

ASBMB research on venom proteins

A look at some ASBMB members whose research focuses on biological toxins, which can be used as models for mammalian systems, as diagnostic tools in the lab or even as potential therapeutic agents. (Titled "Potent science" in print version.)

Whether employed for attack, defense or a little bit of both, biological toxins have evolved throughout the plant, animal and microbial kingdoms. These deadly compounds have been the subject of scientific inquiry for centuries and remain so to this day. In particular, venoms produced by numerous animal species are a subject of great interest. These biological cocktails contain proteins and peptides with exceptionally enhanced activity designed to do a specific job and do it well. Given such potent biochemistry, it certainly makes sense that many American Society for Biochemistry and Molecular Biology members have taken an interest in venom proteins, whether as models for mammalian systems, as diagnostic tools in the lab or even as potential therapeutic agents. Included below are just a handful of our society’s members who brave these venomous waters in their research.

FoxJay Fox

Professor and Assistant Dean of Research Support of Microbiology
University of Virginia Medical School, Charlottesville

Back in the day, working in Jay Fox’s lab was quite an adventure. “We used to house live venomous snakes in our lab, and that typically kept my graduate students on edge,” he says.

These days, most of the venom peptides he needs easily can be synthesized or purchased, so current students don’t need to worry about any slithery companions. However, Fox believes that doesn’t make his research into the molecular biology and proteomics of snake venom any less exciting.

Fox got his first sip of the venom cocktail during his graduate studies with fellow ASBMB member Anthony Tu at Colorado State University, where he used approaches like Raman spectroscopy to identify the toxins present in various snake venoms.

He continued his work in the field of toxinology when he set up his own lab and managed to identify some unusual zinc proteases among the peptides of several crotalid (rattlesnakes and their relatives) venoms. These proteases, termed reprolysins, could break down collagen and other extracellular matrix components and likely are responsible for the hemorrhaging associated with snakebites.

Of course, Fox did realize that his snake venom studies would likely require some applicability for long-term success. “Snakebites are really quite rare, so they’re not perceived as a significant health problem that requires tremendous resources,” he says.

That applicability soon arrived when it was found that the reprolysins were part of a protease subgroup called “a disintegrin and metalloproteinase with thrombospondin motifs.” The multidomain ADAMTS proteases were generating quite a bit of research interest, since many of them were implicated in inflammation, atherosclerosis and cancer.

Since then, Fox and his lab have spent a lot of time in cancer research (although he still makes time for numerous side projects and collaborations in toxinology), particularly examining how cells, the surrounding extracellular matrix and proteases interact in promoting the invasion and metastasis of cancer cells. He’s shown, for example, that melanoma cells can influence the gene expression of nearby fibroblasts in the stromal tissue to produce a more invasion-friendly microenvironment.

“Of course, our studies are just one example of a significant element regarding the repertoire of proteins in snake and other venoms,” Fox says. “Namely that nearly all venom peptides identified to date have human orthologs. The venomous versions are just slightly modified to give them a pronounced effect.”

And that pronounced effect has led to significant payoffs both in research, where venom biology has led the way to understanding the role of certain proteins in mammals, and in medicine, as more than 20 venom-derived drugs and diagnostics have been approved or are in development.

Fox believes that these drugs represent just a fraction of venom’s potential in biomedicine. “I consider venoms to be great natural products libraries (the venom of a single snake can contain up to 50 different proteins and peptides) that probably still hide many secrets,” he says. “We know most of the protein components by now, but what isn’t known is the overall activity profile.”

Once identified, venom peptides usually are assayed for a single, pre-defined toxic activity, whether it’s blocking an ion channel or inducing blood coagulation.

To this end, Fox also has become involved in using proteomic approaches to look into the functionality of venom peptides, such as analyzing post-translational modifications or proteolytic processing. He believes that by using such emerging technologies, researchers can uncover new activities in venom, and that will lead to new drugs.

JBC highlight: Serrano, S. M. T., Kim, J., Wang, D., Dragulev, B., Shannon, J. D., Mann, H. H., Veit, G., Wagener, R., Koch, M., and Fox, J. W. (2006) The cysteine-rich domain of snake venom metalloproteinases is a ligand for von Willebrand factor A domains: role in substrate targeting. J. Biol. Chem. 281, 39746 – 39756.

GelbMichael H. Gelb

Harry and Catherine Jaynne Boand Endowed Professor of Chemistry
University of Washington, Seattle

Snakes may elicit a lot of fear due to their venomous nature, but in fact, the innocuous-looking honeybee kills more people each year in the U.S.

Of course, the threat of a bee sting is not the toxins themselves but rather the allergic reaction (anaphylaxis) that can occur following injection. This is due in large part to the presence of significant amounts of the allergen phospholipase A2, an enzyme that breaks down phospholipids found on cell membranes.

Over the years, bee venom has been a favorite system for allergy research, and scientists have made strides in adapting bee venom as an immunotherapy agent for insect stings and other immune conditions like arthritis.

However, it was in the 1980s, when researchers had found evidence that mammalian secreted phospholipases A2 might be involved in producing arachidonic acid, the precursor to prostaglandins and other eicosanoids (key inflammatory agents), that even more people took notice, including Michael H. Gelb.

Gelb, whose multifaceted interests include medicinal enzymology, drug design and protein prenylation, was interested in the link to inflammation and decided to use bee phospholipase as a model of the mammalian enzymes.

The initial choice was a pragmatic one. “Back then it was quite a challenge to engineer to express these proteins because of their many disulfide bonds,” he says. “But we were able to express the bee PLA2 in E. coli and so we went with that, even though the enzyme is more distant to humans than PLA2 found in snakes.”

The choice proved to be quite fruitful, and since his first experiments some 20 years ago, Gelb’s group has discovered numerous insights into phospholipase biochemistry with the help of the bee model. This work has included structural studies on the enzyme (with renowned structural biologist Paul Sigler of Yale University), which revealed that PLA2 has distinct lipid binding and active sites and that catalysis likely occurs through diffusion of a single phospholipid substrate into the active site slot without a conformational change to the PLA2 upon binding to the membrane surface. His group also has identified mechanisms underlying both membrane binding and antibody binding.

With that fundamental knowledge in hand, Gelb now has turned his attention to understanding the function and regulation of human PLA2 enzymes in the eicosanoid pathway. Together with Gerard Lambeau of IPMC-CNRS in Valbonne, France, he has identified and characterized a number of new and unusual PLA2 proteins, including a catalytically inactive variant that may act as a receptor ligand instead of an enzyme. He’s also taken an interest in the cytosolic phospholipase enzymes and how they interact with the secreted enzymes in various processes.

Some of these novel proteins could hold therapeutic promise, including one (sPLA2-X) that participates in the synthesis of asthma-inducing leukotrienes and another (sPLA2-IIa) that is involved in rheumatoid arthritis.

“We’ve got some exciting results, though it hasn’t been quite enough yet for pharmaceutical companies to take notice,” says Gelb. “I guess they’re being a little cautious these days.”

It’s no big deal, though; with nine different phospholipase A2 groups in humans, along with several variants in each group, Gelb has plenty of work to keep him busy until that day comes along.

JBC highlight: Valentin, E., Ghomashchi, F., Gelb, M. H., Lazdunski, M., and Lambeau, G. (200) Novel human secreted phospholipase A2 with homology to the group III bee venom enzyme. J. Biol. Chem. 275, 7492 – 7496.


LewisRichard Lewis

Professor, Chemistry and Structural Biology Division
Institute of Biomedicine, University of Queensland, Australia

While the United States may not be a major hub for venom studies, it probably comes as no surprise that Australia is an important center for venom research.

As Richard Lewis notes, it’s logical given the abundant source material. “Australia has some of the deadliest spiders, snakes, fish and jellyfish in the world,” he says. “I think the only venomous animal for which we’re not at the top of the list is our scorpions.”

Understanding this deadly fauna has been of great interest to Australian researchers for years, and the country has been a pioneer in areas like developing antivenoms and using compression bandages to treat snakebites.

Now, researchers like Lewis are combining new technologies with established approaches to gain even more insight into the pharmacology of the varied venoms and other bioactive compounds present in the surrounding environment.

Lewis got his own introduction to this field in his doctoral research, where he studied ciguatoxin, a chemical produced by certain dinoflagellates that can accumulate in fish and cause ciguatera, a type of food poisoning. He then spent a few years in industry, working on ways to manage ciguatoxin poisoning, before taking a faculty position at the University of Queensland in 2000.

In his lab, Lewis has turned most of his attention to cone snails, remarkable predatory mollusks that have evolved a diverse set of toxin peptides that typically range from 10 to 30 amino acids long and are stabilized through disulfide bonds (though there are outliers, including a massive 11,000 kd dimer).

“Baldomero Olivera really helped break this field open in the early 1980s when he first isolated some of these peptides and assigned functions to them, and obviously, it would be difficult to compete with him,” Lewis says. “But I thought if I focused on some of our distinct Australian cone sail species, our group could make a unique contribution.”

That certainly has been the case; while most characterized conotoxins have roles in blocking ion channel activity, Lewis’ team has found some more unusual inhibitors, such as peptides that block a G-protein coupled receptor and the norepinephrine (noradrenaline) transporter.

The latter peptide, part of a new family called chi-conopeptides, was particularly exciting. “Norepinephrine is involved in one of the key analgesic pathways in the spinal cord known as descending inhibition,” Lewis notes. “And when we did test the peptide in animals we found that it had some dramatic pain-relieving effects in models of neuropathic pain, which is exciting since neuropathic pain remains poorly treated in humans.”

Along with some colleagues, Lewis spun his discoveries into a startup biotech called Xenome, which is hoping to take his chi-conopeptide (Xen2174) into the clinic along with other leads like new calcium channel-blocking omega-conopeptides recently discovered through a National Health and Medical Research Council program grant. These new omegas have strong analgesic properties but none of the side effects typically seen in this class of peptides.

Recently, Lewis has been looking into integrating mass spectrometry technology and deep sequencing to probe cone snail peptides in even greater detail.

“We’ve been using Edman sequencing to map the amino acid composition of our peptides, but these new approaches have the potential to transform the way we do venom research,” he says. “Each cone snail species has over a thousand unique peptides, not to mention a host of protein machinery that makes and modifies these peptides, and these new technologies are going to be essential in helping us uncover all variations that occur in this amazing group of molecules and give us clues to how they first evolved.”

JBC Highlight: Sharpe, I. A., Palant, E., Schroeder, C. I., Kaye, D. M., Adams, D. J., Alewood, P. F., and Lewis, R. J. (2003) Inhibition of the norepinephrine transporter by the venom peptide χ-MrIA: site of action, Na+ dependence and structure-activity relationship. J. Biol. Chem. 278, 40317 – 40323.

Nick Zagorski (nzagorski@asbmb.org) is a science writer at ASBMB.

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