Researchers identify Botox receptor

Sept. 29, 2003

As doctors tout the toxin found in Botox for its ability to iron out wrinkles, calm muscle spasms and treat migraine headaches, defense agencies condemn it as a weapon that could wipe out large numbers of civilians.

While it is well known that this toxic substance can paralyze the body's muscles, including the ones that help us breathe, how it infiltrates cells to do this has been undetermined.

In a paper published in the Sept. 29 issue of the Journal of Cell Biology, researchers from UW-Madison answer this long-standing question. They identify a receptor - a route of entry - for the Botox toxin that could lead to improved uses of the substance in the medical field and new methods for neutralizing it in the event of biological warfare.

Botulinum neurotoxin - the toxin found in Botox - is the deadliest of all substances. Produced by different strains of a family of bacteria, it comes in seven forms, four of which are reported to cause the paralyzing and potentially fatal disease of botulism. Currently, the family of bacteria that produces these toxins is listed by the Centers for Disease Control and Prevention as having the greatest potential for mass casualties if used as a biological weapon.

"Botulism is an old disease," says Edwin Chapman, a UW-Madison physiology professor and senior author of the new paper. "We know how the toxins block the release of neurotransmitters from neurons, but we didn't know how they entered the neurons in the first place."

Whether inhaled or injected, the deadly toxins head straight for neurons, or nerve cells. Via a binding receptor on the surface of these cells, the toxins are brought inside where they block the release of neurotransmitters, chemicals that control muscle contraction and relaxation.

"The way they kill," explains Chapman, "is by inactivating the diaphragm so you no longer can draw a breath."

The receptor that pulls the toxins inside the nerve cells has puzzled scientists. Chapman says researchers have known that gangliosides - a special type of lipid - and proteins work together as a receptor, but no one until now has been able to identify the specific proteins.

Using a cellular model, Chapman, physiology and neuroscience graduate student Min Dong, and others identified two proteins that function alongside gangliosides as the receptor for botulinum neurotoxin B - one of the four types deadly to humans. The proteins are synaptotagmin, or syt, I and II, which are found in certain types of neurons.

When one of these two proteins extends itself outside the cell during a process called exocytosis, the toxin latches on and then is internalized during the process of endocytosis.

To confirm that this protein and lipid pair is the actual physiological target of the toxin once it enters the body, the Wisconsin researchers set aside cell cultures and turned to a live mouse model. Working with their colleagues, Michael Goodnough and Eric Johnson in the Department of Food Microbiology and Toxicology, the researchers once again found that the toxin binds to and enters the cell via these two proteins and gangliosides.

"Our study is the first to identify a receptor for one of the botulinum neurotoxins and establish its entry route," says Dong, first author of the paper. "This knowledge will improve both the medical application of the [neurotoxin] and the prevention of a [biological] threat."

Knowledge of this receptor already had led to new research findings that suggest a possible antidote for the toxin.

As described in the paper published Sept. 29, Dong has developed decoys that effectively neutralize one type of the neurotoxin. Specifically, he created fragments of the syt II protein that contain the toxin's binding site.

In collaboration with Goodnough and Johnson, these fragments, along with gangliosides, were injected into the bloodstream of mice recently exposed to the toxin. The researchers found that the fragments neutralized most of toxic substance; injecting the fragments one minute prior to exposure neutralized 70 to 80 percent of the toxin.

As Chapman explains, "The fragments are a protective agent - a scavenger - that prevents the toxin from reaching its target."

These findings, says Dong, not only confirm the results from the cell culture studies, but also provide some of the first evidence that identification of the receptor could play an integral role in developing measures that counteract the bacterial toxin, thereby safeguarding human lives against exposure to the lethal poison.

The fragment composed of the syt II protein is being patented by the Wisconsin Alumni Research Foundation, a non-profit agency that manages intellectual property for UW-Madison.

Currently, Dong and others are working to identify receptors for the other types of botulinum neurotoxin fatal to humans.