What do Malaysian Pit Viper snake venom and "resurrected" woolly mammoth hemoglobin have in common?
They represent a sampling of the range of research projects that Jörg Stetefeld, (Canada Research Chair in Structural Biology, Department of Chemistry, and Department of Microbiology), and his team of graduate and undergraduate students have undertaken over the last couple of years.
Stetefeld’s research involves using advanced imaging and biophysical techniques (biological information at the atomic or molecular level) to study the structure, function and regulation of proteins involved with the extracellular matrix (part of the animal tissue outside of the cell). Thanks to funding from the Canada Foundation for Innovation, Stetefeld’s group has access to high-tech equipment, like a Protein X-ray diffractometer and a SAXS (Small Angle X-ray Scattering) system – a combination of sophisticated equipment that is rare for a single institution to have acquired. This equipment allows scientists to be able to actually see protein structure. And seeing the structure opens up a whole new realm of possibility because, according to Stetefeld: “If we know what it looks like, we have a better idea of how it works.”
The investment has been paying off, and as a result of the peer-reviewed, published results coming out of his lab, these somewhat unusual projects have landed on Stetefeld’s door.
The Malaysian Pit Viper (Calloselasma rhodostoma) is a venomous snake, which in Northern Malaysia alone, is responsible for approximately 700 bites per year, with a two per cent mortality rate. Although the mortality rate is not high, victims are often left with dysfunctional or amputated limbs. Further, the rapidity of the venom to afflict its victims has been a mystery that has baffled scientists, and it was this very tricky problem that was presented to the Stetefeld group.
Scientists already knew that exposure to the venom caused systemic bleeding by destroying a victim’s red blood cells, but why did it work so quickly?
In collaboration with a snake farm in Brazil, the Stetefeld group imported sterilized Malaysian Pit Viper venom and started studying its proteins. Snake venom is largely composed of protein, and in this instance, they were searching for an answer among 2,000-3,000 different proteins: that’s a lot of isolating and analyzing. The work, however, paid off.
The heterotetrameric (a protein containing four subunits, where the subunits are not identical) snake venom lectin (a sugar binding protein) Rhodocetin from the venom of the Malayan pit viper forms a unique quaternary structure (a structure that is bonded to four other subunits) with four individual subunits (α, β, γ, δ) each of them showing the classical lectin fold.
In the Stetefeld lab, it was shown that the γδ heterodimer of rhodocetin specifically targets the collagen receptor integrin on the surface of platelets, thereby blocking collagen binding to α2β1 integrin and antagonizing platelet aggregation. Moreover, rhodocetin not only blocks the ligand-binding site, but also changes its conformation into a less active one. To date, rhodocetin is by far the most affine and specific inhibitor for this class of heterodimeric receptors.
In addition, the rhodocetin subunits αβ can bind to the platelet glycoprotein GPlb-IX-V complex, thereby blocking the GPlbα mediated interaction with von Willebrandt factor in a cation-independent manner.
Taken together, heterotetrameric rhodocetin is the only known snake venom lectin that binds both, α2β1 integrin and GPlbα thereby blocking collagen – and vWF induced platelet aggregation much more efficiently than any venom component targeting a single receptor only.
In other words, the same snake venom lectin can block both essential platelet aggregation pathways at the same time. This strategy is unique and very efficient.
The Malaysian Pit Viper has long been identified as one of the medically important poisonous snakes of Malaysia, and the venom has had applications in traditional medicine. However, understanding how the proteins and processes work enables researchers to use the information to potentially create safe compounds that can quickly, effectively, and safely transport drugs in humans. Specifically, these findings could have applications for stroke and heart attack victims where the causal factor is a blood clot. (Blood clots are the main causes of heart attacks and strokes.) By discovering mechanisms that can effectively and safely transport drugs that can inhibit or destroy blood clots, scientists will have more effective means to prevent and treat diseases like heart attack and stroke.
The related publicatons can be found at:
The alpha2beta1 integrin-specific antagonist rhodocetin is a cruciform, heterotetrameric molecule.
Eble JA, Niland S, Bracht T, Mormann M, Peter-Katalinic J, Pohlentz G, Stetefeld J.
FASEB J. 2009 Sep;23(9):2917-27. Epub 2009 Apr 15.
PMID: 19369383 [PubMed - indexed for MEDLINE]
Monoclonal antibodies reveal the alteration of the rhodocetin structure upon alpha2beta1 integrin binding.
Bracht T, Figueiredo de Rezende F, Stetefeld J, Sorokin LM, Eble JA.
Biochem J. 2011 Jul 21. [Epub ahead of print]
PMID: 21774787 [PubMed - as supplied by publisher]
Canada Research Chair in Structural Biology,
Department of Chemistry, and
Department of Microbiology
Malaysian Pit Viper
Stetefeld with student Gregory Cox