Mounting evidence exists for proteins being central to pheromone transport. However, an unresolved question in mammalian olfaction is how these biologically relevant odorants traverse perireceptive olfactory mucous environments to interact with cognate odorant receptors? The extensive dual olfactory system of the macrosmatic Asian elephant (Elephas maximus) offers novel and valid investigative advantages to resolve the source-to-sink molecular trail of two identified pheromones: Z-7-dodecenyl acetate (Z7-12:Ac) and frontalin. The recent discovery of two natural enantiomeric forms of frontalin required in a specific ratio for biological activity is a significant mammalian finding, providing an exquisitely precise chemical model. Our productive international collaboration has described the functional roles of an albumin transporter and an odorant-binding protein sequestrant prior to the final step when the pheromone transits through the long vomeronasal duct before contacting vomeronasal organ (VNO) receptors. Preliminary results support the hypothesis of passage facilitation by a mucous protein.
Three research questions are explored: (1) Is there a particular protein in VNO ductal mucus that specifically binds Z7-12:Ac and frontalin enantiomers, consequently transporting them? The study will employ plentiful synthetic pheromones and innovative radiolabelled and photoaffinity visualization analogs. (2) What is the identity of this vomeronasal ductal protein (VDP) and does its recombinant form retain bioactivity? After the protein has been isolated and identified and its gene cloned and sequenced, sufficient amounts will be expressed recombinantly. (3) Does the VDP with its ligand have appropriate biochemical properties (e.g., binding kinetics) and bioactive attributes to be the VNO ductal transporter?
In elephants, spatial and temporal slowing and separation of individual biochemical events allows entire sequences of perireceptive events to be discerned as discrete steps. In vitro kinetic analyses of VDP-pheromone complexes will be conducted in parallel with in vivo monitoring of discrete-in-time chemosensory responses and behaviors.
This study is multidisciplinary and integrates diverse scientific disciplines, from molecular biology and proteomics through chemosensory bioassays and behavior, integrating the common theme of biochemistry, providing a unique opportunity to quantify responses to pheromones, and elucidating specific and functional protein-ligand associations and transport phenomena.
Investigations by the international team of Drs. Greenwood and Rasmussen will continue the dual tradition of broadly impacting science and practically helping conservation. Advances in knowledge of olfaction in the endangered Asian elephant have proximate utility in practical deterrent development, thereby reducing human-elephant conflict. Ultimately, how binding proteins interact with pheromones at the level of chirality will contribute fundamental insights into vertebrate olfaction. In the broadest sense, this research will benefit human society by helping to preserve an extraordinary species that has interacted with man for thousands of years.
