Reverse chemical ecology: the lost love messages of mammoths

Most animal species, from insects to mammals, communicate through chemical messages. These compounds are called pheromones and carry information about sex, dominance, danger, territory and social ranks. Being generally volatile, they remain in the environment only the short time necessary for the message to be communicated around. It is therefore impossible to analyse pheromones of extinct species, and understand their chemical ecology, that is their relationships with the environment. But, there is a back door to approach this problem, through what we call reverse chemical ecology because it starts from bottom up targeting the proteins that used to bind and carry pheromones. Unlike chemical compounds, that have disappeared with the extinction of the species, we still keep memory of proteins, encoded in the sequences of DNA. The genomes of several extinct species have been sequenced and now we can re-synthesise their proteins and study their structures. This project aims at investigating the structures of pheromone-binding proteins of the mammoth and obtain a detailed picture of their inner pockets, where, as in a jewel moulded case, the pheromone molecules would have been snugly accommodated. Common techniques of molecular biology allow us to make a protein without using biological samples, based only on the knowledge of its encoding gene. We plan to crystallize the mammoth OBP and use X-ray to get the first structure from an extinct animal. Then, following a trial-and-error approach, we shall challenge the protein with the Asian elephant pheromones and structurally related chemicals to eventually find the structure best fitting the binding cavity. This is the first time that such approach is applied to an extinct species, and suffer from the limitation that we shall never be absolutely certain that the structure proposed was indeed the pheromone used by the mammoth. However, we can get an idea on the reliability of our results by performing the same series of experiments on the African elephant, whose pheromones have not yet been discovered, but can be in the future identified starting from biological samples. The choice of the mammoth relies on its close similarity, specifically for the protein sequences of interest, with both elephant species, making this project grounded on solid information and giving confidence to the expected results. This project represents a proof of concept to verify the approach of reverse chemical ecology to extinct species, using a relatively simple model, such as the mammoth. The results will show to what extent the same method can be applied to other extinct species, thus opening a window on the past and enriching our knowledge on the relationships of extinct animals with the environment.

FWF Project P32472 Reverse chemical ecology: the lost love messages of mammoths Public Relations Summary Reverse Chemical Ecology is a relatively new approach to discover pheromones and semiochemicals which mediate communication in animals. Rather than performing chemical analysis of the volatiles produced in specific glands and released in the environment, this approach provides a backdoor from the study of their binding proteins. It is like reconstructing the shape of a foot from the footprint left on the ground by a prehistorical animal. This strategy is particularly useful when chemical analysis of natural pheromones is not feasible, as with extinct species, or when collection of biological samples proves problematic, as with species endangered or difficult to approach. In this project, the strategy of Reverse Chemical Ecology has been applied to answer two different questions. The first regards the hypothetical structure of a human pheromone within a different evolutionary scenario in which humans would use pheromones to communicate with each other. To this aim, we looked at the pheromone-binding proteins in two species of primates, a lemur, known to use pheromonal communication, and a monkey in which pheromones might exist, and compared their properties with a similar protein in humans, which is encoded by a gene silenced before the appearance of our species. The three proteins were very similar in their specific affinity to some large molecules incidentally reported as pheromones of other mammals. We could then conclude that in case humans had retained a sort of chemical communication, their pheromones would have been very similar to those of lemurs and monkeys. The second question was aimed at formulating hypotheses on the mammoth pheromones. Again, we compared the performance of a binding protein encoded in the genome of the mammoth with its orthologue in two species of elephants. We found that this protein was highly conserved from mammoth to elephants with more than 95% of identical amino acids. Moreover, it specifically bound the elephant pheromone (Z)-7-dodecenyl acetate and the semiochemical (E)--farnesene, which were also the best ligands for the elephants' proteins. Therefore, also in this case, we concluded that chemical communication has not changed significantly during evolution from mammoths to elephants. The approach described and tested in this project can be of much wider use and can be easily adapted to the discovery of ligands for any type of binding proteins, receptors and even enzymes. With the wide genomic information currently available, it might be easier in some cases to express the binding proteins and study affinities to a series of chemicals rather than analysing biological samples (often difficult to obtain) in search for a specific ligand or substrate.