Reverse chemical ecology: Olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles

Abstract

The giant panda Ailuropoda melanoleuca belongs to the family of Ursidae; however, it is not carnivorous, feeding almost exclusively on bamboo. Being equipped with a typical carnivorous digestive apparatus, the giant panda cannot get enough energy for an active life and spends most of its time digesting food or sleeping. Feeding and mating are both regulated by odors and pheromones; therefore, a better knowledge of olfaction at the molecular level can help in designing strategies for the conservation of this species. In this context, we have identified the odorant-binding protein (OBP) repertoire of the giant panda and mapped the protein expression in nasal mucus and saliva through proteomics. Four OBPs have been identified in nasal mucus, while the other two were not detected in the samples examined. In particular, AimelOBP3 is similar to a subset of OBPs reported as pheromone carriers in the urine of rodents, saliva of the boar, and seminal fluid of the rabbit. We expressed this protein, mapped its binding specificity, and determined its crystal structure. Structural data guided the design and preparation of three protein mutants bearing single-amino acid replacements in the ligand-binding pocket, for which the corresponding binding affinity spectra were measured. We also expressed AimelOBP5, which is markedly different from AimelOBP3 and complementary in its binding spectrum. By comparing our binding data with the structures of bamboo volatiles and those of typical mammalian pheromones, we formulate hypotheses on which may be the most relevant semiochemicals for the giant panda.odorant-binding proteins | chemical communication | X-ray structure | proteomics | giant panda T he giant panda Ailuropoda melanoleuca is endemic of China and was formerly classified as an endangered species, now as a vulnerable species, but its population has remained rather stable, although very low, during the last centuries (1). Its phylogenetic classification has been a matter of debate for some time, but molecular genetic studies have recently shown that this species belongs to Ursidae, of which it represents an ancestral branch together with the spectacled bears, Tremarctos, and the sloth bear (1-3). The diets of these species are different from those of carnivorous bears: the giant panda is fully herbivorous, the spectacled bears are mainly herbivorous, and sloth bears feed on termites, fruits, and vegetables. The giant panda also shares with spectacled bears and sloth bears the absence of hibernation, an important characteristic that differentiates these species from other Ursidae (4).The obligate bamboo diet of the giant panda, which is not compatible with its carnivorous digestive system, is barely sufficient to provide the energy required for an active life, likely accounting for the slow movements and long periods of rest typical of this species (5). It has been also suggested that the reduced size of the brain, liver, and kidneys of the giant panda relative to other mammals could be a measure to further reduce the use of i...

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“…Competitive binding was measured by titrating a solution of protein and 1‐NPN with 1 mM ligand in methanol. Dissociation constants for 1‐NPN were calculated using Graph Pad Prism (Zhu et al, ). Comparative dissociation constants between olfactory proteins and ligands were calculated by the equation K d = IC50/(1 + [1‐NPN]/ K 1‐NPN ), where IC50 is the concentration of ligands halving the initial fluorescence value of 1‐NPN, [1‐NPN] is the free concentration of 1‐NPN, and K 1‐NPN is the dissociation constant of the protein/1‐NPN complex.…”

“…Dissociation constants for 1-NPN were calculated using Graph Pad Prism (Zhu et al, 2017). Comparative dissociation constants between olfactory proteins and ligands were calculated by the equation…”

From Gas Sensors to Biomimetic Artificial Noses

“…Replacing two or three amino acid residues in the binding pocket of the protein was enough to drastically reduce affinities to long-chain aldehydes and shift the specificity of GOBP2 to terpenoids [84]. In a similar work, a single amino acid mutation in the OBP3 of the giant panda, which binds long-chain aldehydes and several plant-derived terpenoids, narrowed the specificity of this protein to the second class of compounds [77]. Based on the wide and detailed structural information on OBPs and on the role of single amino acid residues, as learned from studies of site-directed mutagenesis, we can easily conceive the possibility of designing completely new proteins tuned to desired ligands.…”

“…Proteins of both classes present a binding cavity where molecules of odorants and pheromones can be accommodated with relatively good affinity (dissociation constants in the micromolar order). Their selectivity to hydrophobic ligands is not as high as known for olfactory receptors, but fine discrimination has been reported in several cases, such as the two enantiomers of carvone bound with different affinities by the pig OBP [76] or the panda OBP5 able to distinguish between stearic, oleic and linoleic acid, as well as isomers of oleic acid differing for the position of the double bond [77].…”

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“…Environmental risks and risks for human and animal health, as well as the development of resistance of many pest species lead to important challenges in the coming decade to discover new physiological targets for pest control. Among emerging ideas for alternative pest management, reverse chemical ecology proposes new specific targets in form of olfactory proteins for pest control or conservation biology (Zhu et al, 2017;Choo et al, 2018). Another emerging target for pest control is to manipulate their digestive enzymes (Zibae, 2012).…”