Abstract 2035 A role for tubular lipid-binding proteins in insect olfaction

A key biochemical issue for olfaction in terrestrial organisms is the hydrophobicity of volatile substances. Olfactory receptor neurons are embedded in an aqueous medium, so for odorants to reach their receptors and to exit from the receptor after signaling, there must be a system of hydrophobic ligand transport. Insect olfactory neurons are encased in sensilla: porous, fluid-filled hair-like projections from the antenna and other chemosensory tissue. Odorant-binding proteins (OBPs) were found to be secreted into the sensillar fluid, and for many years OBPs were assumed to be the needed hydrophobic ligand transporters of odorants. However, recent knock-out experiments raised questions about OBP functions and have re-opened the question of how volatiles are transported from the air to the receptors. We analyzed the proteomes of a variety of insect and tick chemosensory tissues, and in every case we noticed the expression of extracellular tubular lipid-binding proteins (TULIPs). Various forms of these proteins are known to carry out a wide range of intracellular and extracellular functions, including transport of lipids between organelle membranes, transport of lipids between vertebrate serum lipoproteins, transport of juvenile hormone in insect hemolymph, and possible involvement in innate immunity. TULIPs have been overlooked in insect olfaction research, partly because of their high sequence diversity, which impedes identification by sequence searching; and partly because their genes are frequently mis-annotated in databases. In a published antennal transcriptome from Drosophila melanogaster, we found 24 different TULIPs expressed. For a subset of these antennal TULIPs, we used AlphaFold2 models in CASTp, to identify amino acids in the ligand-binding pocket, and MSMS to identify exterior amino acids. The ratio of amino acid substitution frequency in the pocket to the exterior was much higher in the TULIPs than in isoforms of the lipid-binding enzyme ceramide synthase. This higher variability suggests that the antennal TULIPs may bind a comparatively wide range of different ligands. We examined the expression of CG2016, a D. melanogaster antennal TULIP available from the Bloomington Drosophila Stock Center as a Gal4/UAS construct expressing yellow fluorescent protein (YFP). We observed strong YFP fluorescence from the antenna and also the maxillary palps (chemosensory appendages near the mouth). Spinning disc confocal images show that the cells expressing CG2016 are adjacent to the olfactory receptor neurons in basiconic sensilla (which detect general environmental and food odors). These support cells are known to be active in secretion of proteins into the sensillar fluid. Our results are consistent with a role for TULIPs in arthropod olfaction, possibly either in delivery of odorants to the olfactory receptor neurons, or in clearing odorants from the neurons after signaling. This research was supported, in part, by grants from NIH (R21AI151644 ) and USDA (2023-67022-39753 ), and by a gift from the Zas Fund.