BIOLOGICAL OVERVIEW

The lipocalin superfamily constitutes a phylogenetically conserved group of more than 40 proteins that function in the binding and transport of a variety of physiologically important ligands. Members of this family carry out diverse functions as carriers of retinoids (retinol binding protein), odorants (odorant binding proteins or OBPs), chromophores (insecticyanin, INS), pheromones (aphrodisin) and sterols (apolipoprotein D, apoD). OBPs are produced by vertebrate and arthropod chemosensory systems where they are secreted from nonneuronal support cells into the fluid that bathes the olfactory neuron dendrites. Odorants have been shown to bind directly to these proteins in both mammals and insects. In Drosophila, the six previously identified invertebrate OBP members have surprisingly low sequence similarity and are expressed in different, overlapping zones of chemosensory sensilla. No mutants defective for any odorant-binding protein gene have been previously described; therefore the in vivo function of these proteins is unknown. Possible functions include solubilizing or concentrating odorants in the sensillum lymph, or mediating odorant removal. Drosophila Lush is a new member of the invertebrate odorant-binding protein family. lush mutant flies, receiving their name because of their abnormal attraction to high concentrations of ethanol, propanol, and butanol, have normal chemosensory responses to other odorants. Lush protein is expressed in a subset of antennal sensilla and is required for normal olfactory behavioral responses to a small subset of chemically related odorants (Kim, 1998).

Chemosensory hairs or sensilla are located primarily on the third antennal segment (Stocker, 1994). The sensilla are hollow, fluid-filled structures encasing the olfactory neuron dendrites from one to four olfactory neurons; they therefore provide for the anatomical segregation of olfactory neurons. In Drosophila, these sensilla fall into three distinct morphological classes: basiconic, coeloconic, and trichoid (Stocker, 1994 and Riesgo-Escovar, 1997). All three classes are thought to mediate olfactory responses. Odor molecules initially pass through pores or grooves within the cuticle of the sensilla, whereupon they enter the sensillum lymph that bathe the olfactory neuron dendrites. Extracellular recordings of the odor-induced electrical activity from different regions of the Drosophila antenna reveal that different regions have differential sensitivity to specific odorants. However, the molecular mechanisms that confer odor specificity to olfactory neurons in insects are not well understood (Kim, 1998 and references).

In both vertebrates and insects, primary olfactory neurons activated by odorants make their first synapses in the central nervous system where odorant information is processed in complex neural networks called glomeruli. Olfactory information is subsequently delivered to higher brain centers, and ultimately perceived as odor. One destination for this olfactory information is the mushroom bodies, thought to be involved in consolidation of olfactory memory (Davis, 1995). Odorant perception can dramatically influence animal behaviors ranging from attraction to food sources and avoidance of noxious compounds to mediation of reproductive cues (Kim, 1998 and references).

In Drosophila, each of the approximately 2000 antennal olfactory neurons project their axons directly to the bilateral antennal lobes (the Drosophila equivalent of mammalian olfactory bulbs). Each neuron synapses exclusively to one of the 35 glomeruli, either ipsilaterally (on the same side) or bilaterally. Antennal lobe output is routed to higher brain structures, including the mushroom bodies where memory is thought to be consolidated. Different odorants produce different patterns of glomerular activation in Drosophila antennal lobes. Radioactively labelled 2-deoxyglucose autoradiography, a sensitive measure of cellular activity, has revealed foci of cellular activity in the antennal lobe during olfactory stimulation. Labelling in the receptor axons and the lobe interneurons can be observed. Each class of odors stimulates activity in a specific subset of antennal glomeruli. This labelling defines the activity domain, which is recognizably distinct, although overlapping, for different classes of volatile chemicals. The labelling pattern is different for attractant and repellent odors. Stimulation with attractants on one side of the fly results in excitation of the glomeruli on the same side. Collaterals of the receptor neurons to the contralateral (other side) lobe stimulate no visible postsynaptic activity. In contrast, stimulation on one side with repellent odors, such as benzaldehyde, results in equally strong levels of activity in both antennal lobes. There is likely to be the result of a correlation between the odorant specificity of the olfactory neurons and the pattern of glomerular activity in the Drosophila olfactory system (Rodrigues, 1988). A similar coding for olfactory information has been found in vertebrates (Cinelli, 1995).

Lush was identified using the enhancer detection approach as a gene expressed exclusively in the olfactory organs. Single P-transposable elements (P elements) modified to express a tau-LacZ fusion reporter gene were randomly inserted in the genome. Expression of the reporter fusion gene is dependent on enhancer elements near to the site of insertion and can mimic the temporal and spatial expression pattern of individual genes located at or near the integration site. Because the LacZ gene is fused in frame to the gene encoding the microtubule-associated protein tau, expression of the reporter gene in neurons results in LacZ staining of axonal projections when expressed in these cells. Several thousand lines of flies were generated carrying stable, independent P-element insertions. Members of each line were screened for reporter gene expression restricted to the chemosensory structures. One of several lines with adult LacZ expression restricted to a subset of chemosensory sensilla on the third antennal segment turned out to be mutant for lush. In this line LacZ expression was prominent in cells associated with trichoid sensilla on the ventral-lateral surface of the third antennal segment. Based on their relative position in the epithelium, the support cells expressing LacZ in this one line were identified as trichogen support cells, known to secrete the sensillum lymph that bathes the olfactory neuron dendrites (Kim, 1998).

To compare olfactory discrimination between lush and wild-type adults, an olfactory trap assay was used. Briefly, 10 wild-type or mutant flies were placed in a petri plate with a single odorant trap, and the number of flies within the trap was determined after a set time period. A panel of 60 simple volatile organic compounds was screened at different concentrations to test for differences in distribution between control and lush flies. The majority of the compounds attract similar proportions of wild-type and lush flies, indicating there is no global olfactory defect associated with the deletion. However, odor-specific defects in chemosensory behavior are observed in lush flies when challenged with three chemically related odors. A significant increase in the number of mutant flies compared to control flies is observed in traps containing high concentrations of ethanol, propanol, and butanol. Responses of mutant flies to a variety of other alcohols are not different from those of wild type. Interestingly, the apparent increased attraction of lush flies for ethanol, propanol, and butanol is specific to high-odorant concentrations. The extent of the attraction of lush flies to yeast extract, ethyl acetate, and low concentrations of ethanol is similar to that of wild-type. However, the mutant flies display an abnormal attraction to traps containing high concentrations of ethanol. It is concluded that lush flies have odor-specific defects in chemosensory discrimination and are abnormally attracted to high concentrations of a subset of odorants including ethanol, propanol, and butanol (Kim, 1998).

The increased likelihood of lush mutant flies entering traps containing high concentrations of these alcohols could result from either increased attraction to these odorants or a defect in avoidance of high concentrations of these compounds. If there is a defect in chemoavoidance to ethanol in lush mutants, it should be possible to demonstrate this behavioral response in wild-type flies. To determine if wild-type flies have endogenous mechanisms to avoid high-ethanol environments (mechanisms thought to be defective in the lush mutants), the effects of mixing ethanol with yeast extract, a strong chemoattractant, was tested. Wild-type flies are attracted to dilute yeast extract. However, when the same amount of yeast is mixed with concentrated ethanol, wild-type flies are significantly less likely to enter these traps. Therefore, the presence of high levels of ethanol reduces the attraction to yeast in wild-type flies. This demonstrates that there is an active avoidance mechanism in wild-type flies that is stimulated by high concentrations of ethanol. lush mutants are equally attracted to yeast, when compared to wild-type flies but are defective for the avoidance behavioral response. In fact the lush mutants are significantly more attracted to the mixture of yeast and concentrated ethanol than to yeast alone (Kim, 1998).

How does a protein secreted into the fluid that bathes olfactory neuron dendrites (but is not synthesized by olfactory neurons) affect chemosensory behavior? The most likely possibility is that Lush is required to activate a small subset of olfactory neurons in the trichoid sensilla that specifically mediate chemoavoidance. For example, Lush might concentrate or prevent the rapid metabolism of these alcohols in the sensillum lymph thus increasing the steady-state concentration of these odorants in the trichoid sensillum lymph of wild-type flies. This could trigger activation of olfactory neurons mediating avoidance and altering the perception of ethanol so it "smells bad." If true, this model predicts that these Lush-dependent olfactory neurons would not be activated by ethanol in lush mutants, but would be activated in wild-type flies. However, no significant differences were seen in recorded electroantennograms (EAG), again perhaps because these neurons are not detectable. Alternatively, Lush may affect olfactory behavior by regulating processes that occur on a slower time scale apparent in chemosensory behavior assays, but not EAG recordings. Additional experiments will be required to identify the exact biochemical function of the Lush protein and the behavioral specificity of the chemosensory neurons within the trichoid sensilla (Kim, 1998).

Each of the seven members of the Drosophila odorant-binding protein family are expressed in specific zones on the surface of the antenna. Therefore, there is a topographic map on the surface of the antenna defined by zones of odorant-binding protein expression. Lush is required for normal chemosensory responses to specific odorants. This implies a correlation between the odorant-binding protein expression zone and the odor specificity of olfactory neurons. Previous workers have demonstrated a relationship between odorant sensitivity and position on the surface of the antenna; these zones of sensitivity could correspond to odorant-binding protein expression zones. Cobalt backfilling experiments labeling the projections of the olfactory neurons from the Lush expression zone (the ventral-lateral surface) reveal that these olfactory neurons synapse primarily in only 2 of the 35 anatomically identified glomeruli in the antennal lobe: VA-1 and DA-1 (Stocker, 1983). It will be interesting to determine if one or both of these glomeruli specifically function in chemosensory avoidance, and if neurons associated with other odorant-binding protein zones project to common subsets of glomeruli. The lush expression zone overlaps several other Drosophila odorant-binding proteins, specifically PB-PRP-1 and PB-PRP-3/OS-F (McKenna, 1994 and Pikielny, 1994). When mutants defective for these gene products become available, it will be important to determine if they have defective avoidance responses, but to a different subset of odorants. Similarly, it is predicted mutations in OBPs expressed in the other classes of sensilla will have defective attraction to a subset of odorants (Kim, 1998).

It is suggested that chemical specificity of olfactory neurons in Drosophila results from a combination of interaction of odorants with odorant-binding proteins in the sensillum lymph and the specificity of receptor proteins present on the olfactory neurons. A diverse family of odorant-binding proteins could enable a relatively small family of neuronal receptors to respond differentially to a broad range of compounds through a combinatorial mechanism. For example, two neurons in different sensilla expressing the same broadly tuned neuronal receptor might be activated by different subsets of odorants if each sensillum expressed a different repertoire of binding proteins. Elucidation of the relative size of the receptor and binding protein families and determination of their spatial expression relationships will provide further insight into the question of insect chemoreception. Finally, it should be noted that the alcohols that induce abnormal chemosensory reponses in lush mutants are biologically relevant odorants for Drosophila melanogaster. In nature, fruit flies feed and deposit eggs on fermenting plant materials in which ethanol is the most abundant short-chain alcohol. The ability to detect ethanol is important for chemotaxis toward food sources. However, adult flies are also susceptible to intoxication and death in high ethanol environments. Therefore, there is a selective advantage for the ability to avoid environments with dangerously high alcohol concentrations, and Lush is required for this response. The behavioral response to alcohol, therefore, reflects a finely tuned olfactory response (Kim, 1998 and references).

PROTEIN STRUCTURE

Amino Acids - 153

Structural Domains

Database comparison with previously identified proteins reveal significant homology (24% overall identity) with OS-F/PB-PRP3 (McKenna, 1994 and Pikielny, 1994), a Drosophila member of the invertebrate odorant-binding protein family. Lush shares all features of this protein family including a signal sequence to direct polypeptides to the secretory pathway, a chemosensory-specific expression pattern, and six conserved cysteine residues with the spacing between cysteines 2 and 3 and 5 and 6 completely conserved in all members (Kim, 1998).

The high-resolution crystal structures of the Drosophila alcohol-binding protein LUSH has been solved in complex with a series of short-chain n-alcohols. LUSH is the first known nonenzyme protein with a defined in vivo alcohol-binding function. The structure of LUSH reveals a set of molecular interactions that define a specific alcohol-binding site. A group of amino acids, Thr57, Ser52 and Thr48, form a network of concerted hydrogen bonds between the protein and the alcohol that provides a structural motif to increase alcohol-binding affinity at this site. This motif seems to be conserved in a number of mammalian ligand-gated ion channels that are directly implicated in the pharmacological effects of alcohol. Further, these sequences are found in regions of ion channels that are known to confer alcohol sensitivity. It is suggested that the alcohol-binding site in LUSH represents a general model for alcohol-binding sites in proteins (Kruse, 2003).

date revised: 22 February 2022

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