The Interactive Fly

Zygotically transcribed genes

Odorant-binding proteins

• Lush
• Obp19b
• Obp28a
• Obp49a
• Obp56h
• Obp69a
• Obp83ef

A large family of divergent Drosophila odorant-binding proteins expressed in gustatory and olfactory sensilla

A large family of putative odorant-binding protein (OBP) genes has been identified in the genome of Drosophila. Some of these genes are present in large clusters in the genome. Most members are expressed in various taste organs, including gustatory sensilla in the labellum, the pharyngeal labral sense organ, dorsal and ventral cibarial organs, as well as taste bristles located on the wings and tarsi. Some of the gustatory OBPs are expressed exclusively in taste organs, but most are expressed in both olfactory and gustatory sensilla. Multiple binding proteins can be coexpressed in the same gustatory sensillum. Cells in the tarsi that express OBPs are required for normal chemosensation mediated through the leg, since ablation of these cells dramatically reduces the sensitivity of the proboscis extension reflex to sucrose. OBP genes expressed in the pharyngeal taste sensilla are still expressed in the poxneuro genetic background, while OBPs expressed in the labellum are not. These findings support a broad role for members of the OBP family in gustation and olfaction and suggest that poxneuro is required for cell fate determination of labellar but not pharyngeal taste organs (Galindo, 2001).

Seven members of the Drosophila OBP family have been previously identified. With the exception of PBP2, which is secreted by epithelial cells, each is expressed in the sensillum lymph of a subset of olfactory sensilla. How large is the OBP family, and is OBP function restricted to olfactory sensilla? To address these issues, attempts were made to identify all OBP genes encoded in the Drosophila genome. 28 new candidate OBP genes were identified in the Drosophila genome project, making a total of 35 members of this gene family present in the genome of this organism. Each putative member was identified with the tBLASTn algorithm using the previously identified members of the Drosophila OBP family as probes to identify related genes in the Drosophila genome sequence. Candidate genes were screened for features characteristic of the invertebrate OBP family, including low molecular weight (13-16 kD), a predicted signal sequence, and the presence of six conserved cysteines with the invariant spacing between cysteines 2 and 3 (three residues) and cysteines 5 and 6 (eight residues) that define this family. Putative genes meeting these criteria typically are predicted to contain one or two introns with conserved splicing consensus sequences and consensus translation start sequences. Each putative OBP member is named with a number representing its chromosomal location and a letter that designates its order relative to other OBP genes at that position. It is worth noting that 35 OBP genes is a minimum number, since this analysis would miss genes with introns located between conserved cysteine motifs. It is possible, therefore, that additional OBP genes may be encoded in the Drosophila genome that were not identified here (Galindo, 2001).

The genes encoding the OBP family in Drosophila are not randomly distributed in the genome and are often found in large clusters. For example, 14 OBP genes are clustered within 825 kb located at position 56-57 on the second chromosome. A second cluster of 7 genes is located at position 83 on the third chromosome. The localization of so many members of the OBP family in a relatively discrete region of the genome suggests that these genes may have arisen by tandem duplication. Tandem duplication has been suggested to account for the two closely related genes, OBPs 83a and 83b. Several of the OBP genes are tightly linked. For example, OBP56a and OBP56b are located within 2 kb of each other, but are transcribed in opposite directions. Despite this close proximity, these genes appear to have strikingly different expression patterns. This situation is similar to that observed for clustered putative odorant receptor genes, where closely linked, chemosensory-specific genes are expressed in different olfactory organs (Galindo, 2001).

The 35 members of the Drosophila OBP family range from 9.2% to 62.6% amino acid identity, demonstrating the highly divergent nature of this protein family. Indeed this protein family is among the most diverse described. Most frequently there is a single splice immediately downstream from the DNA encoding the signal sequence. There is little or no amino acid sequence conservation among the OBP members at splice junctions. Several of the OBP genes have unique splicing patterns. Three OBP genes have no splices within the coding sequence (OBP28a, 69a, and 83d). This surprising lack of splicing conservation and low sequence similarity suggests that if gene duplication is the mechanism responsible for the large number of these genes, they evolve rapidly. All members are predicted to encode proteins with six alpha-helical domains joined by loops that vary in length. Only the six cysteines are completely conserved among all 35 members. The limited sequence similarity among members is often clustered near the cysteines. For example, all members have a hydrophobic amino acid following cysteines two, three, and six. Overall, however, there is little homology in this OBP family. This diversity is consistent with the notion that different OBPs interact with distinct sets of chemical ligands (Galindo, 2001).

To determine the spatial and temporal expression pattern of each putative OBP gene, several kilobases of upstream regulatory sequence for each OBP gene were fused to a reporter gene encoding a nuclear-localized ß-galactosidase. Transgenic flies were generated that are expected to express the reporter gene in the same cells that normally express that particular OBP. The advantages of this approach over in situ hybridization analysis are that it can detect expression in tissues not amenable to in situ hybridization (like the wing and legs) and it has little background and excellent sensitivity. Similar fusions have been shown to precisely reproduce wild-type gene expression (Galindo, 2001).

Transgenic flies carrying reporter constructs fused to each OBP promoter were generated and the flies were stained for ß-galactosidase activity. Surprisingly, in addition to the expected olfactory expression, a wide variety of gustatory organs were labeled in many lines. On the basis of reporter gene expression, the members of the OBP family can be classified in one of five classes. Class 1 is composed of putative OBP genes (nine members) expressed exclusively in subsets of chemosensory sensilla. Class 2 genes (four members) are expressed exclusively in gustatory sensilla. Class 3 genes (nine members) are expressed in subsets of olfactory and gustatory sensilla. Class 4 (five members) OBP promoters drive LacZ expression in broad areas that include regions that do not contain chemosensory organs. These genes may be functionally related to PB-PRP2, a gene expressed by epithelial cells that may function as a scavenger protein. Class 5 (seven members) includes OBP genes in which no LacZ expression was detectable. These genes may be pseudogenes, or the promoter fragments used may lack essential regulatory elements required for expression. One putative OBP gene, OBP83e, was not analyzed because no appropriate initiation methionine could be identified. This gene may have a large 5' intron or may be a pseudogene (Galindo, 2001).

Surprisingly, only three new class 1 OBP genes were identified -- OBP19a, 57a, and 99a. OBP19a is expressed exclusively in a subset of chemosensory sensilla on the third antennal segment. For OBP19a, a bilaterally symmetric pattern of LacZ staining is visible in the third antennal segment, but not the maxillary palps or larval chemosensory organs. This expression pattern is consistent with previously reported members of the OBP family that are also expressed in subsets of olfactory sensilla. OBP57a and OBP99b are expressed in subsets of sensilla in both olfactory organs, the maxillary palps, and third antennal segments (Galindo, 2001).

Class 3 genes are expressed in both olfactory and gustatory organs. Nine members of this class were identified. For members of this class, LacZ expression is primarily restricted to the olfactory organs, but the genes are also expressed in at least one gustatory organ. Transgenic flies expressing LacZ under control of OBP56d and OBP57c are expressed in all olfactory sensilla, including all sensilla on the antenna and maxillary palp. The expression of these OBPs in the antenna is unique because all other previously reported members are expressed in subsets of sensilla. These lines are not identical, however, since OBP56d is also expressed in the wing and tarsal gustatory sensilla and dorsal organ, and OBP56c is expressed in the wing and the larval olfactory organ, the dorsal organ (Galindo, 2001).

A gustatory-specific, class 2 OBP, OBP19c, is expressed exclusively in six cells, including two cells in the labral sense organ (LSO). The LSO consists of nine sensilla that sample the lumen of the pharynx just distal to the oral opening. Three of the LSO sensilla contain chemosensory neurons; the other six are purely mechanosensory. OBP19c is expressed strongly in a single pair of bilaterally symmetric cells in the LSO and weakly in a single cell in each ventral cibarial sense organ (VCSO) and dorsal cibarial sense organ (DCSO) in the adult. The larval dorsal organ also stains for LacZ in these transgenic flies (Galindo, 2001).

OBP56b is also expressed exclusively in the pharyngeal gustatory organs, including two cells in the LSO and two cells in the DCSO. This expression pattern is very similar to OBP19c in the LSO. These expression patterns are consistent with expression of these OBPs in support cells of gustatory sensilla, although these cells cannot be identified precisely. In Drosophila larvae, volatile odorants are detected by neurons that reside in the dorsal organ, while gustatory responses appear to be mediated by neurons in the terminal organ, in chemosensory neurons located in the ventral pits present on each thoracic hemisegment, and in some of the neurons in the dorsal organ. Both the terminal organ and dorsal organs express LacZ in OBP56b-nlsLacZ transgenic flies (Galindo, 2001).

OBP56g is expressed exclusively in the labellum in the adult. The two outer rows of gustatory bristles are LacZ positive. No other chemosensory organs are labeled in these transgenic flies. The promoter for OBP56h expresses LacZ in approximately five sensilla on each third antennal segment, in the pharyngeal organs and in the dorsal organ, the terminal organ, and the ventral pits of the third instar larvae. This OBP, therefore, may function in both olfactory and gustatory systems (Galindo, 2001).

With the exception of the vaginal plate chemosensory sensilla, members of the OBP family in all chemosensory organs have been identified. Is only one OBP expressed in each gustatory sensillum or can multiple OBPs be coexpressed? Several transgenic lines driving LacZ with different OBP promoters appear to have overlapping expression patterns (Galindo, 2001).

The promoters for OBP57d and 57e drive LacZ expression exclusively in four cells of the tarsi associated with curved chemosensory bristles on each of the six legs in flies expressing nuclear LacZ by the promoter of OBP57d and 57e, respectively. The expression patterns of OBP57d and 57e are also consistent with expression in support cells associated with gustatory sensilla. Interestingly, these genes are located <1 kb apart from each other at the end of the 56-57 gene cluster, have significant amino acid homology to each other in the C-terminal half, and may represent a relatively recent gene duplication event. Data indicate that OBP57d and 57e are coexpressed in the same cells in the tarsi (Galindo, 2001).

The promoters for three OBP genes, OBP56b, OBP56h, and OBP19c, drive LacZ expression in single pairs of cells in the pharyngeal LSO. Are any of the OBPs expressed in the LSO coexpressed in the same cells? OBP56h or 19c are coexpressed in the same cells and OBP56h is expressed in neighboring cells that do not express OBP56b (Galindo, 2001).

Transgenic flies expressing the pro-apoptotic protein Grim exclusively in the OBP57e-positive cells in the tarsi were used to evaluate the biological importance of these cells in gustation. Wild-type and pOBP57eGal4-VP16;UAS Grim-expressing flies were tested for their ability to detect sucrose applied to the tarsi using the proboscis extension reflex (PER). Normally when sucrose is applied to the tarsi, the fly extends the proboscis to feed. Expressing Grim in the cells expressing OBP57d and 57e results in a dramatic loss of sensitivity to sucrose as determined by this assay. Wild-type control flies have strong PERs to sucrose concentrations as low as 10^-6 M sucrose. However, flies lacking the cells that express OBP57d and 57e have dramatically reduced PERs to sucrose concentrations from 10^-6 to 10^-2 M. Interestingly, at concentrations of 10^-1 M, there is no difference in the probability of eliciting the PER between the two groups. This demonstrates that the cells that normally express OBP57d and 57e are important for normal sensitivity of the tarsi to sucrose, but at high sucrose concentrations the loss of these cells has no effect. Therefore the PER reflex is intact in the Grim-expressing animals, but the sensitivity to sucrose is blunted. These results confirm that the cells expressing OBP57d and 57e are required for normal gustatory sensitivity of the tarsi to sucrose (Galindo, 2001).

poxneuro is a paired domain transcriptional regulator. Mutants defective for poxneuro have an abnormal number of leg segments and conversion of labellar gustatory sensilla to mechanosensory bristle phenotype. RT-PCR experiments reveal that poxneuro mutants fail to express most, but not all, putative gustatory receptors. To determine if poxneuro transforms the support cells that make OBPs, it was determined whether reporter genes regulated by the promoter for OBP56g are still expressed in the poxneuro mutant background (Galindo, 2001).

LacZ expression was examined in labellar sensilla in wild-type flies expressing LacZ under control of the OBP56g promoter. When the P element carrying this construct is crossed into the poxneuro genetic background, LacZ expression is completely absent in the labellum. These results indicate that the cells in labellar sensilla do not express OBP56g in the poxneuro genetic background. This supports the notion that poxneuro acts early in the development of chemosensory sensilla to delegate chemosensory identity on all cells in the sensillum, including the cells that synthesize and secrete OBP56g (Galindo, 2001).

To assess the role of poxneuro in the differentiation of the pharngeal chemosensory organs, flies carrying the OBP56b promoter driving LacZ expression in specific pharyngeal organs were crossed into the poxneuro genetic background. poxneuro does not disrupt expression of LacZ regulated by the pharyngeal OBP promoter OBP56b. Together, these data indicate that poxneuro is required for expression of OBP56g in the labellar gustatory sensilla, but not for OBP56b expressed in pharyngeal gustatory organs. This suggests that different developmental mechanisms are required for the proper specification of pharyngeal and labellar gustatory sensilla (Galindo, 2001).

Two features of this gene family are extraordinary: the low degree of sequence similarity among the family members and the location of so many members in large gene clusters. The diversity of the family based on amino acid homology is striking. Only the six conserved cysteines are conserved in all members, probably reflecting a requirement for proper disulfide bonding for functional tertiary structure. Most of the genes have a single splice junction located after the signal sequence. This may reflect a common ancestor for many of these genes. However, many genes have two splices, and often these occur in novel positions. Other than immunoglobulin gene families that underwent an explosive increase in number and diversity in early jawed vertebrates, the other large gene family that has undergone rapid diversification is another chemosensory-specific gene family, the odorant receptors. Seven transmembrane receptor families mediate chemical detection in vertebrates, Caenorhabditis elegans, and Drosophila. These mechanisms could have arisen independently in the three animal groups or, more likely, evolved from a common ancestor early in the animal lineage and diverged. While there is little or no sequence similarity between the receptor families in these three groups of animals, there are similarities between receptor genes within an organism, indicating that diversity within an organism may arise by gene duplication. The OBP family described here has little similarity among the Drosophila members. The presence of most of these genes in clusters suggests these genes arose by tandem duplication. Almost half of the OBP genes are located within 825 kb of genomic DNA located at chromosomal position 56-57 on the right arm of the second chromosome. This clustering is consistent with a gene duplication mechanism for generating the large size of the gene family. OBP83a and OBP83b are closely related and juxtaposed in the Drosophila genome, but there is only a single related gene present in other Drosophila species. This strongly supports a gene duplication mechanism accounting for the size of this family. However, the low sequence identity among the members suggests that there is rapid evolution of these sequences following duplication. Perhaps the OBP genes are evolving more rapidly than the receptor family. If true, this could reflect constraints on the evolution rate of the receptor family, which must interact with other signaling molecules, or there may be unappreciated positive selective pressure to have a diverse OBP population (Galindo, 2001).

The pheromone-binding protein from the moth B. mori undergoes pH-dependent conformational changes thought to reflect a mechanism for loading and unloading the pheromone. In particular it has been suggested that two adjacent histidines present in several pheromone-binding proteins just before alpha-helix 4 may be important for these conformational changes and, thus, for the function of these proteins (references in Galindo, 2001). If this is true, this feature should be conserved among other members of the family. When the Drosophila proteins are aligned with the B. mori pheromone-binding protein, it is found that these histidines are not conserved in the Drosophila sequences. In fact none of the 35 predicted Drosophila proteins contain this motif, although some putative proteins have histidines in this general vicinity. Either the moth pheromone proteins function differently from the other OBP family members or the conformational changes suggested to be important for loading and unloading ligands do not specifically require these residues (Galindo, 2001).

Recent studies analyzing the expression of members of the odorant receptor and gustatory receptor (GR) families suggest that the difference between smell and taste in Drosophila is not based on the receptor family expressed, but on the location and connections of the neurons that express the receptor. Indeed members of the GR family probably mediate olfactory responses. In olfactory and gustatory sensilla, ligands must enter the sensillum lymph to interact with receptor molecules located on the neuronal dendrites. Therefore, in sensilla that mediate both taste and smell the ligand is in solution. Tastants are generally soluble molecules, but many odorants are hydrophobic with low water:oil solubility ratios. Therefore, the role of the OBPs has previously been thought to be odor specific and possibly associated with the special problem of getting odorants into solution. The presence of OBPs in gustatory sensilla forces a rethinking of this issue. Indeed, lush mutants have defective behavioral responses to ethanol, a molecule that should have little difficulty entering aqueous or hydrophobic environments. Perhaps OBPs function to protect chemicals ligands from enzymatic modification in the sensillum lymph, since a variety of enzymes are expressed in the sensillum lymph, including cytochrome P450 enzymes. Whatever the biochemical role of the olfactory OBPs, it seems likely that this function is important for both olfactory and gustatory physiology (Galindo, 2001).

Comparative genomic analysis of the odorant-binding protein family in 12 Drosophila genomes: purifying selection and birth-and-death evolution

Chemoreception is a widespread mechanism that is involved in critical biologic processes, including individual and social behavior. The insect peripheral olfactory system comprises three major multigene families: the olfactory receptor (Or), the gustatory receptor (Gr), and the odorant-binding protein (OBP) families. Members of the latter family establish the first contact with the odorants, and thus constitute the first step in the chemosensory transduction pathway. Comparative analysis of the OBP family in 12 Drosophila genomes allowed the identification of 595 genes that encode putative functional and nonfunctional members in extant species, with 43 gene gains and 28 gene losses (15 deletions and 13 pseudogenization events). The evolution of this family shows tandem gene duplication events, progressive divergence in DNA and amino acid sequence, and prevalence of pseudogenization events in external branches of the phylogenetic tree. The OBP arrangement in clusters is maintained across the Drosophila species, and purifying selection governs the evolution of the family; nevertheless, OBP genes differ in their functional constraints levels. Finally, the OBP repertoire that was detected evolves more rapidly in the specialist lineages of the Drosophila melanogaster group (D. sechellia and D. erecta) than in their closest generalists. Overall, the evolution of the OBP multigene family is consistent with the birth-and-death model. Members of this family exhibit different functional constraints, which is indicative of some functional divergence, and that they might be involved in some of the specialization processes that occurred through the diversification of the Drosophila genus (Vieira, 2007).

Activation of pheromone-sensitive neurons is mediated by conformational activation of pheromone-binding protein

Detection of volatile odorants by olfactory neurons is thought to result from direct activation of seven-transmembrane odorant receptors by odor molecules. This study shows that detection of the Drosophila pheromone, 11-cis vaccenyl acetate (cVA), is instead mediated by pheromone-induced conformational shifts in the extracellular pheromone-binding protein, Lush. Lush undergoes a pheromone-specific conformational change that triggers the firing of pheromone-sensitive neurons. Amino acid substitutions in Lush that are predicted to reduce or enhance the conformational shift alter sensitivity to cVA as predicted in vivo. One substitution, Lush^D118A, produces a dominant-active Lush protein that stimulates T1 neurons through the neuronal receptor components Or67d and SNMP in the complete absence of pheromone. Structural analysis of Lush^D118A reveals that it closely resembles cVA-bound Lush. Therefore, the pheromone-binding protein is an inactive, extracellular ligand converted by pheromone molecules into an activator of pheromone-sensitive neurons and reveals a distinct paradigm for detection of odorants (Laughlin, 2008).

This study has shown that cVA binds to the pheromone-binding protein Lush and induces conformational changes. Mutations predicted to reduce or enhance the conformational changes also reduce or enhance cVA sensitivity in vivo. One Lush mutant, Lush^D118A, is dominantly active, triggering robust action potentials in T1 neurons in the absence of pheromone. This effect is specific to T1 neurons, as basiconic and other trichoid olfactory neurons are unaffected by this protein. Lush^D118A activates T1 neurons through the putative cVA-activated neuronal receptor components, Or67d and SNMP, accounting for the specificity of the dominant Lush. The data reveal that pheromone molecules are not required for activation of T1 neurons and define a novel olfactory signaling paradigm in which the pheromone-induced conformational change in Lush mediates activation of T1 neurons (Laughlin, 2008).

cVA can trigger weak responses in T1 neurons in the absence of Lush when applied at high concentrations. Direct effects of cVA on Or67d/SNMP receptor complexes may mediate these Lush-independent responses, as these two components confer marginal cVA sensitivity to the empty neuron preparation (Benton, 2007). Alternatively, activated Lush may normally dimerize with an unknown cofactor that alone can weakly activate T1 receptors in the presence of cVA. However, the sensitivity for cVA in the absence of Lush is so poor that lush¹ mutants are blind to the pheromone in aggregation assays. In proximity experiments, cVA levels emanating from single male flies are below detection limits in the absence of Lush. Therefore, the Lush-independent activation of T1 neurons is unlikely to play a role in cVA responses in vivo (Laughlin, 2008).

Olfactory neurons are thought to be tuned to odorants exclusively by the odorant receptors they express. Indeed, in Drosophila melanogaster, activation of many odorant receptors results from direct binding of food odorants. Why does cVA reception require a binding protein intermediate? It is suggested that the binding protein may enhance sensitivity and specificity in the pheromone detection process. If a pheromone induces a stable, ligand-specific conformational change in a binding protein, single pheromone molecules could be detectable if the neuronal receptor complex is specifically tuned to that conformation. Further, if the conformation of the binding protein that activates the receptors is specific to the pheromone-bound state, other environmental stimuli are less likely to activate the neurons, even if they interact with the binding protein. Consistent with this idea, Lush increases the sensitivity of T1 neurons to cVA over 500-fold, but, remarkably, does not sensitize the neurons to structurally similar chemicals, such as vaccenyl alcohol or vaccenic acid. Indeed, Lush can bind a large array of chemicals, but only cVA activates T1 neurons. Other OBPs have been shown to bind to a wide range of unnatural compounds, including plasticizers and dyes, and the electrophysiological or behavioral responses to a specific ligand do not correlate with the binding affinity of the OBP for that ligand. Therefore, binding is clearly not sufficient for sensitization. However, by utilizing a ligand-specific conformational shift in a binding protein, detection of rare pheromone molecules is possible with high fidelity and sensitivity by creating an active binding protein species that diffuses within the sensillum lymph until it contacts and activates a receptor on the dendrites (Laughlin, 2008).

Attempted were made to reconstitute the cVA detection pathway in basiconic neurons lacking endogenous receptors. The CD36 homolog SNMP is expressed in most or all trichoid neurons and is required for sensitivity to cVA (Benton, 2007; Jin, 2008). SNMP colocalizes with the odorant receptor complex in T1 neuron dendrites (Benton, 2007), and antiserum to SNMP infused into the lymph of T1 sensilla phenocopies SNMP loss-of-function mutants, suggesting that SNMP directly mediates pheromone sensitivity (Jin, 2008). Expression of SNMP, Or67d, and Lush together in the empty neuron system failed to recapitulate T1 cVA sensitivity. Or67d alone was unresponsive, but adding Lush through the recording pipette did sensitize Or67d receptors slightly to cVA in the absence of SNMP, suggesting that Lush interacts directly with Or67d. Coexpressing SNMP and Or67d enhanced cVA sensitivity, but, surprisingly, adding Lush failed to further enhance sensitivity. These differences between the empty neuron responses and T1 neurons may reflect reduced levels of one or more components when expressed in basiconic sensilla or, more likely, indicate that additional components are missing. Indeed, in a screen for cVA-insensitive mutants, mutations were recovered in the known sensitivity factors as well as three additional unknown genes encoding factors that are essential for cVA sensitivity. It is expected that, when all of these components are identified and expressed in the basiconic neurons, full cVA sensitivity will be conferred (Laughlin, 2008).

OBPs, like Lush, are a large family of soluble proteins secreted into the lymph fluid surrounding the olfactory neurons. Proposed functions for OBPs include transporting ligands to the ORs, protecting the odor from degradation or deactivation by odorant-degrading enzymes (ODEs), and forming a complex with an odor that either directly activates ORs or binds to other accessory proteins, which ultimately results in OR activation. In vitro studies of the pheromone-binding protein (PBP) from Bombyx mori show that the OBP undergoes a conformational change at low pH that prevents ligand binding, suggesting that OBPs may function primarily as passive carriers and changes in the local pH stimulate pheromone release in the vicinity of the neuronal membrane. Furthermore, previous studies reported that high concentrations of moth pheromones can directly activate cognate pheromone receptors expressed in tissue culture and that DMSO is as effective as the pheromone-binding proteins at sensitizing the neurons to pheromone, leading to the conclusion that the binding proteins are pheromone solubilizers/carriers. However, similar studies implicate the binding proteins as factors in receptor specificity. The current data support the latter view. It is noted that Lush homologs in other insects and the 12 Drosophila species have conserved the amino acids predicted to form the salt bridge. Only Drosophila ananassae (D. ana) is predicted to lack the phenylalanine corresponding to F121 in melanogaster (replaced by leucine). A similar activation mechanism, therefore, is likely to occur in these species. Recent work in rodents reveals that vertebrate pheromones can be peptides or protein. It will be interesting to determine whether the conformational activation mechanism identified for Lush is conserved in analogous extracellular binding proteins in other species (Laughlin, 2008).

An odorant-binding protein required for suppression of sweet taste by bitter chemicals

Animals often must decide whether or not to consume a diet that contains competing attractive and aversive compounds. This study used Drosophila to investigate a mechanism that influences this decision. Addition of bitter compounds to sucrose suppressed feeding behavior, and this inhibition depended on an odorant-binding protein (OBP) termed OBP49a. In wild-type flies, bitter compounds suppress sucrose-induced action potentials, and the inhibition is impaired in Obp49a mutants. OBP49a is expressed in accessory cells and acted non-cell-autonomously to attenuate nerve firings in sugar-activated GRNs when bitter compounds were combined with sucrose. These findings demonstrate an unexpected role for an OBP in taste and identify a molecular player involved in the integration of opposing attractive and aversive gustatory inputs (Jeong, 2013).

Uncovering the functional constraints underlying the genomic organization of the odorant-binding protein genes

Animal olfactory systems have a critical role for the survival and reproduction of individuals. In insects, the odorant-binding proteins (OBPs; see Lush, for example) are encoded by a moderately sized gene family, and mediate the first steps of the olfactory processing. Most OBPs are organized in clusters of a few paralogs, which are conserved over time. Currently, the biological mechanism explaining the close physical proximity among OBPs is not yet established. This study conducted a comprehensive study aiming to gain insights into the mechanisms underlying the OBP genomic organization. A total of 31 conserved clusters were identified in Drosophila that include both OBP and other nonhomologous genes. These 31 clusters are maintained, on average, in 5.9 Drosophila species, comprise a mean of 8.3 genes and, more importantly, the study recover most of the OBP clusters defined in previous studies. The OBP clusters were found to be embedded within large conserved arrangements. These organizations also include other non-OBP genes, which often encode proteins integral to plasma membrane. Moreover, the conservation degree of such large clusters is related to the following: 1) the promoter architecture of the confined genes, 2) a characteristic transcriptional environment, and 3) the chromatin conformation of the chromosomal region. These results suggest that chromatin domains may restrict the location of OBP genes to regions having the appropriate transcriptional environment, leading to the OBP cluster structure. However, the appropriate transcriptional environment for OBP and the other neighbor genes is not dominated by reduced levels of expression noise. Indeed, the stochastic fluctuations in the OBP transcript abundance may have a critical role in the combinatorial nature of the olfactory coding process (Librado, 2013).

Obp56h modulates mating behavior in Drosophila melanogaster

Social interactions in insects are driven by conspecific chemical signals that are detected via olfactory and gustatory neurons. Odorant binding proteins (Obps) transport volatile odorants to chemosensory receptors, but their effects on behaviors remain poorly characterized. This study reports that RNAi knockdown of Obp56h gene expression in Drosophila melanogaster enhances mating behavior by reducing courtship latency. The change in mating behavior that results from inhibition of Obp56h expression is accompanied by significant alterations in cuticular hydrocarbon (CHC) composition, including reduction in 5-tricosene (5-T), an inhibitory sex pheromone produced by males that increases copulation latency during courtship. Whole genome RNA sequencing confirms that expression of Obp56h is virtually abolished in Drosophila heads. Inhibition of Obp56h expression also affects expression of other chemoreception genes, including upregulation of lush in both sexes and Obp83ef in females, and reduction in expression of Obp19b and Or19b in males. In addition, several genes associated with lipid metabolism, which underlies the production of cuticular hydrocarbons, show altered transcript abundances. These data show that modulation of mating behavior through reduction of Obp56h is accompanied by altered cuticular hydrocarbon profiles and implicate 5-T as a possible ligand for Obp56h (Shorter, 2016).

Organization and function of Drosophila odorant binding proteins

Odorant binding proteins (Obps) are remarkable in their number, diversity, and abundance, yet their role in olfactory coding remains unclear. They are widely believed to be required for transporting hydrophobic odorants through an aqueous lymph to odorant receptors. This study constructed a map of the Drosophila antenna, in which the abundant Obps are mapped to olfactory sensilla with defined functions. The results lay a foundation for an incisive analysis of Obp function. The map identifies a sensillum type that contains a single abundant Obp, Obp28a. Surprisingly, deletion of the sole abundant Obp in these sensilla does not reduce the magnitude of their olfactory responses. The results suggest that this Obp is not required for odorant transport and that this sensillum does not require an abundant Obp. The results further suggest a novel role for this Obp in buffering changes in the odor environment, perhaps providing a molecular form of gain control (Larter, 2016).

Odorant binding protein 69a connects social interaction to modulation of social responsiveness in Drosophila

Living in a social environment requires the ability to respond to specific social stimuli and to incorporate information obtained from prior interactions into future ones. One of the mechanisms that facilitates social interaction is pheromone-based communication. In Drosophila melanogaster, the male-specific pheromone cis-vaccenyl acetate (cVA) elicits different responses in male and female flies, and functions to modulate behavior in a context and experience-dependent manner. This study explored the functional link between social interaction and pheromone-based communication and discovered an odorant binding protein that links social interaction to sex specific changes in cVA related responses. Odorant binding protein 69a (Obp69a) is expressed in auxiliary cells and secreted into the olfactory sensilla. Its expression is inversely regulated in male and female flies by social interactions: cVA exposure reduces its levels in male flies and increases its levels in female flies. Increasing or decreasing Obp69a levels by genetic means establishes a functional link between Obp69a levels and the extent of male aggression and female receptivity. Activation of cVA-sensing neurons was shown to be sufficient to regulate Obp69a levels in the absence of cVA, and requires active neurotransmission between the sensory neuron to the second order olfactory neuron. The cross-talk between sensory neurons and non-neuronal auxiliary cells at the olfactory sensilla, represents an additional component in the machinery that promotes behavioral plasticity to the same sensory stimuli in male and female flies (Bentzur, 2018).

Odor-specific deactivation defects in a Drosophila odorant binding protein mutant

Insect odorant binding proteins are a large, diverse group of low molecular weight proteins secreted into the fluid bathing olfactory and gustatory neuron dendrites. The best-characterized OBP, LUSH (OBP76a) enhances pheromone sensitivity enabling detection of physiological levels of the male-specific pheromone, 11-cis vaccenyl acetate. The role of the other OBPs encoded in the Drosophila genome is largely unknown. Using CRISPR-Cas9 this study generated and characterized the loss of function phenotype for two genes encoding homologous OBPs, OS-E (OBP83b) and OS-F (OBP83a). Instead of activation defects, these extracellular proteins are required for normal deactivation of odorant responses to a subset of odorants. Remarkably, odorants detected by the same odorant receptor are differentially affected by the loss of the OBPs, revealing an odorant-specific role in deactivation kinetics. In stark contrast to lush mutants, the OS-E/F mutants have normal activation kinetics to the affected odorants, even at low stimulus concentrations, suggesting these OBPs are not competing for these ligands with the odorant receptors. It was also shown that OS-E and OS-F are functionally redundant as either is sufficient to revert the mutant phenotype in transgenic rescue experiments. These findings expand understanding of the roles of OBPs to include deactivation of odorant responses (Scheuermann, 2019).

The Drosophila odorant-binding protein 28a is involved in the detection of the floral odour ss-ionone

Odorant-binding proteins (OBPs) are small soluble proteins that are thought to transport hydrophobic odorants across the aqueous sensillar lymph to olfactory receptors. A recent study revealed that OBP28a, one of the most abundant Drosophila OBPs, is not required for odorant transport, but acts in buffering rapid odour variation in the odorant environment. To further unravel and decipher its functional role, recombinant OBP28a was expressed, and its binding specificity was characterized. Using a fluorescent binding assay, it was found that OBP28a binds a restricted number of floral-like chemicals, including ss-ionone, with an affinity in the micromolar range. The X-ray crystal structure was solved of OBP28a, which showed extensive conformation changes upon ligand binding. Mutant flies genetically deleted for the OBP28a gene showed altered responses to ss-ionone at a given concentration range, supporting its essential role in the detection of specific compounds present in the natural environment of the fly (Gonzalez, 2019).

Robust olfactory responses in the absence of odorant binding proteins

Odorant binding proteins (Obps) are expressed at extremely high levels in the antennae of insects, and have long been believed essential for carrying hydrophobic odorants to odor receptors. Previously work found that when one functional type of olfactory sensillum in Drosophila was depleted of its sole abundant Obp, it retained a robust olfactory response. This study deleted all the Obp genes that are abundantly expressed in the antennal basiconic sensilla. All of six tested sensillum types responded robustly to odors of widely diverse chemical or temporal structure. One mutant gave a greater physiological and behavioral response to an odorant that affects oviposition. These results support a model in which many sensilla can respond to odorants in the absence of Obps, and many Obps are not essential for olfactory response, but that some Obps can modulate olfactory physiology and the behavior that it drives (Xiao, 2019).

A conserved odorant binding protein is required for essential amino acid detection in Drosophila

Animals need to detect in the food essential amino acids that they cannot synthesize. This study found that the odorant binding protein OBP19b, which is highly expressed in Drosophila melanogaster taste sensilla, is necessary for the detection of several amino acids including the essential l-phenylalanine. The recombinant OBP19b protein was produced and characterized for its binding properties: it stereoselectively binds to several amino acids. Using a feeding-choice assay, it was found that OBP19b is necessary for detecting l-phenylalanine and l-glutamine, but not l-alanine or D-phenylalanine. The cells expressing OBP19b were mapped, and the electrophysiological responses of a single taste sensillum were compared to several amino acids: OBP19b mutant flies showed a reduced response compared to control flies when tested to preferred amino acids, but not to the other ones. OBP19b is well conserved in phylogenetically distant species suggesting that this protein is necessary for detection of specific amino acids in insects (Rihani, 2019).

Mutually Exclusive Expression of Closely Related Odorant-Binding Proteins 9A and 9B in the Antenna of the Red Flour Beetle Tribolium castaneum

Olfaction is crucial for insects to find food sources, mates, and oviposition sites. One of the initial steps in olfaction is facilitated by odorant-binding proteins (OBPs) that translocate hydrophobic odorants through the aqueous olfactory sensilla lymph to the odorant receptor complexes embedded in the dendritic membrane of olfactory sensory neurons. The Tribolium OBPs encoded by the gene pair TcasOBP9A and TcasOBP9B represent the closest homologs to Drosophila OBP Lush, which mediates pheromone reception. These two OBPs were shown not to be pheromone-specific but rather enhance the detection of a broad spectrum of organic volatiles. Both OBPs are expressed in the antenna but in a mutually exclusive pattern. A phylogenetic analysis indicates that this gene pair arose at the base of the Cucujiformia, which dates the gene duplication event to about 200 million years ago. Therefore, the high sequence conservation in spite of their expression in different sensilla is potentially the result of a common function as co-OBPs (Montino, 2021).

Odorant Binding Proteins (OBPs) and Odorant Receptors (ORs) of Anopheles stephensi: Identification and comparative insights

Anopheles stephensi is an important vector of malaria in the South Asia, the Middle East, and Eastern Africa. The olfactory system of An. stephensi plays an important role in host-seeking, oviposition, and feeding. Odorant binding proteins (OBPs) are globular proteins that play a pivotal role in insect olfaction by transporting semiochemicals through the sensillum lymph to odorant receptors (ORs). Custom motifs designed from annotated OBPs of Aedes aegypti, Drosophila melanogaster, and Anopheles gambiae were used for the identification of putative OBPs from protein sequences of the An. stephensi Indian strain. Further, BLASTp was also performed to identify missing OBPs and ORs. Subsequently, the presence of domains common to OBPs was confirmed. Identified OBPs were further classified into three sub-classes. Phylogenetic and syntenic analyses were carried out to find homology, and thus the evolutionary relationship between An. stephensi OBPs and ORs with those of An. gambiae, Ae. aegypti and D. melanogaster. Gene structure and physicochemical properties of the OBPs and ORs were also predicted. A total of 44 OBPs and 45 ORs were predicted from the protein sequences of An. stephensi. OBPs were further classified into the classic (27), atypical (10) and plus-C (7) OBP subclasses. The phylogeny revealed close relationship of An. stephensi OBPs and ORs with An. gambiae homologs whereas only five OBPs and two ORs of An. stephensi were related to Ae. aegypti OBPs and ORs, respectively. However, D. melanogaster OBPs and ORs were distantly rooted. Synteny analyses showed the presence of collinear block between the OBPs and ORs of An. stephensi and An. gambiae as well as Ae. aegypti's. No homology was found with D. melanogaster OBPs and ORs. As an important component of the olfactory system, correctly identifying a species' OBPs and ORs provide a valuable resource for downstream translational research that will ultimately aim to better control the malaria vector An. stephensi (Zafar, 2022).

Pleiotropic Fitness Effects of a Drosophila Odorant-binding Protein

Insect odorant-binding proteins (OBPs) are members of a rapidly evolving multigene family traditionally thought to facilitate chemosensation. However, studies on Drosophila have shown that members of this family have evolved functions beyond chemosensation, as evident from their expression in reproductive tissues and the brain. Previous studies implicated diverse functions of Obp56h, a member of the largest gene cluster of the D. melanogaster Obp repertoire. This study examined the effect of CRISPR/Cas9-mediated deletion of Obp56h on two fitness phenotypes, resistance to starvation stress and heat stress, and on locomotion and sleep phenotypes. Obp56h^-/- mutants show a strong sexually dimorphic effect on starvation stress survival, with females being more resistant to starvation stress than the control. In contrast, Obp56h^-/- females, but not males, are highly sensitive to heat stress. Both sexes show changes in locomotion and sleep patterns. Transcriptional profiling of RNA from heads of Obp56h^-/- flies and the wildtype control reveal differentially expressed genes, including gene products associated with antimicrobial immune responses and members of the Turandot family of stress-induced secreted peptides. In addition, differentially expressed genes of unknown function were identified in both sexes. Genes encoding components of the mitochondrial electron transport chain, cuticular proteins, gene products associated with regulation of feeding behavior (Lst and CCHa2), ribosomal proteins, lncRNAs, snoRNAs, tRNAs, and snRNAs show changes in transcript abundances in Obp56h^-/- females. These differentially expressed genes are likely to contribute to Obp56h-mediated effects on the diverse phenotypes that arise upon deletion of this odorant binding protein (Mokashi, 2023).

The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility in Drosophila melanogaster

In Drosophila and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology and behavior. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). This study used RNAi and CRISPR/Cas9 generated mutants to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). Obp56g was found to be required for male fertility and the induction of the PMR, whereas the other six genes had no effect on fertility when mutated individually. Obp56g is expressed in the male's ejaculatory bulb, an important tissue in the reproductive tract that synthesizes components of the mating plug. This study found males lacking Obp56g fail to form a mating plug in the mated female's reproductive tract, leading to ejaculate loss and reduced sperm storage. The evolutionary history of these seminal Obp genes was examined, as several studies have documented rapid evolution and turnover of SFP genes across taxa. Extensive lability was found in gene copy number and evidence of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multiple Drosophila species revealed that Obp56g shows high male reproductive tract expression only in species of the melanogaster and obscura groups, though conserved head expression in all species tested. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time (Brown, 2023).