The lipocalin protein family is a large group of small extracellular proteins. The family demonstrates great diversity at the sequence level; however, most lipocalins share three characteristic conserved sequence motifs (the kernel lipocalins), while a group of more divergent family members (the outlier lipocalins) share only one. Belying this sequence dissimilarity, lipocalin crystal structures are highly conserved and comprise a single eight-stranded continuously hydrogen-bonded antiparallel beta-barrel, which encloses an internal ligand-binding site. Together with two other families of ligand-binding proteins, the fatty-acid-binding proteins (FABPs) and the avidins, the lipocalins form part of an overall structural superfamily: the calycins. Members of the lipocalin family are characterized by several common molecular-recognition properties: the ability to bind a range of small hydrophobic molecules, binding to specific cell-surface receptors and the formation of complexes with soluble macromolecules. The varied biological functions of the lipocalins are mediated by one or more of these properties. In the past, the lipocalins have been classified as transport proteins; however, it is now clear that the lipocalins exhibit great functional diversity, with roles in retinol transport, invertebrate cryptic coloration, olfaction and pheromone transport, and prostaglandin synthesis. The lipocalins have also been implicated in the regulation of cell homoeostasis and the modulation of the immune response, and, as carrier proteins, in the general clearance of endogenous and exogenous compounds (Flower, 1996).

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).

A polymerase chain reaction-based method generated a Drosophila melanogaster antennal cDNA library from which head cDNAs were subtracted. Five cDNAs were identifed that code for antennal proteins containing six cysteines in a conserved pattern shared with known moth antennal proteins, including pheromone-binding proteins. Another cDNA codes for a protein related to vertebrate brain proteins that bind hydrophobic ligands. In all, seven antennal proteins are described that contain potential signal peptides, suggesting that, like pheromone-binding proteins, these proteins may be secreted in the lumen of olfactory hairs. The expression patterns of these putative odorant-binding proteins define at least four different subsets of olfactory hairs and suggest that the Drosophila olfactory apparatus is functionally segregated. Pheromone-binding protein-related protein (Pbprp1) transcripts are detected by in situ hybridization, on the anterior surface of the third antennal segment and appear to be expressed in a subset of sensillum trichodea. Pbprp2 transcripts are detected in antennal segment 3 in regions containing basiconic, coeloconic, and trichoid sensilla. They are also expressed in the maxillary palps, in cells at the bases of the taste hairs on the proboscis and in the internal taste organs of the head. They are also detected in the sheath cells of the taste bristles on the proboscis. Pbprb3 transcripts detected by in situ hybridization are limited to the ventro-lateral region of the third antennal segment, in a pattern consistent with the distribution of sensilla trichodea. In deeper sections, expression is observed beneath sensilla trichodeasensory hairs. Pbprp4 transcripts are expressed in a small number of hairs scattered over the surface of the third antennal segment. Sensilla showing Pbprp5 expression are mostly on the medial and posterior surface of the antenna, a location that correlates well with the position of sensillum basiconica. Antennal protein 10 (a10) transcripts are primarily found in cells in the largest cavity of the sacculus. They are also expressed in a few cells on the anterior and posterior surfaces of the antennae, in sensilla that are probably coeloconic. Antennal protein 5 (a5) transcripts are restricted to a few scattered sensilla on the posterior surface of the antenna that are probably coeloconic sensilla (Pikielny, 1994).

Four genes expressed in the olfactory system of Drosophila melanogaster have been identified by subtractive hybridization. Two of these genes, OS-E and OS-F, are related to genes encoding moth pheromone-binding proteins. The OS-E and OS-F genes are tightly linked and are expressed in a subregion of the antenna (the primary olfactory organ). A protein sequence analysis suggests the possibility that pheromone-binding proteins are members of a larger class of proteins, extending beyond the olfactory system. The predicted product of a third gene, OS-D, shares features common to vertebrate odorant-binding proteins, but has a primary structure unlike odorant-binding proteins. The fourth gene, OS-C, encodes a novel 13-kDa protein that contains a putative nuclear import sequence and an acid-rich region. The expression patterns of these genes differ within the antenna; their transcript distributions support the notion of specialized roles for different olfactory sensilla. The functions of the OS gene products have not been demonstrated; however, the potential identification of pheromone-binding proteins in Drosophila, a species with well characterized genetics, may offer a means of analyzing the function of these molecules that is not available in other systems (McKenna, 1994).

The genomic organization and expression patterns of two olfactory-specific Drosophila genes (OS-E and OS-F) were examined. The products coded for by these genes are members of a protein family in Drosophila sharing sequence similarity with moth OBPs. The OS-E and OS-F transcription units are located <1 kb apart. They are oriented in the same direction and display a similar intron-exon organization. Expression of both OS-E and OS-F proteins is restricted spatially to the ventrolateral region of the Drosophila antenna. Within this region both OS-E and OS-F proteins are expressed within two different types of sensory hairs: in most, if not all, sensilla trichodea, and in ~40% of the interspersed small sensilla basiconica. Although the resolution of this analysis does not allow a precise identification of the cells labeled, it is noted that the location of the label directly beneath the cuticle corresponds to the position of the sensillar auxiliary cells and the sensillum lymph cavities; neuronal cell bodies are located further below the cuticle. In some cases staining extends into the sensory hair itself. No discernible staining was observed in the brain or other chemosensory organs (maxillary palps and proboscis) with either the anti-E or anti-F antiserum. OS-E and OS-F consistently observed to be coexpressed, indicating that an individual sensillum can contain more than one odorant-binding protein (Hekmat-Scafe, 1997).

During adult metamorphosis, the moth olfactory neurons and their glia-like support cells pass through a coordinated and synchronous development. By 60% of development, the olfactory system is anatomically complete, but functional maturation does not occur until about 90% of development. Maturation is characterized by the onset of odorant sensitivity in the sensory neurons and the expression of certain antennal-specific proteins including odorant binding proteins (OBPs) and odorant degrading enzymes (ODEs). The OBPs have been cloned and sequenced, and are thus useful models for investigating the molecular mechanisms coordinating final maturation of the developing olfactory system. The ecdysteroid hormones have been observed to regulate many cellular level neuronal changes during adult metamorphosis. In particular, the late pupal decline in ecdysteroids is known to influence programmed death of nerves and muscles at the end of metamorphosis. Indications are that this decline in ecdysteroids also induces the expression of the OBPs. Normal OBP expression occurs 35-40 h before adult emergence. In culture, OBP expression can be induced at least 90 h before adult emergence by the premature removal of ecdysteroid. This premature expression is blocked by culturing tissue in the presence of the biologically active ecdysteroid 20-hydroxyecdysone. These findings suggest that maturation of the olfactory system is regulated by the decline in ecdysteroids, and support the view that olfactory development, in general, may be coordinated by changing levels of pupal ecdysteroids (Vogt, 1993).

According to precise molar mass determined by mass spectrometry and N-terminal sequence, some 25 odorant-binding-like proteins were characterized from the antennae and legs of worker and drone honeybees. Antennal specific proteins, composed of six different molecules, were classified into three subclasses according to N-terminal sequence homology. The major sexual difference lies in the relative abundance of these antennal specific proteins and in the occurrence of a drone-specific isoform. At least 19 other related proteins are found to occur in antennae and legs, forming another class showing homology with insect OBP. Genotype comparison of two honeybee races reveals a variability limited to this second class. Provided that these odorant-binding-like proteins are indeed able to bind odorants or pheromones, the question of whether their peculiar multiplicity contributes to the remarkable capacity of the honeybee to discriminate among a wide range of odor molecules is raised (Danty, 1998).

Odorant-binding proteins (OBPs) are small abundant extracellular proteins thought to participate in perireceptor events of odor-pheromone detection by carrying, deactivating, and/or selecting odor stimuli. The honeybee queen pheromone is known to play a crucial role in colony organization, in addition to drone sex attraction. For the first time in a social insect, a binding protein called antennal-specific protein 1 (ASP1), has been identified that binds at least one of the major queen pheromone components. ASP1 was characterized by cDNA cloning, expression in Pichia pastoris, and pheromone binding. The highest amino acid sequence identity of ASP1 to other similar proteins was observed for antennal binding protein X from Antheraea pernyi (28%), most of the other members of the family presenting approximately 16%-24% amino acid sequence identity with ASP1. The six cysteines and their interval spacing are the most striking features shared by proteins belonging to this family. In situ hybridization shows that ASP1 is specifically expressed in the auxiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence reveals it as a divergent member of the insect OBP family. The recombinant protein presents the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chromatography shows that recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major components, just the opposite of another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation starts at day 1 before emergence, in concomitance with the functional maturation of olfactory neurons. The ASP1b isoform appears simultaneously with a second isoform (ASP1a) in workers, but only ~2 weeks after its emergence in drones. Comparison of in vivo and heterologous expressions suggests that the difference between ASP1 isoforms might be because of dimerization, which might play a physiological role in relation to mate attraction (Danty, 1999).

The antennae are involved in the detection of a variety of environmental odorous stimuli. In the honeybee, olfactory receptor neurons are ensheathed in sensilla, namely the sensilla placodea and the sensilla trichodea, more precisely of type A. In drones and in workers, sensilla placodea are innervated by a large number of sensory neurons responding to various odor stimuli, including queen pheromone or its isolated major component 9-ODA. In holometabolous insects such as honeybee, each sensillum is formed by a set of auxiliary cells and sensory neurons. In workers, sensilla placodea are intermingled with sensilla trichodea, which represent, respectively, 55% and 45% of the olfactory sensilla, whereas in drones, sensilla placodea represent 94% of olfactory sensilla. Compared with ASP2, which is expressed specifically in sensilla trichodea, ASP1 expression seems to be restricted to groups of cells corresponding to auxiliary cells of sensilla placodea, strongly suggesting a role of olfactory-binding protein for ASP1. This data are consistent with previous reports on moth PBPs and Drosophila Lush, indicating that OBPs are secreted by auxiliary cells in the sensillum lymph (Danty, 1999 and references).

In drones the majority of drone sensilla placodea express ASP1, and olfactory neurons responding to the queen pheromone are ensheathed in sensilla placodea. The higher sensitivity of drones than workers to queen pheromone or to 9-ODA suggests that the drones possess many more sensory neurons responding to these odors. Thus, it might be expected that ASP1 is secreted by auxiliary cells associated with sensory neurons responding to queen pheromone in sensilla placodea, because ASP1 can interact with 9-ODA and/or 9-HDA. In the honeybee worker, olfactory neuron functional maturation starts at ~2 d before emergence, just before the beginning of ASP1 and ASP2 production, which thus might reflect the maturation of olfactory sensilla. In drones, the electrophoretic profile of ASP1 is modified in greater than 1-week-old drones, even though the main difference is detected in older insects. Compared with in vitro ASP1 production, ASP1a might be a monomeric form first appearing at low concentrations when sensilla maturation occurs, and ASP1b might be the homodimer formed when the concentration increases in adults older than 1 week. Sexual maturation occurs between E+9 and E+23, and queen pheromone does not become behaviorally relevant for mate attraction until this maturation process. When comparing samples from adults, it has been estimated that, in 6- to 17-d-old adults, drones possess approximately five times more ASP1 than workers, as drones possess also approximately five times more sensory neurons than workers. Between approximately 1 d before emergence and at least 2 d after emergence, ASP1 is still much more abundant in drones than in workers. However, the putative dimer is detected at all ages in workers, although only as a putative monomer during this period in drones. Because the dimerization of ASP1 is spontaneously observed by in vitro production, one might expect that, in drones, a physiological mechanism might affect the putative dimerization of ASP1 up to the time of sexual maturity, possibly by controlling the concentration or the biochemical environment of ASP1 in the sensillum lymph (Danty, 1999 and references).

In a given species, several members of the insect OBP family are known to present differential binding properties and/or associate with distinct olfactory cells. Such a conclusion can be drawn about the honeybee in which a pheromone binding protein (ASP1) able to bind components of the queen pheromone, has been detected that can be contrasted with ASP2, a putative general odorant-binding protein. At present, there is only one demonstration of a physiological role for a general odorant-binding protein, the alcohol-binding protein Lush in D. melanogaster. A disruption of the protein gene causes a drastic effect on behavior, which is restored when a wild-type copy of the gene is introduced into mutant fruit flies. It has been suggested that Lush might be required to activate a small subset of olfactory neurons mediating chemoavoidance rather than odorant solubilization or desensitization. In light of these data, the existence of finely tuned sensory neuron responses to 9-ODA could be related to the role of this molecule in the honeybee society, with different behavioral effects on workers and drones. How this sexual diversity is encoded by the olfactory system is now under current investigation by analyzing the properties of ASP1 using both in vitro and in vivo approaches (Danty, 1999 and references).

Lygus antennal protein (LAP) is an olfactory-related protein of the tarnished plant bug Lygus lineolaris (Hemiptera, Heteroptera: Miridae), a hemimetabolous insect. In previous work, a polyclonal antiserum was generated against the N-terminal sequence of LAP; LAP immunoreactivity is strongest in the antennae of adult males, but is also present in antennae of adult females and of nymphs. In the current study, LAP immunoreactivity was examined to determine the species specificity and the tissue and cellular localization of LAP expression. Western blot analysis indicates that LAP immunoreactivity is present in the antennae of the male congeners L. lineolaris and L. hesperous, but is not detectable in male antennae of the more distant relatives: Podisus maculiventris or Nezara viridula (Hemiptera, Heteroptera: Pentatomidae). Western blot analysis further confirms that LAP expression is restricted to antennal tissue. Histological analyses show that LAP expression within the antennae is specifically associated with chemosensory sensilla on the antenna. Within the sensilla, LAP immunoreactivity is distributed throughout the extracellular lumen and is concentrated in dense granules within the cytoplasm of sensillar support cells. LAP immunoreactivity is restricted to a subset of antennal chemosensory sensilla, specifically the multiporous olfactory sensilla. These findings suggest that LAP has an important olfactory function in Lygus, possibly related to that of odorant-binding proteins (OBP) found in other insect orders. If so, LAP would be the first OBP-like protein characterized outside the Endopterygota (Dickens, 1998).

Olfactory sensilla show a large diversification of sensillum types even in the same species. Thus, double-walled and single-walled sensilla with highly different wall pores are usually found on the same antenna, and these may appear in the form of long slender hairs, pore plates or pit pegs. The selective constraints leading to this diversification are evident only in a few cases, e.g. the demand for extreme sensitivity in moth pheromone communication supports the evolution of long sensilla trichodea with high efficiency for capturing odor molecules. The structural diversity continues with the odorant-binding proteins (OBPs) in the sensillum lymph surrounding the sensory dendrites. These proteins may be subdivided into pheromone-binding proteins and two classes of general odorant-binding proteins according to their primary sequence. Different sensilla of the same morphological type may contain different OBPs of the same or of different subclasses. However, OBPs of different subclasses are not co-localized in the same individual sensory hair. The presence of a given OBP is related more to the functional specificity of the receptor cells than to the morphological type of the sensillum, suggesting a role for OBPs in stimulus recognition (Steinbrecht, 1996).

The genome of Drosophila encodes a variety of predicted odorant-binding proteins (OBPs), each of which is expressed in a characteristic portion of the antenna. Although most of these OBPs differ markedly from one another, those encoded by the olfactory-specific genes OS-E and OS-F show substantial sequence similarity: 69% amino acid identity for the mature proteins. The OS-E and OS-F genes are located <1 kb apart and are suggested to have arisen by gene duplication. They are coexpressed within two morphological types of olfactory sensilla that are located in the same region of the antenna, the ventrolateral region (Hekmat-Scafe, 2000).

To address the functional significance and evolution of the OS-E and OS-F proteins in Drosophila, OS-E and OS-F gene homologs were examined in a variety of Drosophila species, with particular emphasis on D. virilis, a species thought to have shared a common ancestor with D. melanogaster ~40 million years ago (mya). This analysis uncovered an OS-F homolog in D. virilis, but no D. virilis counterpart to the OS-E gene. D. melanogaster and D. virilis OS-F proteins show remarkable conservation but diverge notably in two regions: the N terminus and a C-terminal region that exhibits heterogeneity in other insect OBPs. OS-F transcripts are expressed in a different spatial pattern within the antenna of D. virilis than in D. melanogaster, possibly reflecting the presence of this OS-F protein in an additional class of olfactory sensilla in D. virilis. The OS-F intron shows a surprisingly high degree of sequence conservation; a putative regulatory element has been identified within it (Hekmat-Scafe, 2000).

This examination of OS-E and OS-F homologues in a variety of Drosophila species suggests that the duplication that gave rise to OS-E and OS-F is an ancient one. These studies also highlight regions of potential functional importance in the OS-E and OS-F proteins, one of which might mediate binding to odorant receptor proteins. Finally, this phylogenetic analysis illustrates that OS-E and OS-F are members of a diverse and ancient family of OBP-related insect proteins (Hekmat-Scafe, 2000).

D. virilis OS-F and D. melanogaster OS-E and OS-F are small proteins, with a predicted primary translation product of ~16-17 kD. All carry an N-terminal signal sequence, and all have the six aligned cysteine residues that are diagnostic of insect OBPs. D. virilis OS-F protein shows 76% sequence identity to D. melanogaster OS-F and 57% identity to D. melanogaster OS-E. The sequence identity between D. virilis and D. melanogaster OS-F is greater than that seen when comparing D. melanogaster OS-E and OS-F (62%) (Hekmat-Scafe, 2000).

Two regions of the protein exhibit a high degree of sequence divergence. The majority of the amino acid substitutions between mature D. virilis and D. melanogaster OS-F occur in a 22-amino-acid stretch, which is called the 'heterogeneous region' and which extends from L¹⁰⁷ to H¹²⁸ in D. virilis OS-F. D. virilis and D. melanogaster OS-F are only 55% identical within this 22-amino-acid stretch, whereas the remaining portions of the mature proteins are 86% identical. D. melanogaster OS-E and OS-F show an even greater degree of heterogeneity in this region: 39% identity, as compared to 76% identity in the remaining portions of the mature proteins. A second region of heterogeneity is at the N terminus. Much of the N-terminal heterogeneity resides within the signal sequence, but the N-terminal region of the mature proteins is predicted to exhibit substantial heterogeneity as well (Hekmat-Scafe, 2000).

The D. virilis and D. melanogaster OS-F genes display a similar intron-exon structure. There are three small introns within the D. virilis OS-F coding region. They are located between N⁴³ and Y⁴⁴ (76 bp), between E⁶⁸ and A⁶⁹ (78 bp), and between K¹⁵⁴ and H¹⁵⁵ (67 bp). These three introns are present at positions corresponding to those of the three introns within the coding region of D. melanogaster OS-F; the first two of these intron insertion sites also correspond to those of the two introns in D. melanogaster OS-E (Hekmat-Scafe, 2000).

The first intron in the coding region of D. virilis OS-F shows a surprisingly high degree of nucleotide sequence identity to that of D. melanogaster OS-F. Overall, the two introns are 76% identical. This similarity suggests that the intron harbors conserved regulatory sequences needed for appropriate gene expression in the olfactory system. One possibility for such a regulatory element is the sequence GCCACGC, which is also present in the first intron within the coding region of the pheromone-binding protein-related protein, PBPRP-1. PBPRP-1 encodes a predicted OBP, which, like OS-F, is expressed in regions of the D. melanogaster antenna rich in trichoid sensilla (Hekmat-Scafe, 2000).

In situ hybridization has revealed that D. virilis OS-F transcripts are expressed predominantly, perhaps exclusively, in the antenna, as has been observed previously for D. melanogaster OS-E and OS-F. Interestingly, the distribution of D. virilis OS-F transcripts within the antenna is different from that observed for D. melanogaster OS-E and OS-F. D. melanogaster OS-E and OS-F are coexpressed specifically in the ventrolateral region of the antenna in a pattern that mimics the distribution of one morphological class of sensory hairs, the trichoid sensilla. However, an antisense probe for D. virilis OS-F mRNAs shows a broader distribution, extending to include a portion of the antenna immediately ventral to the sacculus, a chamber lined with sensory hairs. Visual inspection by light microscopy revealed no major differences in the distribution of trichoid sensilla between the two species, suggesting the possibility that the distribution of OS-F among the different morphological classes of sensilla is different between the two species. No hybridization was observed with the D. virilis OS-F sense probe (Hekmat-Scafe, 2000).

To expand the analysis of OS-E and OS-F genes, OS-E and OS-F homologs were isolated from a wide variety of Drosophila species. Specifically, a ~170-bp fragment of OS-E- and/or OS-F-related genes was amplified from the genomic DNA of other Drosophila species, using PCR conditions similar to those used to amplify the D. virilis OS-F sequences. The amplification products extend between the amino acids corresponding to C⁶⁸ and W¹²⁵ of D. melanogaster OS-E. This region was analyzed because it includes the heterogeneous stretch of 22-23 amino acids, which represents a region of great sequence divergence both between OS-F in different species (D. melanogaster and D. virilis) and between OS-E and OS-F in D. melanogaster (Hekmat-Scafe, 2000).

Parsimony analysis of OS-E- and OS-F-related protein sequences from the various Drosophila species yields 16 minimum-length trees. This tree groups OS-E-related proteins in one cluster and OS-F-related proteins in a sister cluster. Interestingly, in three species, D. simulans, D. mauritiana, and D. virilis, an OS-F gene was found but not an OS-E gene, and in one species, D. lebanonensis, two genes closely related to OS-E were found (which were named OS-E1 and OS-E2), but none to OS-F. Two other species, D. teissieri and D. willistoni, are like D. melanogaster in that they contain both an OS-E gene and an OS-F gene. The simplest interpretation of all these results taken together is that not all Drosophila species carry both an OS-E and an OS-F gene (Hekmat-Scafe, 2000).

D. lebanonensis, which diverged from D. melanogaster ~40 mya, also has two E/F genes, although both of them are closely related to OS-E (OS-E1 and OS-E2). D. teissieri and D. willistoni, which diverged from D. melanogaster more recently, have both an OS-E and an OS-F gene. By contrast, an OS-F gene was identified but no evidence has been found for an OS-E gene in D. simulans and D. mauritiana, which diverged from D. melanogaster more recently than did D. teissieri and D. willistoni. This apparent lack is also the case in D. virilis, which diverged from D. melanogaster less recently than did D. teissieri and D. willistoni. An alignment of the various Drosophila OS-E and OS-F protein sequences reveals the marked sequence conservation of these proteins. In particular, all contain the motif -HPEGDTL following the fourth conserved cysteine, suggesting that this region is functionally important in both the OS-E and OS-F proteins of Drosophila.

In contrast, certain amino acid residues appear to distinguish OS-E from OS-F unambiguously. The serine at position 27 is present in all OS-F, but no OS-E sequences. Similarly, three residues (G²⁵, L²⁸, and I³¹) are present in all OS-E, but not in OS-F sequences. An additional residue, N²¹, is present in all OS-E sequences except for D. lebanonensis OS-E2. These diagnostic residues may underlie OS-E- or OS-F-specific functions (Hekmat-Scafe, 2000).

A broader phylogenetic analysis of the insect OBP family was carried out. Two major clusters were revealed. The first major cluster includes the moth OBPs. It has two major subdivisions, corresponding to the various moth PBPs and GOBPs, respectively. The second major cluster corresponds to all other insect OBPs and related proteins. In both trees, OS-E and OS-F are grouped with antennal proteins from a large variety of insect species. These include PBPRP-1 from D. melanogaster; Rpa12 and Rpa12', two closely related presumptive OBPs from the beetle Rhynchophorus palmarum; antennal binding proteins of unknown function (ABPXs) from a variety of species of moth; closely related PBPs from the beetles Popillia japonica and Anomala osakana; and LAP, an antennal protein from the hemipteran Lygus lineolaris (Hekmat-Scafe, 2000).

Also included in the second major cluster are a number of proteins expressed in tissues other than the olfactory organs. These include sericotropin, which is present in the brain of the wax moth Galleria mellonella; the B1 and B2 proteins, which are present in the secretions of the male accessory sex gland of the beetle Tenebrio molitor; the T. molitor antifreeze protein precursor; the male-specific protein MSSP, which is present in the hemolymph of the medfly Ceratitis capitata; a variety of D7-related proteins, which are found in saliva of the mosquitoes Anopheles gambiae and Aedes aegypti, and the SL1 protein, which is present in the saliva of the fly Lutzomyia longipalpis (Hekmat-Scafe, 2000).