Putative Odorant-binding Proteins Research Articles

Expression patterns of two putative odorant-binding proteins in the olfactory organs of Drosophila melanogaster have different implications for their functions.

The aqueous medium bathing the dendrites of olfactory neurons contains high concentrations of odorant-binding proteins (OBPs) whose role is still unclear. OBPs may facilitate interactions between odorants and their membrane-bound receptors, perhaps by increasing the water solubility of hydrophobic molecules. Alternatively, OBPs may be involved in the inactivation of odorants and other volatile molecules, preventing desensitization and/or protecting olfactory neurons from toxic chemicals. We report here novel features of the localization of two putative OBPs, PBPRP2 and PBPRP5, that have important and different implications for their role in olfaction. Unlike several other putative OBPs of Drosophila melanogaster that are only found in adult olfactory organs, PBPRP5 is also expressed in the larval olfactory organs, suggesting that it plays a common role in olfaction at both stages. In the adult, PBPRP5 expression is restricted to the sensillum lymph that bathes the olfactory dendrites of a subset of olfactory hairs, the basiconic sensilla. Since individual basiconic sensilla differ in olfactory specificity, PBPRP5 may be able to bind to and mediate olfactory responses to a wide range of odorants. In contrast, PBPRP2 is present in the space immediately below the antennal cuticle and in the outer cavity of approximately 30% of the double-walled coeloconic sensilla on the antennal surface. In neither case is PBPRP2 in contact with the dendritic membranes of olfactory neurons, making a carrier function unlikely for this protein. Instead, PBPRP2 may act as a sink, binding to odorants and other volatile chemicals and limiting their interactions with olfactory neurons.

Cloning and Expression of a Queen Pheromone-Binding Protein in the Honeybee: an Olfactory-Specific, Developmentally Regulated Protein

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. We identified, for the first time in a social insect, a binding protein called antennal-specific protein 1 (ASP1), which binds at least one of the major queen pheromone components. ASP1 was characterized by cDNA cloning, expression in Pichia pastoris, and pheromone binding. In situ hybridization showed that it is specifically expressed in the auxiliary cell layer of the antennal olfactory sensilla. The ASP1 sequence revealed it as a divergent member of the insect OBP family. The recombinant protein presented the exact characteristics of the native protein, as shown by mass spectrometry, and N-terminal sequencing and exclusion-diffusion chromatography showed that recombinant ASP1 is dimeric. ASP1 interacts with queen pheromone major components, opposite to another putative honeybee OBP, called ASP2. ASP1 biosynthetic accumulation, followed by nondenaturing electrophoresis during development, starts at day 1 before emergence, in concomitance with the functional maturation of olfactory neurons. The isobar ASP1b isoform appears simultaneously to ASP1a in workers, but only at approximately 2 weeks after 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 with mate attraction.

O-Crystallin, arginine kinase and ferritin from the octopus lens

Three proteins have been identified in the eye lens of the octopus, Octopus dofleini. A 22 kDa protein comprising 3–5% of the soluble protein of the lens is 35–43% identical to a family of phosphatidylethanolamine-binding proteins of vertebrates. Other members of this family include the immunodominant antigen of the filarial parasite, Onchocerca volvulus, putative odorant-binding proteins of Drosophila and a protein with unknown function of Caenorhabditis elegans. We have called this protein O-crystallin on the basis of its abundance in the transparent lens. O-Crystallin mRNA was detected only in the lens. Two tryptic peptides of another octopus lens protein, less abundant than O-crystallin, showed 80% identity to arginine kinase of invertebrates, a relative of creatine kinase of vertebrates. Finally, ferritin cDNA was isolated as an abundant cDNA from the octopus lens library. Northern blots showed that ferritin mRNA is not lens-specific.

Perireceptor Events in Pheromone Perception in Scarab Beetles

Despite the remarkable diversity of the sex pheromone chemistry in scarab beetles, various species utilize a common type of γ-lactones in their chemical communication channels. These compounds differ primarily in length of the alkenyl side chain and the stereochemistry at the chiral center. Two species, Anomala osakana and Popillia japonica, utilize the opposite enantiomers of japonilure as sex pheromones. Each species produces only one of the enantiomers that functions as its own sex pheromone and as a behavioral antagonist to the alloreceiver. Pheromone binding proteins (PBPs) have been characterized, which are present in these and several other scarab species. In most cases there was only one class of PBP, which was expressed in both sexes. A. osakana and P. japonica possess each one single PBP with high homology to each other. In each species the same PBP seems to recognize both enantiomers of japonilure, i. e., the pheromonal and the “stop” signals. Based on the N-terminal sequences, the antennae-specific proteins from various other species were highly conserved within the family and showed moderate homology to putative odorant binding protein from Drosophila melanogaster (47%), Lygus lineolaris (45∼50%) and the ABPX protein from Bombyx mori (30∼35%). From analysis of extracts of soluble antennal proteins from several species, significant degradation of the γ-lactones (buibuilactone, japonilure) was detected, essentially in all of them, even in species that do not use these compounds as pheromones. Recently a peculiar pheromone with a diamide moiety [1, 3-dimethyl-2, 4-(1 H, 3 H)-quinazolinedione] was isolated from Phyllopertha diversa, which was rapidly degraded by antennal enzymes from this species. Beetles that utilize lactones as their pheromones possess little or no ability to metabolize this compound.

Separation, Characterization and Sexual Heterogeneity of Multiple Putative Odorant-binding Proteins in the Honeybee Apis mellifera L. (Hymenoptera: Apidea)

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 was shown to lie in the relative abundance of these antennal specific proteins and in the occurrence of a drone-specific isoform. At least 19 other related proteins were found to occur in antennae and legs, forming another class showing homology with insect OBP. Genotype comparison of two honeybee races revealed 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.

Intergeneric distribution and immunolocalization of a putative odorant-binding protein in true bugs (Hemiptera, Heteroptera).

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 was strongest in antennae of adult males, but was 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 indicated that LAP immunoreactivity was present in the antennae of the male congeners L. lineolaris and L. hesperous, but was not detectable in male antennae of the more distant relatives Podisus maculiventris or Nezara viridula (Hemiptera, Heteroptera: Pentatomidae). Western blot analysis further confirmed that LAP expression was restricted to antennal tissue. Histological analyses showed that LAP expression within the antennae was specifically associated with chemosensory sensilla on the antenna. Within the sensilla, LAP immunoreactivity was distributed throughout the extracellular lumen and was concentrated in dense granules within the cytoplasm of sensillar support cells. LAP immunoreactivity was 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 sp., 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.

Biochemical characterization, molecular cloning and localization of a putative odorant-binding protein in the honey bee Apis mellifera L. (Hymenoptera: Apidea)

A honey bee antennal water-soluble protein, APS2, was purified and characterized as the first Hymenoptera putative odorant-binding protein. Comparison of its measured M r (13 695.2±1.6) to that of the corresponding cDNA clone shows it does not undergo any post-translational modification other than a 19-residue signal peptide cleavage and formation of three disulfide bridges. These biochemical features are close to those of Lepidoptera odorant-binding proteins. In situ hybridization experiments demonstrated its specific expression in olfactory areas. Based on its higher expression in the worker than in the drone, ASP2 might be more involved in general odorant than in sex pheromone detection.

Putative odorant-binding protein in antennae and legs of carausius morosus (Insecta, Phasmatodea)

A 19 kDa protein has been purified by gel filtration and anion-exchange chromatography from the antennae of Carausius morosus. Its amino terminal amino acid sequence shows significant similarity (30% identity) with another putative odorant-binding protein, the so called OS-D protein isolated from the antennae of Drosophila melanogaster; only 20% of its amino acids are shared with some members of Lepidoptera pheromone-binding proteins. Polyclonal antibodies, raised against a synthetic amino terminal peptide cross-react with 19 kDa band in the legs extracts, but not with soluble proteins from other parts of the body. The amino terminal sequence of this protein, purified from the legs was identical with that of the antennal protein.

Members of a family of drosophila putative odorant-binding proteins are expressed in different subsets of olfactory hairs

A polymerase chain reaction-based method was used to generate a Drosophila melanogaster antennal cDNA library from which head cDNAs were subtracted. We identified five cDNAs 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, we describe seven antennal proteins which contain potential signal peptides, suggesting that, like pheromone-binding proteins, they 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 funnctionally segregated.

Molecular cloning of putative odorant-binding and odorant-metabolizing proteins

Olfactory reception occurs via the interaction of odorants with the chemosensory cilia of the olfactory receptor cells located in the nasal epithelium. The cDNA clones from mRNA specific to olfactory mucosa were studied. One of these clones, OBPII, encodes a secretory protein with significant homology to odorant-binding protein (OBP), a protein with broad odorant-binding ability, and is expressed in the lateral nasal gland, which is the site of expression of OBP. The OBPII sequence also shows significant homology to the VEG protein, which is thought to be involved in taste transduction. OBPII is a new member of the lipophilic molecule carrier protein family. The second cDNA clone encodes a novel homologue of glutathione peroxidase, an enzyme involved in cellular biotransformation pathways. Its expression appears to be localized to the Bowman's glands, the site of several previously identified olfactory-specific biotransformation enzymes.