Reverse chemical ecology: Olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles.

The giant panda Ailuropoda melanoleuca belongs to the family of Ursidae; however, it is not carnivorous, feeding almost exclusively on bamboo. Being equipped with a typical carnivorous digestive apparatus, the giant panda cannot get enough energy for an active life and spends most of its time digesting food or sleeping. Feeding and mating are both regulated by odors and pheromones; therefore, a better knowledge of olfaction at the molecular level can help in designing strategies for the conservation of this species. In this context, we have identified the odorant-binding protein (OBP) repertoire of the giant panda and mapped the protein expression in nasal mucus and saliva through proteomics. Four OBPs have been identified in nasal mucus, while the other two were not detected in the samples examined. In particular, AimelOBP3 is similar to a subset of OBPs reported as pheromone carriers in the urine of rodents, saliva of the boar, and seminal fluid of the rabbit. We expressed this protein, mapped its binding specificity, and determined its crystal structure. Structural data guided the design and preparation of three protein mutants bearing single-amino acid replacements in the ligand-binding pocket, for which the corresponding binding affinity spectra were measured. We also expressed AimelOBP5, which is markedly different from AimelOBP3 and complementary in its binding spectrum. By comparing our binding data with the structures of bamboo volatiles and those of typical mammalian pheromones, we formulate hypotheses on which may be the most relevant semiochemicals for the giant panda.

Recombinant expression, detergent solubilisation and purification of insect odorant receptor subunits.

Insect odorant receptors (ORs) are seven transmembrane domain proteins that comprise a novel family of ligand-gated non-selective cation channels. The functional channel is made up of an odour activated ligand-binding OR and the OR co-receptor, Orco. However, the structure, stoichiometry and mechanism of activation of the receptor complex are not well understood. Here we demonstrate that baculovirus-mediated Sf9 cell expression and wheat germ cell-free expression, but not Escherichia coli cell-based or cell-free expression, can be used successfully to over-express a selection of insect ORs. From a panel of 19 detergents, 1%w/v Zwittergent 3-16 was able to solubilise five Drosophila melanogaster ORs produced from both eukaryotic expression systems. A large-scale purification protocol was then developed for DmOrco and the ligand-binding receptor, DmOr22a. The proteins were nickel-affinity purified using a deca-histidine tag in a buffer containing 0.2 mM Zwittergent 3-16, followed by size exclusion chromatography. These purified ORs appear to form similarly sized protein-detergent complexes when isolated from both expression systems. Circular dichroism analysis of both purified proteins suggests they are folded correctly. We also provide evidence that when DmOrco is expressed in Sf9 cells it undergoes post translational modification, probably glycosylation. Finally we show that the recombinant ORs can be incorporated into pre-formed liposomes. The ability to recombinantly express and purify insect ORs to homogeneity on a preparative scale, as well as insert them into liposomes, is a major step forward in enabling future structural and functional studies, as well as their use in OR based biosensors.

Identification and characterization of odorant binding proteins in the diamondback moth, Plutella xylostella.

Odorant binding proteins (OBPs) are a group of soluble proteins functioning as odorant carriers in insect antennae, mouth parts and other chemosensory organs. However, multiple insect OBPs have been detected in other tissues and various functions have been proposed. Therefore, a detailed expression profile including stages, tissues and sexes where OBPs are expressed will assist in building the links to their potential functions, enhancing the functional studies of insect OBPs. Here, we identified 39 putative OBP genes from its genome and transcriptome sequences of diamondback moth (DBM), Plutella xylostella. The expression patterns of identified PxylOBPs were further investigated from eggs, larvae, pupae, virgin adults, mated adults, larval midgut, larval heads, adult antennae, adult heads and adult tarsi. Moreover, P. xylostella larvae and adults with and without host plants for 5 h were utilized to study the interactions between OBP expression and host plants. The results showed that most PxylOBPs were highly expressed in male and female adult antennae. The expression levels of certain PxyOBPs could be regulated by mating activities and feeding host plants. This study advances our knowledge of P. xylostella OBPs, which may help develop new strategies for more environmentally sustainable management of P. xylostella.

Expression, solubilization and purification of a human olfactory receptor from Escherichia coli.

Olfactory receptors pertaining to G protein-coupled receptor (GPCR) are integral membrane proteins composed of seven transmembrane spanning domains. It has been reported that these receptor proteins are difficult to overexpress, solubilize, and purify because of their complicated structures and strong hydrophobicity. In this study, full-length human olfactory receptor (hOR) 2AG1 was overexpressed in E. coli as a fusion protein with a glutathione S-transferase (GST) tag mainly as an inclusion body without any mutations or deletions in the gene. This protein was difficult to solubilize with detergents and chaotropic agents, and only N-lauroyl sarcosine was found to be suitable for solubilizing it. In contrast, Triton X-100 was found to solubilize most of the impurity proteins from the insoluble fraction in E. coli. Based on this observation, we applied a simple and efficient column-free method using these two detergents for the purification of the olfactory receptor protein. In this method, the insoluble fraction of the cell lysate was first treated with Triton X-100 to remove impurity proteins. The remaining insoluble fraction was then further treated with N-lauroyl sarcosine to solubilize the olfactory receptor protein. Milligram quantity of the human olfactory receptor was produced. This is the first report to produce full-length of the olfactory receptor from E. coli.

Cell-based high-throughput odorant screening system through visualization on a microwell array.

The development of a cell-based high-throughput screening system has attracted much attention from researchers who study drug screening mechanisms and characterization of G-protein coupled receptors (GPCRs). Although olfactory receptors (ORs) constitute the largest group of GPCRs that play a critical role recognizing and discriminating odorants, only a few ORs have been characterized, and most remain orphan. The conventional cell-based assay system for characterizing GPCRs, including ORs, is very laborious, time consuming, and requires an expensive assay system. In this study, we developed a simple, low-cost miniaturized odorant screening method by combining Micro-Electro-Mechanical system (MEMs) technique and visualization technique for detecting an odorant response. We fabricated PEG microwell from a photocrosslinkable polyethylene glycol diacrylate (PEGDA) solution and applied it to cell culture and a reverse transfection platform for cell-based high-throughput screening. For the first time, the olfactory receptors were expressed on the microwell platform using reverse transfection technique. The various olfactory receptors can be expressed simultaneously using this technique and the microwell spotted with olfactory receptor genes can be used as a high-throughput screening platform. The odorant response was detected via fluorescence analysis on the microwell using a cAMP response element (CRE) reporter assay. We tested this platform using four de-orphaned ORs. This new cell-based screening method not only reduced numerous time-consuming steps but also allowed for simple, efficient, and quantitative screening and patterning of large numbers of GPCRs including ORs, which can help to visualize the OR response to odorants on a microwell.

Deciphering activation of olfactory receptors using heterologous expression in Saccharomyces cerevisiae and bioluminescence resonance energy transfer.

Hetero- and homo-oligomerization of G protein-coupled receptors (GPCRs) has been addressed in the past years using various approaches such as co-immunoprecipitation, fluorescence resonance energy transfer and bioluminescence resonance energy transfer (BRET). Here, we report the methodological details from a previously published study to investigate the relationships between oligomerization and activation states of olfactory receptors (ORs). This methodology combines heterologous expression of ORs in Saccharomyces cerevisiae and BRET assays on membrane fractions, in particular, upon odorant stimulation. We have demonstrated that ORs constitutively homodimerize at the plasma membrane and that high odorant concentrations promote a conformational change of the dimer, which becomes inactive. We proposed a model in which one odorant molecule binding the dimer would induce activation, while two odorant molecules, each binding one protomer of the dimer, would blunt signaling.

Designer lipid-like peptides: a class of detergents for studying functional olfactory receptors using commercial cell-free systems.

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.

Proteinaceous precursors of human axillary odor: isolation of two novel odor-binding proteins.

The characteristic odor which arises in the human axillary region consists of volatile C6-C11 acids with the most abundant being (E)-3-methyl-2-hexenoic acid (E-3M2H). This acid, as well as several other components of the characteristic axillary odor, can be liberated from the odorless, aqueous soluble components of apocrine secretion by either saponification or bacteriolysis. It is therefore likely that a major characteristic odor is being carried to the skin surface bound to a water soluble precursor where it is liberated by axillary bacteria. The individual proteins found in apocrine secretions were separated, isolated and hydrolyzed with the resultant hydrolyzates analyzed by gas chromatography/mass spectrometry. These studies demonstrated that 3M2H was liberated from 2 proteins with apparent molecular mass of 26 and 45 kilodaltons: Apocrine Secretion Odor-Binding Protein 1 and 2, respectively (ASOB1 and ASOB2). Antisera to these proteins were prepared and used to examine a variety of other body fluids. Several fluids contained an immunoreactive protein with the same electropheretic migration pattern as the 45 KDa protein. Three of these body fluids (tears, nasal secretions and saliva) were separated into aqueous and organic soluble fractions and hydrolyzed to demonstrate that 3M2H could be liberated from the aqueous soluble materials. These results suggest interesting parallels between non-human mammalian odors used as chemical signals and human axillary odor. Previous studies have suggested the axillae as a source of human primer-type pheromones; consequently, if the odors which characterize the underarm are responsible for the pheromonal activity, then the chemistry involved may be similar to that in other mammalian chemical communication systems where proteins act as carriers of one or more chemical signals.

Overexpression, solubilization and purification of rat and human olfactory receptors.

The superfamily of olfactory receptor genes, whose products are thought to be activated by odorant ligands, is critical for odor recognition. Two olfactory receptors, olp4 from rat and OR17-4 from human, were overexpressed in Sf9 insect cells. The presence of the proteins in cell membranes was monitored by immunoblotting with peptide-specific polyclonal antibodies directed against the C-terminal sequences of these receptors and with a mAb against an N-terminal octapeptide epitope tag. A DNA sequence that codes for a His6 tag, which binds tightly to a Ni2+-chelate-affinity column, was incorporated into the N-termini of both genes. The expressed olfactory receptors were found mainly in the cell-membrane fraction. The proteins were difficult to solubilize by many detergents and only lysophosphatidylcholine was found to be both suitable for efficient solubilization of the overexpressed olfactory receptors and compatible with the purification system used. After solubilization, the olfactory receptors were purified to near homogeneity by affinity chromatography on nickel nitrilotriacetic acid resin and by cation-exchange chromatography. Electrophoresis of the purified proteins and visualization with Coomassie Blue staining or by immunoblotting with specific antibodies, revealed bands of 32, 69 and 94 kDa, which were identified as the monomeric, dimeric and trimeric forms of the receptor proteins. The oligomeric forms were resistant to reduction and alkylation, and are therefore thought to be held together by non-covalent hydrophobic interactions that are resistant to SDS. This finding is similar to previous observations for other guanine-nucleotide-binding-regulatory-protein-coupled receptors. Reconstitution in phospholipid vesicles showed that the purified olfactory receptors insert specifically into the lipid bilayer. This provides a means to study functional reconstitution with putative transduction components such as olfactory guanine-nucleotide-binding-regulatory protein.