Chemical Senses Advance Access published online on November 20, 2007
Chemical Senses, doi:10.1093/chemse/bjm073
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Proteomic Analysis of a Membrane Preparation from Rat Olfactory Sensory Cilia
1 Department of Molecular Physiology, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany 2 Functional Proteome Analysis, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
Correspondence to be sent to: Frank Möhrlen, Department of Molecular Physiology, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany. e-mail: moehrlen{at}uni-hd.de
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Abstract |
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The cilia of mammalian olfactory receptor neurons (ORNs) represent the sensory interface that is exposed to the air within the nasal cavity. The cilia are the site where odorants bind to specific receptors and initiate olfactory transduction that leads to excitation of the neuron. This process involves a multitude of ciliary proteins that mediate chemoelectrical transduction, amplification, and adaptation of the primary sensory signal. Many of these proteins were initially identified by their enzymatic activities using a membrane protein preparation from olfactory cilia. This so-called "calcium-shock" preparation is a versatile tool for the exploration of protein expression, enzyme kinetics, regulatory mechanisms, and ciliary development. To support such studies, we present a first proteomic analysis of this membrane preparation. We subjected the cilia preparation to liquid chromatography-electrospray ionisation (LC-ESI-MS/MS) tandem mass spectrometry and identified 268 proteins, of which 49% are membrane proteins. A detailed analysis of their cellular and subcellular localization showed that the cilia preparation obtained by calcium shock not only is highly enriched in ORN proteins but also contains a significant amount of nonciliary material. Although our proteomic study does not identify the entire set of ciliary and nonciliary proteins, it provides the first estimate of the purity of the calcium-shock preparation and provides valuable biochemical information for further research.
Key words: olfactory receptor neurons, proteomic analysis, signal transduction, sensory cilia
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Introduction |
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In mammals, odor perception begins when odorants interact with the sensory surface of the olfactory epithelium in the nose. This surface contains a dense layer of cilia, the sensory organelles of olfactory receptor neurons (ORNs). Exposed to the inhaled air, this ciliary layer represents the only known structure where neurons are in direct contact with the external environment. The cilia detect odorants by exposing metabotropic odorant receptor proteins to the air within the nasal cavity (Buck and Axel 1991
). All components of the signal transduction cascade initiated by these receptors are associated with the ciliary membrane (reviews: Schild and Restrepo 1998; Frings 2001), so that the generation of the olfactory receptor potential is indeed a function of the cilia themselves. Moreover, the constant exposure of the cilia to environmental compounds may require an effective xenobiotic biotransformation metabolism, which protects the olfactory epithelium and the ORNs from cytotoxic compounds and terminates the sensory response (reviews: Marini et al. 1998; Ling et al. 2004). The expression of xenobiotic-metabolizing enzymes in ORNs is supported by the finding that cytochrome oxidase staining, which is correlated with xenobiotic functional activity, was not only observed in epithelial supporting cells but also in ORN perikarya, dendrites and their ciliated knobs, as well as within the mucus layer that contains parts of the ORN knobs, their cilia, and supporting cell microvilli (Pataramekin and Meisami 2005). However, highest activity levels of xenobiotic-metabolizing enzymes were generally found in supporting cells, suggesting that protection of the entire sensory neuroepithelium from environmental compounds is mainly a task of supporting cells (reviews: Dahl and Hadley 1991; Thornton-Manning and Dahl 1997; Marini et al. 1998). ORNs have a very short life span. After operating for only a few weeks, the neurons undergo apoptosis and are replaced by newly differentiating stem cells. This fascinating example of adult neurogenesis is a subject of intense research activity (reviews: Beites et al. 2005; Nicolay et al. 2006; Gheusi and Lledo 2007; Henion and Schwarting 2007) and may also profit from our proteomic analysis.
Biochemical exploration of the olfactory cilia largely relies on a membrane preparation protocol that was established by Chen and Lancet (1984). Ciliary membranes are detached from the olfactory epithelium by a "calcium-shock" and are subsequently collected by ultracentrifugation. The resulting preparation was partially characterized by electron microscopy and analysis of glycoprotein expression (Anholt et al. 1986). It was accepted as an in vitro model for olfactory biochemistry and, from that time on, successfully used for research in olfactory transduction. Virtually, all components of the signal transduction cascade—apart from odorant receptors and transduction channels—have been initially discovered using this preparation. In particular, adenylate cyclase type III (AC III), its stimulatory guanosine triphosphate (GTP)–binding protein Golf, and the cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase PDE1C were characterized by their enzymatic activity which leads to subsequent identification by molecular cloning (e.g., Pace et al. 1985; Sklar et al. 1986; Lowe et al. 1989; Pfeuffer et al. 1989; Yan et al. 1995). Furthermore, the kinetics of odor-induced second messenger signaling (Breer et al. 1990; Boekhoff and Breer 1992), as well as various molecular mechanisms for feedback inhibition and adaptation (e.g., Boekhoff et al. 1992; Schleicher et al. 1993; Wei et al. 1998), were described based on biochemical experimentation with the cilia preparation. Finally, evidence was found for other odor-induced signal transduction cascades, which operate with IP3, Ca2+, or cyclic guanosine monophosphate as second messengers (e.g., Breer and Boekhoff 1992; Moon et al. 1998). Thus, the examination of the cilia preparation yielded many of the molecular details which support—in conjunction with the biophysical identification of transduction channels in the ciliary membrane (Nakamura and Gold 1987; Kleene and Gesteland 1991)—the current concept for vertebrate olfactory signal transduction.
For further investigations, it would be helpful to have an overview of the specific set of proteins present in the sensory cilia. Possible functions of such proteins in signal transduction, adaptation, xenobiotic metabolism, and ORN maturation could be investigated on the grounds of such information. Here, we present a first proteomic analysis of the cilia preparation from rat olfactory epithelium. We have tested the degree of cilia purification using marker proteins for the ciliary and nonciliary cell compartments. To characterize proteins present in the preparation, we have isolated membrane integral and membrane-associated proteins using cetyl trimethyl ammonium bromide (CTAB) to solubilize the proteins. After separating the proteins by two-dimensional (2D) gel electrophoresis, we identified 268 different proteins by LC-ESI-MS/MS tandem mass spectrometry. These proteins were classified according to their expected cellular and subcellular locations and functions. Our data extend the view on biochemical signal processing in olfactory cilia and promote systematic studies of olfactory signal transduction, xenobiotic metabolism, and other functions of ORNs.
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Animals
Preparations of olfactory epithelium and olfactory cilia were obtained from 3- to 6-month-old Wistar rats. For immunohistochemical staining, the olfactory marker protein (OMP)–green fluorescent protein (GFP) mouse line, which contains a gene-targeted insertion of the GFP reporter in the OMP locus (Potter et al. 2001), was kindly provided by Dr Jörg Strotmann (University of Hohenheim). All experiments were performed in accordance with the Animal Protection Law and the guidelines and permissions of the University of Heidelberg, Germany. Animals were killed by CO2 inhalation and decapitation. For immunohistochemical treatment, the rostral part of the skull, containing the nasal cavity, was dissected. For membrane preparations, the head capsule was opened by a sagittal section to remove the olfactory epithelium from the dorsal posterior part of the nasal septum.
Immunohistochemistry and microscopy
Immunohistochemistry was performed as previously described (Bönigk et al. 1999). Rabbit anti-AC III (Santa Cruz Biotechnology, Inc., Heidelberg, Germany, #sc-588, 1:400) was used as primary antibody and visualized by donkey anti-rabbit Alexa 568 (Molecular Probes, Karlsruhe, Germany, A-11057, 1:500) secondary antibody. A 1:300 dilution of a 90-µM 4',6-diamidino-2-phenylindole solution (Molecular Probes, C-7509) was used to stain nuclei. Sections were analyzed using a Nikon TE2000-E Confocal Microscope C1 system at the Nikon Imaging Center at the University of Heidelberg.
Isolation of olfactory ciliary membranes
Olfactory cilia were isolated using the established Ca2+-shock method as described in Bönigk et al. (1999) and Delgado et al. (2003). Briefly, the olfactory epithelium of 5 rats was washed in ice-cold Ringer's solution (120 mM NaCl, 5 mM KCl, 1.6 mM K2HPO4, 25 mM NaHCO3, 7.5 mM D-glucose, protease-inhibitor-Mix M [Serva, Heidelberg, Germany], pH 7.4), transferred to Ringer's solution containing 10 mM CaCl2, and gently stirred for 20 min at 4 °C. The detached cilia were isolated by 3 sequential centrifugation steps for 5 min at 7700 x g. The supernatants were collected and loaded on top of a 45% sucrose solution in Ringer's solution with 10 mM CaCl2 and concentrated at 100 000 x g for 30 min at 4 °C using a Beckman L-70 ultracentrifuge. Ciliary membranes represented by a yellowish layer at the sucrose–supernatant interface were diluted in their 10-fold volume of Ringer's solution with 10 mM CaCl2 and centrifuged at 100 000 x g for 30 min at 4 °C. Routinely, the resulting ciliary membrane pellet was resuspended in 10 mM Tris, 3 mM MgCl2, 2 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid (EGTA), pH 7.4, and stored at –70 °C. Protein yield was measured using the amido black assay (Schaffner and Weissmann 1973) and resulted in 
250 µg per 5 rats.
Whole-tissue membrane protein purification
Olfactory epithelium was homogenized in a glass/Teflon homogenizer in 10 ml ice-cold hypotonic solution (20 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA], 0.1 mM EGTA, 1 mM dithiothreitol [DTT], 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES], 0.02% o-phenanthrolin, complete protease inhibitor cocktail [Roche, Mannheim, Germany], pH 7.4). The suspension was washed by centrifugation at 300 x g for 5 min at 4 °C to separate the membranes from nuclei. The cellular and ciliary membranes were washed and collected in hypotonic solution by 2 centrifugation steps at 100 000 x g for 30 min at 4 °C. The resulting membrane pellet was resuspended in ice-cold hypertonic solution (500 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 20 mM HEPES, 0.02% o-phenanthrolin, complete protease inhibitor cocktail [Roche], pH 7.4), centrifuged (100 000 x g, 30 min, 4 °C), and resuspended in 100 µl of isotonic solution (150 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 20 mM HEPES, 0.02% o-phenanthrolin, complete protease inhibitor cocktail [Roche], pH 7.4).
Gel electrophoresis
1D sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970) using a polyacrylamide concentration of 10% in the separation gel. For the detergent-based solubilization and separation of the ciliary membrane proteins, we applied a technique for 2D gel electrophoresis according to Hartinger et al. (1996) and Navarre et al. (2002). This protocol combines an acidic buffer and the cationic detergent CTAB in the first dimension with an SDS-PAGE in the second dimension. In both dimensions, a vertical gel with a separation length of 11 cm was used and electrophoresis was performed at 18 °C. For the first dimension, the 8% T 1.04% C resolving gel (3 M urea, acrylamide/bisacrylamide, 0.1% CTAB, 4 mM ascorbic acid, 75 mM KH2PO4, pH 2.1 [H3PO4]) and the 8% T 2.56% C stacking gel (1.66 M urea, 0.1% CTAB, 4 mM ascorbic acid, 125 mM KH2PO4, pH 4.1 (H3PO4)) were degassed and polymerized with 0.0125 mM FeSO4 and 0.0012% H2O2. For sample preparation, 1 volume of a 2-fold sample buffer (6 M urea, 0.2% CTAB, 10% glycerol, 75 mM DTT, 0.05% pyronin Y) was transferred to 1 volume of protein solution corresponding to 80 µg protein. The denaturation occurred for 15 min at 70 °C. Electrophoresis toward the cathode was performed at 25 mA in the stacking gel and 45 mA in the separation gel. The resulting gel stripe was washed 4 times for 5 min in deionized water and then equilibrated 4 times for 15 min in an adequate volume of SDS-PAGE lysis buffer. For the second-dimension run, the stripe was positioned in a large well of the 3.8% stacking gel of a 10% Laemmli SDS gel. Electrophoresis toward the anode was performed at 45 mA.
Immunoblot
In total, 10 or 60 µg of proteins from ciliary membranes or whole olfactory tissue were loaded on 1D-SDS gels or 2D-CTAB/SDS gels, respectively, and electroblotted to polyvinylidene difluoride membranes (Machery & Nagel, Dueren, Germany) according to Towbin et al. (1979) using a semidry blotting apparatus. Evenly, protein loading of each probe was confirmed by densitometric quantification of colloidal coomassie CBB G250 stained gels. Nonspecific binding sites were blocked with 5% milk powder dissolved in phosphate-buffered saline (PBS) (8.1 mM Na2HPO4, 1.9 mM NaH2PO4, 130 mM NaCl, pH 7.4) overnight at 4 °C and incubated with primary antibody in MPBST (1% milk powder, 0.05% Tween 20, PBS) for 90 min. The following antibodies and dilutions were used: rabbit anti–AC III 1:100 (Santa Cruz Biotechnology, Inc., #sc-588), rabbit anti-CNGA2 1:100 (Sigma, Steinheim, Germany, #N7529), mouse anti-CNGA4 (mAB7B11, monoclonal, directed against c-terminal residues 392–575; developed by Bradley et al. (1997) and obtained from the Developmental Studies Hybridoma Bank, University of Iowa), rabbit anti-G
olf 1:2000 (Santa Cruz Biotechnology, Inc., #sc-383), goat anti-occludin 1:200 (Santa Cruz Biotechnology, Inc., #sc-8145), rabbit anti-prohibitin 1:200 (Abcam Limited, Cambridge, UK, #ab2996), and goat anti-ezrin 1:50 (Santa Cruz Biotechnology, Inc., #sc-6409). The blots were washed 3 times with 0.1% Triton X-100 in PBS and incubated with adequate horseradish peroxidase–conjugated secondary antibodies (Sigma) in a dilution range of 1:20000–1:80000 in MPBST for 60 min. The blots were washed 3 times with 0.1% Triton X-100 in PBS and once in water. The ECL plus enhanced chemoluminescence system (GE Healthcare, Munich, Germany) was used to monitor bound antibodies.
Mass spectrometry
For mass spectrometric analysis, the ciliary membranes were resuspended in 300 µl of 10 mM Tris, 100 µM phenylmethylsulfonyl fluoridephenylmethylsulfonyl fluoride, 10 mM beta-mercaptoethanol, 3 mM MgCl2, pH 8.0, and purified from nucleic acids with Benzonase (Merck, Germany). After an incubation step for 30 min at 37 °C, the suspension was diluted in 10 mM Tris, 3 mM MgCl2, 2 mM EGTA, pH 7.4, and centrifuged at 100 000 x g for 30 min at 4 °C. A total of 80 µg of ciliary proteins were separated by 2D-CTAB/SDS-PAGE as described above and stained by colloidal coomassie CBB G250 according to Neuhoff et al. (1990). The resulting gel was cut into 63 pieces and digested with trypsin (Promega, Madison, WI). The supernatant from the tryptic digestion and the extracts from steps 1 (acetonitrile [MeCN]/H2O/formic acid [FA], 50.0/49.9/0.1, v/v/v), 2 (MeCN 100%), 3 (H2O/FA, 99.9/0.1 v/v), and 4 (MeCN/H2O/FA, 50.0/49.9/0.1, v/v/v) were combined, evaporated, and dissolved in H2O/FA, 99.9/0.1, v/v.
Nanoscale LC-ESI-MS/MS analysis of the extract aliquots was performed using the CapLC capillary LC system (Waters, Eschborn, Germany) coupled to a hybrid quadruple orthogonal acceleration time-of-flight tandem mass spectrometer (Micromass, Manchester, UK). The LC-ESI-MS/MS device was adjusted with a PicoTip Emitter (New Objective, Woburn, MA) fitted on a Z-spray (Micromass) interface. Peptides were trapped on a Symmetry 300 NanoEase C18 column (Waters). Chromatographic separations were performed on a reversed-phase capillary column Atlantis C18, 3 µm particle size, 75 µm i.d., 15 cm length (Waters), with a flow rate of 200 nl/min.
For weakly stained gel slices (1–3, 5, 14, 17, 24, 28, 29, 33, 34, 37–39, 42–46, 49–51, 53, 55, 58–63; Figure 3A), the chromatography was carried out using a short linear gradient from 5% to 40% solvent B in 35 min and from 40% to 95% solvent B in 55 min (solvent A: H2O/FA, 99.9/0.1, v/v; solvent B: H2O/acetonitrile/FA, 5.0/94.9/0.1, v/v/v). For intensely stained gel slices (4, 6–13, 15, 16, 25–27, 30–32, 35, 36, 40, 41, 47, 48, 52, 54, 56, 57; Figure 3A), the chromatography was carried out using a long linear gradient from 0% to 55% solvent B in 2.3 h and from 55% to 100% solvent B in 3.8 h. Eluted peptides were ionized using a nanoelectrospray ion source. The capillary voltage was set to 2400 V, and the cone voltage was set to 80 V. Data acquisition was controlled by MassLynx 4.0 software (Waters). Low-energy collision-induced dissociation was performed using argon as a collision gas (pressure in the collision cell was set to 5 x 10–5 mbar), and the collision energy was optimized for all precursor ions dependent on their charge state and molecular weight in the range of 25–40 eV. MassLynx raw data files were processed with Protein Lynx Global Server 2.2 software (Waters). Deisotoping was performed using the MaxEnt3 algorithm.
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Protein identification
Processed data were searched against the rat protein subdatabase (67 996 protein sequences) of the National Center for Biotechnology Information (NCBI) nonredundant database (version 071507; 5 269 953 protein sequences) using the Mascot algorithm version v2.1.0 (Matrix Science Ltd, London, UK). The mass tolerance was set to 200 ppm for precursor ions and 0.1 Da for fragment ions. No fragment ions score cutoff was applied when performing the search. Protein hits were taken as identified if 1) the sum Mascot ion score exceeded the significance threshold of 49, 2) a minimum of 1 peptide had an individual ion score exceeding the Mascot identity threshold, and 3) a minimum of 2 nonredundant peptide matches were detected. Under the applied search parameters, a sum Mascot score of >49 typically refers to a match probability of P 
0.0005, where P is the probability that the observed match is a random event. If peptides matched to multiple members of a protein family, we routinely assigned redundant peptide matches to the highest scoring protein. Proteins whose percentage of redundant peptide matches was lower than 40% were considered as identified. The following search parameters were selected: fixed cysteine modification with the carbamidomethyl side chain, variable modification due to methionine oxidation, and one missed cleavage site in the case of incomplete trypsin hydrolysis.
Bioinformatics
For annotation of protein sequences, BLAST (Basic Local Alignment Search Tool) searches (Altschul et al. 1990; Altschul and Gish 1996) at NCBI and sequence-based Harvester searches (Liebel et al. 2004) at FZK were used. Prediction of transmembrane helices was performed by using TMHMM Server v2.0 (Krogh et al. 2001) and SOSUI (Hirokawa et al. 1998). For subcellular localization, the subcellular localization database LOCATE was used (Fink et al. 2006). For analysis of the cellular source of the identified proteins, we compared the respective genes to the expression profiling of ORNs recently published by Sammeta et al. (2007).
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Results |
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Preparation of ciliary membrane proteins
The sensory cilia contribute only a small fraction to the total membrane material of the olfactory epithelium. This is illustrated by displaying the immunosignal of AC III (Figure 1), a protein that is specifically expressed in the sensory cilia. To detach the cilia from the apical surface of the epithelium, we used the "calcium-shock" method that produces the membrane preparation that is routinely used in olfactory biochemistry. We obtained roughly 50 µg protein per animal by this method and first characterized the preparation by a membrane protein enrichment assay (Bönigk et al. 1999). The rationale for this assay is to compare the immunosignals of a protein on 2 western blots made with the same amount of protein (10 µg, verified by amido black assay) from either membranes of the entire olfactory epithelium, including cilia (Figure 2, lane I), or from the cilia preparation gained by calcium-shock treatment (Figure 2, lane II). If the calcium-shock protocol leads to a significant enrichment of ciliary membranes in the preparation, a cilia-specific protein should give rise to a much stronger signal in lane II, whereas proteins with nonciliary localization should be prominent in lane I. Accordingly, we found that 3 proteins which are known to be expressed almost exclusively in the cilia—the cyclic nucleotide-gated channel subunits CNGA2 and CNGA4 as well as AC III—display strong immunosignals in lane II and only weak signals in lane I. Golf, a protein which is homogeneously distributed throughout ORNs (Belluscio et al. 1998) yielded signals of similar intensities in both lanes, thus serving as a control for nonpolarized expression. The protein occludin, which is a constituent of the tight junction complex between epithelial supporting cells and ORNs, was less prominent in the cilia preparation as was prohibitin, a marker protein for intracellular membranes (Mishra et al. 2006). Ezrin is a protein localized in the microvilli of epithelial cells (Elsaesser et al. 2005). Although microvilli are located in direct proximity to olfactory cilia and are in contact with agents used in the cilia-detaching procedure, ezrin was merely detected with similar intensities in both lanes. This finding indicates that the cilia preparation contains a small but detectable amount of microvilli membrane. To estimate the degree of enrichment of the analyzed marker proteins densitometrically, we used the software ImageJ 1.37S. We found that CNGA2, CNGA4, and AC III were enriched about 30-, 20-, and 50-fold, respectively, in the ciliary preparation. With occludin or prohibitin, a 50- and 30-fold depletion was measured. For Golf and ezrin, neither enrichment nor depletion was observed, as shown by the immunoblots. The finding that the ciliary marker proteins are 20–50 times enriched in the cilia preparation, while the microvilli marker ezrin is not detectably enriched, indicates that the preparation contains 20–50 times more ciliary material than microvillar material. In other words, the microvillar contamination of the cilia preparation is only 2–5%. Taken together, these results demonstrate that the preparation obtained by the calcium-shock method is indeed strongly enriched in ciliary membrane but contains a significant amount of nonciliary material.
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2D gel electrophoresis and mass spectrometry
The cilia preparation was subjected to 2D gel electrophoresis using the cationic detergent CTAB in the first and SDS in the second dimension. CTAB is particularly suitable for the solubilization of membrane proteins (Navarre et al., 2002; Rais et al., 2004; Helling et al., 2006), whereas the conventional separation method by isoelectric focusing tends to precipitate membrane proteins (Santoni et al. 2000). Because the signal transduction molecules of the ciliary membrane are of critical interest for olfactory biochemistry, we tried to optimize the yield of membrane proteins. Figure 3A shows a 2D gel, stained with colloidal coomassie, that was obtained from the cilia preparation. Western blots showed that the ciliary marker proteins CNGA2 and CNGA4, as well as Golf, were present on the gel (Figure 3B–D). To obtain an overview of the protein content of the cilia preparation, we divided the entire stained area of the 2D gel into 63 areas and prepared each gel block separately for mass spectrometry by in-gel digestion with trypsin. Data obtained from LC-ESI-MS/MS tandem mass spectrometry of the 63 samples were searched against the NCBInr database using the Mascot algorithm for protein identification. The significance threshold for unambiguous protein identification was a minimum of 2 unique peptide fragments with a sum Mascot score of 
50. In total, 268 distinct rat proteins could be identified in the cilia preparation (Table 1). The identified proteins were grouped according to their expected subcellular localization, and each group is ranked according to their score and to the number of identified peptides per protein. These parameters are an approximate measure for the relative quantity of each identified protein in the samples analyzed by the mass spectrometer (Corbin et al. 2003; Ishihama et al. 2005). They do, however, not reflect the relative amount of proteins in the original cilia preparation prior to CTAB treatment and electrophoresis. The original protein mix can be substantially altered due to specific interactions of each protein with the solvents used in the experiments. The complete absence of the highly glycosylated >210 kDa AC III protein from the protein list is a case in point. The complete protein list including sum Mascot ion scores per number of scored peptides, protein sequence coverage, predicted transmembrane helices, gene annotations, literature references, and short description of the cellular function is available at the authors' Web site (http://www.zoo.uni-heidelberg.de/prot/UM1031). In addition, all individual Mascot search results can be downloaded from this site.
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Bioinformatic analysis of the identified proteins
Figure 4 summarizes distinctive properties of the identified protein set. Molecular weights of 10 to >100 kDa were detected (Figure 4A), illustrating that the CTAB/SDS gel covers a range that represents a considerable fraction of relevant protein sizes. Figure 4B shows that the protein set contains acidic, neutral, and basic proteins with isoelectric point (IP) values ranging from 4 to 12. Importantly, there was no systematic codependence of molecular weight and IP (Figure 4C), demonstrating that no IP range was excluded from any range of protein sizes by the separation method. To identify membrane proteins, we used the prediction algorithms for transmembrane helices of the TMHMM Server v2.0 (Krogh et al. 2001) and the SOSUI (Hirokawa et al. 1998) server for classification of integral membrane proteins. Of the 268 proteins identified in the cilia preparation, 83 (31%) are integral membrane proteins with 1–10 predicted membrane-spanning domains (Figure 4D).
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Cellular source and subcellular localization of the identified proteins
To predict the cellular source of the 268 proteins identified in this proteomic study, we compared the genes encoding these proteins to the list of genes recently published in Sammeta et al. (2007), which predicts with great accuracy whether a gene is expressed primarily by ORNs. For 57 proteins (21%), no corresponding gene entry could be found. Of the remaining proteins, 190 (90%) were found to be expressed in ORNs with, respectively, >99% (P = 0.0001, 110 proteins) and >83% (P = 0.05, 80 proteins) confidence (Table 1, column "In ORN"). This clearly demonstrates that ORNs are the primary cellular source of the cilia preparation.
Using the curated subcellular localization database LOCATE (Fink et al. 2006) and peer-reviewed publications, we categorized all proteins identified in the cilia preparation according to the classification criteria of the Gene Ontology Consortium (Ashburner et al. 2000). We assigned the identified proteins to 3 main groups that are subdivided into different categories (Figure 5A). Of these proteins, 49% could be assigned to the membrane fraction, which includes integral membrane proteins as well as membrane-associated proteins. The remaining 51% are composed of soluble proteins (41%) and proteins belonging to the cytoskeleton (10%). The high percentage of membrane proteins clearly demonstrates the suitability of the calcium-shock method combined with 2D-CTAB/SDS-PAGE for purification of membrane proteins. Within the membrane protein fraction, components of the olfactory signal transduction cascade were robustly identified, including GTP-binding proteins and 2 subunits of the cAMP-gated transduction channel (CNGA2 and CNGA4). The high identification scores of these components are in good accordance with the immunoblot analysis and demonstrate the effective enrichment of ciliary membranes. The absence of AC III from the protein list most likely indicates that the predominant glycosylated form of this protein (>210 kDa, Figure 2) did not enter the separation gel. In addition to ciliary proteins, we also detected proteins belonging to organelle membranes, indicating contributions from the endoplasmic reticulum (ER), mitochondria, the Golgi system, and cytosolic vesicles. Although density-gradient centrifugation was applied to the isolated cilia, a high proportion of soluble proteins were identified which originated from mitochondria, ER, ribosomes, and cytosol. These proteins are mainly involved in metabolism, xenobiosis, protein biosynthesis, and signal transduction processes. The latter category contains the strongly enriched cAMP-dependent phosphodiesterase PDE1C, which is a key component of the signal transduction cascade in olfactory cilia. Thus, our data demonstrate that the enriched cilia preparation resulting from calcium-shock treatment contains a number of proteins that are not associated with the primary transduction process.
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Discussion |
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Sensory cells with polarized protein expression patterns present a special challenge to research. The proteins involved in signal transduction are usually restricted to specific sensory organelles, such as the photoreceptor outer segment or the olfactory sensory cilia. Biochemists need to separate these organelles from the rest of the cells in order to dissect and to study the relevant proteins. Based on deciliation procedures for gill tissue of the mollusc Aequipecten irradians (Link 1973
), Rhein and Cagan (1980) developed an isolation method for ORN sensory cilia that uses a calcium-shock treatment followed by centrifugation to detach and harvest the cilia from the olfactory epithelium. Chen and Lancet (1984) and Anholt et al. (1986) improved this technique and established the cilia preparation for biochemical experimentation with ciliary membranes. Examination of membrane structures by electron microscopy revealed that the ciliary membrane was largely detached from the axoneme and reorganized in vesicles of 100–500 nm diameter (Anholt et al. 1986). This preparation was considered to be contaminated to some extent with membranes from other organelles. But, in the absence of specific ciliary markers, it was not possible to determine its purity. Nevertheless, the cilia preparation was successfully applied in many biochemical studies and yielded information of critical importance for olfactory research. Further biochemical investigations will benefit from a catalogue of proteins present in the cilia preparation because these proteins represent a significant part of the protein inventory involved in sensory function, biotransformation, and other tasks performed by ORNs.
Of our identified proteins, 211 (78%) could be assigned to the GeneChip expression profiling of OMP–GFP cells (GFP+) versus all nonfluorescent olfactory cell types (GFP–) (Sammeta et al. 2007). One hundred and ninety (90%) of them are in fact expressed in ORNs with 83% confidence. Many of the proteins listed in the specific subcellular categories "cilia membrane," "plasma membrane," "intracellular membrane," "rough ER," "ER/Golgi," "peroxisomal," or "mitochondrial" even meet more stringent criteria (GFP+/GFP– ratios 1.3, GFP+ signals >150; Sammeta et al. 2007), where ORN expression is supported with 99% confidence. In contrast, most of the biotransformation enzymes, ribosomal proteins, cytosolic proteins, or cytoskeleton proteins have intermediate GFP+/GFP– ratios (0.5–1.2) and GFP+ signals above 150, indicating that these genes are expressed in ORNs as well in other cell types. In other words, some of these proteins may, therefore, partly originate from contaminating epithelial supporting cells or other olfactory cell types. Ezrin, for example, a cytoskeletal protein expressed in supporting cells but not in ORNs (Elsaesser et al. 2005; GFP+/GFP– ratio = 0.1), points to the presence of small amounts of microvillar material in the preparation. In addition, the xenobiotic-metabolizing enzymes are thought to be expressed in ORNs and epithelial supporting cells but with much higher expression levels in the latter (Dahl and Hadley 1991; Thornton-Manning and Dahl 1997; Pataramekin and Meisami 2005). Nevertheless, in conjunction with the immunoblot experiments it could be stated that the preparation is strongly enriched in ORN membranes and the contribution of other cell types appears to be small.
The detection of odorants involves odorant-binding proteins, olfactory receptor proteins, GTP-binding proteins, AC III, transduction channels, as well as various regulatory proteins (reviews: Frings 2001; Ronnett and Moon 2002). Many of the signal transduction molecules could be identified in our data set with robust peptide signals. They have been localized to the distal segment of the cilia (Menco 1997). Apart from the axoneme, the distal cilia contain no discernible structures, suggesting that this largest part of the cilia is specialized for the transduction process. In contrast, the proximal segment and the ciliary knobs show various subcellular structures (Figure 5B; Andres 1969) that are important for the interpretation of biochemical data obtained from the cilia preparation. Knobs and proximal cilia contain smooth endoplasmic reticulum (sER), and about 50 of our identified 268 proteins are localized to that compartment (sER/microsomal fraction). Most of these proteins are biotransformation enzymes involved in the xenobiotic metabolism as well as various stress-induced proteins (e.g., 4 heat-shock proteins). This collection of cytoprotective proteins may reflect the precarious situation of a neuron, which is exposed to every chemical compound present in the inhaled air. Mitochondria within the ciliary knob supply adenosine triphosphate for the transduction process because no mitochondria are present in the distal segments of the cilia themselves. Notably, the gene expression profiling (Sammeta et al. 2007) indicated that most of the 30 mitochondrial proteins (GFP+/GFP– ratios 1.3, GFP+ signals >150) found in the cilia preparation originated most likely from ORNs. Ultrastructural and light microsopic studies have demonstrated vesicle trafficking in the ciliary knobs (e.g., Mashukova et al. 2006). Thus, sER, endosomes, multivesicular bodies, and other vesicular structures are present in the apical regions of both ORNs and supporting cells (Andres 1969) and are possible sources for the microsomal and peroxisomal proteins in our data set. But, again, the gene expression profiling of these proteins points most likely to an ORN origin. The presence of 35 cytosolic proteins, mainly enzymes of the glycolytic metabolism and various signal transduction processes, may be interpreted as a consequence of vesicle formation and trapping of cytosol during the calcium-shock treatment (Anholt et al. 1986). Finally, a small number of nucleus- or rough ER–associated proteins (in total 19 proteins; 7%) was detected. Considering that the olfactory epithelium undergoes a continuous turnover and that a sizeable fraction of ORNs is in the process of apoptosis at any time, this contamination with somatic proteins appears small.
In summary, this study revealed that the cilia preparation routinely used in olfactory biochemistry is indeed highly enriched in ciliary over nonciliary membranes. Our proteomic analysis provides a window into the set of proteins present in the cilia preparation and suggests that the cilia are detached close to their base at the dendritic endings of the ORNs. Many proteins originating from sER and mitochondria (in total 80 proteins), but much fewer from rough ER and nucleus (in total 19 proteins), are present in the preparation. This suggests that part of the apical segment of the ORNs (ciliary knobs plus cilia) is collected in the preparation, whereas somatic and dendritic materials are largely excluded. For biochemical analyses, it should be taken into account that the preparation contains proteins related to different physiological tasks, which include signal transduction, biotransformation, metabolism, neurogenesis, and apoptosis.
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Deutsche Forschungsgemeinschaft (Fr 937/8); Studienstiftung des Deutschen Volkes (D.K.).
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We are grateful to Dr Tore Kempf (DKFZ Heidelberg) for valuable help with the protein analysis and to Dr Urban Liebel (Karlsruhe Research Center) for assistance with the FZK Harvester. We thank the Nikon Imaging Center at the University of Heidelberg for access to confocal microscopy.
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Accepted 4 October 2007
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