JP2003319781A - Olfactory cell-specific protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a stomatin related olfactory (SRO) protein, an antibody thereto and an sro gene useful as an odor signal transmission controlling chemical or for elucidating an odor signal transmission function thereof. <P>SOLUTION: The protein is the following (a) or (b). (a) a protein having an amino acid sequence represented by a specific sequence or (b) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted, added or inserted in the specific sequence (a) and forming a complex with adenylyl cyclase type III. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel protein involved in the control of odor signal transduction in the cilia of olfactory nerve cells and a gene encoding the same.

[0002]

2. Description of the Related Art Odor is received by olfactory cells in the olfactory epithelium located on the upper wall of the nasal cavity. These olfactory cells are bipolar nerve cells that extend a dendrite and a nerve axon from a cell body with a nucleus. The tip of the dendrite protrudes from the olfactory tissue, and the cilia, which is a motile cilia that serves as a place for transducing neural information, is derived from the tip. The initial process of odor information conversion in the cilia of olfactory cells begins with the binding of odor molecules to the 7-transmembrane protein. This information is transmitted to the olfactory specific G protein (Golf). Go
The α-subunit of lf has high homology with Gsα, which is a G protein, and is therefore of the adenylate cyclase type.
It is believed to be linked to III. Since adenylate cyclase in cilia samples is activated by many odorants, it is considered that this adenylate cyclase-cAMP system commonly mediates information conversion to any odor stimulus. That is, it is said that cAMP in olfactory cilia by adenylate cyclase increases the cation conductance of ciliated cell membrane and converts chemical information into an electrical signal.

[0003]

However, there are still many unclear points regarding the mechanism of olfactory information conversion,
Elucidation of new proteins and genes involved in odor signal transduction is desired. Therefore, the present invention is to provide a novel protein involved in the transmission of odor signals and its gene.

[0004]

[Means for Solving the Problems] Therefore, the present inventor has conducted various studies to elucidate the function of the protein present in the cilia of olfactory nerve cells. It was found that it is a protein encoded by a novel gene having high homology to stromatin, and is mainly present in the lipid rafts of the cilia. Furthermore, antibodies against the protein that is the expression product of the gene include adenylate cyclase type III and caveolin (c
aveolin) was found to be co-immunoprecipitated. Furthermore, it was also found that when the antibody was added to the ciliary membrane and then stimulated with forskolin, the cAMP producing activity was doubled as compared with the case where the control standard serum was added. Therefore, this novel protein was found to be adenylate cyclase-type in olfactory neurons.
Forming odor signaling complex with III and caveolin,
The inventors have elucidated that the protein is a novel protein that controls the transmission of odor signals through adenylate cyclase, and completed the present invention.

That is, the present invention provides the following protein (a) or (b). (A) a protein having the amino acid sequence represented by SEQ ID NO: 1, (b) an amino acid sequence in which one or several amino acids in SEQ ID NO: 1 have been deleted, substituted, added or inserted, and an adenylate cycler A protein that forms a complex with zetype III.

[0006] The present invention also provides a gene encoding the above protein.

The present invention further provides an antibody against the above protein.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The protein of the present invention (hereinafter referred to as SR
O protein (stomatin related olfactoryprotein)
Abbreviated) means (a) a protein having the amino acid sequence represented by SEQ ID NO: 1, and (b) 1 in SEQ ID NO: 1.
Alternatively, it is a protein having an amino acid sequence in which several amino acids have been deleted, substituted, added or inserted, and which forms a complex with adenylate cyclase type III (hereinafter abbreviated as ACIII). Here, one of the amino acids in the amino acid sequence shown in SEQ ID NO: 1 in the protein (b)
Alternatively, the degree of deletion, substitution, addition or insertion of several amino acids is not limited as long as the modified protein forms a complex with ACIII. For example, the amino acid sequence of SEQ ID NO: 1 is 80% or more, and 90% or more. % Or more, and particularly preferably 95% or more.

Further, the SRO protein of the present invention also includes those having a sugar bound thereto. Of the SRO proteins of the present invention, the protein having the amino acid sequence represented by SEQ ID NO: 1 is derived from mouse, but the SRO protein of the present invention also includes proteins other than mouse as long as the condition (b) above is satisfied. . That is, proteins derived from spinal animals such as rat, pig, cow, human, and rabbit are also included, as well as proteins expressed in Escherichia coli and the like by a recombinant technique using the gene described below.

The gene of the present invention is not particularly limited as long as it is a gene encoding the protein (a) or (b) described above, but a gene comprising the following DNA (A) or (B) is particularly preferable. (A) a DNA having the nucleotide sequence represented by SEQ ID NO: 2,
(B) A DNA that hybridizes with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 2 under stringent conditions and encodes a protein that forms a complex with adenylate cyclase type III.

In the present invention, a gene includes not only a double-stranded DNA but also a single-stranded DNA of each of a sense strand and an unsense strand constituting the double-stranded DNA. In addition, the stringent condition in the present invention means, for example, 0.1% SD.
50 ° C in 0.2xSSC containing S, or 0.1% SDS
And 60 ° C. in 1 × SSC. As a DNA that hybridizes under such stringent conditions, a DNA having 70%, 80%, and particularly 90% identity with the nucleotide sequence represented by SEQ ID NO: 2 is preferable.

The SRO gene of the present invention can be easily produced and obtained by a general genetic engineering method [Mo.
lecular Cloning 2d Ed, Cold Spring Harbor Lab. Pre
ss (1989); Continuation Biochemistry Laboratory "Gene Research Methods I, II, I"
II ", edited by the Japanese Biochemical Society (1986), etc.]. Specifically, a cDNA library is prepared from an appropriate source in which the gene of the present invention is expressed by a conventional method, and a desired clone is prepared from the library using an appropriate probe or antibody specific to the sro gene of the present invention. It can be carried out by selecting [Proc. Natl. Acad. Sci., USA., 78, 6613 (198
1); Science, 222, 778 (1983), etc.]. In the above,
Examples of the origin of cDNA include various cells and tissues expressing the sro gene of the present invention, cultured cells derived from these, and the like. Also, separation of total RNA from these, m
Separation and purification of RNA, acquisition of cDNA and cloning thereof, etc. can all be carried out according to conventional methods.
Further, cDNA libraries are commercially available, and in the present invention, those cDNA libraries, for example, various cDNA libraries marketed by Clontech Lab. Inc. and the like can also be used. The method for screening the sro gene of the present invention from a cDNA library is not particularly limited, and can be a conventional method. Specifically, for example, cDNA
A method for selecting a corresponding cDNA clone by immunological screening using a specific antibody of the protein, a plaque hybridization using a probe that selectively binds to a target DNA sequence, colony high Examples thereof include hybridization and combinations thereof. As the probe used here, DNA chemically synthesized based on the information on the nucleotide sequence of the sro gene of the present invention can be generally used, but the already obtained gene of the present invention or a fragment thereof can also be favorably used. Available. Further, a sense primer or antisense primer set based on the nucleotide sequence information of the sro gene of the present invention can also be used as a screening probe. For obtaining the sro gene of the present invention, a PCR method [Science, 230, 1350 (1985)]
The DNA / RNA amplification method by can be preferably used. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method [Rapid amplification of cDN
A ends; Experimental Medicine, 12 (6), 35 (1994)], especially 5'-RA
CE method [MA Frohman, et al., Proc. Natl. Acad. Sc
i., USA., 8, 8998 (1988)] is preferable.
The primer used in adopting such a PCR method can be appropriately set based on the sequence information of the sro gene of the present invention clarified by the present invention, and can be synthesized by a conventional method. The amplified DNA / RNA fragment can be isolated and purified according to a conventional method as described above, for example, gel electrophoresis. Further, the sro gene of the present invention or various DNA fragments obtained above can be prepared by a conventional method,
For example, the dideoxy method [Proc. Natl. Acad. Sci., USA.,
74, 5463 (1977)] and the Maxam-Gilbert method [Methods
in Enzymology, 65, 499 (1980)] and the like, or simply using a commercially available sequencing kit or the like, the nucleotide sequence can be determined. According to the sro gene of the present invention thus obtained, the presence or absence of the expression of the sro gene of the present invention in an individual or various tissues can be specifically detected by utilizing, for example, part or all of the nucleotide sequence of the gene. can do. Such detection can be performed according to a conventional method, for example, RT-PCR [Reve
rse transcribed-Polymerase chain reaction; ES Ka
wasaki, et al., Amplification of RNA. In PCR Proto
col, A Guide to methods and applications, Academic
Press, Inc., San Diego, 21-27 (1991)] RNA amplification and Northern blotting analysis [Molecular Cloning,
Cold Spring Harbor Lab. (1989)], in situ RT-P
CR [Nucl. Acids Res., 21, 3159-3166 (1993)] and i
Measurement at cell level using n situ hybridization, NASBA method [Nucleic acid sequence-b
ased amplification, Nature, 350, 91-92 (1991)] and various other methods. Suitably, the detection method by RT-PCR can be mentioned.
The primer used in the PCR method is not particularly limited as long as it is unique to the gene capable of specifically amplifying only the sro gene of the present invention. It can be set appropriately based on the above. Examples of the normal primer include those having a partial sequence of the gene of the present invention having a length of about 10 to 35 nucleotides, preferably about 15 to 30 nucleotides. According to the sro gene of the present invention, SR can be obtained by using an ordinary genetic engineering technique.
O protein or a protein containing the O protein can be easily produced in a large amount and stably. Based on the sequence information of the sro gene of the present invention, the SRO protein of the present invention has a gene recombination technique of a conventional method [eg Science, 224,
1431 (1984); Biochem. Biophys. Res. Comm., 130, 6
92 (1985); Proc. Natl. Acad. Sci., USA., 80, 5990.
(1983), etc.]. The production of said protein is more particularly carried out by recombinant DN in which the gene encoding said desired protein is expressed in host cells.
It is carried out by preparing A (expression vector), introducing this into a host cell for transformation, culturing the transformant, and then recovering from the resulting culture. As the host cell, both prokaryotic and eukaryotic organisms can be used. For example, as a prokaryotic host, commonly used ones such as Escherichia coli and Bacillus subtilis are widely mentioned, preferably Escherichia coli, especially Escherichia coli (Es
Examples include those contained in the cherichia coli) K12 strain. In addition, eukaryotic host cells include cells such as vertebrates and yeasts. The former include, for example, monkey cells such as COS cells [Cell, 23: 175 (1981)] and Chinese hamster ovary cells. And its dihydrofolate reductase-deficient strain [Proc. Natl. Acad. Sci., USA., 77: 4216
(1980)] and the like, but as the latter, Saccharomyces yeast cells and the like are preferably used. Of course, it is not limited to these. The SRO protein of the present invention can also be produced by a general chemical synthesis method according to the amino acid sequence shown in SEQ ID NO: 1. The method includes a conventional liquid phase method and a solid phase method for peptide synthesis.
The SRO protein of the present invention can be suitably used as an immune antigen for producing its specific antibody, and by using this antigen, desired antiserum (polyclonal antibody) and monoclonal antibody can be obtained. The method of producing the antibody itself is well understood by those skilled in the art, and these conventional methods can be followed in the present invention as well (for example, sequel biochemistry experiment course "immunobiochemistry research method", edited by the Japanese Biochemical Society). (1986) etc.]. The antibody thus obtained can be used, for example, for purification of SRO protein and measurement or detection thereof by an immunological method.

[0013]

EFFECTS OF THE INVENTION The SRO protein of the present invention (1) is specific to olfactory nerve cells, and (2) a group of proteins involved in odor recognition (odorant receptor, G-olf, adenylyl cyclase).
Type III (ACIII), CNG channel, etc.) is mainly present in the cilia structure. (3) SRO protein was mainly present in lipid rafts in cilia. Lipid rafts are microdomains that exist in biological membranes that are rich in glycosphingolipids and cholesterol, and are known to function as a field for transduction of various signals across lipid bilayers. Further, (4) immunoprecipitation of olfactory epithelial membrane protein solubilized by a nonionic surfactant with anti-SRO antibody can regulate ACIII and odor signal, which play a major role in the generation of odor signal in cilia. It was shown to co-precipitate with the known caveolin. This indicates that the SRO protein forms a complex with ACIII and caveolin in lipid rafts. Further, (5) cAMP is produced when the isolated olfactory nerve cell cilia are stimulated with forskolin. If SR
If the O protein forms an odor signal transduction complex with ACIII, it is possible that the cAMP production activity is changed by the action of anti-SRO antibody on the cilia. Based on such a hypothesis, it was shown that when anti-SRO antibody was added to the ciliary membrane and then stimulated with forskolin, the cAMP producing activity was increased about 2-fold as compared with the case where the control standard serum was added. From these results S
It is considered that the RO protein forms an odor signal transduction complex with ACIII and caveolin in the lipid rafts of the olfactory nerve cell cilia, and controls the transduction of odor signals via ACIII.

Therefore, the SRO protein of the present invention, its antibody and sro gene are useful as agents for controlling odor signal transduction or for elucidation of these odor signal transduction functions.

[0015]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1 (Cloning of sro gene) Fluorescent differential display (FDD) was performed according to the protocol described in the literature (Ito et al., FEBS Lett. 351, 231-6, 1994). The prospective cDNA is used as a plasmid vector pGEM-T.
(Promega). Using the sro cDNA obtained by the FDD screening as a probe, the full-length cDNA was transformed into the phage vector λZAPII.
Olfactory epithelium of mouse (Stratagene)
epithelium, OE) cDNA library. The inserted cDNA was isolated by in vivo excision using ExAssist helper phage (Stratagene). Using the 5'-upstream region of the sro gene as a probe, sro cDNA was used for
It was isolated from a mouse genomic library using L3-SP6 / T7 (Clonetech). The nucleotide sequence of the obtained sro gene is shown in SEQ ID NO: 2, and the amino acid sequence predicted from the sequence is shown in SEQ ID NO: 1.

Example 2 (SR in HEK293 cells
Expression of O-GFP fusion protein) Primers 5'-gaattcatggattcaccggagaaact-3 '(SEQ ID NO: 3) and 5'-gtcgacttggctttagcagtgaccttct-3' (SEQ ID NO: 4) were used to prepare EcoRI site (5 'side) and Sal.
The sro coding sequence added with I site (3 'side) was amplified. The amplified fragment was cloned into the plasmid vector pEGFP-N1 (Clontech),
HEK293 cells were transfected with the lipofetamine plus reagent (Invitrogen). After incubation for 3 days, total protein was extracted from transfected and non-transfected cells, dissolved in SDS sample buffer and subjected to SDS-PAGE.

Example 3 (Preparation of antibody) N-terminal peptide of SRO protein (SRO protein, 1-19 amino acid residues and cysteine-binding peptide:
SR using MDSPEKLEKNNLVGTNKSR-C (SEQ ID NO: 5)
A polyclonal antibody against O protein was prepared.
Inject Maleimicle-Activated mcKLH Kit (PIERCE)
Was used to bind keyhole limpet hemocyanin to the peptide and injected into 2 guinea pigs. Booster immunization was performed three more times, and whole blood was collected from all animals. From the obtained blood, HiTrap affinity column (Amersham
Anti-SRO antibody was purified using

Example 4 Comparison of Stomatin Family Proteins (FIGS. 1 to 4) a) The deduced amino acid sequences of SRO and proteins belonging to the stomatin family were compared. (FIG. 1) Amino acid sequences are shown in single letter code. Putative transmembrane domains are shaded. Stomatine signatures are boxed. Preserved, potentially palmitated, 23,4
The cysteine residues at the 6th and 80th residues are shown in bold type. The asterisk indicates the position where the same amino acid residue is conserved. The dots indicate where amino acid residues with similar properties are conserved. The gene is derived from C. elegan
s) (C), mouse (M), human (H). b) The dendrogram of stromatin family proteins is shown in FIG. Mouse (M), human (H), nematode (C)
The protein that belongs to the stromatin family of origin is C
It was analyzed by the LUSTAL algorithm. The length of the branches in the figure is proportional to the difference in arrangement. Mouse and human SRO and stomatin are present in the nematode UNC-1 and MEC-2 clusters. c) The hydrophobic profiles of mouse SRO, stomatin, and nematode UNC-1 were analyzed by the Kyte-Doolittle algorithm (FIG. 3). The vertical axis represents the degree of hydrophobicity. The number of amino acid residues is shown on the horizontal axis. d) The secondary structure of SRO of mouse, stomatin, and UNC-1 of nematode was predicted (FIG. 4). Regions presumed to be rich in β-sheet and α-helices are indicated by gray and white squares, respectively. The number of amino acid residues is shown on the horizontal axis.

Example 5 Expression of sro gene specific to olfactory epithelium a) Northern blot analysis of sro transcript was performed (FIG. 5). 3 μg of polyA + RNA was extracted from various mouse tissues, separated by agarose gel electrophoresis, transferred to a nylon membrane, and ³² P-labeled sr was added.
o Hybridization with cDNA was performed. After washing off the probe, apply the same nylon membrane to β.
-Hybridization with actin probe was performed. b) The sro transcript was detected by the RT-PCR method (Fig. 6). Total-RNA was extracted from the olfactory epithelium (OE), vomeronasal epithelium (VNE), retina (retina), tongue (tongue), treated with RNase-free DNaseI, and then reverse-transcribed using random hexamer primers. , CDNA was synthesized. The PCR reaction was performed using primers for sro, stomatin and control tubulin. c) The Olf-1 binding sequence upstream of the sro gene is shown (FIG. 7). Adenylate cyclase type III, Gol
f, olfactory nerve-specific CNG channel (olfactory-specif
ic cyclic nucleotide-gated channel) (OcNC),
The upstream genomic sequences of OMP (olfactory marker protein) and sro were compared. The Olf-1 binding sequence is shaded. The common sequence is shown in the top row.

Example 6 Expression of the sro gene in the olfactory epithelium and vomeronasal epithelium was analyzed using the in situ hybridization method. The results are shown in Fig. 8. OMP (olfactorymar) labeled with digoxigenin in sections of olfactory epithelium and vomeronasal epithelium
ker protein) (a, e, i, m), stomatin (stoma
tin) (b, f, j, n), and sro (c, g, k,
Hybridization was performed with the antisense RNA probe of o). Sro as a negative control
Of the sense probe of (d,
h, l, p). Low magnification (ad, i-1) and high magnification (e
-H, mp) images are shown. Expression of the sro gene was found only in mature olfactory neurons. Stomatin and OMP gene expression was detected in vomeronasal neurons, but sro
No gene expression was seen.

Example 7 Western blotting analysis of SRO a) The detection results of SRO-GFP (green fluorescent protein) fusion protein are shown (FIG. 9a). HEK29
3 cells (mock) and HEK293 cells (SRO-G) transfected with the sro-GFP fusion gene.
FP) -derived cell extract is separated by SDS-PAGE,
Western blotting was performed using an antibody against the N-terminal peptide of SRO. 60kDa SRO-G
FP fusion protein and trace amounts of degradation products were detected from the transfected cells. b) shows the results of detection of endogenous SRO protein (Fig. 9)
b). Soluble tissue extract of olfactory epithelium (OE), cell membrane,
The olfactory cilia were fractionated, separated by SDS-PAGE, and detected by Western blotting using SRO antibody. An endogenous SRO band having a molecular weight of about 30 kDa was detected in the cell membrane and the olfactory cilia fraction. c) SR of 30 kDa molecular weight specific for olfactory epithelium (OE)
The detection result of O is shown (FIG. 9c). Cell extracts from various mouse tissues were separated by SDS-PAGE and detected by Western blotting using SRO antibody. Thirty
The SRO band of kDa was detected only in the olfactory epithelium (OE).

Example 8 The results of SRO protein detection using immunohistochemistry are shown (FIG. 10). Mouse olfactory epithelium (OE), (a, b),
Vomeronasal epithelium (VNE), (e, f), olfactory bulb (OB),
SRO protein in (g, h) 16 μm thick tissue sections was detected using SRO antibody. As a negative control, the antigen peptide was added to the SRO antibody solution and immunohistochemistry was performed (+ Ag). The low-magnification image is shown in the upper row, and the high-magnification image is shown in the lower row. By immunohistochemistry, SRO protein was detected in olfactory cilia (cilia) and (ci) of olfactory nerve cells in the olfactory epithelium, but not in villus (microville) (mv) of vomeronasal epithelium.

Example 9 One olfactory nerve cell cilia isolated from mouse olfactory epithelium (OE)
% Triton X-100. The obtained lysate was used for Optiprep flotation gradient (AXIS-SHIE
It fractionated using LD company. Seven fractions from top to bottom were collected and subjected to SDS-PAGE. Stain one gel with Coomassie Brilliant Blue (CBB),
The rest was subjected to Western blotting. Blots are anti-SRO antibody (α-SRO), anti-caveolin-1 antibody (α-cav) or anti-transferrin receptor antibody (α
-TrfR). Caveolin-1 and transferrin receptor are lipid raft-bound and non-lipid raft-bound membrane proteins, respectively. As a result, as shown in FIG. 11, both SRO protein and caveolin-1 were present in the low specific gravity fraction (lanes 1 and 2). From this result, SRO
It was revealed that the protein exists in the lipid raft part of the olfactory nerve cell cilia.

Example 10 Triton X-100 insoluble fraction in olfactory epithelium (OE) membrane sample
-Solubilized with octyl glucopyranoside, anti-SRO antibody, anti-SRO antibody
Immunoprecipitation was performed using either caveolin-1 antibody or anti-ACII antibody. Immunoprecipitation was performed using guinea pig serum (IgG) as a control. Immunoprecipitation products are anti-SRO antibody, anti-cav
Analysis was carried out by Western blotting using any of the eolin-1 antibody, anti-ACII antibody and anti-transferrin receptor antibody (Fig. 12). Olfactory epithelium (OE) without immunoprecipitation
Membrane proteins were similarly analyzed by Western blotting. The antibody used for immunoprecipitation is shown in the top row. The antibody used for Western blotting is shown on the left side. As a result, it was confirmed that ACIII and caveolin-1 were immunoprecipitated using the anti-SRO antibody. Therefore, SRO was shown to form a complex with ACIII and caveolin in lipid rafts.

Example 11 Cilia of olfactory nerve cells were pre-incubated with anti-SRO antibody (white bar (+ SRO-Ab) in FIG. 13 or pre-immune guinea pig IgG (grey bar, + pre-immune) in ice water for 15 minutes. The group in which the cilia were not treated with any antibody was used as a control (black bar, control).
Stimulation with TPγS or 5 μM forskolin at 37 ° C. for 20 minutes induced the production of cAMP. After the reaction c
The production of AMP was quantified. The results are shown in Fig. 13. The vertical axis shows the amount of cAMP per 1 mg cilia protein (pmol / mg protein). The control of cAMP concentration produced by GTPγS stimulation was 2400 ± 110 pmol
/ Mg protein, 6200 ± 1100 pmol / mg protein when pre-incubated with anti-SRO antibody, and 1950 ± 8 for pre-immune guinea pig IgG
It was 10 pmol / mg protein. The cAMP concentration produced by forskolin stimulation was 2730 ± 450 pmol / mg protein in the control and 5800 ± 930 pmol in the case of preincubation with anti-SRO antibody.
/ Mg protein, 310 in the case of preimmune guinea pig IgG
It was 0 ± 410 pmol / mg protein. The cAMP production activity by the anti-SRO antibody was significantly higher than that in the pre-immune guinea pig IgG (by the Student t test, p <0.004 in the case of GTPγS and p <0. 000001)).

Thus, when SRO antibody was added to the ciliary membrane and then stimulated with forskolin, the cAMP producing activity was doubled as compared with the case where the control standard serum was added. Therefore, it was revealed that SRO regulates odor signal transduction by forming an odor signal transduction complex with ACIII.

[0028]

[Sequence list]                                SEQUENCE LISTING        <110> Japan Science and Technology Corporation <120> Olfactory specific protein <130> P02031405 <140> <141> <160> 5 <170> PatentIn Ver. 2.1 <210> 1 <211> 287 <212> PRT <213> mouse <400> 1 Met Asp Ser Pro Glu Lys Leu Glu Lys Asn Asn Leu Val Gly Thr Asn   1 5 10 15 Lys Ser Arg Leu Gly Val Cys Gly Trp Ile Leu Phe Phe Leu Ser Phe              20 25 30 Leu Leu Met Leu Val Thr Phe Pro Ile Ser Val Trp Met Cys Leu Lys          35 40 45 Ile Ile Lys Glu Tyr Glu Arg Ala Val Val Phe Arg Leu Gly Arg Ile      50 55 60 Gln Ala Asp Lys Ala Lys Gly Pro Gly Leu Ile Leu Val Leu Pro Cys  65 70 75 80 Ile Asp Val Phe Val Lys Val Asp Leu Arg Thr Val Thr Cys Asn Ile                  85 90 95 Pro Pro Gln Glu Ile Leu Thr Arg Asp Ser Val Thr Thr Gln Val Asp             100 105 110 Gly Val Val Tyr Tyr Arg Ile Tyr Ser Ala Val Ser Ala Val Ala Asn         115 120 125 Val Asn Asp Val His Gln Ala Thr Phe Leu Leu Ala Gln Thr Thr Leu     130 135 140 Arg Asn Val Leu Gly Thr Gln Thr Leu Ser Gln Ile Leu Ser Gly Arg 145 150 155 160 Glu Glu Ile Ala His Ser Ile Gln Thr Leu Leu Asp Asp Ala Thr Glu                 165 170 175 Leu Trp Gly Ile Arg Val Ala Arg Val Glu Ile Lys Asp Val Arg Ile             180 185 190 Pro Val Gln Leu Gln Arg Ser Met Ala Ala Glu Ala Glu Ala Thr Arg         195 200 205 Glu Ala Arg Ala Lys Val Leu Ala Ala Glu Gly Glu Met Asn Ala Ser     210 215 220 Lys Ser Leu Lys Ser Ala Ser Met Val Leu Ala Glu Ser Pro Val Ala 225 230 235 240 Leu Gln Leu Arg Tyr Leu Gln Thr Leu Thr Thr Val Ala Thr Glu Lys                 245 250 255 Asn Ser Thr Ile Val Phe Pro Leu Pro Met Asn Ile Leu Glu Gly Ile             260 265 270 Gly Gly Ile Ser Tyr Gly Asn Asn Lys Lys Val Thr Ala Lys Ala         275 280 285 <210> 2 <211> 1835 <212> DNA <213> mouse <400> 2 ccttcctctc cacacaccac tgagctgatg cctgctgcca ggctcgaatc ccgacccagg 60 agcacctgca agatttgaaa ggcacctcaa gaatgagatg gattcaccgg agaaactgga 120 gaagaacaat ttagtgggca ccaacaaaag ccggcttggt gtgtgcggct ggatcctgtt 180 tttcctttcc ttcctgctga tgctcgttac cttcccaatc tcagtatgga tgtgcttgaa 240 gatcattaag gagtatgagc gcgctgttgt cttcagactg ggacgcatcc aagctgataa 300 agccaaggga ccaggtttga tcctggtcct gccttgcatt gacgtgtttg tcaaagtaga 360 tcttcgaacg gttacttgta acattcctcc acaagagatc ctcacgagag actctgtgac 420 cacccaagtg gatggagtcg tctattacag aatctacagc gccgtctcag ccgtggccaa 480 tgtgaacgat gttcaccagg ccacgttcct gctggctcag accactctga gaaatgtctt 540 agggacacag accttgtccc agatcttgtc tgggcgagaa gagatcgcac atagcatcca 600 gaccttgctt gatgatgcca ccgagctctg gggaatccga gtggcccggg tggaaatcaa 660 agatgtccgg attcctgtgc agctgcagag gtccatggcg gctgaggctg aggctacccg 720 ggaagccaga gccaaggtcc ttgcagcaga gggagaaatg aatgcttcaa agtctctgaa 780 gtcagcttcc atggtgctgg cagagtcccc cgtcgccctc caacttcggt acctgcagac 840 cttgaccaca gtggccacgg agaaaaattc caccattgtg tttcccttgc cgatgaacat 900 acttgagggc attggtggca tcagctatgg caacaataag aaggtcactg ctaaagcctg 960 aggtcttcca cgtaacagcc tgctacttag aaaggccttt ctgtaccatg gagaagtcag 1020 ccagtagaga ccaatgcacg cgcacacaca cacacacaca cacacacaca cacacacaca 1080 cacacacaca cactcatgca ccatgaataa aatacggtaa ctccacagct agcagaattc 1140 ccagagagac atagaagcta cacaaacaaa aacaaaatcg agagccatgc actgctttga 1200 ttttaaaggc ataaccaata ttcaatattc atatataatt agttagtggt tgtccctgac 1260 tatcaacaga gaatctctca gggtgggcgg agactctagg tgactgataa gaactttaga 1320 atttaagctg tgttggagaa ggaccctagg tcatttgtaa agaaaaagca gatcccacat 1380 ttatgatatg catttgatcc actgttccaa taaaacgact tgctttagtt caagtaagaa 1440 aacgactcaa ggctgaaact taggacggat ttggatgtag aaaataaaaa accgctgcag 1500 catagcagca acacggggca aaggtaccag gtcaggccca gctctcttga caccctggat 1560 gtcctgttaa agtgctggcg ttctcagcac cctcacacct ttgtcagggc cagaggcaca 1620 gcaaggcagc ctagcacagg gtaatttgct tactagtaag accttcagag ccactgggga 1680 ggtaggtctg tgaattgcat gttttcactc tgtaagtccc aagagccata gtgattaagg 1740 gctgtttgta atctttcccc agctcaaaca acttgttgca aataaaaaaa aaaaggaagt 1800 cataaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1835 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <400> 3 gaattcatgg attcaccgga gaaact 26 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <400> 4 gtcgacttgg ctttagcagt gaccttct 28 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <400> 5 Met Asp Ser Pro Glu Lys Leu Glu Lys Asn Asn Leu Val Gly Thr Asn   1 5 10 15 Lys Ser Arg Cys              20

[Brief description of drawings]

FIG. 1 is a diagram showing a comparison of the deduced amino acid sequences of SRO and proteins belonging to the stomatin family.

FIG. 2 is a diagram showing a dendrogram of stromatin family proteins.

[Fig. 3] Mouse SRO, stomatin, and nematode UNC-
It is a figure which shows the result of having analyzed the hydrophobicity profile of 1 by the Kyte-Doolittle algorithm.

FIG. 4 Mouse SRO, stomatin, and nematode UNC-
It is a figure which shows the secondary structure prediction of 1.

FIG. 5 shows Northern blot analysis of sro transcripts.

FIG. 6 shows the detection results of sro transcripts by RT-PCR.

FIG. 7 shows an Olf-1 binding sequence upstream of the sro gene.

FIG. 8: sr by in situ hybridization method
It is a figure which shows the analysis result of the expression in the olfactory epithelium and vomeronasal epithelium of o gene.

FIG. 9 is a diagram showing the results of Western blotting analysis of SRO.

FIG. 10 shows detection of SRO protein using immunohistochemistry.

FIG. 11: CBB staining of protein extracted from cilia of olfactory nerve cells, fractionated by density centrifugation, and anti-SR
O antibody (α-SRO), anti-caveolin antibody (α-ca
v), anti-transferrin receptor antibody (α-TrfR)
It is a figure which shows the result of the western blotting using.

FIG. 12: Anti-SRO antibody (α-SRO), anti-caveolin antibody (α-cav), anti-ACIII antibody (α-ACII)
I), anti-transferrin receptor antibody (α-Trf
It is a figure which shows the result of the immunoprecipitation of the protein extracted from the cilia of an olfactory nerve cell using R).

FIG. 13 shows GTPγ of the ciliary membrane of olfactory nerve cells preincubated with anti-SRO antibody (+ SRO-Ab) or pre-immune guinea pig IgG (+ pre-immune) and the control ciliary membrane to which no antibody was added.
It is a figure which shows the amount of cAMP production when stimulated with S or forskolin.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/88 C12N 15/00 ZNAA (72) Inventor Akio Tsuboi 1446-1 Mabashi, Matsudo-shi, Chiba Bruce House AF Term (Reference) 4B024 AA01 AA11 BA07 BA63 CA04 CA07 DA02 EA03 EA04 GA11 GA18 HA03 HA11 4B050 CC03 DD11 KK18 LL01 LL03 4H045 AA10 AA11 BA10 BA41 CA40 DA75 DA86 DA89 EA20 EA50 FA74

Claims (4)

[Claims] 1. The following protein (a) or (b): (A) a protein having the amino acid sequence represented by SEQ ID NO: 1, (b) an amino acid sequence in which one or several amino acids in SEQ ID NO: 1 have been deleted, substituted, added or inserted, and an adenylate cycler A protein that forms a complex with zetype III. 2. A gene encoding the following protein (a) or (b). (A) a protein having the amino acid sequence represented by SEQ ID NO: 1, (b) an amino acid sequence in which one or several amino acids in SEQ ID NO: 1 have been deleted, substituted, added or inserted, and an adenylate cycler A protein that forms a complex with zetype III. 3. The gene according to claim 2, which consists of the following DNA (A) or (B). (A) a DNA having the nucleotide sequence represented by SEQ ID NO: 2,
(B) A DNA that hybridizes with a DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 2 under stringent conditions and encodes a protein that forms a complex with adenylate cyclase type III. 4. An antibody against the protein according to claim 1.