WO2012005379A1

WO2012005379A1 - Transformant obtained by introducing gm1 gene and crmp2 gene

Info

Publication number: WO2012005379A1
Application number: PCT/JP2011/065918
Authority: WO
Inventors: 康彦高橋; 正樹片岡
Original assignee: 住友化学株式会社
Priority date: 2010-07-07
Filing date: 2011-07-06
Publication date: 2012-01-12
Also published as: JP2012016303A

Abstract

The present invention provides: a transformant which is obtained by introducing a Gm1 gene and a CRMP2 gene; a screening method for a signaling modulator substance mediated by a Gm1 protein, said method using the transformant; and the like.

Description

Gm1 gene and CRMP2 gene-introduced transformant

The present invention relates to a transformant into which a Gm1 gene and a CRMP2 gene have been introduced and use thereof.

Gm1 protein is a kind of G-protein α subunit, and is known to be involved in intracellular signal transduction by G-protein coupled receptor (GPCR) stimulation. The Gm1 gene is a Splicing variant of the Golf gene, and has a unique base sequence only in the first exon region, and shares the base sequence with the Golf gene after the second exon region. The Gm1 protein is a kind of G-protein α subunit represented by the registration number “NM — 182978” in the NCBI database. Both guanine nucleotide binding protein (G protein), alpha activating lipid ligand, olfactory ligand, The N-terminal region has a characteristic structure that is about 80 amino acids longer than other G proteins (see, for example, US2005-0260595).
The CRMP2 protein is a collapse response mediator protein 2 (Collapsin Response Mediator Protein 2, CRMP2), which is a protein identified as a factor that mediates information on semaphorin, which is a nerve guidance factor (eg, Nature (1995), 376). , 509-514). It is known that CRMP2 protein has a function of extending axon by directly or indirectly binding to molecules such as tubulin, Numb, Sra-1, TrkB, and transporting these molecules to the axon tip. (Nat. Cell Biol., (2002) 4: 583-591, Nat. Cell Biol., (2003) 5: 819-826, Nat. Rev. Neurosci., 8: 194-205 (2007), Developmental Cell, 16, 675-686 (2009)). CRMP2 protein is phosphorylated by a kinase such as cdk5, GSK3β, Rho kinase, etc., and its activity is regulated. Specifically, non-phosphorylated form has activity and is phosphorylated by kinase It is known that it is inactivated (Cell, 120, 137-149, (2005), Mol. Cell. Biol., 25, 9973-9984 (2005)). It has also been reported that introduction of a non-phosphorylated mutant CRMP2 gene into neurons impairs the axon retraction, and inhibition of CRMP2 protein biological function in neurons inhibits axon elongation. (Cell, 120, 137-149, (2005), J. Biol. Chem., 275, 2397-23980 (2000)). Overexpression of CRMP2 protein after nerve injury promotes nerve axon regeneration (eg, J. Neurochem., 86, 1042-50, (2003)), and CRMP2 protein mutation is a risk factor for schizophrenia (For example, Biol Psychiatry., 53, 571-576, (2003)) has been reported.

Accumulation of knowledge and development of techniques useful for ameliorating the symptoms of diseases that appear to be due to abnormal signaling through the CRMP2 protein, such as Alzheimer's disease, schizophrenia and axon injury There is an urgent need, and development of related testing tools is expected.
The present invention is based on the newly discovered finding that activation of the biological function of Gm1 protein suppresses phosphorylation of CRMP2 protein, that is, the biological function of CRMP2 protein is activated. .
That is, the present invention is 1. A transformant in which both of the following genes (1) and (2) are introduced into a host cell (hereinafter sometimes referred to as the transformant of the present invention):
(1) a foreign gene having a base sequence encoding the amino acid sequence of Gm1 protein, and (2) a foreign gene having a base sequence encoding the amino acid sequence of CRMP2 protein;
2. [2] The transformant according to [1] above, wherein the foreign gene is a nucleic acid in the form of a plasmid containing a base sequence encoding the amino acid sequence of the protein;
3. 3. The transformant according to

item

1 or 2, wherein both of the proteins encoded by the genes (1) and (2) are co-expressed in the cells;
4). (A) a first step of bringing a test substance into contact with the transformant according to the preceding

item

1, 2, or 3,
(B) a second step of measuring the phosphorylation state of CRMP2 protein in the transformant after the first step or an index value correlated therewith;
(C) a test substance that varies the phosphorylation state of CRMP2 protein in the transformant or the index value correlated therewith by comparing the phosphorylation state measured in the second step or the index value correlated therewith with a control. A screening method for a substance that regulates signal transduction via Gm1 protein, which comprises a third step to be selected (hereinafter also referred to as the screening method of the present invention);
5. 4. The screening method according to item 4, wherein the control in the third step is a phosphorylation state of CRMP2 protein in the transformant according to

item

1, 2 or 3 not contacted with the test substance or an index value correlated therewith;
6). A substance that regulates signal transduction via Gm1 protein obtained by the screening method according to 4 or 5 above;
7. A therapeutic or prophylactic agent for diseases caused by abnormal signaling via CRMP2 protein, which contains as an active ingredient a signal transduction regulator via Gm1 protein according to item 6 (hereinafter sometimes referred to as the drug of the present invention). ;
According to the present invention, it is possible to provide a screening method for a signal transduction regulator via Gm1 protein, a test tool including a transformant used for the screening method, and the like.

FIG. 1 is a diagram showing the results of Western blotting with an anti-CRMP2 antibody of a two-dimensional electrophoresis sample. The upper diagram shows the results for the control cells that express the CRMP2 protein and does not express the activated Gm1 protein, and the lower diagram shows the results for the cells that co-express the CRMP2 protein and the activated Gm1 protein.
FIG. 2 is a diagram showing the results of quantifying each spot of the Western blot shown in FIG. The intensity of each phosphorylated CRMP2 protein spot when the intensity of the non-phosphorylated CRMP2 protein spot (No. 1) is “1” is shown. Black bar graphs are the results for control cells, and white bar graphs are the results for cells that co-express CRMP2 protein and activated Gm1 protein.
FIG. 3 is a diagram showing the results of Western blotting with an anti-CRMP2 antibody of a two-dimensional electrophoresis sample. The upper graph shows the results for the control (DMSO), and the lower graph shows the results using SKF81297 as the test substance.
FIG. 4 is a diagram showing the results of quantifying each spot of the Western blot shown in FIG. The intensity of each phosphorylated CRMP2 protein spot when the intensity of the non-phosphorylated CRMP2 protein spot (No. 1) is “1” is shown. The black bar graph is the result for the control (DMSO), and the white bar graph is the result using SKF81297 as the test substance.
FIG. 5 is a diagram showing the results of comparing the quantitative values of each spot shown in FIG. 4 between the solvent control and the test substance. The black bar graph is the result for the control (DMSO), and the white bar graph is the result using SKF81297 as the test substance.

In this specification, genetic engineering techniques such as DNA preparation and plasmid preparation, and protein engineering techniques such as protein extraction and western blotting, unless otherwise specified, are described in Molecular Cloning, A Laboratory Manual (T. Maniatis). et al., Cold Spring Harbor Laboratory (1982)), New Chemistry Laboratory (Edited by The Japanese Biochemical Society; Tokyo Kagaku Dojin), etc., or a method analogous thereto.
As described in the background art, the “Gm1 protein” in the present invention is a G-protein α known to be involved in intracellular signal transduction by G-protein coupled receptor (GPCR) stimulation. A type of subunit. The Gm1 protein is a known protein described in, for example, US2005-0260595.
As an amino acid sequence which Gm1 protein has, the amino acid sequence shown by sequence number 1, 25, or 26 described in the said publication | presentation gazette etc. can be mentioned, for example. The Gm1 protein is a kind of G-protein α subunit represented by the registration number “NM — 182978” in the NCBI database. Both guanine nucleotide binding protein (G protein), alpha activating lipid ligand, olfactory ligand, The N-terminal region has a characteristic structure that is about 80 amino acids longer than other G proteins.
The Gm1 gene is a Splicing variant of the Golf gene, and has a unique base sequence only in the first exon region, and shares the base sequence with the Golf gene after the second exon region. Examples of the base sequence encoding the amino acid sequence of the Gm1 protein include the base sequence represented by SEQ ID NO: 2, 27 or 28 described in the above-mentioned publication.
In US2005-0260595, the amino acid sequence shown by SEQ ID NO: 1 is shown in SEQ ID NO: 1, and in US2005-0260595, the base sequence shown by SEQ ID NO: 2 is shown in SEQ ID NO: 2.
The “Gm1 protein” in the present invention includes a part thereof as long as the biological function of the protein is maintained. For example, the amino acid sequence represented by SEQ ID NO: 1 described in the above publication has sequence identity with the amino acid sequences of “GTP binding site” and “GTPase activation site” conserved among G protein α subunits. Has a high amino acid sequence portion. These portions are the regions of amino acid numbers 126 to 133, 287 to 292, 353 to 359, and 428 to 435 in the amino acid sequence represented by SEQ ID NO: 1 described in the publication. These amino acid regions are amino acid sequences of “GTP binding site” and “GTPase activation site” in “Gs” and “Golf” that have already been identified as G protein α subunits (NATURE, 117-127 (1991), vol.349). The amino acid sequence represented by SEQ ID NO: 1 is the same as the characteristic sequence highly conserved among “Gs” and “Golf” belonging to the Gs family among G protein α subunits (shown by SEQ ID NO: 1). This sequence has amino acid numbers 119 to 126) and can take an α-helical structure conserved between G protein α subunits.
An index indicating how many amino acid residues of the Gm1 protein can be substituted, deleted, or added without losing the biological function of the Gm1 protein should be found by the method described in JP-A-2003-193330, etc. Can do. The modification that does not lose the biological function may be performed, for example, on a portion having low sequence identity with the amino acid sequences of various G protein α subunits that have already been identified.
Examples of the “Gm1 protein” include proteins described in US2009-0042789.
As described in the background art, “CRMP2 protein” in the present invention is collapsin response mediator protein 2, CRMP2, and is a factor that mediates information on semaphorin, which is a nerve guidance factor. The identified protein. CRMP2 protein is a known protein described in, for example, Goshima et al., Nature (1995), 376, 509-514.
Examples of the amino acid sequence possessed by CRMP2 protein and the base sequence encoding the amino acid sequence include the amino acid sequences and base sequences described in the above documents. The CRMP2 protein is a protein represented by the registration number “NP — 001377” in the NCBI database, and is sometimes called dihydropyrimidinase-like 2, dihydropyrimidinase-like 2, or DPYSL2.
The transformant of the present invention is obtained by introducing both of the following genes (1) and (2) into a host cell:
(1) a foreign gene having a base sequence encoding the amino acid sequence of Gm1 protein protein, and (2) a foreign gene having a base sequence encoding the amino acid sequence of CRMP2 protein protein.
That is, the transformant of the present invention is a transformed cell containing a polynucleotide having a base sequence encoding the amino acid sequence of Gm1 protein protein and a polynucleotide having a base sequence encoding the amino acid sequence of CRMP2 protein protein.
As the host cell of the transformant of the present invention, any known host cell can be used. For example, bacteria such as Escherichia coli (for example, K12), Bacillus bacteria (for example, MI114), yeast (for example, AH22) Etc.), insect cells (eg, Sf cells, etc.), animal cells (eg, COS7 cells, etc.), and the like. Among these, cultured cells derived from mammals (for example, COS7 cells, CHO cells, HEK293 cells, Hela cells, PC12 cells, etc.) can be preferably mentioned.
The transformant of the present invention contains a nucleic acid in the form of a plasmid containing a base sequence encoding the amino acid sequence of Gm1 protein and a base sequence encoding the amino acid sequence of CRMP2 protein for such a host cell. It can be prepared by performing an artificial operation to introduce both nucleic acids in the form of plasmids to be introduced exogenously. The transformant of the present invention is subjected to an artificial operation for exogenously introducing a nucleic acid in the form of a plasmid containing both the base sequence encoding the amino acid sequence of Gm1 protein and the base sequence encoding the amino acid sequence of CRMP2 protein. Can also be produced.
As the artificial operation, a normal genetic engineering technique corresponding to the host cell to be used may be used. Specifically, for example, a commercially available transfection reagent can be used according to the method described in the instructions attached to the reagent. Moreover, when using the nucleic acid which is the form of a plasmid as a foreign gene, the calcium phosphate method, the electroporation method, the lipofection method, the DEAE dextran method etc. can be mentioned.
Here, examples of the “plasmid” include a plasmid capable of co-expressing both Gm1 protein and CRMP2 protein in a host cell. Such a plasmid is prepared according to a conventional genetic engineering technique, in which both a nucleic acid containing a base sequence encoding the amino acid sequence of the Gm1 protein and a nucleic acid containing a base sequence encoding the amino acid sequence of the CRMP2 protein are respectively present. It can be prepared by linking to a position where expression is possible downstream of the promoter of a known expression plasmid.
Here, examples of the “expression plasmid” include pBR322, pUC12, pUC119, pBluescript, etc. when Escherichia coli is used as a host cell, and pUB110, pC194, etc., when Bacillus bacterium is used as a host cell. When cells are used, Yip5, Yep24, etc., when insect cells are used as host cells, AcNPV, etc. When animal cells are used as host cells, pUC18, pcDNA3.1, etc. should be mentioned. Can do.
In the transformant of the present invention, the foreign gene having the base sequence encoding the amino acid sequence of Gm1 protein and the foreign gene having the base sequence encoding the amino acid sequence of CRMP2 protein are temporarily retained in the host cell. It may be made, or may be stably held. The transformant in which the foreign gene is stably retained is an indicator of a selection marker such as a drug resistance marker (for example, a drug resistance marker contained in an expression plasmid) introduced into the host cell simultaneously with the foreign gene. You can make a selection.
In order to co-express both Gm1 protein and CRMP2 protein in the cells of the transformant of the present invention, for example, the transformant of the present invention may be cultured in a culture solution.
The culture conditions for the transformant of the present invention may be appropriately selected according to the type of the transformant of the present invention.
For example, when the transformant of the present invention is a microorganism, it may be cultured using a liquid medium or a plate medium usually used for culturing microorganisms. The culture temperature only needs to be within the temperature at which microorganisms can grow, and examples thereof include about 15 to 40 ° C. The pH of the medium may be within the range in which microorganisms can grow, and examples thereof include about pH 6 to 8. The culture time varies depending on other culture conditions, but is usually about 1 to 5 days, particularly about 1 to 2 days. When an induction type expression plasmid such as a temperature shift type or an IPTG induction type is used, the induction time may be within one day, particularly about several hours.
When the transformant is an insect cell, culture conditions suitable for the type of insect cell may be used. For example, the culture may be performed using an insect cell medium such as Grace's medium containing FBS and yeast. The culture temperature can be about 25 to 35 ° C. The pH of the medium may be within the range in which insect cells can grow, and examples thereof include about pH 6-8. Although the culture time varies depending on other culture conditions, it is usually about 1 to 5 days, particularly about 2 to 3 days.
In the case of a transformant containing a virus such as Baculovirus as an expression plasmid, the culture time is preferably set until the cytoplasmic effect appears and the cells die (for example, about 3 to 7 days, particularly about 4 to 6 days). .
When the transformant is a mammalian cell, culture conditions suitable for the type of mammalian cell may be used. For example, a DMEM medium (Nissui, etc.) supplemented with FBS may be used in the presence of 5% CO ₂ while being replaced with a new medium every few days. Once the mammalian cells have grown to confluence, for example, add trypsin solution to disperse them into individual cells, dilute the resulting cell suspension several times, seed in a new petri dish, and continue to pass. Good.
The culture temperature can be about 36 to 38 ° C. The pH of the medium may be within the range in which mammalian cells can grow, and examples thereof include about pH 6-8. The culture time varies depending on other culture conditions, but is usually about 2 to 5 days, particularly about 2 to 3 days.
As a method for confirming that both Gm1 protein and CRMP2 protein are co-expressed in the cells of the transformant of the present invention, an ordinary protein engineering method may be used. Specific examples include a method in which a protein extract prepared from the transformant of the present invention is subjected to Western blotting using an anti-Gm1 protein antibody and an anti-CRMP2 protein antibody.
The screening method of the present invention comprises (a) a first step in which a test substance is brought into contact with the transformant described in the

preceding item

1, 2 or 3, and (b) phosphorylation of CRMP2 protein in the transformant after the first step. A second step of measuring a state or an index value correlated therewith, and (c) comparing the phosphorylation state measured in the second step or an index value correlated therewith with a CRMP2 protein in the transformant, And a third step of selecting a test substance that varies the phosphorylation state or an index value correlated therewith.
The “test substance” in the screening method of the present invention is not particularly limited, and examples thereof include proteins (including antibodies), peptides, non-peptidic compounds (nucleotides, amines, carbohydrates, lipids, etc.), organic low molecular compounds, inorganic Examples thereof include low molecular weight compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts and the like. The form of the test substance is not particularly limited, and examples thereof include a solid, a liquid, a mixture with a base, a suspension or a solution. In the case of a suspension or solution, water, pH buffer solution, methylcellulose solution, physiological saline, organic solvent aqueous solution (ethanol or dimethyl sulfoxide is usually used as the organic solvent) and the like may be used.
The contact amount, the number of times of contact, and the contact time of the test substance may be set within a range that does not seriously affect the transformant of the present invention, for example.
The “contact” in the first step of the screening method of the present invention can be performed by a method widely used by those skilled in the art. When the test substance is brought into contact with the transformant of the present invention, the contact method is not particularly limited. For example, the concentration is about 0.001 to 10 μM in a culture solution / agar medium containing the transformant of the present invention. The test substance may be added so that
In the second step of the screening method of the present invention, the “measurement of the phosphorylation state of CRMP2 protein or an index value correlated therewith” comprises the preparation of a protein extract, electrophoresis, transcription onto a membrane, detection of CRMP2 protein A method or the like can be used. In these methods, analysis may be performed using a normal biochemical technique, protein engineering technique, or the like. For example, it may be performed by combining two-dimensional electrophoresis and Western blotting. Specifically, for example, a protein extract is first prepared from the transformant of the present invention in contact with a test substance. The prepared protein extract is subjected to the first dimension electrophoresis and then the second dimension electrophoresis. Transfer the separated CRMP2 protein onto a suitable membrane. Next, the CRMP2 protein transferred onto the membrane is detected with an anti-CRMP2 protein antibody or the like. As a result of these manipulations, CRMP2 protein is separated according to the degree of phosphorylation.
Non-phosphorylated CRMP2 is detected on the more alkaline side, and phosphorylated CRMP2 is detected on the more acidic side as the degree of phosphorylation increases. Separation of CRMP2 based on the degree of phosphorylation can be performed according to a commonly used method known per se.
As a method for quantifying the phosphorylation state of CRMP2, a known quantification method may be used. For example, a signal detected by Western blot may be quantified with an image analyzer or the like. Specifically, for example, the spot intensity of non-phosphorylated CRMP2 in the CRMP2 spot detected by Western blot after two-dimensional electrophoresis in the transformant of the present invention is “1”, and each spot of phosphorylated CRMP2 Calculate the intensity. Similarly, regarding the intensity of the CRMP2 spot in the control, the spot intensity of the non-phosphorylated CRMP2 is “1”, and the spot intensity of each phosphorylated CRMP2 is calculated.
In comparison with the control in the third step of the screening method of the present invention, for example, the phosphorylation state measured in the second step of CRMP2 protein in the transformant of the present invention contacted with the test substance or an index value correlated therewith, What is necessary is just to compare with the phosphorylation state measured at the 2nd process of CRMP2 protein in the transformant of this invention which is not contacting the test substance, or the index value correlated with it. Here, the “transformant of the present invention not in contact with the test substance” may be, for example, the transformant of the present invention in which only the solvent is contacted.
Such a control may be obtained by measuring the phosphorylation state of CRMP2 protein as a control or an index value correlated therewith in parallel with the phosphorylation state of CRMP2 protein in the transformant of the present invention or an index value correlated therewith. However, it may be determined by measuring separately.
For example, the measured value of the CRMP2 protein phosphorylation state in the transformant of the present invention brought into contact with the test substance or an index value correlated therewith correlates with the phosphorylation state of CRMP2 protein in the control transformant of the present invention. If it is lower than the measured value of the index value, it means that the contact with the test substance has an effect of activating CRMP2 protein by suppressing the phosphorylation state of CRMP2 protein, and the test substance has Gm1 It can be selected as a signal transduction regulator. Specifically, for example, each spot in the control is defined as 100%, and the percentage of each spot of CRMP2 detected by the transformant of the present invention in contact with the test substance is calculated. A test substance in which one of the percentages of each spot of CRMP2 detected in the transformant of the present invention contacted with the test substance is, for example, 70% or less, preferably 60% or less, more preferably 50% or less May be selected as a candidate substance for a signal transduction regulator. Also preferably, in a highly phosphorylated spot, a test substance in which the percentage of the spot is, for example, 70% or less, preferably 60% or less, more preferably 50% or less is selected as a candidate for activating signal transduction. What is necessary is just to select as a substance.
By carrying out the screening method of the present invention using a positive control or negative control that can be a “control substance” instead of the test substance, the above-mentioned control can be used as the case may be.
Here, “positive control” represents any substance having an effect of controlling the phosphorylation state of CRMP2 protein, and “negative control” includes a solvent contained in the test substance, a test system solution serving as a background, etc. Is mentioned.
When the “control substance” is a negative control, if the “effect of suppressing the phosphorylation state of CRMP2 protein” possessed by the test substance is greater than the “effect of suppressing the phosphorylation state of CRMP2 protein” possessed by the control substance, What is necessary is just to evaluate that the said test substance has "the effect which suppresses the phosphorylation state of CRMP2 protein." On the other hand, if the “effect of suppressing the phosphorylation state of CRMP2 protein” possessed by the test substance is similar to or smaller than the “effect of suppressing the phosphorylation state of CRMP2 protein” possessed by the control substance, “CRMP2 It can be evaluated that it does not have an “effect of suppressing the phosphorylation state of a protein”.
When the control substance is used as a positive control, compare the “effect of suppressing the phosphorylation state of CRMP2 protein” possessed by the test substance with the “effect of suppressing the phosphorylation state of CRMP2 protein” possessed by the control substance. Thus, the “effect of suppressing the phosphorylation state of CRMP2 protein” possessed by the test substance may be evaluated.
Thus, when it is evaluated that it has an effect, the said to-be-tested substance can be selected as a signal transduction regulator via Gm1.
The drug of the present invention contains, for example, the “signal transduction modulator via Gm1 protein” selected as described above as an active ingredient. The agent of the present invention is useful as a therapeutic or prophylactic agent for diseases caused by abnormal signaling through the CRMP2 protein.

EXAMPLES Hereinafter, although the Example of this invention is shown and demonstrated in detail, this invention is not limited to these Examples.
Example 1 (Inhibition of CRMP2 phosphorylation by activated Gm1)
(1) Acquisition of Gm1 gene Using a method according to the method described in the literature (Japanese Patent Laid-Open No. 2004-350672), using 20 ng of human brain-derived cDNA library (Takara Shuzo) as a template and 10 μM forward primer prGm1ATG (5′-ATGGGTCTGTGCTACAGTCTGGCGG; SEQ ID NO: 3) 10 μM reverse primer prGNAL 3 ′ (5′-TCACAAGAGCTCATACTGCTT; SEQ ID NO: 4) and TAKARA LA Taq polymerase (manufactured by TAKARALA Taq with GC Buffer, Takara Shuzo) . The PCR conditions were 95 ° C for 30 seconds, then 60 ° C for 30 seconds, and then 72 ° C for 2 minutes, with one cycle being 35 cycles. The obtained DNA fragment was subjected to agarose gel electrophoresis, then a band of about 1.5 kb detected on the gel was cut out, DNA was recovered from the cut out gel, and the DNA was pCR2.1-TOPO vector (manufactured by Invitrogen). To pCR-Gm1.
(2) Construction of pcDNA-AFGm1 Using a method according to the method described in the literature (US2009-0042789), using 10 ng of the plasmid pCR-Gm1 prepared in Example 1 as a template and a 10 μM forward primer prGm1ATG ( 5′-ATGGGTCTGTGCTACAGTCTGGCGG; SEQ ID NO: 5) PCR was performed using 10 μM reverse ply hAFGm1AS (5′-TCCATTTTCTCCTCTCATCCCTTAAGCCACCAACATCAAACAT; manufactured by Takara Shuzo Co., Ltd.). The PCR was performed at 95 ° C. for 30 seconds, then at 60 ° C. for 30 seconds, and then at 72 ° C. for 1 minute for one cycle. The obtained DNA was subjected to agarose gel electrophoresis, the band detected on the gel was cut out, the DNA fragment was recovered from the cut out gel, and this DNA fragment was designated as AF5 ′.
Similarly, using 10 ng of the plasmid pCR-Gm1 as a template and 10 μM of the reverse primer prGNAL3 ′ (5′-TCACAAGAGCTCATACTGCTT; SEQ ID NO: 6), 10 μM of the forward ply hAFGm1S (5′-ATGTTTGATGTGTGTGTGTAGAGGATGA) PCR was performed using the same product. The PCR was performed at 95 ° C. for 30 seconds, then at 60 ° C. for 30 seconds, and then at 72 ° C. for 1 minute for one cycle. The obtained DNA was subjected to agarose gel electrophoresis, the band detected on the gel was cut out, the DNA fragment was recovered from the cut out gel, and this DNA fragment was designated as AF3 ′.
Next, 10 ng of each of the recovered DNA fragments AF5 ′ and AF3 ′ was used as a template, and 10 μM forward primer prGm1ATG (5′-ATGGGTCTGTGCTACAGTCTGCGGG; SEQ ID NO: 5), 10 μM reverse primer prGNAL3 ′ (5′-TCACAGAGCTCACTACTGCTT; SEQ ID NO: PCR was performed using 6) and Pyrobest polymerase (Takara Shuzo). The PCR conditions were 30 cycles of 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, and then 72 ° C. for 2 minutes. After the obtained DNA fragment was subjected to agarose gel electrophoresis, a band of about 1.5 kb detected on the gel was excised, DNA was recovered from the excised gel, and the DNA was cloned into a pDrive vector (Q1AGEN). pDr-AFGm1 was produced. After double-digesting the prepared plasmid pDr-AFGm1 with EcoRV and SpeI, this DNA fragment was subjected to agarose gel electrophoresis, a band of about 1.5 kb detected on the gel was excised, and DNA was excised from the excised gel. The DNA was recovered and used as insert DNA. Plasmid pcDNA-AFGm1 was prepared by ligating pcDNA3.1 (manufactured by Invitrogen) with EcoRV and XbaI as a vector and ligating the insert DNA with ligase.
(3) Construction of pcDNA-CRMP2 Using 20 ng of human brain-derived cDNA library (Takara Shuzo) as a template and 10 μM forward primer hCRMP2 (ATG) (5′-CACCCATGTCTTATCGGGGGAAGAAAA; SEQ ID NO: 9), 10 μM reverse primer hCRMP2 (-STOP) PCR was performed using rev (5'-GCCCAGGCTGGTGATGTTGGCACGGCCA; SEQ ID NO: 10) and KOD DNA polymerase (manufactured by Toyobo). PCR was performed at 35 ° C. for 30 seconds at 95 ° C., then at 60 ° C. for 30 seconds, and then at 68 ° C. for 2 minutes. After agarose gel electrophoresis of the obtained DNA, a band of about 1.7 kb detected on the gel was cut out, DNA was recovered from the cut out gel, and the DNA was pENTR / D-TOPO vector (manufactured by Invitrogen) Was cloned into pENTR-CRMP2. Subsequently, 4 μl pENTR-CRMP2, 1 μl pcDNA6.2 · V5-DEST and 2 μl LR clonase II (manufactured by Invitrogen) were mixed and kept at room temperature for 1 hour to prepare plasmid pcDNA-CRMP2.
(4) Preparation of pcDNA3.1 For pcDNA3.1, a commercially available product sold by Invitrogen Corporation was purchased and used.
(5) Production of the transformant of the present invention COS7 cells (purchased from ATCC) were seeded in a 10 cm dish at 5 × 10 ⁶ cells / dish, and 37% in DMEM medium (Invitrogen) containing 10% FCS. The cells were cultured for about 24 hours at 5 ° C. under the conditions of 5 ° C. Both the “activated Gm1 expression plasmid (pcDNA-AFGm1; 12 μg)” obtained in (2) above and the “CRMP2 expression plasmid (pcDNA-CRMP2; 6 μg)” obtained in (3) above, The cells were mixed with 45 μl of Lipofectamine 2000 (Invitrogen) and maintained for 20 minutes to transfect the cells. The medium was completely changed 3 hours after the transfection, and further cultured for about 21 hours. As a control, control cells were prepared by performing the same operation except that the expression plasmid (pcDNA3.1; 12 μg) described in (4) above was used instead of the activated Gm1 expression plasmid.
(6) Confirmation of inhibitory action on phosphorylation of CRMP2 protein by activated Gm1 protein The cells prepared in (5) above were used as test cells in the following tests.
The cells prepared in (5) above were lysed using a PhosphoProtein Purification kit (Qiagen) after the medium was removed to obtain a cell extract. A protein extract (purified protein) was prepared from the cell extract using 2-D Clean-Up kit (GE Health Science).
The obtained protein extract (800 μg of purified protein) was subjected to first-dimensional electrophoresis using Immobiline DryStripe (GE Health Science). Next, second-dimensional electrophoresis was performed using 7.5% SDS-PAGE gel. The gel after two-dimensional electrophoresis was transferred to Hybond-P (GE Health Science). Subsequently, the CRMP2 protein transferred onto the membrane was detected by Western blotting using an anti-CRMP2 antibody (C4G) (Immuno-Biological Laboratories, Inc.) (see FIG. 1). In FIG. No. 1 is a spot of non-phosphorylated CRMP2 protein, No. 1 located on the acidic side of it. 2-6 is a spot of phosphorylated CRMP2 protein. Moreover, the spot located on the more acidic side, that is, No. The spot located on the 6 side is a CRMP2 protein spot with a higher degree of phosphorylation.
Next, the signal intensity of each spot detected by the Western blot shown in FIG. 1 was measured using LAS-4000 (Fuji Film). The intensity of the phosphorylated CRMP2 protein spot was calculated when the intensity of the non-phosphorylated CRMP2 protein spot was “1” (see FIG. 2). As is clear from the results shown in FIG. 2, the signal intensity of the phosphorylated CRMP2 protein spots (spots No. 2 to No. 6) in the cells in which the activated Gm1 protein was co-expressed was determined to be the activated Gm1 protein. Decreased compared to the signal intensity of the phosphorylated CRMP2 protein spot in control cells not co-expressing.
From the above results, it was confirmed that activation of the Gm1 protein suppresses phosphorylation of the CRMP2 protein.
Example 2 (Screening method of the present invention: screening of a signal transduction regulator through Gm1 protein) (Part 1)
(1) Construction of pcDNA-Gm1 The plasmid pCR-Gm1 prepared in Example 1 was double digested with restriction enzymes XbaI and KpnI using a method according to the method described in the literature (Japanese Patent Laid-Open No. 2004-350672). Thereafter, this DNA fragment was subjected to agarose gel electrophoresis, a band of about 1.5 kb detected on the gel was cut out, DNA was recovered from the cut out gel, and the DNA was used as insert DNA. A plasmid pcDNA-Gm1 was prepared by ligating pcDNA3.1 (manufactured by Invitrogen) with XbaI and KpnI as a vector and ligating the insert DNA with ligase.
(2) Screening for signal transduction modulators COS7 cells (purchased from ATCC) were seeded in 10 cm dishes at 5 × 10 ⁶ cells / dish, and 37 ° C. in DMEM medium (manufactured by Invitrogen) containing 10% FCS. The cells were cultured for about 24 hours under 5% CO2. Both “Gm1 expression plasmid (pcDNA-Gm1; 12 μg)” obtained in (7) above and “CRMP2 expression plasmid (pcDNA-CRMP2; 6 μg)” obtained in (3) above were combined with 45 μl of lipoprotein. The cells were mixed with Perfectamine 2000 (Invitrogen) and kept for 20 minutes to transfect the cells. The medium was completely changed 3 hours after the transfection, and further cultured for about 21 hours.
Next, the test substance SKF81297 ((±) -6-Chloro-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzopine hydrobromide; C ₁₆ H ₁₆ ClNO ₂ .HBr) was added to a final concentration of 10 μM. After being added to the cell culture medium, the obtained culture was allowed to stand at 37 ° C. for 30 minutes.
Next, after removing the medium containing SKF81297 from the obtained culture, the cell extract was obtained by lysis using PhosphoProtein Purification kit (Qiagen). A protein extract (purified protein) was prepared from the cell extract using 2-D Clean-Up kit (GE Health Science). As a control, a culture in which only the DMSO solvent was added to the cell culture medium instead of SKF81297 was prepared, and a protein extract (purified protein) was prepared in the same manner.
The obtained protein extract (800 μg of purified protein) was subjected to first-dimensional electrophoresis using Immobiline DryStripe (GE Health Science). Next, second-dimensional electrophoresis was performed using 7.5% SDS-PAGE gel. The gel after two-dimensional electrophoresis was transferred to Hybond-P (GE Health Science). Subsequently, the CRMP2 protein transferred onto the membrane was detected by Western blotting using an anti-CRMP2 antibody (C4G) (Immuno-Biological Laboratories, Inc.) (see FIG. 3). In FIG. No. 1 is a spot of non-phosphorylated CRMP2 protein, No. 1 located on the acidic side of it. 2-6 is a spot of phosphorylated CRMP2 protein. Moreover, the spot located on the more acidic side, that is, No. The spot located on the 6 side is a CRMP2 protein spot with a higher degree of phosphorylation.
Next, the signal intensity of each spot detected by the Western blot shown in FIG. 3 was measured using LAS-4000 (Fuji Film). The intensity of the phosphorylated CRMP2 protein spot was calculated when the intensity of the non-phosphorylated CRMP2 protein spot was “1” (see FIG. 4). As is clear from the results shown in FIG. 4, the signal intensity of the “higher phosphorylated spots (spots No. 6 and No. 5)” in the cells contacted with SKF81297 was contacted only with DMSO solvent. In comparison with the signal intensity of “higher phosphorylated spots (spots No. 6 and No. 5)” in the cells. On the other hand, the signal intensity of “spots with low phosphorylation (spots No. 2 and No. 3)” in cells contacted with SKF81297 is “spot with low phosphorylation” in cells contacted only with DMSO solvent. (Spot No. 2 and No. 3) ”and increased.
From the above results, in the cells contacted with SKF81297, as a result of the suppression of the degree of phosphorylation, CRMP2 protein in a more highly phosphorylated state decreased, and accordingly, the CRMP2 protein having a low degree of phosphorylation It was confirmed that the amount increased relatively.
Next, in order to quantify the degree of signal change at each spot, the value of each spot in cells contacted with DMSO solvent alone is taken as 100%, and the percentage of each spot in cells contacted with SKF81297 is Calculated (see FIG. 5). As a result, in the “highly phosphorylated CRMP2 spots (spots No. 6 and No. 5)” of the cells contacted with SKF81297, the percentage of the spot signal was 28.4% and 35. respectively. It was confirmed to be 5%.
From the above results, it was found that SKF81297 is a signal transduction regulator via Gm1 protein.
Example 3 (screening method of the present invention: screening of a signal transduction regulator through Gm1 protein) (Part 2)
COS7 cells (purchased from ATCC) were seeded in a 10 cm petri dish at 5 × 10 ⁶ cells / petri dish, and about 10% FCS-containing DMEM medium (manufactured by Invitrogen) under conditions of 37 ° C. and 5% CO 2. Incubate for 24 hours. Both “Gm1 expression plasmid (pcDNA-Gm1; 12 μg)” obtained in (7) above and “CRMP2 expression plasmid (pcDNA-CRMP2; 6 μg)” obtained in (3) above were combined with 45 μl of lipoprotein. The cells are mixed with Perfectamine 2000 (Invitrogen) and kept for 20 minutes to transfect the cells. The medium is completely changed 3 hours after transfection, and further cultured for about 21 hours.
Subsequently, various test substances are added to the cell culture solution so as to have a final concentration of 10 μM, and the obtained culture is allowed to stand at 37 ° C. for 30 minutes.
Next, after removing the medium containing the test substance from the obtained culture, a cell extract is obtained by lysis using a PhosphoProtein Purification kit (Qiagen), and then 2-D Clean from the cell extract. -A protein extract (purified protein) is prepared using Up kit (GE Health Science). As a control, a culture in which only the DMSO solvent is added to the cell culture medium instead of the test substance is prepared, and a protein extract (purified protein) is prepared in the same manner.
The obtained protein extract (800 μg of purified protein) is subjected to first-dimensional electrophoresis using Immobiline DryStripe (GE Health Science). Next, second-dimensional electrophoresis is performed using 7.5% SDS-PAGE gel. The gel after two-dimensional electrophoresis is transferred to Hybond-P (GE Health Science). Subsequently, the CRMP2 protein transferred onto the membrane is detected by Western blotting using an anti-CRMP2 antibody (C4G) (Immuno-Biological Laboratories, Inc.).
Next, the signal intensity of each spot detected by Western blotting was measured using LAS-4000 (Fuji Film). The intensity of the phosphorylated CRMP2 protein spot is calculated when the intensity of the non-phosphorylated CRMP2 protein spot is “1”.
Next, in order to quantify the degree of change in the signal at each spot, the value of each spot in the cells contacted only with DMSO solvent is set to 100%, and the value of each spot in the cells contacted with the test substance is determined. Calculate the percentage. As a result, in the phosphorylated CRMP2 protein spot in any of the cells contacted with the test substance, the test substance in which the percentage of the spot signal is 50% or less is defined as a “signal transduction modulator via Gm1 protein”. select. A drug containing the selected “signal transduction modulator via Gm1 protein” as an active ingredient (that is, a therapeutic or prophylactic agent for a disease caused by abnormal signal transduction via CRMP2 protein) is prepared.

According to the present invention, it is possible to provide a screening method for a signal transduction regulator through Gm1 protein, a test tool including a transformant used for the screening method, and the like.

SEQ ID NO: 3
Oligonucleotide primer designed for PCR SEQ ID NO: 4
Oligonucleotide primer designed for PCR SEQ ID NO: 5
Oligonucleotide primer designed for PCR SEQ ID NO: 6
Oligonucleotide primer designed for PCR SEQ ID NO: 7
Oligonucleotide primer designed for PCR SEQ ID NO: 8
Oligonucleotide primer designed for PCR SEQ ID NO: 9
Oligonucleotide primer designed for PCR SEQ ID NO: 10
Oligonucleotide primers designed for PCR

Claims

A transformant in which both of the following genes (1) and (2) are introduced into the host cell:
(1) a foreign gene having a base sequence encoding the amino acid sequence of Gm1 protein, and (2) a foreign gene having a base sequence encoding the amino acid sequence of CRMP2 protein.
The transformant according to claim 1, wherein the foreign gene is a nucleic acid in the form of a plasmid containing a base sequence encoding the amino acid sequence of the protein.
The transformant according to claim 1 or 2, wherein both of the proteins encoded by the genes (1) and (2) are co-expressed in the cells.
(A) a first step of bringing the test substance into contact with the transformant according to claim 1, 2, or 3,
(B) a second step of measuring the phosphorylation state of CRMP2 protein in the transformant after the first step or an index value correlated therewith;
(C) a test substance that varies the phosphorylation state of CRMP2 protein in the transformant or the index value correlated therewith by comparing the phosphorylation state measured in the second step or the index value correlated therewith with a control. A method for screening a substance that regulates signal transduction via Gm1 protein, comprising a third step of selecting.
The screening method according to claim 4, wherein the control in the third step is the phosphorylation state of CRMP2 protein in the transformant according to claim 1, 2 or 3, which is not contacted with the test substance, or an index value correlated therewith.
A signal transduction modulator via Gm1 protein obtained by the screening method according to claim 4 or 5.
A therapeutic or prophylactic agent for a disease caused by abnormal signal transduction via CRMP2 protein, comprising as an active ingredient the substance that regulates signal transduction via Gm1 protein according to claim 6.