Patent Application
20080085859
For a more complete understanding of the present invention and the advantages thereof, reference is now made to the following detailed description taken in conjunction with the accompanying drawings, in which:
A drug of the present invention is composed of hCERT protein having an amino acid sequence of SEQ ID NO:1, hCERTL protein having an amino acid sequence of SEQ ID NO: 2, cCERT protein having an amino acid sequence of SEQ ID NO:3, or cCERTL protein having an amino acid sequence of SEQ ID NO:4, or the recombinant protein of these described above as an effective component. Domain deletion structures of hCERT protein, cCERT protein, hCERTL protein and cCERTL protein are shown in
Human CERT (hCERT) protein can be obtained by the following way. Complementary deoxyribonucleic acid (cDNA) that recovers SM content of LY-A strain is isolated and identified in accordance with the following procedure. After cDNA library derived from human cultured cell has been frequently and stably introduced into LY-A strain using retrovirus vector, the function recovery strain is isolated. The method of selecting a functionally recovered strain can be performed on the basis of the knowledge that the cell whose SM content has been decreased is highly sensitive to cholesterol drawing reagent/methylcyclodextrin (MCD), and the cell whose SM content has been recovered recovers the resistance against MCD. Subsequently, cDNA which has been introduced into the isolated functionally recovered strain was amplified by a genomic polymerase chain reaction (PCR) method, and retrieved. The retrieved cDNA changes the MCD sensitivity of LY-A strain into the level of a wild strain. The protein coded by cDNA described in SEQ ID NO: 5 obtained in this way was the same with the protein published before as human GPBPΔ 26 (hGPBPΔ 26). The function within the cell of GPBPΔ 26 has not been elucidated. As described in detail in the present specification, we have found that the function within the cell of GPBPΔ 26 is ceramide trafficking, therefore, as it is made nomenclature representing the function within a cell, the protein having substantially the same sequence with GPBPΔ 26 was named as CERT protein. Moreover, the protein having substantially the same with GPBP was named as CERTL (a large splicing variant of CERT). CERT protein can be obtained by expressing the isolated and identified cDNA, for example in bacterium such as E. coli, yeast and the like, insect cultured cell such as Sf9 cell and the like, mammal animal cultured cell such as CHO cell, HeLa cell, HEK293 cell and the like by a known method.
Human GPBP (hGPBP) has been reported as a protein for binding to carboxyl end sequence of α 3 chain of human collagen 4 type and phosphorylating it in cell-free system. Moreover, in the non-patent document 2, it has been reported that the separate splicing type product/hGPBPΔ 26 which is smaller than hGPBP by 26 amino acid also exists. From the analysis of transcription RNA level, it is indicated that both of hGPBP and hGPBPΔ 26 express in a variety of organs, however, it has been indicated that hGPBPΔ 26 dominantly expresses. However, hGPBP Δ 26 and hGPBP are localized mainly in cytoplasm, this localization is contradictory to the function expected first that collagen, that is an extracellular molecule is modified. Hence, it is considered that the physiological function of these proteins within a cell has not been elucidated from the studies achieved in the past, however, it is indicated that it is effective as a drug for promoting ceramide transport by the present inventors.
Moreover, as a recombinant protein of CERT protein, by making amino acid at N-end the first residue, START domain, that is, a fragment containing 371 residue-598 residue of SEQ ID NO:1 or 3, or 397 residue-624 residue of SEQ ID NO:2 or 4 can be listed, and as such a fragment, recombinant proteins obtained by expressing a DNA fragment obtained by an in vitro recombinant DNA method, a method of synthesizing, and in vivo recombination/gene recombination are listed. As such a protein, domain deletion protein such as PH domain deleted CERT Δ PH protein, MR domain deleted CERTΔ MR protein, ST CERT protein having only START domain and the like can be exemplified. It should be noted that it is estimated that lysine residue which is 370th amino acid of CERT protein corresponds to carboxyl end of MR domain. However, since there is a possible that the protein becomes unstable by the existence of lysine residue at the carboxyl end, in a domain deleted protein, the relevant lysine residue is incorporated at the amino end of START domain, and it is removed from the MR domain.
Furthermore, hGABPΔ 26 protein, that is, a recombinant protein whose amino acid base sequence is essentially the same with hCERT protein can be also obtained by expressing in accordance with a method of Raya, A et al (J. Biol. Chem. 274, 12642-12649, 1999) using plasmid vector obtained by a method of Raya, A et al (J. Biol. Chem. 275, 40392-40399, 2000) and purifying the generated product.
The sequence of cDNA of hGPBP, that is, a recombinant protein which is substantially the same with hCERTL protein can be obtained by a known method (GenBank No: AF136450). Hence, DNA sequence of SEQ ID NO: 6 coding hCERTL can be obtained by adding DNA sequence which is shorted to hCERT sequence by a PCR method. Then, hCERTL protein can be obtained by expressing using plasmid vector prepared from the obtained cDNA and purifying the generated product. Moreover, the relevant hCERTL protein can be also obtained by performing the expression using a plasmid vector prepared from cDNA in accordance with a method of Raya, A. et al (J. Biol. Chem. 274, 12642-12649, 1999).
Furthermore, the full length cDNA of CERT (cCERT) derived from CHO cell corresponding to base sequence of SEQ ID NO:7 can be determined by performing rapid amplification of cDNA ends (RACE) of cDNA end using SMART RACE cDNA amplifier kit manufactured by Clontech, Co., Ltd. Then, ORF of cCERT can be obtained by performing a PCR that CHO cell cDNA library has been made template. Moreover, DNA sequence for coding CERTL (cCERTL) derived from CHO cell corresponding to base sequence of SEQ ID NO:8 can be also amplified and cloned by this PCR. The amino acid sequence of cCERTL is indicted in SEQ ID NO:4.
A drug of the present invention can be used as antitumor agent, anti-inflammatory agent, organoregenesis agent, or anti-infective agent by promoting or suppressing a cell death. Moreover, in cosmetics, as a distribution promoting agent of ceramide it can be used. Furthermore, it can be also utilized for inhibitor research of ceramide transport protein.
A drug of the present invention can be administered patenterally and orally by injection, rapid injection, nasopharynx absorption and percutaneous absorption. As a carrier preparation which is acceptable in a pharmaceutical preparation for parenteral administration, sterilized or aqueous or non-aqueous solution, suspended liquid and emulsion are listed. As an example of non-aqueous solvent, propylene glycol, pylyethylene glycol, a plant oil, for example, an olive oil, an injectable organic ester, for example ethyl oleate. A carrier for occluded bandage increases dermal permeability, then, it can be used for enhancing an antigen absorption. A liquid medication form for oral administration can be, in general, can include liposome solution containing a liquid medication form. As a suitable form for suspending liposome, inactive diluting agent generally used in the art, for example, an emulsion, a suspension, a solution a syrup and an elixir containing a purified water. Besides inactive diluting agent, such a composition can also contain an adjuvant, a wetting agent, an emulsified agent and a suspension stabilizer, and an edulcorant, a flavoring agent and perfume.
A drug of the present invention is also capable of containing an adjuvant. An adjuvant is a substance which can be used for non-specifically increasing the specific immune response. An adjuvant is classified into largely some groups.
As these groups, oil adjuvant (for example, Freund's complete and incomplete adjuvant), inorganic salt (for example, AlK(SO4)2), AlNH4(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, caolin and carbon), polynucleotide (for example, poly IC and poly AU acid), and a certain species of natural substance (for example, wax D from Mycobacterium tuberculosis and substance found in members of Bordetella pertussis and Brucella family) are listed.
The present inventors have found that after a lipid membrane containing ceramide has been incubated with CERT protein, when it is centrifuged, ceramide remaining in a lipid membrane is sedimented, ceramide released from the lipid membrane transfers to the supernatant, and have invented a method of measuring the activity for promoting release of ceramide. Hereinafter, a method of measuring the activity for promoting ceramide release of the present invention will be explained. As a lipid membrane containing ceramide, it is not particularly limited, however, a lipid membrane in which ceramide has been added to the mixed lipid of phosphatidylcholine derived from egg yolk and phosphatidylethanolamine, or the mixed lipid of a synthetic phosphatidylcholine and a synthetic phosphatidylethanolamine and prepared can be exemplified. When a lipid membrane containing ceramide obtained in this way is sprayed by an inert gas such as nitrogen, argon or the like, or by drying under vacuum pressure, the exsiccation can be done. Moreover, it is also possible that a supersonic treatment can be performed by a method of performing the supersonic treatment by a bath type supersonic generator by adding a buffer such as Hepes-NaOH buffer in which NaCl and EDTA have been added to a lipid membrane or a lipid membrane dried under vacuum pressure, Tris-hydrochloric acid buffer or the like. As supersonic treatment conditions, for example, the conditions under which conditions of at 20 to 25° C. for 3 to 6 minutes it is performed can be listed.
As ceramide that is added to the foregoing mixed lipid, a ceramide which has not been radioactively labeled may be available, however, if the convenience of measurement is considered, ceramide which has been radioactively labeled is preferable, as such a ceramide, ceramide which is radioactively labeled by 14C, 3H, 13N, 15O and the like can be listed, among these, ceramide radioactively labeled by 14C is preferable when its easy availability, stability and that the double labeling experiment with a separate lipid which has been 3H labeled can be performed.
As a drug for promoting ceramide release, it is not particularly limited, however, a drug for promoting ceramide transport of the above-described present invention can be exemplified.
In an incubating process, as a method of mixing a lipid membrane containing ceramide and a drug for promoting ceramide release, it is not particularly limited, however, a method that the foregoing lipid membrane is added to a buffer such as Hepes-NaOH buffer, Tris-hydrochloric acid buffer or the like into which the foregoing drug is dispersed can be listed. Moreover, as a method of incubating, a method of incubating at 15 to 42° C. for 10 to 250 minutes by thermostatic bath, hot water bath or the like can be listed.
In a separate process, the supernatant is obtained by centrifuging the mixture after it has been incubated for 30 to 60 minutes at 50,000×g.
In a quantifying process, in the case where ceramide is radioactively labeled, ceramide contained in the supernatant can be quantified by measuring the radioactivity of ceramide contained in the supernatant by means of liquid scintillation counter. Moreover, after a lipid contained in the supernatant has been separated by TLC, it can be quantified by analyzing the radioactivity of ceramide using an image analyzer. In the case where ceramide has not been radioactively labeled, ceramide contained in the supernatant can be quantified by measuring by means of analysis using mass spectrometer or by means of a ceramide quantification method using E. coli diacylglycerol.
A method of measuring the activity for promoting the intermembrane transfer of ceramide of the present invention will be described below. As a donating membrane containing ceramide, it is not particularly limited, however, a donated membrane prepared by adding ceramide to the mixed lipid of phosphatidyl choline derived from egg yolk, phosphatidylethanolamine and lactocylceramide derived from pig tissue, or phosphatidylcholine, phosphatidylethanolamine and lactocylceramide which have been chemically synthesized can be exemplified. It is also possible that biotinated phosphatidylethanolamine is utilized instead of lactocylceramide. A donated membrane containing ceramide obtained in this way can be exsiccated by spraying an inert gas such as nitrogen, argon or the like or dried under vacuum pressure.
Moreover, it is also possible that a supersonic treatment can be performed by a method of performing the supersonic treatment by a bath-type supersonic generator by adding a buffer such as Hepes-NaOH buffer in which NaCl and EDTA have been added to a lipid membrane or a lipid membrane dried under vacuum pressure, Tris-hydrochloric acid buffer or the like. As supersonic treatment conditions, for example, the conditions under which conditions of at 20 to 25° C. for 3 to 6 minutes it is performed can be listed.
As ceramide for adding to the foregoing mixed lipid, ceramide similar to a method of measuring the activity for promoting the above-described ceramide release can be listed.
As a receiving membrane, the mixed lipid of phosphatidylcholine derived from egg yolk, and phosphatidylethanolamine derived from egg yolk, or the mixed lipid of synthesized phosphatidylcholine and synthesized phosphatidylethanolamine or the like can be used.
As a method of mixing a drug for promoting ceramide transport and a donating membrane containing ceramide and a method of incubating the obtained mixture, it can be performed similar to the incubating process by a method of measuring the activity for promoting ceramide release.
As a selective membrane aggregating agent which is added to the mixture obtained in the incubating process, a castor seed lectin can be listed. Since a castor seed lectin is bound to galactocyl group contained in lactocyl ceramide, an aggregate consisting of a donating membrane and a castor seed lectin is formed, and only the donating membrane can be selectively precipitated by centrifuging at a low speed. In the case where biotinated phosphatidylethanolamine is utilized instead of lactocylceramide, biotin binding reagent such as avidin, streptoavidin and the like can be used as a selective membrane aggregating agent. Moreover, low-temperature insulation maintenance can be performed for 5 to 10 minutes at 0 to 4° C. using ice bath, refrigerator or the like.
In a separate process for separating the mixture obtained in the cooling and maintaining process, the supernatant and precipitated donating membrane is obtained by centrifuging the mixture cooled after a selective membrane aggregating agent has been added for 3 to 10 minutes at 12,000×g. Moreover, the obtained donating membrane can be used by dissolving in SDS, octyl glucoside, chloroforme or the like in the quantifying process.
The quantifying process can be performed similar to a method of measuring the activity for promoting ceramide release.
Hereinafter, the present invention will be further explained by exemplifying Examples, however, this is only exemplification, the present invention is not limited. It should be noted that in the following Examples, as far as the basic molecular biological operation is not particularly clearly indicated, it was performed by a method described in “Molecular Cloning”, second edition, written by Sambrook, J., Fritsch, E. F. and Maniatis, T., published by Cold Spring Harbor Laboratory Press, in 1989, or “Current Protocol in Molecular Biology” written by Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J. and Struhl, K., having been continuously published from 1987 up to the present by Wiley and Sons, or it was used in accordance with the instruction of the product which is commercially available in the market in the case where a reagent or a kit which is commercially available in the market.
PCR primers used in the present experiment are indicated in Table 1.
CERT protein and its recombinant protein were manufactured by the following procedure.
Since as for CHO cell, positive amino acid transporter (mCAT-1) which has been derived from mouse and is a mouse retrovirus receptor is not expressed, if it remains as it is, a mouse retrovirus vector cannot be infected. Then, the strain in which mCAT-1 cDNA has been stably expressed in LY-A strain (named as LY-A2 strain) was established as the followings. First, mCAT-1 cDNA which has been cut out by treating the restricted endonucleotide Stu I and Bam HI from plasmid pJET (donated from Dr. James Cunningham of Brigham & Women's Hospital in U.S.A.) described in Albritton, L. M. et al (Cell 57, 659-666, 1989) was inserted into Eco RV/Bam HI site of mammal animal cell expressing plasmid/pcDNA 3.1/Hyg(+) and mCAT-1/pcDNA 3.1/Hyg was prepared. After it has been treated with mCAT-1/pcDNA3.1/Hyg with Bg1 II and linearized, it was introduced into LY-A cell using lipofectAMINE PLUS reagent. After it has been cultured in F12/NBS medium in which hygromycin B (250 μg/mL) has been added and hygromycin B resistant cell has been selected, 6 pieces of hygromycin B resistant strain was purified by a limited-dilution method. The mCAT-1 expression level and its stability of these strains have been tested by making radioactive arginine uptake as an index, and the strain that expresses in the most stable state was made LY-A2 strain. The radioactive arginine uptake assay method carried out was in accordance with a method by Wang et al (J. Biol. Chem. 267, 23617-23624, 1992). However, as an uptake reaction liquid, a reaction liquid in which 115 mM KCl, 0.9 mM CaCl2, 0.81 mM MgSO4, 5 mM D-glucose and 0.1 mM L-[3H] arginine (1.25 μCi/mL) have been added was used, and the reaction was performed at 25° C. for 30 seconds.
A plasmid of a retrovirus library constructed from cDNA derived from HeLa cell was introduced into a Plat-E cell which is a packaging cell using a Fu′ Gene transfection reagent. On the next day of the transfection treatment initiation, the medium was exchanged and further after it has been cultured for 24 hours, the medium was collected. The collected medium was, first, centrifuged at 150×g for 5 minutes, and subsequently, the supernatant in which the supernatant has been centrifuged at 1350×g for 5 minutes was made virus particle liquid. The prepared virus particle liquid has been cryopreserved at −80° C., and immediately before it was used, it was melted and used in an infection experiment.
A plasmid in which the cDNA desired to express has been incorporated in a pLIB vector was introduced into a Plat-E cell by a method similar to the method described above. In order to express a green fluorescene protein (GFP), a virus particle liquid was prepared by introducing pLIB-EGFP into Plat-E cell.
As a NBS used in a cell culture of infectious experiment, NBS which has been treated at 58° C. for 30 minutes was used for the purpose of inactivating complement factor. 2 million pieces of LY-A2 cells were inoculated in a culture dish having the diameter of 100-mm and it has been cultured overnight at 37° C. in 10 mL of F12/NBS. On the next day, human cDNA library expression retrovirus particle liquid was 1:10 diluted in F 12/NBS, and the liquid in which polybrene (800 μg/mL PBS) which has been filtered and sterilized was added so that the final concentration is 4 μg/mL was made infectious medium. The culture liquid of cell was exchanged with 5 mL per a culture dish of infectious medium and cultured at 37° C. for 6 hours. The medium was exchanged again with 10 mL of F12/NBS, and further it has been cultured overnight. The cells were collected by a trypsin treatment 2 millions cells per a dish was inoculated in a 150-mm diameter culture dish. After it has been cultured at 33° C. overnight in 10 mL of F12/NBS, it was subjected to MCD selection described in the next paragraph. It should be noted that from the preliminary experiment using a retrovirus particle liquid prepared from pLIB-EGFP vector, it was estimated that about 50% of LY-A2 cells are infected under the infectious conditions performed herein.
The method of selecting function recovery strain used herein was performed on the basis of the knowledge that a cell whose SM content has been reduced becomes highly sensitive to cholesterol drawing reagent/methylcyclodextrin (MCD), and the cell whose SM content has been recovered recovers the resistance to MCD. As described in the foregoing paragraph, after the infected cell that has been seeded and cultured at 33° C. overnight was washed twice in 10 mL of serum-free F12 medium, it has been at 37° C. for one hour in 25 mL. After the culture dish was washed three times with 12 mL of PBS, 25 mL of F12/NBS was added and cultured at 33° C. for 7 to 10 days. The surviving cell was inoculated by performing a trypsin treatment, and subjected to a similar MCD treatment. However, in the MCD treatments after the second treatment, the number of cells is small, therefore, the cell culture scale was made 60-mm diameter culture dish. Concerning with the still survived cell after the total 4 times of MCD treatments, the cell was purified by performing the limited-dilution. From the purified cells, the cell strain/LY-A2R cell whose MCD resistance, lysenin sensitivity and SM biosynthesis has been recovered to the level found in CHO-K1 cell was obtained.
The cDNA introduced into LY-A2R cell was amplified by genomic PCR, and cloned. Specifically, by making genomic DNA prepared from LY-A2R cell as a template, and by making a synthetic oligonucleotide corresponding to a sequence in a pLIB vector (primers #1 and #2 indicated in Table 2) as a primer, PCR was performed. DNA having about 2.4 kilo base pair (kbp) which has been amplified was inserted into pcDNA3.1/V5-His-TOPO vector. When the obtained plasmid was introduced into LY-A cell using LipofectAMINE PLUS reagent, the cell recovers to the CHO-K1 cell level was observed. When the sequence having the relevant 2.4 kbp DNA was determined, it was clarified that it is substantially the same with cDNA sequence (GenBank No: AF232930) of human GPBP Δ 26 protein which has been already reported by Raya, A et al (J. Biol. Chem. 275, 40392-40399, 2000). As already described in detail in the present specification, since it has been clarified that the function of this gene product within the cell is involved in ceramide transport, the present inventors have named the protein having essentially the same sequence with GPBP Δ 26 protein as CERT protein.
ORF of human CERT protein was cloned by the following method. PCR was performed by making human HeLa retrovirus library manufactured by Clontech, Co., Ltd. as a template, and using the primers #3 and #5 indicated in Table 1. It should be noted that Eco RI site was added to the primer #3 which is a prospective primer and Xho I site has been added to the primer #5 which is a reversed primer, respectively. After DNA having about 1.8 kbp which has been amplified was treated with Eco RI and Xho I, the cloning was carried out by inserting it into Eco RI-Xho I site of pBS vector. The obtained plasmid was named as pBS/hCERT. It was admitted that the sequence of cDNA cloned herein is the same with ORF sequence of human GPBPΔ 26 protein (GenBank No: AF232930) already reported by Raya, A et al (J. Biol. Chem. 275, 40392-40399, 2000).
It has been known that as a separate splicing type of GPBP Δ 26 protein (that is, substantially, CERT protein), exon having 78 bp has been further added and as a result of this, GPBP (that is, substantially, CERTL) which is a product larger by 26 amino acid residue has expressed in a variety of species of cells. ORF sequence corresponding to human CERTL (hCERTL) was prepared as the followings, and the cloning was carried out. First, PCR was carried out by making pBS/hCERT as a template and using the primers #3 and #5 indicated in Table 1, and the amplified DNA having about 1.2 kbp was obtained. On the other hand, PCR was carried out by making pBS/hCERT as a template and using the primers #8 and #5 indicated in Table 1, and the amplified DNA having about 0.7 kbp was obtained. Next, PCR was carried out by making the protein in which these DNA having 1.2 kbp and DNA having 0.7 kbp have been mixed in the equimolar ratio as a template and using the primers #3 and #7 indicated in Table 1, and the amplified DNA having about 1.9 kbp was obtained. The plasmid cloned by inserting Eco RI and Xho I treatment products of this DNA having 1.9 kbp into Eco RI-Xho I site of pBS vector was named as pBS/hCERTL. It was admitted that the sequence of DNA having 1.9 kbp is corresponded with the ORF sequence of hGPBP protein (GenBank No: AF136450) which has been already reported by Raya, A et al (J. Biol. Chem. 274, 12642-12649, 1999).
PCR was carried out by making pBS/hCERT as a template, and combining the primers indicated in Table 1 as the following, and the amplified DNA for coding a variety of deleted variants of CERT protein was obtained. For the purpose of obtaining the amplified DNA for coding PH domain deleted variant (hCERTΔ PH protein), and for the purpose of obtaining the amplified DNA for coding START domain deleted variant (hCERTΔ ST protein), the primers #3 and #10 are used, respectively. Moreover, for the purpose of obtaining the amplified DNA for coding MR domain deleted variant (hCERTΔ MR protein), after the product having about 350 bp by carrying out PCR using the primers #3 and #11 and the product having about 770 bp by carrying out using the primers #12 and #5 were obtained, respectively, PCR was carried out by making the one in which these PCR product DNAs have been mixed using the primers #3 and #5, the amplified DNA having about 1.1 kbp for coding hCERT A MR protein was obtained. The DNA for coding these deled variants was treated with Eco RI and Xho I and the cloning was carried out by inserting it into Eco RI-Xho I site of pBS vector. For the purpose of obtaining the amplified DNA for coding deleted variant having only PH domain (PHhCERT protein), for the purpose of obtaining the amplified DNA for coding deleted variant having only MR domain (MRhCERT protein), PCR product was obtained using the primers #15 and #16, for the purpose of obtaining the amplified DNA for coding deleted variant having only START domain (SThCERT protein), PCR product was obtained using the primers #17 and #18, respectively. The cloning was carried out by inserting Hind III and Xho I treatment product of these amplified DNA into HindIII-Xho I site of pBS/nFL vector.
For the purpose of adding Asp-Tyr-Lys-Asp-Asp-Asp-Lys sequence (FL sequence) which is capable of admitting by anti-FLAG antibody at amino end of CERT, PCR was carried out by making pBS/hCERT as a template and using the primers #4 and #5 indicated in Table 1. The plasmid cloned by inserting Eco RI and Xho I treatment products of this amplified DNA into Eco RI-Xho I site of pBS vector was named as pBS/FL-hCERT. At the time when FL sequence is added to amino acid end of CERTL, a similar PCR was carried out using pBS/hCERTL as a template, and a plasmid in which the relevant product has been cloned was named as pBS/FL-hCERTL-<
For the purpose of expressing via retrovirus vector infection, a variety of DNAs cloned at Eco RI-Xho I site of pBS vector was cut out by performing Eco RI-Xho I treatment, and the plasmid obtained by inserting these into Eco RI-Sal I site of pLIB vector for expressing retrovirus expression was used. This method can be also applied to a recombinant protein of CERT protein. On the other hand, for the purpose of expressing via lipofection, plasmid obtained by inserting DNA in which a variety of DNAs have been cloned into Eco RI-Xho I site of pBS vector was cut out by performing Eco RI-Xho I treatment into Eco RI-Xho I site of pcDNA3.1/Neo(+) vector was used. ORF of hCERT cloned by performing PCR amplification from HeLa cell cDNA library was inserted into pcDNA 3.1/Neo(+) vector and the plasmid pcDNA 3.1/hCERT was obtained.
100 thousand pieces of cells per each well of a 12-well culture dish were inoculated in 1 mL of F12/NBS culture medium and cultured at 33° C. overnight. After Cells existed on the culture dish was washed twice in 1 mL of serum-free F12 medium, 1 mL of 10 mM MCD/F12 or lysenin (25 ng/mL)/F12 was added. It should be noted that culture medium in which 1 mL of only serum-free F12 has been added was made control. After it has been incubated at 37° C. for one hour, it was washed twice in 1 mL of phosphate buffered saline (PBS). Subsequently, 1 mL of MTT (0.5 mg/mL)/F12 was added and incubated at 37° C. for one hour. The relative amount of the generated reduced form of MTT was measured by a method of reference literature written by Hanada et al (J. Biol. Chem. 273, 33787-33794, 1998).
The relevant gene was expressed in LY-A2 cell by making virus particle derived from retrovirus vector infect it prepared in the foregoing paragraph. Specifically, the relevant gene was expressed in LY-A2 cell by making virus particle derived from pLIB/hCERT. In the case where hCERT was expressed, the purified transformed strain was also separated by a ultradilution method, and this strain was named as LY-A2/hCERT. After it has been cultured for days, the sensitivity to MCD and lysenin was examined by the above-described method. As a control, also concerning with the cell that virus particle derived from pLIB vector in which cDNA has not inserted infects LY-A2 cell, the sensibility of it to MCD and lysenin was examined. Furthermore, also concerning with CHO-K1 cell, the sensitivity of it to MCD and lysenin was examined. The results of the sensitivity measurement to MCD and lysenin are indicated in
Moreover, the relevant gene was expressed by introducing the expressed plasmid derived from pcDNA 3.1/Neo(+) prepared in the foregoing paragraph in LY-A cell using LipofectAMINE PLUS reagent. Specifically, the relevant gene was expressed in LY-A cell by introducing pcDNA 3.1/hCERT. For the purpose of purifying the stably expressed strain of hCERT, first, G418 resistant cell was selected and it was purified by a ultradilution method. Then, the sensibility of it to MCD and lysenin was examined by the above-described method, and the strain whose MCD sensibility has been recovered to the wild type level was separated. This transformed strain was named as LY-A/hCERT.
From LY-A2 cell infected with virus particle derived from pLIB/hCERT, transformed strain was purified and the reactivity of LY-A2/hCERT cell which is a purified strain with respect to MCD and lysenin was further examined in detail. The results are indicated in
3×106 cells per culture dish having the 150-mm diameter of CHO-K1 cell, LY-A2 cell, or LY-A2/hCERT cell was inoculated in 20 mL of F12/NBS medium, and cultured at 33° C. overnight. After it was washed twice in 10 mL of serum-free F12 medium, it was further cultured for 2 days in 20 mL of Nutridoma BO medium. After it was washed in PBS, the cells were collected by a method of scaping, suspended again in PBS, and lipid was extracted by a method described in Bligh et al (Can. J. Biochem. Physiol. 37, 911-917, 1959). The extracted lipid was separated using TLC whose development solvents were chloroform/methanol/acetic acid/H2O (volume ratio, 25:15:4:2). After the separated phospholipid on the TLC plate was colored by iodine vapor, the band of the respective separated phospholipid was scraped out from the plate. The scraped separated phospholipid was quantified by measuring phosphorus content by a method of Rouser et al (Lipids 1, 85-86, 1966). The results are indicated in
CHO-K1 cell, LY-A2 cell, and LY-A2/hCERT cell were inoculated at the cell density of 1.0×106 per culture dish having the 60-mm diameter, and cultured at 33° C. overnight. Subsequently, it was exchanged with 15 mL of Nutridoma medium, after [14C] serine (0.75 μCi) or [14C] choline (1.0 μCi) have been added, in the case where [14C] serine is added, it was cultured for 2 hours, and in the case where [14C] choline is added, it was cultured at 33° C. for 5 hours. However, in the case where the influence on (1R, 3R) HPA-12 is analyzed, after it was pretreated at 4° C. for 15 minutes in 1.5 mL of Nutridoma medium to which 1 μM of (1R,3R) HPA-12 has been added (in control experiment, dimethylsulfoxide used for dissolving a drug was added at the concentration corresponding to 0.01% of the final concentration), [14C] serine was added, and cultured at 33° C. for 2 hours. After the cell was washed twice in 2 mL of cooled PBS, dissolved in 900 μL of sodium dodecyl sulfate (SDS), then, 800 μL and 20 μL of dissolved matter were used for determining lipid extraction and protein concentration. For the purpose of analyzing lipid labeled with [14C] serine, the lipid was separated by TLC by making acetic methyl/n-propanol/chloroform/methanol/0.25% KCl (25:25:25 10:9, volume ratio) development solvent. The radioactive lipid separated on the plate was detected and analyzed by an image analyzer. The results using [14C] serine are indicated in
The assay of SM synthase activity was carried out using C6—NBD-Cer as a substrate by a method of Hanada et al (Biochim. Biophys. Acta 1086, 151-156, 1991). However, the membrane fractions prepared respectively from CHO-K1 cell, LY-A2 cell and LY-A2/hCERT cell were used as an enzyme source. The results are indicated in
The present inventors have already made it clear that the activity of ATP dependent transport from the endoplasmic reticulum of natural ceramide (hereinafter, also referred to as ER) to Golgi apparatus can be qualitatively evaluated from the analysis of reallocation from ER of C5-DMB-Cer which is a fluorescent ceramide analogue to Golgi apparatus (J. Cell Biol. 144,673-685, 1999). As described in detail, the cell was exposed to C5-DMB-Cer at 4° C. for 30 minutes for the purpose of performing the pulse labeling of a variety of organelle membranes within a cell containing ER, after washing, it was traced at 33° C. for 15 minutes by a fluorescene microscopy observation method. A method of performing cell labeling using C5-DMB-Cer and a method of performing an observation by a fluorescence microscopy were carried out by a method of Yasuda et al (J. Biol. Chem. 276, 43994-44002, 2001) and a method of Fukasawa et al (J. Cell Biol. 144, 673-685, 1999). Specifically, after CHO-K1 cell, LY-A2 cell and LY-A2/hCERT cell grown on a coverslip made of glass were treated at 4° C. for 30 minutes in F12 medium containing 1 micro-M C5-DMB-Cer, it was washed three times in 1 mL of F12 medium. In the case where 1 mL of Nutridoma medium is added and not traced, immediately washed in PBS, on the other hand, in the case where it is traced, it was washed in PBS after it was cultured at 33° C. for 15 minutes. Next, it was fixed at 4° C. for 5 minutes in PBS solution of 0.125% glutaraldehyde. The sample was observed under the fluorescence microscopy immediately after it has been fixed and photographed with a digital CCD camera. The results are indicated in
Furthermore, the present inventors have already made it clear that in the transport pathway through which ceramide transfers from ER to the place of SM synthesis, there are at least two pathways that is dependent on ATP and is not dependent on ATP, and in LY-A cell, ATP dependent transport pathway is deleted. For the purpose of examining whether or not ceramide transport pathway recovered by introducing hCERT is ATP dependent transport pathway, the influence of ATP depletion with respect to transfer within a cell of C5-DMB-Cer was analyzed. It should be noted that in the case where ATP depletion conditions are used, after it was washed three times in 1 mL of F12 medium, pretreated at 33° C. for 15 minutes in 1 mL of Nutridoma medium in which energy inhibitor (50 mM deoxyglucose and 5 mM sodium azide) has been added, it was C5-DMB-Cer treated, washed in PBS, fixed similarly, observed and shot. Moreover, in the case where it is traced, after it was C5-DMB-Cer treated, and further after it was cultured at 33° C. for 15 minutes in the energy inhibitor added medium, it was washed in PBS, similarly fixed, observed and shot. The results are indicated in
The present inventors have already found a selective inhibitor with respect to ATP dependent ceramide transport pathway (J. Biol. Chem. 276,43994-44002, 2001). For the purpose of examining whether or not ceramide transport pathway recovered by introducing hCERT is a transport pathway which is sensitive to (1R,3R) HPA-12, the analysis of the influence of (1R, 3R) HPA-12 treatment with respect to SM biosynthesis was performed by performing a lipid metabolism labeling experiment using radioactive serine. The results are indicated in
Also in the original LY-A cell to which retrovirus receptor has not been introduced, it has been ascertained as described below that ceramide transport function is recovered by hCERT expression. hCERT inserted pcDNA3.1/Neo plasmid was introduced into LY-A cell by performing lipofection, after G418 resistant cell was selected, transformed strain LY-A/hCERT was purified by a limited-dilution. Subsequently, the measurement of the sensitivity to MCD, metabolism labeling experiment of lipid using [14C] serine and [14C] choline and cell labeling using C5-DMB-Cer and fluorescene microscopy observation were similarly performed. The results are indicated in FIG. 9(A)-(C), respectively.
It has been validated whether or not CERT protein and CERTL which is a large separate splicing type product having 26 amino acid residue complements the deletion of LY-A cell. LY-2A cell were infected with retroviruses for expressing CERT protein and CERTL protein that FL sequence has been added to amino end, respectively. The MCD resistance of the obtained (LY-A2+FL-hCERT) cell and ((LY-A2+FL-hCERTL) cell were measured by counting the number of survived cells in accordance with the method described in <Method of selecting MCD resistance recovery strain>. Moreover, the MCD resistance of (LY-A2+empty vector) cell, CHO-K1 cell and LY-A2 cell obtained by infecting LY-A2 cell using pLIB vector in which gene has not been inserted were also similarly measured. The results are indicated in FIG. 10(A).
The method of preparing cell lysate and Western blot method were performed in accordance with a method of Hanada et al (J. Biol. Chem. 273, 337787-33794, 1998) and a method of Bejaoui et al (J. Clin. Invest. 110, 1301-1308, 2002). Specifically, (LY-A2+FL-hCERT) cell, (LY-A2+FL-hCERTL) cell (LY-A2+empty vector) cell which have been washed in chilled PBS were collected by a method of scraping out, respectively, after it was suspended in HSEI buffer [in which 250 mM sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA) and protease inhibitor cocktail have been added to 10 mM Hepes-NaOH buffer (pH 7.5)], the cells were lysed by performing supersonic treatment using an immersion supersonic generator. This cell lysate was subjected to SDS-polyacrylamide gel electrophoresis, and transcribed to a PVDF membrane. The immunological reaction protein were detected by performing a blocking treatment, the primary antibody treatment and its washing treatment, the secondary antibody treatment and its washing treatment, and then ECL treatment with respect to this blot membrane. For the purpose of detecting GFP or GFP fusion CERT protein, BD Living Colors™ A.v. monoclonal antibody was used as the primary antibody, respectively. As the secondary antibody, HRP binding goat anti-mouse immunoglobulin G was used. The results are indicated in
<cDNA Cloning Method of CERT Protein Derived from CHO-K1 Cell>
cDNA of CERT protein derived from CHO-K1 cell was cloned by performing rapid amplification of cDNA ends (RACE) of cDNA ends using SMART RACE cDNA amplification kit manufactured by Clontech, Co., Ltd. Specifically, by making 5′-RACE Ready CHO cDNA prepared by utilizing the relevant kit template, and by making the primer #19 indicated in Table 1 gene specific primer, the PCR was carried out. At the time of carrying out this, Pyrobest DNA polymerase was used as a DNA polymerase. On the other hand, by making 3′-RACE Ready CHO cDNA template, and by making the primer #20 indicated in Table 1 gene specific primer, the PCR was carried out. The amplification products having about 1.2 kbp and about 1.4 kbp were obtained, respectively. After these products have been cloned in pCR-Blunt II-TOPO vector, the DNA sequence was determined. Considering overlapping region in the determined sequence, the sequence having the full length of cDNA, sequence 2473 bp was determined. Next, for the purpose of obtaining DNA containing the complete ORF of cCERT, the PCR was carried out by making CHO cell cDNA library template and by utilizing the primers #21 and #22 indicated in Table 1. The amplified DNA having about 1.9 kbp was treated by Eco RI and Xho I, the plasmid inserted into Eco RI-Xho I site of pBS vector was named as pBS/cCERT.
The total RNA was prepared from CHO-K1 cell and LY-A cell using isogen manufactured by Nippon Gene, Co., Ltd. Moreover, as a probe, a probe in which DNA having about 1.2 kbp obtained by the above-described cCERT 5′-RACE has been 32P labeled using [α-32P] dCTP and megaprime DNA labeling kit was used. Concerning with the washing conditions after the hybridization was carried out, it was performed under the stringent conditions. The analysis of radioactive pattern on the blot membrane was performed using BAS 2000 image analyzer. Moreover, the transcription membrane re-probing experiment for internal standard was performed by making a fragment that β-actin DNA fragment which has been commercially obtained from Wako Junyaku Kogyo, Co., Ltd. was 32P labeled a probe. The results are indicated in
The total RNA was prepared from LY-A cell using isogen. Next, cDNA was prepared from this RNA using the superscript first chain synthesizing system. Next, the PCR was carried out by making the present cDNA a template and by utilizing the primers #21 and #22 indicated in Table 1. The amplified DNA having about 1.9 kbp was treated with Eco RI and Xho I, the plasmid inserted into Eco RI-Xho I site of pBS vector was named as pBS/cCERT (G67E). As described below, it was clarified that in cCERT derived from LY-A cell, a missense mutation that 67 glycine residue has been substituted by glutamic acid occurs, therefore, this protein was named as cCERT (G67E).
The PCR was carried out for the purpose of adding a FL sequence which is capable of admitting it with an anti-FLAG antibody by making pBS/cCERT template and by utilizing the primers #23 and #6 indicated in Table 1. DNA having about 300 bp obtained by treating this amplification product with Cla I and Xho I was purified. Then, DNA for coding CERT that FL sequence has added at carboxyl end was prepared by inserting the previously purified DNA having about 300 bp into the DNA having about 3.7 kbp obtained by treating pBS/cCERT and pBS/cCERT (G67E) with Cla I and Xho I and was cloned on pBS. After the sequence was ascertained, the cloned DNA was transferred into pLIB vector.
Moreover, whether or not the nature of LY-A2 cell is recovered to the wild-type was analyzed by measuring the MCD resistance of (Ly-A2+cCERT-FL) cell and (LY-A2+cCERT (F67E) cell in which cDNA for coding FL sequence addition type (named as cCERT-FL and cCERT (G67E)-FL) of cCERT and cCERT (G67E) has been introduced into LY-A2 cell by means of counting the number of survived cells in accordance with the method described in <Method of selecting MCD resistance recovery strain>. The results are indicated in
Plasmid for expressing a fusion protein that GFP has been added at carboxyl end of hCERT, cCERT and cCERT (G67E) was prepared as the following.
The PCR was carried out by making pBS/hCERT, pBS/cCERT or pBS/cCERT (G67E) a template and by utilizing the primers #3 and #24 indicated in Table 1. After the amplified DNA having about 1.9 kbp was treated with Eco RI and Xho I and was inserted into Eco RI-Xho I site of pBS vector and cloned, the base sequence was admitted. Then, these cloned DNA was cut out by treating it with Eco RI and Xho I and inserted into Eco RI-Sal I site of pEGFP-N3 vector. The CERT-GFP fusion protein expression plasmids prepared in this way were systematically named as phCERT-GFP, pcCERT-GFP, pcCERT (G67E)-GFP and the like, respectively. It should be noted that the base sequence of the primers #3 and #25 are designed on the basis of DNA sequence of hCERT and is partially different from DNA sequence of the relevant portion of cCERT. However, since the amino acid sequences of the relevant portion are identified between hCERT and cCERT, and these primers were effective in PCR with respect to cCERT, these were used.
pEGFP-N3, phCERT-GFP, pcCERT-GFP or pcCERT (G67E)-GFP plasmid was introduced into CHO-K1 cell using FuGene reagent. The obtained cells were named as (CHO-K1+pEGFP-N3) cell, (CHO-K1+phCERT-GFP) cell, (CHO-K1+pcCERT-GFP) cell, and (CHO-K1+pcCERT (G67E)-GFP) cell, respectively. After it has been cultured for 24 hours from the introduction initiation of DNA, the inoculation was performed again, and further after it has been cultured for 2 days, it was subjected to the observation of fluorescence or the preparation of cell lysate.
In (CHO-K1+pEGFP-N3) cell, (CHO-K1+phCERT-GFP) cell, (CHO-K1+pcCERT-GFP) cell, and (CHO-K1+pcCERT (G67E)-GFP) cell, in which CERT fusion GFP has been transiently expressed, indirect immunocyte staining using antibody with respect to GS28 protein locating in Golgi apparatus, and the distributions of GFP and GS28 within a cell were compared. The immunocytochemical method was performed in, accordance with a method of Yasuda et al (J. Biol. Chem. 278, 4176-4183, 2003). Specifically, after 3.7% formaldehyde fixation treatment, 0.1 M ammonia chloride treatment, 0.2% Triton X-100 treatment, blocking treatment, the primary antibody treatment and its washings, and the secondary antibody and its washings were performed with respect to a cell cultured on the coverslip, the cell was mounted on the slide glass, and the fluorescene microscopic observation was performed. However, a mouse anti-GS28 monoclonal antibody was used for the primary antibody, and alexa fluoro 594 goat anti-mouse immunoglobulin G was used for the secondary antibody. The results are indicated in
A method of preparing a cell lysate and Western blot method were performed in accordance with a method of Hanada et al (J. Biol. Chem. 273, 33787-33794, 1998) and a method of Bejaoui et al (J. Clin. Invest. 110, 1301-1308, 2002). Specifically, cell which have been washed in cooled PBS were collected by a method of scraping out, after it was suspended in HSEI buffer [in which 250 mM scrose, 1 mM ethylenediaminetetraacetic acid (EDTA) and protease inhibitor cocktail have been added to 10 mM Hepes-NaOH buffer (pH 7.5)], the cells were lysed by performing supersonic treatment using an immersion supersonic generator. This cell lysate were subjected to SDS-polyacrylamide gel electrophoresis, and transcribed to a PVDF membrane. The immunological reaction protein were detected by performing a blocking treatment, the primary antibody treatment and its washing treatment, the secondary antibody treatment and its washing treatment, and then ECL treatment with respect to this transcription member. For the purpose of detecting GFP or GFP fusion CERT protein, BD Living Colors™ A.v. monoclonal antibody was used as the primary antibody, respectively. As the secondary antibody, HRP binding goat anti-mouse immunoglobulin G was used. The results are indicated in
For the purpose of expressing hCERT protein and its related proteins that histidine tag sequence has added in E. coli, the plasmid in which DNA fragment prepared and cloned in the paragraph from 0064 to 0066 of the specification of the present application has been transferred to multi-cloning site of pET28a (+) vector so that it is in-frame was prepared. Specifically, hCERT, hCERTL, hCERTΔ PH, hCERTΔ MR, and hCERTΔ ST were collected by Eco RI-Xho I treatment, inserted it into Eco RI-Xho I site of pET-28a(+). On the other hand, PHhCERT, MRhCERT and SThCERT that have been cloned in pBS/nFL were collected by Hind III-Xho I treatment, and inserted into Hind III-Xho I site of PET-28a(+).
The pET-28a(+) plasmid in which DNA for coding hCERT protein and its related protein (hCERT, hCERTL, hCERTΔ PH, hCERT A MR, hCERTΔ ST, PHhCERT, MRhCERT and SThCERT) has been introduced into E. coli BL21 (DE3) strain by a heat shock method, and transformed as a resistant bacterium with respect to kanamycin. The transformed cell has been cultured at 37° C. in the Luria broth medium until the cell turbidity reaches to 0.6 at the absorbance in the wavelength of 600 nm. At the time when the absorbance has reached to 0.6, IPTG (isopropyle-1-thio-beta-D-galactopyranoside) was added so that the final concentration becomes 250 μM, and further cultured at 25° C. overnight. After the cultured liquid was centrifuged and suspended in a buffer for lysing the precipitated E. coli {25 mM Tris (pH7.4), 1% Triton X-100, 1 mM orthovanadic acid, 50 mM sodium fluoride, 5 mM sodium pyrophosphoric acid, 2.5 mM 2 mercaptoethanol, 0.27 M sucrose, and protease inhibitor mixture (Roche #1873580) one tablet/50 mL}, the bacterium was lysed using an immersion supersonic generator. This has been centrifuged at 100,000 g for one hour, and the supernatant fraction was used for purification. For purification, TALON (cobalt ion chelate resin) manufactured by Clontech, Co., Ltd. was used. The purification using TALON was performed in accordance with the manual. After the targeted protein eluted by 150 mM of imidazol was dialyzed overnight, substituted with a buffer consisting of 10 mM Tris (pH 7.4) and 250 mM sucrose, or a buffer consisting of 10 mM Tris (pH 7.4) and 150 mM of sodium chloride, it was stored at −80° C. As shown in
The followings were clarified in
As shown in
As shown in
As shown in
As shown in
As shown in
As shown in
The sequence of the full length cDNA with respect to CERT mRNA derived from CHO-K1 cell was determined by RACE or the like. The sequence having the full length of 2473 bp contained ORF having 1794 bp for coding the product (cCERT) of 598 amino acid (Sequence tables 3 and 7). CERT is 598 amino acid in all of human, mouse and Chinese hamster, and the homology of about 90% exists at DNA sequence level and the homology of about 98% exists between cCERT and hCERT. The results indicate the strong conservation of CERT among mammals. Moreover, cDNA library derived from CHO cell, ORF for coding protein homologous to hCERTL (named as cCERTL) was also amplified by a PCR (Sequence tables 4 and 8). cCERTL is a product larger than cCERT by 26 amino acid similar to the relationship between hCERTL and hCERT.
As shown in
As indicated in the foregoing paragraph, it has been found that also in LY-A cell, CERT mRNA was expressed. Then, CERT cDNA was cloned from LY-A cell by reverse transcription PCR. It has been clarified that CERT cDNA of LY-A cell has the sole mutation comparing to sequence derived from CHO-K1 cell. This mutation is single base substitution in which 67th codon of ORF is changed from GGA to GAA, as a result of this, it was a missense mutation in which 67 glycine residue of cCERT protein is substituted by glutamic acid residue. Then, this ORF product was named as cCERT (G67E). It should be noted that in the present experiment, two independent PCR experiments were carried out and the identical sequence was obtained. Hence, the missense mutation previously described is not an artificial mutation by PCR.
<cDNA Introduction Expression Effect of cCERT or cCERT (G67E) in LY-A2 Cell>
As shown in
It should be noted that in the results of Western blot analysis shown in
As shown in
When hCERT cDNA was introduced into LY-A strain, the SM synthesis was recovered to the wild type level. Moreover, it has been also clarified by an assay using fluorescent ceramide analogue that ceramide transfer from endoplasmic reticulum to Golgi apparatus was recovered. hCERTL corresponding to a separate splicing type also has made LY-A strain recovered. Hence CERT protein is a factor involving in ceramide selective transport within a cell.
From the results described above, it has been clarified that (1) CERT protein has been selected from cDNA library by a method of searching the function recovery of LY-A cell having the default involving in ceramide transport, (2) CERT protein complements all of the known defects of LY-A cell, (3) in LY-A cell, missense mutation is occurring in an endogenous gene for CERT protein, (4) CERT protein having the mutation occurring in LY-A cell damages the defect-complementing ability. Therefore, from these results, it has been clarified for the first time that the mutation in the gene for coding the relevant CERT protein is a causal mutation of the defect of LY-A cell, and CERT is a factor specifically involving in ceramide selective transport within a cell.
Next, whether or not hCERT protein and its recombinant protein (hCERT protein, hCERTL protein, hCERTΔ PH protein, hCERT Δ MR protein, and hCERTΔ ST protein, and further, PHhCERT protein, MRhCERT protein and SThCERT protein) indicates the activity for releasing ceramide from lipid membrane was considered. In this assay, hCERT protein and its recombinant protein (hCERT protein, hCERTL protein, hCERTΔ PH protein, hCERTΔ MR protein, and hCERT Δ ST protein, and further, PHhCERT protein, MRhCERT protein and SThCERT protein) which were expressed by E. coli described above, used in <Preparation of vector for expressing hCERT protein and its related protein to which histidine tag sequence has been added by E. coli> and <Method of purifying hCERT from recombinant E. coli> were purified and used.
Hereinafter, the standard method will be described in detail, however, even if the described conditions are changed, a method of detecting ceramide release promotion activity having means for separating releasing ceramide by a centrifugation principally belongs to the present invention. The lipid membrane containing ceramide was prepared so that it contains 12.5 nCi (225 pmol) per sample of [palmitoyl-1-14C]N-palmitoyl-D-ethyro-sphigosine (hereinafter, may be referred to as 14C-ceramide) on the basis of the mixed lipid consisting of phosphatidylcholine and phosphatidylethanolamine derived from egg yolk at the ratio of 4:1 and its concentration becomes 2.5 mg/mL. This lipid membrane is required at the rate of 20 μL per one sample of the activity measurement. After the amount of lipid required for activity measurement was dispensed in Eppendorf tube, it was dried by spraying nitrogen gas. After the buffer 1 [20 mM Hepes-NaOH buffer (pH7.4) to which 50 mM NaCl and 1 mM EDTA have been added] was added to the dried lipid membrane so that the concentration becomes 2.5 mg/mL, the supersonic treatment was gently performed using bath type supersonic generator [Model 2210 manufactured by Branson, Co., Ltd.]. The supersonic treatment was performed at 25° C., and the procedure that the supersonic treatment for 3 minutes, the vortex for 30 seconds and the supersonic treatment for 3 minutes were performed in this order. The lipid membrane prepared in this way was used in ceramide release experiment. The ceramide release reaction for lipid membrane and its detection were performed as the followings.
hCERT protein or its recombinant protein described above as a protein sample to be targeted (under the standard conditions, the amount of protein corresponding to 450 picomoles, which is 2-fold molar equivalent amount of ceramide contained in the donating membrane was used) were messed up to 30 μL using the buffer 2 [50 mM Hepes-NaOH buffer (pH7.4) to which 100 mM NaCl and 0.5 mM EDTA have been added]. Here, the reaction was initiated by adding 20 μL of lipid membrane containing ceramide. The final concentration of phospholipids became 1 mg/mL, and ceramide was contained at the ratio of about 0.3% comparing to the total phospholipid amount. After the mixture of these has been incubated at 37° C. for 30 minutes, it was centrifuged at 50,000×g for 30 minutes (centrifuging machine: himac CS120EX manufactured by HITACHI Engineering Machine, Co., Ltd.: centrifuging rotor: RP100AT3-200; centrifugation tube, 0.23PC tube) and the lipid membrane was precipitated. In the case where hCERT protein and its recombinant protein which has been purified from the recombinant E. coli is used, most of the protein remains in the supernatant under these centrifugation conditions, therefore, when 14C-ceramide binds to hCERT protein, it releases from the lipid membrane and transfers to the supernatant fraction. The activity for promoting ceramide release with hCERT was calculated by measuring the radioactive activity of 14C in the supernatant fraction using a liquid scintillation counter. The results of measurement of the activity for promoting ceramide release using hCERT protein are indicated in
The activity for promoting ceramide release was measured using the purified recombinant hCERT. When phospholipid multilamellar vesicle containing a trace of ceramide was centrifuged, ceramide was almost completely precipitated accompanying with phospholipid vesicle. However, in the case where hCERT co-exists, ceramide released from the membrane and allocated to the supernatant fraction accompanying with hCERT while phospholipid multilamellar vesicle was completely precipitated. Specifically, as it is understood from
Up to now, as a lipid that protein having START domain except for CERT admits, phosphatidylcholine and cholesterol are known. However, as shown in
As shown in
From the results described above, it has been clarified that CERT and CERTL have the activity for specifically releasing ceramide from the lipid membrane via its START domain.
Subsequently, as described in detail below, whether or not hCERT protein and recombinant protein promotes the transfer between ceramide lipid membrane was considered, the purified hCERT protein and its recombinant protein were used in this assay were used. The present method was constructed on the basis of the principle of method of measuring the activity for promoting the intermembrane transfer of phosphatidylinositol by phosphatidylinositol transfer protein already reported by Kasper et al (Biochim. Biophys. Acta 664, 22-32, 1981) so that it becomes a new method of measuring the activity for promoting the ceramide intermembrane transfer. For the purpose of measuring the activity for promoting the intermembrane transfer, a donating membrane in which radioactive ceramide has been contained and a receiving membrane not containing ceramide were prepared. For the purpose of separating the lipid membrane, lactocylceramide was contained in the donating membrane. After the donating membrane and receiving membrane have been incubated with the protein sample to be targeted, castor seed lectin was added. Ceramide intermembrane-transferred can be quantified by measuring the radioactive activity of receptor membrane which has not been precipitated by a low speed centrifugation. Hereinafter, the standard method will be described in detail, however, principally, all of the method of introducing selective recognition marker into either of ceramide donating membrane or receiving membrane detecting the activity for promoting the intermembrane transfer having means for separating ceramide donating membrane and receiving membrane by utilizing the marker are belonged to the present invention.
The donating membrane was prepared by adding 12.5 nCi per one sample and 125 nCi of [2-palmitoyl-9, 10−3H(N)] L-α-dipalmitoyl phosphatidylcholine (hereinafter, may be referred to as 3H-DPPC) so that the total lipid concentration becomes 1.77 mg/mL. The substance amount of ceramide to be targeted was achieved by adding the non-radioactive ceramide to 14C-ceramide. This lipid membrane is required at the rate of 20 μL per one sample of the activity measurement. After the amount of lipid required for activity measurement was dispensed in polypropylene microtube, it was dried by spraying nitrogen gas. After the buffer 1 [20 mM Hepes-NaOH buffer (pH7.4) to which 50 mM NaCl and 1 mM EDTA have been added] was added to the dried lipid membrane so that the concentration becomes 1.77 mg/mL, the supersonic treatment was performed using an immersion supersonic generator (for example, UP50H manufactured by KUBOTA, Co., Ltd.). The supersonic treatment was performed at 25° C. for 10 minutes in water bath. Even if the donating membrane prepared in this way is centrifuged under the conditions of 4° C., 12,000×g and 30 minutes (centrifuging machine: MRX-150 manufactured TOMY, Co., Ltd.; centrifugation tube, 1.5-ml micro-test-tube manufactured by Eppendorf, Co., Ltd.), 90% or more of the lipid membrane is collected in the supernatant without precipitation.
The lipid membrane remained in the supernatant and the lipid membrane before the centrifugation was measured by a liquid scintillation counter, and it has been admitted that the radioactive activity of 3H is not changed between both in the respective experiments (normally, 90% or more was collected in the supernatant). Moreover, it has been admitted that the ratio of precipitation/non-precipitation found in radioactive activity of 3H indicated approximately the same value as the value calculated by phosphorus quantitative determination and it is possible that the allocation amount of the total donating membrane from the allocation amount of 3H-DPPC is estimated.
The receiving membrane, which is a mixed lipid consisting of phosphatidylcholine and phosphatidylethanolamine derived from egg yolk at the ratio of 4/1, was prepared so that the total lipid concentration becomes 5.33 mg/mL. This lipid membrane is required at the rate of 60 μL per one sample for activity measurement. After the amount of lipid required for measuring the activity has been dispensed in a 1.5-mL micro-test-tube (manufactured Eppendorf, Co., Ltd.), nitrogen gas was sprayed to it and dried. After the buffer 1 has been added the dried lipid membrane so that the concentration becomes 5.33 mg/mL, the supersonic treatment was performed using an immersion supersonic generator (for example, UP50H manufactured by KUBOTA, Co., Ltd.). The conditions of the supersonic treatment, subsequent centrifuging conditions and the like were performed similar to the preparation of the donating membrane.
hCERT protein, hCERTL protein, hCERTΔ PH protein, hCERT Δ MR protein, or hCERTΔ ST protein, or PhCERT protein, MRhCERT protein or SThCERT protein (under the standard conditions, a protein corresponding to 2 picomoles corresponding to 0.4% molar equivalent amount of ceramide in the donating membrane was used) which is a protein sample to be targeted, and 60 μL of receiving membrane was messed up to 80 μL using the buffer 1 in 1.5-mL micro-test-tube (manufactured by Eppendorf, Co., Ltd.). Here, the reaction was initiated by adding 20 μL of donating membrane, and it has been incubated at 37° C. for 15 minutes. The reaction was terminated by adding 30 μL of castor seed lectin and agitating by means of pipetting. For the purpose of sufficiently forming an aggregate, after it was cooled further for 15 minutes in ice bath, it was centrifuged under the conditions of 4° C., 12,000×g, 3 minutes. The supernatant was collected by a pipette and the precipitated donating membrane was dissolved in 130 μL of 0.1% SDS. The activity for promoting ceramide intermembrane transfer that the sample has was measured by measuring the radioactive activity of 14C of the supernatant and the donating membrane. Even in the case where the sample is not contained, ceramide extremely slightly intermembrane-transfers, therefore, it is subtracted as background by free diffusion. It has been admitted in each experiment that the donating membrane and receiving membrane are selectively separated by admitting that the most part of the radioactive activity derived from 3H-DPPC that has been added to the donating membrane (usually, 90% or more) is precipitating accompanying with the donating membrane. The results that the activity for promoting ceramide intermembrane transfer are indicated in
The protein was quantified using BCA assay reagent of Pierce, Co., Ltd. BSA was used for the reference.
As shown in
As shown in
The present invention can provide a novel drug for promoting ceramide transport. Moreover, the present invention can provide base sequence used for producing a drug of the present invention. Moreover, the present invention can provide a method of measuring the activity for promoting a novel ceramide release. Furthermore, the present invention can provide a method of measuring the activity for promoting a novel ceramide intermembrane transfer.
SEQ ID NOs: 1-4 indicate an amino acid sequence of hCERT protein, an amino acid sequence of hCERTL, an amino acid sequence of cCERT protein, and an amino acid sequence of cCERTL protein, respectively. SEQ ID NOs: 5-8 indicate ORF sequence of hCERT mRNA, ORF sequence of hCERTL mRNA, the full length cDNA sequence of cCERT mRNA and DNA sequence of cCERTL ORF. SEQ ID NOs: 9-32 indicate sequences of primers.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-274232 | Jul 2003 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP04/04455 | 3/29/2004 | WO | 00 | 11/30/2006 |
