WO2006011485A1

WO2006011485A1 - Protein inhibiting amyloid peptide aggregation and effect of the same

Info

Publication number: WO2006011485A1
Application number: PCT/JP2005/013658
Authority: WO
Inventors: Yoshihiro Urade; Masako Taniike; Kosuke Aritake; Takahisa Kanekiyo; Yuji Goto; Tadato Ban
Original assignee: Osaka Bioscience Institute; Osaka University; Kansai Technology Licensing Organization Co., Ltd.
Priority date: 2004-07-27
Filing date: 2005-07-26
Publication date: 2006-02-02
Also published as: JP2007284351A

Abstract

It is intended to regulate and ameliorate the symptoms of Alzheimer’s disease. A drug containing as the active ingredients: 1) lipocalin-type prostaglandin D synthase (L-PGDS); 2) an L-PGDS mutant having substitution of at least one cysteine residue in L-PGDS by another amino acid residue; and a polypeptide selected from the group consisting of: 3) β-lactoglobulin; and 4) a substance enhancing the expression of the L-PGDS protein selected from the group consisting of estrogen, glucocorticoid, interleukin-1β and thyroid hormone; is administered. This polypeptide binds to β-amyloid peptide and controls the interaction between lipid components on the bran cell membrane surface and β-amyloid peptide, thereby inhibiting the aggregation.

Description

Specification

Proteins that suppress amyloid peptide aggregation and their effects

Technical field

[0001] The present invention relates to an agent that suppresses or improves the symptoms of Arno and Imah's disease. More specifically, the present invention relates to a drug that suppresses and improves the symptoms of Alzheimer's disease by inhibiting aggregation of amyloid peptide and interacting with a glycolipid component present on the surface of brain cell membrane.

Background art

[0002] With the advent of an aging society, the number of dementia patients has increased, and about 5% of those over the age of 65 are said to have dementia. It is an important disease that accounts for most of the dementia. The first stage of the pathogenesis is that the A peptide forms a sheet structure in the brain parenchyma and aggregates and deposits, and is thought to be caused by the binding with lipid components present on the brain cell surface. It causes plaque formation and neurogenic changes in euron. Recently, in order to inhibit the aggregation and deposition of A j8 peptide, administration of A j8 vaccine · ¾Α | 8 antibody and j8 sheet breaker has been considered (Hock C et al. Antibodies against beta-amyloid slow cognitive decline in Alzneimer s disease. Neur on. 2003 38: 547—54., Janus C et al. A beta peptide immunization reduces behavioral impairment and plaques in a model of Alzheimer's disease. Nature. 2000 408: 979- 82., Soto C et al. Beta-sheet breaker peptides inhibit nbnllogenesis in a rat brain model of amyloidosis: implications for Alzheimer's therapy. Nat Med. 1998 4: 822-6.). However, both are non-physiological substances and may have side effects.

Non-Patent Document 1: Neuron. 2003 38: 547-554

Non-Patent Document 2: Nature. 2000 408: 979-982

Non-Patent Document 3: Nat Med. 1998 4: 822-826

Disclosure of the invention

Problems to be solved by the invention

[0003] The present invention suppresses the aggregation of amyloid peptide and is a sugar present on the surface of brain cell membrane The purpose is to provide a method for suppressing and improving Alzheimer's disease symptoms by interacting with lipid components.

Means for solving the problem

[0004] The inventor who achieves the above-mentioned object has conducted extensive research and has completed the present invention based on the following findings!

1) Alzheimer's disease patient's autopsy brain and Alzheimer's disease model mouse! Amyloid plaques-PGDS deposited!

2) Lipocalin-type prostaglandin D synthase (L-PGDS) has a strong affinity for human amyloid protein, and also exhibits a strong aggregation inhibitory effect in the aggregation test with thioflavin T (ThT) atsey.

3) Lipocalin-type prostaglandin D synthase (L-PGDS) has a strong affinity for the glycosphingolipid group, a glycolipid component present on the surface of brain cell membranes. Furthermore, there is a strong affinity between glycosphingolipid and human amyloid protein. The binding inhibitory action is shown at a very low concentration.

4) The effects of 2) and 3) above are caused not only by the lipocalin type prostaglandin D synthase but also by its mutants. Such mutants include those in which the cysteine residue at the active site of plastaglandin D synthase is substituted.

[0005] That is, the present invention is a drug used for the suppression and improvement of the symptoms of Arno and Imah's disease,

1) Lipocalin-type prostaglandin D synthase (L—PGDS);

2) L—PGDS variants in which at least one cysteine residue of L—PGDS is replaced by another amino acid residue, eg alanine or serine;

3) β-Lactoglobulin

4) Substances that enhance the expression of L PGDS protein in vivo, especially in the brain

A gist is a drug containing a compound selected from the group consisting of as active ingredients.

Substances that enhance the expression of LPGDS protein in vivo, particularly in the brain, include estrogen, darcocorticoid, interleukin-1 j8, and thyroid hormone. The invention's effect

[0006] According to the agent of the present invention, the suppression of amyloid peptide aggregation and the interaction with a glycolipid component present on the surface of brain cell membrane can suppress or improve the symptoms of Alzheimer's disease. You can do good.

Brief Description of Drawings

FIG. 1 is a micrograph of a micrograph in which binding of L PGDS to senile plaques in an autopsy brain of an Arno-Ima disease patient is detected by immunohistochemical staining. * Indicates amyloid 'plaque, and arrow indicates LPGDS expressed in oligodendrocytes.

[Fig. 2] Alzheimer's disease model mouse Tg2576 amyloid 'plaque located in L

— A photomicrograph of PGDS detected by immunohistochemical staining.

FIG. 3 is a graph showing the results of examining the concentration-dependent binding of human L-PGDS (Ala2.3.4) to human amyloid 'beta 1-40 peptide by surface plasmon resonance.

[Fig. 4] Α | 8 This is a copy of a photograph showing the results of observing the inhibitory effect of Ala2.3.4 on seed-dependent aggregation of 1-40 peptides with a laser fluorescence microscope.

[Fig.5] Ala2, 3 for spontaneous aggregation of A j8 1-40 peptide or A j8 1-42 peptide

, 4 is a graph showing the results of investigating the dose-dependent inhibitory effect of 4 by ThT Atsay.

[Fig. 6] Α | 8 A copy of a photograph showing the results of observing the inhibitory effect of Ala2.3.4 on seed-dependent aggregation of 1-40 peptides with an atomic force microscope (AFM).

[Fig. 7] A graph showing the results obtained by examining the inhibitory effect of Ala2.3.4 on Aβ1-40 peptide whose aggregation is promoted in the presence of Gandarioside GM-1 and ribosome by ThT Atsei.

[FIG. 8] A graph showing the results of examining the dose-dependent inhibitory effect of trace on the seed-dependent aggregation of Α | 8 1-40 peptide and A | 8 1-42 peptide by ThT analysis.

[Fig.9] Α | 8 (1-40) ThT Atsey showed the antagonism of two mouse anti-human LPGDS monoclonal antibodies against the inhibitory effect of Ala2.3.4 peptide on seed-dependent aggregation of peptides. It is a graph to show.

FIG. 10 is a graph showing the results of examining the binding ability of several mouse L PGDS mutants to A | 8 (1-40) peptide by surface plasmon resonance.

[Fig. 11] Wild-type mouse or LPGDS administered human A j8 1-42 peptide into the ventricle FIG. 5 is a photocopy showing the results of detecting the human A1-42 peptide remaining in the brain of a knockout mouse in a ligamentous and woven manner. For detection, human Α | 81-42 peptide labeled with piotin was used.

BEST MODE FOR CARRYING OUT THE INVENTION

[0008] Inhibition of the symptoms of the Arno-Ima disease of the present invention 'The first embodiment of the drug used for improvement is L

PGDS is the active ingredient. L PGDS is derived from mammals, from human (SEQ ID NO: 1, amino acids 1 to 21 are signal peptides) and from mouse (SEQ ID NO: 2, amino acids 1 to 24 are signal peptides) Those derived from humans are preferred.

[0009] The second embodiment of the drug used for suppressing and improving the symptoms of the Arno-i-maima disease of the present invention comprises an L-PGDS mutant as an active ingredient. The L PGDS mutant in the present invention is a substitution mutant of L PGDS, wherein at least one of the three cysteine residues is replaced with another amino acid residue, preferably glycine, alanine, serine, norin, histidine, treasure. A relatively small variant of L-PGDS, including substitutions with amino acids such as nin, asparagine, aspartate, etc.

1) Proteins of human L—PGDS (SEQ ID NO: 1) with the cysteines at positions 65 and 167 substituted with alanine or serine (hereinafter, may be abbreviated as “Alal. 3” and “Serl. 3”, respectively);

2) Human L—PGDS (SEQ ID NO: 1) proteins with 89, 167 and 186th cysteine replaced with alanine or serine (hereinafter referred to as “Ala2.3.4” and “Ser2.3.4”, respectively) May be abbreviated);

3) Protein obtained by substituting the 65th cysteine of mouse L PGDS (SEQ ID NO: 2) with alanine or serine (hereinafter abbreviated as “Alal” and “Serl”, respectively);

4) Mouse L—A protein in which cystines 89 and 186 of PGDS (SEQ ID NO: 2) are substituted with alanine or serine (hereinafter abbreviated as “Ala2.3” or “Ser2.3”, respectively). .

Among these substitution mutants, those in which one to several amino acid residues are further substituted with other amino acid residues can also achieve the object of the present invention.

[0010] Mammal-derived L PGDS is a force gene that can also obtain the tissue power of mammals. It is convenient to manufacture by recombinant technology. The amino acid sequence of human or mouse-derived lipocalin-type prostaglandin D synthase (L-PGDS) is known. The amino acid sequence of human-derived L—PGDS is shown in SEQ ID NO: 1 (Hoffinann A et al., J. Neurochem. 1993 Aug 61 (2) 45-46) (amino acid residues 1 to 21 of SEQ ID NO: 1) Is a signal peptide). The amino acid sequence of L PGDS derived from mouse is shown in SEQ ID NO: 2 (Hoffinann A et al.,

Dev Dyn. 1996 Nov. 207 (3) 332-43; Irikura D et al "J. Biochem. (Tokyo) 2003, Jan.

133 (1) 29-32) (The amino acid residues 1 to 24 of SEQ ID NO: 1 are signal peptides). The nucleotide sequence of cDNA encoding L—PGDS is also known. The nucleotide sequence of cDNA encoding human L-PGDS is shown in SEQ ID NO: 3 (nucleotide residues 1 to 63 of SEQ ID NO: 3 encode a signal peptide). The nucleotide sequence of cDNA encoding mouse-derived LPG DS is shown in SEQ ID NO: 4 (nucleotide residues 1 to 72 of SEQ ID NO: 4 encode a signal peptide). Therefore, an expression vector that expresses recombinant L-PGDS can be constructed in an appropriate host system. Recombinant LPGDS can be produced by transforming host cells with the obtained expression vector and culturing the transformant under conditions suitable for expression of DNA encoding LPGDS.

[0011] An expression vector containing DNA encoding L-PGDS of the present invention can be constructed by methods known to those skilled in the art. A vector suitable for L-PGDS expression would have a promoter for transcription initiation immediately upstream of the DNA insertion site. Appropriate promoters are also known in the art and can be selected according to their functional properties in the host cell. For example, the promoter of the SV40 virus early gene, the peptide chain extension factor EF-1a promoter, the metamouthoneine gene promoter, the 13-cutin promoter, the CMV vinores promoter, etc. can be expressed in animal cell systems, Merase promoters and beta-galactosidase gene promoters can be used for expression in bacteria and E. coli. It is desirable that there is a transcription termination signal downstream of the insertion site of human hematopoietic PGD synthase DNA.

[0012] Desirably, a selectable marker such as a drug resistance marker is present in the vector. Alternatively, an expression vector containing a polynucleotide encoding L-PGDS Transformation may be performed simultaneously using a plasmid encoding drug resistance such as antibiotics separate from Kuta.

[0013] To construct an expression vector, DNA encoding L-PGDS is inserted into an appropriate vector. A suitable vector is selected from those known in the art in consideration of the promoter, transcription termination signal, selection marker and other conditions. As a DNA vector that can be used for the purpose of expressing this cDNA by inserting LPGDS cDNA and introducing it into cultured cells, for example, pKCR, pEF-BOS, CDM8, pCEV4 In E. coli, such as papillomavirus DNA, pGEMEX, pUC, etc. can be mentioned.

[0014] The cells that can be used for the expression of LPGDS may be any cells that can replicate and can express DNA encoding LPGDS. For example, prokaryotic microorganisms such as E. coli, eukaryotic microorganisms such as S. cerevisiae, and mammalian cells are used. Tissue culture cells include avian or mammalian cells such as murine, rat and monkey cells. Selection and use methods of suitable host cell-vector systems are known to those skilled in the art, and a system suitable for the expression of cDNA encoding their internal L-PGDS can be arbitrarily selected.

A desired protein can be obtained by culturing the transformed cells according to a conventional method. The medium used for the culture can be appropriately selected depending on the properties of the host. For example, when the host is E. coli, LB medium or TB medium is used. When the host is a mammalian cell, RPMI 1640 medium. Etc. can be used as appropriate.

The isolation and purification of the protein used in the present invention by the culture force obtained by this culture can be carried out by conventional methods. For example, the culture can be obtained by using various physical and chemical properties of the protein. This processing operation can be performed. Specifically, treatment with a protein precipitating agent, ultrafiltration, high performance liquid chromatography, centrifugation, electrophoresis, affinity chromatography and the like can be used alone or in combination. The obtained protein solution can be powdered by lyophilization if necessary. In lyophilization, stabilizers such as sorbitol, mannitol, dextrose, maltose, and glycerol can be added. [0015] Preferably, a site-directed mutagenesis method is used to produce a mutant of LPGDS. This method is well known, and kits for implementation (for example, Quick Change (registered trademark) Site-Directed Mutagenesis Kit manufactured by STRATAGENE) are commercially available.

In order to introduce a mutation by the site-directed mutagenesis method, a primer pair of about 30 to 40 bases containing mismaticodone at the target site was synthesized, and a wild-type plasmid was transformed into a saddle type using these primers. As a whole PCR amplification with Pfu polymerase. After completion of the PCR, the reaction mixture contains both the plasmid and the synthesized mutant plasmid. Treat the plasmid with Dpnl endonuclease, purify the mutant plasmid by decomposing and removing the plasmid.

[0016] In the third embodiment of the drug used for suppressing and improving the symptoms of the Arno-Ima disease of the present invention, j8 lactoglobulin is used as an active ingredient. Lactoglobulin is a protein that is contained in milk and belongs to the lipocalin family. | 8 A commercially available lactoglobulin (eg, Sigma) can be used.

[0017] In the fourth aspect of the drug used for improvement of the present invention,

A substance that enhances the expression of PGDS is used as an active ingredient. As a substance that enhances the expression of L PGDS, estrogen (Otsuki M et al. Specific regulation of lipocalin—type prostaglandin D synthase in mouse heart by estrogen receptor beta. Mol Endocrinol. 2003 Sep; 17 (9): 1844-55 Epub 2003 Jun 26.), Darciacorticoid (Garcia- Fernand ez LF et al. Dexamethasone induces lipocalin— type prostaglandin D synthase gene e xpression in mouse neuronal cells. J Neurochem. 2000 Aug; 75 (2): 460-70. ), Inter ~ Leukine 1 β (Fujimori K et al. Regulation of lipocalin— type prostaglandin D synt hase gene expression by Hes— 1 through E— box and interleukin— 1 beta via two NF— k appa B elements in rat leptomeningeal J Biol hem. 2003 Feb 21; 278 (8): 6018 -26. Epub 2002 Dec 17.), thyroid hormone (Leone MG et al. Lipocalin type prost aglandin D—synthase: which role in male fertility Contraception 2002 Apr; 65 (4): 29 3-5.). These substances are known to enhance the expression of LPGDS in vivo, particularly in the brain.

[0018] When the active ingredient of the drug of the present invention is a protein, the protein is mixed with a carrier known per se It can be diluted and formulated as a liquid, for example. The liquid preparation can be prepared using, for example, purified water, physiological saline, alcohols such as ethanol “propylene glycol” glycerin ”polyethylene glycol, and a solvent such as triacetin. The liquid thus prepared can be used by diluting, for example, a lactate Ringer's solution, a maintenance solution comprising an infusion electrolyte solution, a postoperative recovery solution, a dehydration replenisher, a drip physiological saline solution, or the like. Additives appropriately selected for use in normal liquids, for example, buffering agents for pH adjustment (for example, phosphate buffer, borate buffer, citrate buffer, tartrate buffer, acetate buffer, etc.), isotonic Agents (eg sorbitol, glycerin, polyethylene glycol, propylene glycol, glucose, sodium chloride, etc.) Such preparations may further include pharmaceutically acceptable salts, sodium benzoyl alcohol, paraoxybenzoates, etc. , Benzyl alcohol, «lachlormethaxinol, chlorcresol, phenethyl alcohol, sorbic acid or salts thereof, suitable antiseptic disinfectants such as thimerosal, chlorobutanol, humectants such as talc, emulsifiers such as polyethylene glycol monooleate, Auxiliary agents such as dispersants, stabilizers such as sulfites, bisulfites, metabisulfites It may be added. It is also preferable to administer as a suspending agent using gum arabic, kaolin, methylcellulose, sodium carboxymethylcellulose, etc. as a suspending agent.

[0019] Non-oral administration is preferred as the route of administration, for example, including intravenous administration, transcerebral spinal fluid administration, transarterial local administration by catheter, or surgical local administration. The dosage of the active ingredient of the present invention is 0.01 to 20.0 mgZkgZ days, preferably 0.5 to 5 mgZkgZ days.

[0020] Even when the active ingredient of the drug of the present invention is a low molecular compound such as estrogen or darcocorticoid, it can be mixed with a pharmaceutically acceptable carrier to produce the drug. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. As a route of administration, parenteral administration and oral administration! /, Or deviation can be used. Powders, pills, capsules and tablets for oral administration are excipients such as ratatoses, glucose, sucrose, and maltol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc. Agents, binders such as polybulal alcohol, hydroxypropylcellulose, and gelatin, surfactants such as fatty acid esters, and plasticizers such as glycerin Can be manufactured. The dose of the active ingredient of the present invention is 0.001 to 10 mgZkgZ day, preferably 0.01 to LmgZkgZ day.

[0021] Furthermore, the present invention uses LPGDS or a mutant gene thereof for gene therapy, and locally expresses LPGDS or a mutant in the brain, thereby producing Α | 8 protein aggregation 'deposition. Provided is a method for use in the prevention, suppression, or improvement of symptoms of Alzheimer's disease by inhibiting and interacting with glycolipid components present on the surface of brain cell membrane.

The gene of the present invention, PGDS or a variant thereof, is incorporated into a vector and introduced into the brain. Vectors used for gene therapy include retrovirus vectors, any virus vectors including adenovirus vectors, and ribosome vectors. More specifically, a viral vector is a gene in which a pathogenic gene is cut out using a part of the virus shell and a gene, and an L PG DS gene modified to be expressed in brain membrane or oligodendrocyte is inserted instead. is there. A ribosome vector is a microsphere composed mainly of artificial lipid bilayers, which incorporates the L-PGDS gene modified so that it is expressed in brain membranes or oligodendrocytes (lipofection method). A ribosome called phosphatidylserine is usually used. Instead of this negatively charged phospholipid, a cation called DOTMA (N— [1,1- (2,3 dioleoxy) pill] —N, N, N trimethylammo-um chloride) that makes it easier to produce more stable ribosomes Lipids are now on sale. When positively charged, the ribosome is negatively charged, adsorbs the cells to the surface, and fuses with the cell membrane to introduce DNA into the cells. This vector is administered by intravenous injection, arterial injection, intramuscular injection, intrathecal administration, or local administration.

EXAMPLES The present invention will be further described below with reference to examples, but it is obvious that the present invention is not limited to the examples.

Example

[0022] ¾fine

Human notification of lipocalin-type prostaglandin D synthase

The human L—PGDS gene from Ala21 to C-terminus was obtained by PCR amplification from the cDNA library, the signal peptide was deleted, and the expression vector pGEX—2T (Amersham) from the 67th to the 3 ′ end of the DNA sequence of SEQ ID NO: 3 was obtained. · Harumacia · Biotech) Then, Escherichia coli DH5a was transformed with the expression vector. pGEX-2T is an expression vector for inserting the target gene into the downstream of the dartathione-S-tonsphrase (GST) gene and expressing the target protein as a GST fusion protein. Subsequently, the vector was transformed into Escherichia coli BL21 strain. After culturing the E. coli strain transformed with the expression plasmid in LB medium (containing 50 μg ZmL ampicillin) at 37 ° C, IPTG (isopropyl 1-1-thio-13-D-galactoside) was brought to a final concentration of 0. By adding 4 forces to 0.6 mM, the expression of the fusion protein of GST and L-PGDS was induced and further cultured for 4-6 hours.

After completion of the culture, E. coli was recovered from the culture solution, and proteins were extracted from the cells by ultrasonic disruption. Unnecessary substances were removed by centrifugation, and then retained on a dartathione sepharose 4B column. The fusion protein retained on the column was extinguished with thrombin, and the L-PGDS fraction was eluted. Subsequently, it was purified by gel filtration column chromatography to obtain a human-derived lipocalin-type prostaglandin D synthase.

[0023] Jewelery 12

Manufacture of lipocalin-type prostaglandin D synthase

In Example 1, except that human-derived PGDS-encoding cDNA was replaced with mouse-derived L PGDS-encoding cDNA (up to the 3rd end at the 70th force of the DNA sequence of SEQ ID NO: 4). Similarly, a mouse-derived lipocalin-type prostaglandin D synthase was obtained.

[0024] Example 3

Manufacture of a mutant (Ala l. 3) in which human L—PGDS 65th and 167th cysteines are replaced with alanine

To create a mutant in which Cys65 of human L-PGDS is substituted with alanine, a pair of synthetic oligomers containing the mismatch codon at the target site (Cys65) using the expression vector obtained in Example 1 as a saddle type:

0 — gcgttgtccatgGCCaagtctgtggtggcc— 3, (eyes ti 歹 U number 5)

5 -ggccaccacagactt GL, catggacaacgc-j (Tsubaki series b)

And use STRATAGENE's QuickChange Site-Directed Mutagenesis Kit Thus, a Cys65Ala expression vector was prepared.

Furthermore, a pair of synthetic oligomers containing a mismatch codon at the target site (Cysl67) using the obtained expression vector as a saddle type:

5-attcaccgccttc C and aaggcccagggct-3

5 -agccctgggccttGGCgaaggcggtgaat-3

And Cys65 and 167Ala expression vectors were prepared using the QuickChange Site-Directed Mutagenesis Kit.

[0025] An expression vector in which Cys65 and 167 were substituted with alanine was transformed into Escherichia coli BL21 strain. The E. coli strain transformed with the expression plasmid was cultured in LB medium (containing 50 gZ mL ampicillin) at 37 ° C, and then IPTG (isopropyl-1-thio-13-D-galactoside) was added at a final concentration of 0. The mixture was added to a force of 0.6 mM to induce expression of the fusion protein of GST and L—PG DS, and further cultured for 4 to 6 hours.

After completion of the culture, E. coli was recovered from the culture solution, and proteins were extracted from the cells by ultrasonic disruption. Unnecessary substances were removed by centrifugation, and then retained on a dartathione sepharose 4B column. The fusion protein retained on the column was extinguished with thrombin, and the L-PGDS fraction was eluted. Subsequently, purification was performed by gel filtration column chromatography to obtain a human PGDS mutant (Alal. 3) in which 65 and 167 cysteines of human L-PGDS were substituted with alanine.

[0026] Example 4

Manufacture of a mutant (Serl. 3) in which human L—PGDS 65 and 167 cysteines are replaced with serine

To create a mutant in which Cys65 is substituted with serine, a pair of synthetic oligomers containing the mismatch codon at the target site (Cys65) using the expression vector obtained in Example 1 as a saddle type

(Synthetic oligomer for Cys-65- Ser):

5 — gcgttgtccatgTCCaagtctgtggtggcc— 3, (eyes ti 歹 U number 9)

5 -ggccaccacagacttGLrAcatggacaacgc-3

The Cys65Ser expression vector was prepared using the QuickChange Site-Directed Mutagenesis Kit manufactured by STRATAGENE. Further, a pair of synthetic oligomers containing the mismatched codon at the target site using the obtained expression vector as a saddle type:

5-attcaccgccttc fC and aaggcccagggct-3 (Megumi ¾ · No.11)

0 -agccctgggccttGGAgaaggcggtgaat-3 '(2)

A Cyso5, 16 / ¾ΘΓ expression vector was prepared using the Quick; hange; te-Directed Mutagenesis Kit.

[0027] An expression vector in which Cys65 and 167 were substituted with serine was transformed into Escherichia coli BL21 strain. The Escherichia coli strain transformed with the expression plasmid was cultured in LB medium (containing 50 gZmL ampicillin) 37. After culturing in C, add IPTG (Isopropyl-1-thio-β-D-galatatoside) to a final concentration of 0.4 to 0.6 mM and add GST and L-PGDS fusion protein. Expression was induced and further cultured for 4-6 hours.

After completion of the culture, E. coli was recovered from the culture solution, and proteins were extracted from the cells by ultrasonic disruption. Unnecessary substances were removed by centrifugation, and then retained on a dartathione sepharose 4B column. The fusion protein retained on the column was extinguished with thrombin, and the L-PGDS fraction was eluted. Subsequently, it was purified by gel filtration column chromatography to obtain a mutant (Serl. 3) in which the 65th and 167th human L-PGDS were substituted with serine.

[0028] Decoration 15

Human L— PGDS 89. 167 Manufacture of a mutant (Ala 2. 3. 4) with 186th cysteine in alanine

In Example 4, except that the design of the synthetic oligomer was changed, a mutant (Ala 2.3.4) in which the 89, 167 and 186th cysteines of human L-PGDS were replaced with alanine was the same as in Example 3. Manufactured.

[0029] Example 6

Mouse L— Production of a mutant (Alal) in which the 65th cysteine of PGDS is replaced with alanine

Using the STRATAGENE QuickChange Site-Directed Mutagenesis Kit, a mutant in which the mouse L PGDS expression plasmid obtained in Example 2 was used as a saddle and the 65th cysteine of mouse L PGDS was replaced with alanine (Alal) Was manufactured as follows. A pair of synthetic oligomers containing mismatched codons to replace Cys65 with Ala

5 '-gctgtattgtatatgGCAaagacagtggta-3' (Self column number 13)

5-taccactgtcttT Ccatatacaatacagc-3 (Eye G Row ¾ · No. 14)

This was used to PCR-amplify the Cys65Ala mutant plasmid using the L-PGDS expression plasmid as a saddle. The obtained mutant expression plasmid was transformed into Escherichia coli BL21 strain, and a mutant (Alal) in which the 65th cysteine of mouse LPGDS was replaced with alanine was produced in the same manner as in Example 2.

[0030] Example 7

Mouse L— ¾¾ report of mutant (Ala2.3) in which cystine at positions 89 and 186 of PGDS were replaced with alanine

In Example 6, a mutant (Ala2.3) in which murine L-PGDS 89 and 186th cysteines were substituted with alanine was produced in the same manner as in Example 6 except that the design of the synthetic oligomer was changed.

[0031] ms

Mice L— PGDS 89 186th cysteine and 54th tryptophan dressed as alanine

In Example 6, as a synthetic oligomer

5 ― gcctccaactcaagc CAttccgggagaag—3 (歹 ϋ¾ · 号 l o ノ

5-cttctcccggaa TGCgcttgagttggaggc -3 (up to ¾)

In the same way as in Example 6, the following is a mutant (A1 a2. 3) in which mouse L-PGDS 89 and 186th systemine and 54th tributophane are replaced with alanine. Manufactured.

[0032] Example 9

Mouse L— Production of mutants in PGDS with 89 and 186th cysteine replaced with alanine and 54th tryptophan with phenylalanine

In Example 6, as a synthetic oligomer

5-gcctccaactcaagcTTL, ttccgggagaag S '(Self number 1

ΰ-cttctcccggaaGAAgcttgagttggaggc -3 '(Tatsumi column number 18) In the same manner as in Example 6, the following mutants were prepared by substituting the 89th and 186th systems of mouse L-PGDS with alanine and the 54th tributophane with phenylalanin.

[0033] Example 10

Mouse L—A variant made of PGDS 89 and 186th cysteine and 111th histidine in alanine.

In Example 6, as a synthetic oligomer

5-acctacagcagccccG Ctcgggcagcatc -3

0-gatgctgcccgagGCGgggctgctgtaggt -3 '(酉己歹 U number 20)

In the same manner as in Example 6, mutants were prepared by substituting the 89th and 186th systems of mouse L-PGDS and the 111th tributophane with alanine.

[0034] Example 11

Mouse L — PGDS 89 186th Cystein and a 43rd tryptophan placed in a phenylalanine

In Example 6, except that the design of the synthetic oligomer was changed, in the same manner as in Example 6, a mutant was prepared in which mouse L-PGDS 89 and 186th cysteine were replaced with alanine and 43th tryptophan was replaced with feluluranine. did.

[0035] Example 12

Binding of lipocalin-type prostaglandin D synthase to amyloid 'plaques in Alzheimer's disease model mice

Alzheimer's disease autopsy brain and Alzheimer's disease model mouse Tg2576 (Hsia 0 K et al., Orrelative memory deficits, Abeta elevation, ana amyloid plaques in tran sgenic mice. Science. 274: 99-102.1996; Hsiao KK et al. Age- related CNS disorder and early death in transgenic FVB / N mice overexpressing Alzheimer amyloid precur sor proteins. Neuron.l5: 1203— 18. 1995) (See Fig. 1 and Fig. 2) L-PGDS was identified to bind to the amyloid 'plaque site in both the autopsy brain of Alzheimer's disease patients and the Arno-Ima disease model mouse. [0036] Example 13

Binding ability of L PGDS to human amyloid beta peptide

Using surface plasmon resonance (Biacore), we analyzed the binding ability of recombinant protein (Ala2.3.4) of LPGDS to human amyloid 'beta 140 peptide (see Fig. 3). Ala2. 3. 4ί and human amyloid 'beta 1-40, 1 42 peptides, and also ί, 1- 28, 2 5-35 peptide fragments showed strong binding, but 1-16 fragments I did not admit. It also showed strong binding ability to fibrotic human amyloid 'beta 1-40 and 1-42 peptides. Table 1 shows the dissociation constants of Ala2.3.4 for various Α | 8 peptides by surface plasmon resonance.

[0037] [Table 1]

A β peptide dissociation constant (η Μ)

Α β (1-40) 2 0

Α β (1-42) 2 7

A β (1-16) N D

A β (1-28) 1 9

A β (25-35) 1 8

A β (1-40) Fiber 3 1

A β (1-42) fiber 2_6

N D: Below detection limit (> 1 μΜ)

[0038] Example 14

L PGDS and β-trace violently styling human amyloid 'beta bept ¾ for production

Using the ThT / 'assay (Ban T et al., Direct ooservation of amyloid fibril growth monitored by thioflavin T fluorescence. J Biol Chem. 278: 16462—5. 2003) The effects of L-PGDS (β TRACE) and human L-PGDS mutant (Ala2.3.4) purified from the solution were analyzed (see Figures 4-8).

ThT is a fluorescent substance that specifically recognizes the cross-beta sheet structure, and it is known that the fluorescence intensity of ThT increases with the growth of amyloid fibrils in vitro. Therefore, a mixed solution of phosphate buffer (50 mM, pH 7.5) and NaCl (lOOmM) ゝ A j8 1-40 (50 M) was prepared and kept at 37 ° C. At that time, a group in which the concentrations of L-PGDS analogues were adjusted and mixed was also set. Mix 5 μl of each with lml of ThT (5 μ と) and glycine buffer (5 OmM, pH 8.5) and use a fluorimeter (excitation wavelength: 446 nm, fluorescence wavelength: 490 nm). The light intensity was measured over time. The effect of human L-PGDS mutant (Ala2. 3. 4) on the spontaneous aggregation of Α | 8 1-40 and 144 peptides was analyzed using ThT assay. As shown (see Figure 5).

The aggregation reaction of human amyloid beta is promoted by adding fibrotic human amyloid beta as a seed. The effect of human L-PGDS mutant (Ala2. 3. 4) on seed-dependent aggregation of A | 8 1 -40 peptide was analyzed by laser fluorescence microscopy and atomic force microscopy. (See Figures 4 and 6).

Aggregation reaction of human amyloid beta can also be promoted by adding gandarioside GM1 ribosome as a seed. By ThT assay, the mutant of human L-PGDS (Ala2.3.4) also showed strong aggregation inhibitory action against GM1 liposome-dependent aggregation of A j81-40 peptide (see Fig. 7).

The effect of i8-trace purified from human cerebrospinal fluid on seed-dependent aggregation of A j8 1-40 and 1-42 peptides was analyzed using the ThT assay described above. Indicated. (See Fig. 8)

[0039] Example 15

Mouse ¾ pile L PGDS human amyloid beta ί 40 モノクローナル 40

Using the ThT assay described above, the effects of mouse monoclonal antibodies (1B7 and 9A6) and pretreated human Ala2.3.4 on A j8 1-40 peptide on seed-dependent aggregation were analyzed. L— 1B7 antibody that recognizes the vicinity of Tyr ¹¹⁶ Thr ^{123 of} PGDS (Oda et.al Development and evaluation of a practical JJ¾A for human urinary lipocalin—type pro staglandin D synthase. Clin Chem. 2002 Sep; 48 (9): 1445 -53.) showed inhibition of the suppression effect (see Figure 9).

[0040] Example 16

Binding ability of mutant L PGDS to human amyloid beta peptide

For the mutant mouse L-PGDS (Examples 3-11) prepared by recombinant technology, the surface plasmon resonance method (Biacore) described above was used to detect mutant L-PGDS for human amyloid 'beta 140 peptide. The binding ability was analyzed (see Fig. 10). Dissociation constant As for Cys ⁸⁹ ′ ¹⁸⁵ Ala and His ^m Ala, the binding ability was ^13.2 nM, for Cys ^89, ¹⁸⁵ Ala and Trp ⁵⁴ Ala 4. OnM Cys ⁸⁹ ′ ¹⁸⁵ Ala was 3.5 nM. A positive relationship was observed between the enzyme activity of mutant L-PGDS and affinity for human amyloid 'beta 1-40 peptide (Table 2).

[Table 2]

[0041] Example 17

Piotin-labeled human amyloid beta against wild-type mice and L-PGDS knockout mice (Eguchi N. et al. Proc. Natl. Acad. Sci., USA, Jan 19, 96 (2) 726-730) An intraventricular administration experiment was conducted. FIG. 11 is a photomicrograph of a brain section taken 1 hour after 50 μ1 intraventricular administration of 100 μΜ piotin-labeled human amyloid beta 1-42 to wild-type mice and L-PGDS knockout mice, respectively. Compared to wild-type mice (left), L PGDS knockout mice (right) showed aggregation of amyloid beta in the ventricle (upper center), and more intense deposition in the brain parenchyma (lower). The color intensity shows that the clearance of amyloid beta, which is significantly 4.82 times stronger in L-PGDS knockout mice than in wild-type mice, is attenuated.

Industrial applicability

[0042] Suppression of symptoms of Alzheimer's disease Can be used in the manufacture of a drug used for improvement.

Claims

Claims [1] Arno, a drug used for the suppression and improvement of symptoms of Imah's disease, 1) lipocalin type prostaglandin D synthase (L-PGDS); 2) substitution mutant of L-PGDS A substitution mutant of LPGDS comprising a substitution wherein at least one of the three cysteine residues is replaced by another amino acid residue; 3) β-lactoglobulin; and 4) estrogens, darcocorticoids, interleukins—1 a substance that enhances the expression of LPGDS protein, selected from the group consisting of j8 and thyroid hormone; [2] The drug according to claim 1, wherein the lipocalin-type prostaglandin D synthase (L—PGDS) is derived from human (SEQ ID NO: 1) or mouse (SEQ ID NO: 2). [3] Substitutional power of L—PGDS L—At least one of the three cysteine residues of PGDS was selected as a group force consisting of glycine, alanine, serine, parin, histidine, threonine, asparagine, and asnoginic acid. 2. The drug according to claim 1, wherein the drug is substituted with an amino acid residue. [4] L—The substitution mutant of PGDS is

(1) a protein obtained by substituting the cysteines at positions 65 and 167 of human L-PGDS of SEQ ID NO: 1 with alanine or selenium;

(2) a protein obtained by substituting the cysteines at positions 89, 167 and 186 of human L-PGDS of SEQ ID NO: 1 with alanine or serine;

(3) a protein obtained by substituting the 65th cysteine of mouse L-PGDS of SEQ ID NO: 2 with alanine or serine; and

(4) mouse L-PGDS of SEQ ID NO: 2, a protein obtained by substituting cysteine at positions 89 and 186 with alanine or serine;

The drug according to claim 1 or 2, which is selected.

[5] The drug according to claim 1, wherein one or several amino acid residues of the substitution mutant according to claim 3 or 4 are further substituted with other amino acid residues.

[6] Inhibition of symptoms of Alzheimer's disease · Used in gene therapy for improvement 1 A nucleic acid encoding LPGDS and a substitution variant thereof according to any one of -5.