JP3324611B2 - Method for phosphorylating tau protein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for phosphorylating a tau protein, a partial peptide thereof or a peptide similar thereto using a phosphorylase as a catalyst.
It can be used for searching for a drug that inhibits or promotes the phosphorylation of tau protein.

[0002]

2. Description of the Related Art Alzheimer's disease is in old age (45-65).
Years of age, progressive dementia, pathologically manifesting numerous senile plaques and neurofibrillary tangles in the brain. As for the senile dementia due to the so-called natural aging that occurs in the senile period of 65 years or older, there is no essential difference in pathological condition.
It is called Alzheimer's disease. The number of patients with this disease has increased with the increase in the elderly population, and has become a socially important disease. However, although the causes of this disease have various theories, the results are still unclear, and early elucidation is desired.

Alzheimer's disease and Alzheimer's type
The amount of the two pathological changes characteristic of senile dementia was
It is known to correlate well with the degree of harm. Therefore,
Insoluble accumulated substances that cause these two pathological changes are
Research to elucidate with the bell and reach the etiology of this disease
Has been around since the early 1980s. This pathological change
Neurofibrillary tangles, which is one of the processes of
(Paired helical filament: paired
helical filament)
Spiral fibrous material accumulates. recent years,
Its component is a brain-specific microtubule-associated protein.
Tau protein and ubiquitin, one of the qualities, have been identified
(Ihara et al., J. Biochem.,99, 1807-1
810 (1986); Grundke-Iqbal et al.
Proc. Natl. Acad. Sci. USA,8
3, 4913-4917 (1986)). Of which tau
Protein (hereinafter sometimes referred to as “tau”) is usually S
Molecular weight 48 to 6 by DS polyacrylamide gel electrophoresis
A family of closely related proteins that form several bands at 5K
It is known to promote the formation of microtubules. Further
In addition, tau incorporated in PHF is stronger than normal tau
The phosphorylation of PHF
Antibody (anti-ptau antibody: Ihara et al., J. Bioch)
em. ,99, 1807-1810 (1986)),
Monoclonal antibody against tau (tau-1 antibody: G
runke-Iqbal et al., Proc. Natl. A
cad. Sci. USA,83, 4913-4917
(1986)).

The present inventors have isolated an enzyme that catalyzes this abnormal phosphorylation and named it tau protein kinase I (Uchida et al., Biochemistry, Vol. 64, No. 5, p. 308).
(1992)). However, the primary structure of tau protein kinase I is unknown, and tau protein kinase I
At present, the conditions that can be used as substrates and details of phosphorylation have not been clarified.

[0005]

SUMMARY OF THE INVENTION The present inventors have developed PHF
Is thought to induce neuronal degeneration and subsequent death in Alzheimer's disease brain, and tested a response corresponding to abnormal phosphorylation of tau protein, which causes PHF accumulation
By revealing a method of performing within the tube (in vitro), it is intended to be obtained means of treatment and prevention of Alzheimer's disease, more therapy and other diseases caused by these phosphorylated abnormal It seeks preventive measures.

[0006]

Means for Solving the Problems In order to achieve the above object, the present inventors have determined the phosphorylation site of phosphorylated tau having the same epitope as phosphorylated tau incorporated in PHF, and determined the phosphorylation site thereof. By elucidating the structure of a phosphorylating enzyme that catalyzes oxidation, the necessary conditions for such a phosphorylation reaction were elucidated, and the present invention was reached.

[0007] That is, the gist of the present invention resides in a method for phosphorylating tau protein characterized by phosphorylating tau protein or a partial peptide thereof by the action of a phosphorylase I having the following physicochemical properties. (A) Action: Using ATP as a phosphate donor, phosphorylates serine and threonine in tau protein, and introduces a phosphate group of 4 residues or less per one molecule of tau protein. (B) Substrate specificity: specifically phosphorylates tau protein and MAP2, which are microtubule-associated proteins, in proteins of brain extract. Does not phosphorylate histones. Slightly phosphorylates α-casein. (C) Optimum pH: The optimum pH in the absence of tubulin is 6.5, and the optimum pH in the presence of tubulin is 6.5.
0. (D) Suitable temperature for action: 37 ° C

(E) Molecular weight: The measured value obtained by gel filtration in the presence of 0.5 M sodium chloride is about 50,000. In the absence of sodium chloride, a complex is formed with tau protein, and the complex is formed by gel filtration. The measured value is about 100,000. S
The measured value determined by DS-polyacrylamide gel electrophoresis is about 45,000. (F) Stability: pH 6.5 in 25% glycerol and 0.02% polyoxyethylene sorbitan monolaurate.
5. Stable at -20 ° C for at least one year. (G) Activation: cA, a known kinase activator
It is not activated by MP, cGMP, Ca ²⁺ , calmodulin and phospholipids. Under tubulin polymerization conditions,
The phosphorylation of tau protein is activated. (H) Inhibition: 90 mM NaCl or KCl halves the activity.

Hereinafter, the present invention will be described in detail. Phosphorylase I (hereinafter abbreviated as "tau protein kinase I") used in the present invention can be obtained from, for example, brain extract of mammals such as rats and cows by temperature-dependent polymerization, depolymerization and the like. Fractions can be obtained and purified by a combination of phosphocellulose column chromatography, gel filtration, hydroxyapatite column chromatography, S-sepharose column chromatography, heparin column chromatography, and the like (Uchida et al., Biochemistry , Vol. 64, No. 5,
p. 308 (1992)). The purified tau protein kinase I has the physicochemical properties described in the above (a) to (h). The partial primary structure of this purified protein can be obtained by performing amino acid sequence analysis by a conventional method, but the entire primary structure can be known by cloning the cDNA of the protein. For the cloning of cDNA, a method based on the above information on the partial primary structure, a method using an antibody specific to the protein, a method using detection of a function or action specific to the protein, and the like can be applied. Further, using a synthetic nucleotide oligomer corresponding to the partial amino acid sequence as a primer, only the mRNA encoding tau protein kinase I is amplified from the brain mRNA fraction of a rat or the like using a gene amplification method (PCR method), and the corresponding cDNA is amplified. May be cloned.

The entire primary structure of rat- derived tau protein kinase I thus obtained is represented by, for example, the amino acid sequence of SEQ ID NO: 1 in the sequence listing. In addition,
The entire primary structure of rat- derived tau protein kinase I is based on the enzyme known as GSK-3β (glycogen synthase kinase 3β) (JR Woodwood).
t t, The EMBO J. , 9 (8), 2431-
2438 (1990)).

[0011] Tau protein kinase I can be obtained by purifying from a mammalian brain extract as described above. However, in order to obtain a larger amount and stably, cDNA obtained by cloning must be obtained. Tau protein kinase I-producing cells can be prepared by incorporating the vector into a suitable vector and introducing the vector into a host on which the vector can act, and it is preferable to purify the target protein from the culture.

In the present invention, the tau protein serving as a substrate is
From mammalian brain tissue, for example, the method of Ihara et al. (J. Bi)
ochem. , 86 , 587-590 (1979)) and the method of Grundke-Iqbal et al. (J. Biol.
Chem. , 261 , 6084 @ 6089 (198
It can be extracted and purified according to 6)). Further, its entire primary structure can be determined by the same method as that for tau protein kinase I described above. The total primary structure of the tau protein thus obtained includes, as human-derived ones, those represented by the amino acid sequence of SEQ ID NO: 2 in the sequence listing (Goedert et al, P
rc. Natl. Acad. Sci. USA, 85 ,
4051-4055 (1988)).

The tau protein extracted and purified from brain tissue has been phosphorylated to some extent, and serine 144 and / or serine 315 in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing is partially phosphorylated. (See Example 1 described later). In addition, tau protein is converted to another type of kinase II having the following physicochemical properties (tau protein kinase II: Uchida et al., Biochemistry, Vol. 64, No. 5,
p. 308 (1992)),
In the amino acid sequence of SEQ ID NO: 2 in the above sequence listing, 14
In addition to the fourth serine and the 315th serine, 147
Threonine and / or serine at position 177 are phosphorylated (Uchida et al., Biochemistry, Vol. 64, No. 5,
p. 308 (1992); Ishiguro et al., Neurosie.
nceLetters, 128 , 195 (199
1)).

(I) Action: Using ATP as a phosphate donor, phosphorylates serine and threonine having a proline adjacent to the C-terminal side in tau protein, and has a phosphate group of 4 residues per molecule of tau protein. Is introduced. (J) Substrate specificity: specifically phosphorylates tau protein and MAP2, which are proteins associated with microtubules, in proteins of brain extract. Histone H1 and the neurofilament H subunit also phosphorylate. Slightly phosphorylates β-casein. (K) Optimum pH: The optimum pH in the absence of tubulin is 6.5, and the optimum pH in the presence of tubulin is 6.5.
0. (L) Suitable temperature for action: 37 ° C (m) Molecular weight: The measured value obtained by gel filtration in the presence of 0.5 M sodium chloride is about 50,000, and a complex with tau protein is formed in the absence of sodium chloride. Its measured value by gel filtration is about 100,000. The measured value obtained by SDS-polyacrylamide gel electrophoresis is about 30,000, and there are 23,000 other proteins, and the two proteins are combined to be about 50,000 by gel filtration. (N) Stability: 25% glycerol and 0.02% polyoxyethylene sorbitan monolaurate, pH 6.0.
5. Stable at -20 ° C for at least one year. (O) Activation: cA, a known kinase activator
It is not activated by MP, cGMP, Ca ²⁺ , calmodulin and phospholipids. Under tubulin polymerization conditions,
The phosphorylation of tau protein is activated. (P) Inhibition: Activity is halved with 40 mM NaCl or KCl.

This is because tau protein kinase II has
/ TP (serine / threonine-proline) sequence, which is an enzyme that specifically phosphorylates serine and / or threonine located at the N-terminal side of proline. However, the partially phosphorylated tau protein did not react with the aforementioned anti-ptau antibody. On the other hand, extracted from brain tissue as described above,
If the purified tau protein is dephosphorylated by treating with alkaline phosphatase, the phosphorylation reaction of the present invention does not proceed to an appreciable degree, whereas the tau protein extracted and purified from brain tissue Alternatively, it was confirmed that the phosphorylation reaction of the present invention proceeds when tau protein partially phosphorylated using tau protein kinase II is used as a substrate (see Examples described later). From the above facts, it was considered that the phosphorylation of tau protein proceeds by the following pathway.

[0016]

Embedded image As the substrate of the present invention, any tau protein or a partial peptide thereof which can be a substrate of tau protein kinase I can be used. For example, those having a primary structure represented by the amino acid sequence of SEQ ID NO: 2 in the sequence listing, those having a spliced primary structure represented by the amino acid sequence of SEQ ID NO: 2 in the sequence listing, and those of the sequence listing And a partial peptide of serine and / or threonine, preferably 1 to 4 residues phosphorylated. Specific examples of tau proteins include Goe
Neuron, 3 , 519- of dert et al.
526 (1989).
Tau protein having a primary structure of one amino acid residue and the like. When the tau protein whose primary structure is represented by the amino acid sequence described in SEQ ID NO: 2 in the sequence listing is used as a substrate, it is selected from 141st serine, 173th threonine, 307th serine and 324th serine of the same sequence. One or more amino acids are phosphorylated. Therefore, the partial peptide of the tau protein preferably used in the present invention is preferably a peptide comprising the amino acid sequence of SEQ ID NOs: 3 to 29 in the sequence listing. Preferred substrates and their phosphorylation sites are shown below. A peptide comprising the amino acid sequence of SEQ ID NO: 3 in the sequence listing, serine at position 4, threonine at position 7, serine at position 37 and 1
One or more amino acids selected from the 75th serine are phosphorylated in advance, and one or more amino acids selected from the 1st serine, the 33rd threonine, the 167th serine and the 184th serine are phosphorylated. .

A peptide comprising the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing, wherein one or more amino acids selected from serine 4th, 7th threonine, 37th serine and 175th serine are Oxidized, the first serine, 33
One or more amino acids selected from th-threonine and serine at position 167 are phosphorylated.

A peptide comprising the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing, wherein one or more amino acids selected from serine 4th, threonine 7th and serine 37th are phosphorylated in advance; One or more amino acids selected from the first serine, the threonine 33, and the serine 167 are phosphorylated. A peptide containing the amino acid sequence of SEQ ID NO: 6 in the sequence listing, wherein one or more amino acids selected from serine 4th, threonine 7 and serine 37 are phosphorylated in advance, and Serine and / or threonine at position 33 are phosphorylated.

A peptide comprising the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing, wherein the 4th serine and / or the 7th threonine are phosphorylated in advance, and the 1st serine and / or the 33rd Threonine is phosphorylated. -Peptide containing the amino acid sequence described in SEQ ID NO: 8 in the sequence listing Serine at the fourth position and / or threonine at the seventh position are phosphorylated in advance, and serine at the first position is phosphorylated.

A peptide comprising the amino acid sequence of SEQ ID NO: 9 in the sequence listing. Serine 4 is phosphorylated in advance, and serine 1 is phosphorylated. -A peptide comprising the amino acid sequence of SEQ ID NO: 10 in the sequence listing, wherein one or more amino acids selected from the first serine, the fourth threonine, the thirty-fourth serine and the 172th serine are phosphorylated in advance. In addition, at least one amino acid selected from threonine at position 30, serine at position 164, and serine at position 181 is phosphorylated.

A peptide comprising the amino acid sequence of SEQ ID NO: 11 in the sequence listing, wherein one or more amino acids selected from the first serine, the fourth threonine, the thirty-fourth serine and the 172th serine are previously phosphorylated It is oxidized, and threonine at position 30 and / or serine at position 164 are phosphorylated. A peptide comprising the amino acid sequence of SEQ ID NO: 12 in the sequence listing, wherein one or more amino acids selected from the first serine, the fourth threonine and the thirty-fourth serine have been phosphorylated beforehand, Threonine and / or 164
The second serine is phosphorylated.

A peptide comprising the amino acid sequence of SEQ ID NO: 13 in the sequence listing, wherein one or more amino acids selected from the first serine, the fourth threonine and the thirty-fourth serine are phosphorylated in advance, Threonine at position 30 is phosphorylated. -Peptide containing the amino acid sequence of SEQ ID NO: 14 in the sequence listing The first serine and / or the fourth threonine has been phosphorylated in advance, and the threonine in the thirtyth is phosphorylated.

A peptide comprising the amino acid sequence of SEQ ID NO: 15 in the sequence listing, wherein one or more amino acids selected from the first threonine, the 31st serine and the 169th serine are phosphorylated in advance, One or more amino acids selected from threonine 27, serine 161 and serine 178 are phosphorylated.

A peptide comprising the amino acid sequence set forth in SEQ ID NO: 16 in the sequence listing, wherein one or more amino acids selected from the first threonine, the 31st serine and the 169th serine have been phosphorylated in advance, Threonine and / or 1 at position 27
Serine at position 61 is phosphorylated. A peptide comprising the amino acid sequence of SEQ ID NO: 17 in the sequence listing, wherein the first threonine and / or the thirty-first serine has been phosphorylated in advance, and the threonine at the 27th position and / or the serine at the 161th position are Oxidized.

A peptide comprising the amino acid sequence of SEQ ID NO: 18 in the sequence listing The first threonine and / or the thirty-first serine are phosphorylated beforehand, and the threonine at the 27th is phosphorylated. -Peptide containing the amino acid sequence described in SEQ ID NO: 19 in the sequence listing The first threonine is phosphorylated in advance, and the threonine at position 27 is phosphorylated.

A peptide comprising the amino acid sequence of SEQ ID NO: 20 in the sequence listing, wherein serine 5 and / or serine 143 are phosphorylated in advance, and threonine 1 and serine 135 One or more amino acids selected from the 152nd serine are phosphorylated. A peptide comprising the amino acid sequence of SEQ ID NO: 21 in the sequence listing, wherein serine 5 and / or serine 143 is phosphorylated in advance, and threonine 1 and / or serine 135 is Oxidized.

Peptide containing the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing. Serine 5 is phosphorylated in advance, and threonine 1 and / or serine 135 are phosphorylated. -Peptide containing the amino acid sequence of SEQ ID NO: 23 in the sequence listing The 5th serine is phosphorylated in advance, and the 1st threonine is phosphorylated.

A peptide comprising the amino acid sequence of SEQ ID NO: 24 in the sequence listing, wherein the first serine and / or the 139th serine are phosphorylated in advance, and the 131st serine and / or the 148th serine Serine is phosphorylated. -Peptide containing the amino acid sequence of SEQ ID NO: 25 in the sequence listing The first serine and / or the 139th serine are phosphorylated in advance, and the 131st serine is phosphorylated.

A peptide containing the amino acid sequence of SEQ ID NO: 26 in the sequence listing The first serine is phosphorylated in advance, and the 131st serine is phosphorylated. -Peptide containing the amino acid sequence of SEQ ID NO: 27 in the sequence listing The ninth serine is phosphorylated in advance, and the first serine and / or the eighteenth serine are phosphorylated.

Peptide containing the amino acid sequence shown in SEQ ID NO: 28 in the sequence listing The ninth serine is phosphorylated in advance, and the first serine is phosphorylated. -Peptide containing the amino acid sequence of SEQ ID NO: 29 in the sequence listing The first serine is phosphorylated in advance, and the tenth serine is phosphorylated. Further, in the present invention,
As described above, a protein phosphorylated by tau protein kinase II or a peptide thereof can also be used as a substrate for tau protein kinase I.

As a specific method for preparing tau protein, for example, Journal of Biological C
hemistry, 261 , 6084-6089 (19
According to the method described in 86), a three-fold volume of a solution having the following composition is added to the microtubule protein of the brain.

Table 1 100 mM MES (pH 2.7) 0.75 M NaCl 0.5 mM MgCl _{2 2} mM DTT 1 mM EGTA 0.1 mM PMSF 0.1 mM EDTA Heat this solution at 95 ° C. for 5 minutes, then cool at 0 ° C. for 15 minutes After centrifugation at 10,000 G for 20 minutes, a supernatant is obtained. The supernatant is concentrated with an ultrafiltration device such as an Amicon PM-10 membrane, and dialyzed against RB at a protein concentration of about 7 mg / ml. This fraction contains tau protein with almost 100% purity.

The partial peptide of the tau protein can be synthesized by the solid phase synthesis method of Merrifield et al. (J. Am. Chem. So.
c. , 85 , 2149-2154 (1964)) using a peptide synthesizer such as a Biosearch model 9500. As a method for dephosphorylating tau protein, for example, 1M of pH 8.0 is used.
Tau protein can be dephosphorylated by adding bovine intestinal alkaline phosphatase (typeVII-NT, Sigma) in Tris-HCl buffer and reacting at 37 ° C for about 5 to 8 hours. Alkaline phosphatase may be inactivated by heat treatment for about 10 minutes after completion of the reaction.

Next, the phosphorylation reaction of the present invention will be described. The enzyme (tau protein kinase I) used in the present invention has an optimum pH of 6.4 and an optimum temperature of 37 ° C., and does not require a second messenger known as an activator.
Activated by tubulin, requires nucleoside triphosphate as phosphate donor. Further, the reaction is inhibited by a high concentration of sodium chloride, and the concentration of sodium chloride corresponding to 50% inhibition is about 100 mM. Accordingly, the phosphorylation reaction conditions in the present invention are not particularly limited as long as the conditions are determined in consideration of the above properties,
More preferably, 100 mM 2- (N-morpholine)
An appropriate amount of enzyme and substrate are added to a pH 6.4 buffer containing sodium ethanesulfonate, 0.9 mM magnesium acetate, and 0.4 mM ATP, and incubated at 37 ° C. Conditions such as the type, pH, and temperature of the buffer can be arbitrarily changed. For example, guanosine triphosphate may be used instead of ATP as a phosphate donor. That is, in the range of pH 3 to 10 and temperature of 0 to 70 ° C.,
The phosphorylation reaction of the present invention proceeds by allowing the above-mentioned substrate enzyme to act in the presence of nucleoside triphosphate.

The phosphorylation reaction according the, (1) 0.5~5μC the [.gamma. ³² P] ATP at quantitative phosphorylation when (a) phosphorylated amount of the protein as a substrate
Add i / ml and label the phosphorylated protein with ³² P. The reaction solution is adsorbed on a 2 cm × 2 cm square filter paper, and immersed in 5% TCA-0.25% Na ₂ WO ₄ -0.5% sodium pyrophosphate (pH 2) to stop the reaction. Wash thoroughly and remain [γ- ³² P] A
The TP is removed and the radioactivity remaining on the filter paper is counted with a scintillation counter. As a result, the amount of phosphate groups incorporated into the protein can be determined.

(B) Determination of phosphorylation site The reaction solution was electrophoresed on a Laemmli system (Natur
e, 227 , 680-685 (1970)), the protein is transferred to a nitrocellulose membrane, and the presence or absence of staining is examined by immunoblotting using an antibody that specifically reacts with a specific phosphorylated peptide. (2) In the case of using a peptide as a substrate (a) Quantification of phosphorylated amount At the time of the phosphorylation reaction, [γ- ³² P] ATP was added at 0.5 to 5 μC
Add i / ml and label the phosphorylated peptide with ^32P . After adding TCA to this reaction solution to precipitate a high-molecular-weight protein, the phosphorylated peptide in the supernatant is adsorbed to a circular square phosphocellulose paper, and washed with a phosphate buffer to remain. After removing [γ- ³² P] ATP, the radioactivity remaining on the filter paper is counted with a scintillation counter. Thereby, the amount of the phosphate group incorporated in the peptide can be quantified.

(B) Determination of phosphorylation site After adding PCA to the reaction solution to precipitate a high molecular weight protein, the supernatant is subjected to reversed phase high performance liquid chromatography. Whether or not the phosphorylation site differs from the standard product can be determined based on whether or not the retention time matches the retention time of the standard phosphorylated peptide prepared in advance. Further, the peak fraction was collected and subjected to Neuroscience Letters, 1
28 , 195-198 (1991), the phosphorylation site can be determined by performing protein chemical analysis according to the method reported by Ishiguro et al. It can be confirmed by such a method.

Specifically, a tau protein serving as a substrate or a partial peptide thereof is added to an RB buffer (see below).
A composition containing 100-600 μg / ml, 0.1-1 mM ATP and tau protein kinase is usually added in a quantity of 3
Incubate at 7 ° C for 1 to 16 hours, and stop the reaction by TCA treatment or heat treatment. Thereafter, the phosphorylation reaction is confirmed by the above method. When quantifying the amount of phosphorylation, [γ- ³² P] ATP was added at 0.5 to
It is preferable to carry out the reaction by adding 5 μCi / ml. In order to partially partially phosphorylate the substrate (tau protein) used in the present invention with tau protein kinase II or the like, the method described in Ishiguro et al. Biochem. , 104 , 319
(1988).

In the description of the present invention, the symbols RB,
RBG, MES, EGTA, EDTA, PMSF, PT
H-amino acid, ATP, PC, SB, DTT, Tween
n20, TCA, and KLH are the following abbreviations, respectively. RB: Buffer consisting of 100 mM MES, 0.5 mM magnesium acetate and 1 mM EGTA (pH 6.
5) RBG: Buffer solution obtained by adding 1 mM GTP to RB MES: 2- (N-morpholino) ethanesulfonic acid EGTA: Ethylene glycol-bis (β-aminoethyl ether) N, N, N ′, N′-tetraacetic acid EDTA: Ethylenediaminetetraacetic acid PMSF: Phenylmethylsulfonylfluoride PTH-amino acid: Phenylthiohydantoin amino acid ATP: Adenosine triphosphate PC: Buffer consisting of 20 mM MES, 0.5 mM magnesium acetate and 1 mM EGTA (pH 6.
8) SB: 20 mM N- [2-hydroxyethyl] piperazine-N ′-[2-ethanesulfonic acid], 0.5 mM
Buffer consisting of magnesium acetate and 1 mM EGTA (pH 8.2) DTT: dithiothreitol Tween 20: polyoxyethylene sorbitan monolaurate TCA: trichloroacetic acid KLH: keyhole limpet hemocyanin

[0039]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to reference examples and examples, but the present invention is not limited to the following without departing from the gist thereof. Reference Example 1 Preparation of tau protein kinase (1) Preparation of microtubule protein Immediately after decapitation of 15 adult cattle, the brain was taken out, blood clots were quickly removed, and 1 ml of 0.5 mM MgCl ₂ , 1 g / g of brain
mM EGTA-NaOH (pH 7) was added, and the mixture was gently agitated, the liquid was discarded, and the blood was washed away.

Next, 1 ml of RB was added to 1 g of the brain, homogenized three times for 5 seconds in a Waring blender (18000 rpm), and centrifuged at 30,000 G for 1 hour to collect a supernatant. To this supernatant (brain extract), １ ／ volume of glycerol and GTP were added to make the total GTP concentration in the solution 1 mM, and the mixture was heated to 37 ° C. and held for 30 minutes.
The precipitate was collected by centrifugation at 100,000 G for 30 minutes at the same temperature.

This precipitate was suspended in 1/5 volume of RB of the above-mentioned brain extract, kept at 0 ° C. for 30 minutes, and then centrifuged at 4 ° C. at 100,000 G for 30 minutes. RB containing 1/4 volume of 50% glycerol was added and 12.5 mM phosphoenolpyruvate, 12.5 μg / ml pyruvate kinase and 25%
1/25 volume of 0 μM GTP was added and heated at 37 ° C. for 30 minutes. The solution was then washed with R containing 25% glycerol.
B and 30 ° C. at 100,000 G for 30
The precipitate was collected by centrifugation in a swing rotor for minutes. The precipitate was suspended in 1/25 volume of RBG of the brain extract, kept at 0 ° C. for 30 minutes, and then suspended at 100,000 G for 30 minutes.
After centrifugation for minutes, the microtubule protein was obtained as a supernatant.

The following operation was performed using 0.02% Tween2
Performed in the presence of 0, 10% glycerol. (2) Ammonium sulfate fraction The microtubule protein obtained in the above (1) was diluted with RB to a protein concentration of 10 mg / ml, a powder of ammonium sulfate was added to make a 35% saturated ammonium sulfate solution, and the mixture was allowed to stand for 30 minutes. The supernatant was collected by centrifugation for 20 minutes. Ammonium sulfate powder was added again to the supernatant, and the mixture was allowed to stand as a 50% saturated ammonium sulfate solution for 30 minutes. The precipitate obtained by centrifugation at 20,000 G for 20 minutes was dissolved in RB, dialyzed into RB, and subjected to ammonium sulfate fractionation. I got (3) follower scan ammonium sulfate fraction obtained in follower cellulose column chromatography above (2) was added 1/4 volume of RB containing 50% glycerol. This was converted to protein 30 in the ammonium sulfate fraction.
A column of P11 cellulose, containing 1 ml of P11 cellulose (manufactured by Whatman) per mg and pre-equilibrated with PC, was charged with 5 volumes of 0.1 M NaCl.
-Wash the column with PC, then 0.1-0.8M
A developing solution having a linear concentration gradient of NaCl-PC was applied to the column, and a PC fraction eluted with 0.2 to 0.4 M NaCl was collected. (4) Gel Filtration The PC fraction obtained in the above (3) was concentrated to a protein concentration of 5 mg / ml or more with an ultrafiltration device of Amicon PM-10 membrane, and this was previously concentrated with 0.3 M NaCl-RB buffer. G equilibrated
3000SW column (manufactured by Tosoh Corporation; silica gel column,
21 mm × 60 cm), and the mixture was allowed to flow at a flow rate of 2 ml / min to perform gel filtration. A tau protein kinase fraction (a fraction with a molecular weight of 50,000) was collected and concentrated with an ultrafiltration device of Amicon PM-10 membrane. (5) Hydroxyapatite column chromatography The fraction obtained in the above (4) was applied to a hydroxyapatite column (7.5 mm x 100 mm, manufactured by Tonensha Co., Ltd.) previously equilibrated with 12 mM sodium phosphate buffer (pH 6.8). The HA fraction was charged, developed with a 10 ml linear gradient of 12 to 400 mM sodium phosphate buffer, and the HA fraction eluted at a concentration of 150 mM was collected. (6) S-Sepharose column chromatography The fraction of the above (5) was dialyzed with PC, and the S-Sepharose column (Pharmacia; 5 mm ×
20mm), 7.5m linear gradient between PC and SB
l followed by a 5 ml linear gradient from 0 to 200 mM NaCl in SB, eluting at a concentration of 150 mM.
One fraction and the S2 fraction eluted at a concentration of 50 mM were collected. The S2 fraction was dialyzed against RB to obtain a tau protein kinase II fraction. (7) Heparin column chromatography The fraction of S1 was charged on a TSK gel AF-heparin column (manufactured by Tosoh Corporation; 4 mm x 8 mm) equilibrated with 20 mM sodium phosphate buffer (pH 7.6),
In 0 mM sodium phosphate buffer (pH 7.6),
The heparin fraction eluted with a linear gradient of 0 to 200 mM NaCl and eluted with 80 mM NaCl was collected. This fraction was dialyzed against RB to obtain a tau protein kinase I fraction.

The tau protein kinase I thus obtained had the following physicochemical properties. (A) Action: Using ATP as a phosphate donor, phosphorylates serine and threonine in tau protein, and introduces a phosphate group of 4 residues per molecule of tau protein. (B) Substrate specificity: specifically phosphorylates tau protein and MAP2, which are microtubule-associated proteins, in proteins of brain extract. Does not phosphorylate histones. Slightly phosphorylates α-casein.

(C) Optimum pH: The optimum pH in the absence of tubulin is 6.5, and the optimum pH in the presence of tubulin is 6.0. (D) Appropriate temperature for action: 37 ° C. (e) Molecular weight: The measured value obtained by gel filtration in the presence of 0.5 M sodium chloride is about 50,000, and a complex with tau protein is formed in the absence of sodium chloride. Its measured value by gel filtration is about 100,000. The measured value obtained by SDS-polyacrylamide gel electrophoresis was about 45,000.
Thousands.

(F) Stability: 25% glycerol and 0.1%
02% polyoxyethylene sorbitan monolaurate
Medium, pH 6.5, temperature -20 ° C for at least one year
Stable for a while. (G) Activation: cA, a known kinase activator
MP, cGMP, Ca ²⁺, Calmodulin and phospholipids
It is not activated by quality. Under tubulin polymerization conditions,
The phosphorylation of tau protein is activated.

(H) Inhibition: 90 mM NaCl or K
The activity is reduced by half with Cl. In addition, tau protein kinase II had the following physicochemical properties. (I) Action: Using ATP as a phosphate donor, phosphorylates serine and threonine having proline next to the C-terminal side in tau protein, and introduces a phosphate group of 4 residues per molecule of tau protein. .

(J) Substrate specificity: Among proteins of the brain extract, tau protein and MAP2, which are microtubule-associated proteins,
Is specifically phosphorylated. Histone H1 and the neurofilament H subunit also phosphorylate. Slightly phosphorylates β-casein. (K) Optimum pH: The optimum pH in the absence of tubulin is 6.5, and the optimum pH in the presence of tubulin is 6.5.
0.

(L) Suitable temperature for action: 37 ° C. (m) Molecular weight: The measured value obtained by gel filtration in the presence of 0.5 M sodium chloride is about 50,000, and complexed with tau protein in the absence of sodium chloride. The body is made and its gel filtration measurement is about 100,000. The measured value obtained by SDS-polyacrylamide gel electrophoresis is about 30,000, and there are 23,000 other proteins, and the two proteins are combined to be about 50,000 by gel filtration.

(N) Stability: 25% glycerol and 0.1%
02% polyoxyethylene sorbitan monolaurate
Medium, pH 6.5, temperature -20 ° C for at least one year
Stable for a while. (O) Activation: cA, a known kinase activator
MP, cGMP, Ca ²⁺, Calmodulin and phospholipids
It is not activated by quality. Under tubulin polymerization conditions,
The phosphorylation of tau protein is activated.

(P) Inhibition: 40 mM NaCl or K
The activity is reduced by half with Cl. Reference Example 2 Cloning of Tau Protein Kinase I Approximately 60 pmoles of tau protein kinase I purified from 15 cows was ligated to endoprotease Lys-C.
(Boehringer Mannheim Gmb
Was digested with H), column C ₈ (RP-300, Appl
ied Biosystems Inc. ) Was used for reverse phase high performance liquid chromatography. The following eight peptides were fractionated by elution with a gradient of 0% to 70% acetonitrile in 0.1% trifluoroacetic acid, and a part thereof was again purified by chromatography to obtain a sequencer (A).
Applied Biosystems Inc. , 47
The amino acid sequence was determined using 7A).

2K06: SEQ ID NO: 30 in the Sequence Listing 2K13: SEQ ID NO: 31 in the Sequence Listing 2K19: SEQ ID NO: 32 in the Sequence Listing 2K20: SEQ ID NO: 33 in the Sequence Listing 2K33: SEQ ID NO: 34 in the Sequence Listing 2K35: SEQ ID NO in the Sequence Listing 35 2K37: SEQ ID NO: 36 in Sequence Listing Based on the amino acid sequences of 2K35 and 2K37 among the above eight peptides, a sense primer described in SEQ ID NO: 37 and an antisense primer described in SEQ ID NO: 38 in the Sequence Listing were prepared. C synthesized and prepared from bovine brain tissue
Using DNA as a template, the polymerase chain reaction method (PCR method) developed by Saiki et al.
Gene amplification was performed according to Nature, 324 , 126 (1986)). As a result, SEQ ID NO: 39 in the sequence listing
The main products shown in the following were obtained. In the same product, 15 nucleotides sandwiched between the two primers were sequences expected from the above peptide.

[0052] Based on the sequence further shown in SEQ ID NO: 39 in Sequence Listing, were synthesized antisense primer new 41 nucleotide chromatography (SEQ ID NO: 40 of Sequence Listing), 5 'end of ³²
P was used as a probe for screening cDNA clones. A commercially available cDNA library (Clonetech) was used. This is obtained by inserting cDNA synthesized from rat cerebral cortex mRNA by random priming into λgt11. Hybridize at 48 ° C. according to a conventional method, and give × 1 SSC (0.3M
By washing at 35 ° C. with a buffer solution consisting of sodium chloride and 0.03 M sodium citrate, one positive clone (# 3
1) was obtained. # 31 has an insert of about 1.3 kb, which was subcloned into pUC19 to determine the DNA sequence. The amino acid sequences of all the eight peptides were confirmed on the same frame of this sequence, but no termination codon was present at the 3 'end. Therefore, a new clone (# 11) was obtained by using a part of this sequence as a probe. The # 11 insert started approximately halfway through the sequence of clone # 31 and was approximately 1.2 kb in size. Both sequences were ligated and the presence of an open reading frame (ORF) of about 420 amino acid residues was confirmed. The amino acid sequence and the base sequence are shown in SEQ ID NO: 1 in the sequence listing.
FIG. 1 shows a restriction map. Example 1 Tau protein extracted and purified from bovine brain was phosphorylated with tau protein kinase II, and then pH containing 1 M urea, 20 mM methylamine and 50 mM sodium phosphate was added.
1/20 of tau protein (weight ratio) in 8.5 buffer
Endoprotease Lys-C (Boehring
er Mannheim GmbH) and 37
Enzymatic digestion was performed at 2 ° C for 2 hours. The resulting peptides were separated by reversed-phase high-performance column chromatography. The time column C ₄ column (Bakerbond, 4.6mm × 5c
m) was used as the eluent, and the mixed solvent of acetonitrile and isopropanol (mixing ratio 3: 7) in 0.1% trifluoroacetic acid was increased in a linear gradient from 0% to 70% of the total solvent.

Next, the tau protein was phosphorylated in the presence of [γ- ³² P] ATP. Three peaks of the ³² P-labeled peptide were detected, and pK1, pK2 and pK3 in the elution order.
("P" indicates phosphorylated). Tau protein was similarly phosphorylated using non-radioactive ATP. Each fraction of the pK1, pK2 and pK3 fractions was collected from a gas phase protein sequencer (Appli).
The amino acid sequence of the peptide was analyzed using ed Biosystems 477A). As a result, K1 is a peptide from valine at position 168 to lysine at position 182 in the amino acid sequence of human-derived tau protein described in SEQ ID NO: 2 in the sequence listing, and K2 is a sequence at position 166 from serine at position 133. K3 is a peptide from serine at position 307 to lysine at position 349 (Goe
dert et al., Neuron, 3, 519-526 (19
89)) , the phosphorylation sites for pK1, pK2 and pK3 were also determined as follows. That is, in amino acid sequence analysis, phosphorylated serine is DTT
It is known to produce DTT adducts via PTH-dehydroalanine in the presence and not PTH-serine (Meyer et al., FEBS Lett., 20) .
4 , 61-66 (1986)). Similarly, phosphorylated threonine also produces DTT adducts, but does not produce PTH-threonine. This revealed that serine 177 in the amino acid sequence described in SEQ ID NO: 2 in pK1 was phosphorylated, serine 144 and serone 147 in pK2 were phosphorylated, and serine 315 in pK3 was phosphorylated. did.

Based on this result, SEQ ID NO: 2 in the sequence listing
From the 140th serine to 151th amino acid in the amino acid sequence of
In the peptide leading to the arginine at position 144, only serine at position 144, only threonine at position 147, and 14
Three peptides (SEQ ID NOs: 41, 42 and 43 in the sequence listing) having a phosphate group introduced into both the fourth serine and the 147 threonine, and 31 peptides in the peptide from lysine at 306 to arginine at 317
Synthesis of a peptide (SEQ ID NO: 44 in the sequence listing) in which a phosphate group has been introduced into the fifth serine, binding of each to KLH, immunization of rabbits, and purification of the obtained antiserum for specificity Thus, an anti-PS144 antibody that specifically recognizes the 144th serine, an anti-PT147 antibody that specifically recognizes the 147th threonine, and an anti-PS315 antibody that specifically recognizes the 315th serine were obtained.

On the other hand, tau protein dephosphorylated and unphosphorylated tau protein kinase II were subjected to electrophoresis and immunoblotting (Toubin et al., Proc. Natl.
Acad. Sci. USA, 76 , 4350-4354.
(1979)). As a result, in the electrophoresis, the mobility was lower in the order of dephosphorylated tau protein, untreated tau protein, and tau protein phosphorylated by tau protein kinase II. In addition, anti-PS144 antibody, anti-PT147
Regarding the reactivity with the antibody and the anti-PS315 antibody, the dephosphorylated tau protein did not react with any antibody,
Tau protein phosphorylated by tau protein kinase II reacted with any antibody, and untreated tau protein was expressed by anti-P
The reaction showed a relatively weak but sufficiently recognized reaction with the S144 antibody and the anti-PS315 antibody.

Using these various tau proteins as substrates, phosphorylation by tau protein kinase I was attempted as follows. First, tau protein kinase I acts on untreated tau protein, dephosphorylated tau protein, and tau protein phosphorylated with tau protein kinase II after dephosphorylation in the presence and absence of [γ- ³² P] ATP. As a result, in the case of the dephosphorylated tau protein, there was no change in the electrophoretic mobility, and the incorporation of ³² P was extremely small, but in the case of the other two tau proteins, the electrophoretic mobility was high. It became clear that phosphorylation progressed due to the incorporation of a large amount of ³² P. Next, in order to compare the effects of tau protein kinases I and II, the unreacted tau protein was phosphorylated with each kinase, and the reactivity with the antibody was examined by immunoblotting. The tau-1 antibody used here is an antibody which has a strong reaction with normal tau protein present in the brain and a weak reaction with abnormally phosphorylated tau protein in PHF. The ptau antibody is an antibody that specifically reacts with abnormal phosphorylated tau protein in PHF. As a result of immunoblotting, the reactivity with the tau-1 antibody was the strongest for the unphosphorylated tau protein, and the phosphorylation with tau protein kinase I or II weakened the reaction.
In particular, the band which was phosphorylated by tau protein kinase I and had the lowest electrophoretic mobility did not react with the tau-1 antibody. In contrast, reactivity with anti-ptau antibodies was first observed by phosphorylation with tau protein kinase I.

From the above results, serine at position 144 and 147 in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing were obtained.
Tau protein kinase I using a tau protein phosphorylated at one or more amino acids selected from threonine, 177th serine and 315th serine as a substrate
It was found that phosphorylation, similar to abnormal phosphorylation in PHF, progressed when was made to act. Example 2 Tau protein having a primary structure represented by the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing was phosphorylated with tau protein kinase II, and then tau protein kinase I was further added in the presence of [γ- ³² P] ATP. After phosphorylation, it was digested with endoprotease Lys-C. The peptide peak incorporating ³² P was identified in the same manner as in Example 1, and the corresponding unlabeled peptide was collected in the same manner as in Example 1. These peptides were analyzed by amino acid sequence analysis.
It was confirmed that they corresponded to pK1, pK2 and pK3 of Example 1, and it was concluded that these were further phosphorylated by tau protein kinase I. These peptides were designated as ppK1, ppK2 and pp, respectively.
Called K3. In each amino acid sequence analysis, the phosphorylation site was determined based on whether or not the PTH-amino acid was a DTT adduct of a PTH derivative of dehydrated serine or threonine in the same manner as in Example 1. 1 in the amino acid sequence of SEQ ID NO: 2
It was revealed that threonine 73, serine 141 in ppK2, serine 307 and serine 324 in ppK3 were amino acids phosphorylated by tau protein kinase I. Example 3 A peptide represented by the amino acid sequence of SEQ ID NO: 45 in the sequence listing was synthesized and phosphorylated with tau protein kinase I in the presence of [γ- ³² P] ATP.
³² P was not incorporated. However, by pre-phosphorylate the peptide in tau protein kinase II, could be phosphorylated in data U protein kinase I. Comparison of the amino acid sequence analysis of the peptide before and after phosphorylation with tau protein kinase I revealed that the PTH-amino acid corresponding to threonine at position 6 in the amino acid sequence described in SEQ ID NO: 45 in the sequence listing was PTH-threonine before phosphorylation. In contrast, after phosphorylation, it was a DTT adduct of PTH-dehydro-α-aminobutyric acid. The PTH-amino acids corresponding to the tenth serine in the same sequence were all DTT adducts of PTH-dehydroalanine.

From these results, phosphorylation of the peptide represented by the amino acid sequence of SEQ ID NO: 45 by tau protein kinase I requires phosphorylation of the tenth serine by tau protein kinase II. And the site of phosphorylation by tau protein kinase I was found to be threonine at position 6. Example 4 A peptide represented by the amino acid sequence of SEQ ID NO: 46 in the sequence listing was synthesized and phosphorylated with tau protein kinase I in the presence of [γ- ³² P] ATP.
³² P was not incorporated. However, by pre-phosphorylate the peptide in tau protein kinase II, could be phosphorylated in data U protein kinase I. Comparison of amino acid sequence analysis of the peptide before and after phosphorylation by tau protein kinase I revealed that the PTH-amino acid corresponding to the ninth serine in the amino acid sequence described in SEQ ID NO: 46 in the sequence listing was PTH-serine before phosphorylation. However, after phosphorylation, PT
It was a DTT adduct of H-dehydroalanine. In addition,
The PTH-amino acid corresponding to the 12th serine in the same sequence is the DTT of PTH-dehydroalanine.
PTH which is an adduct and corresponds to threonine at position 15
-The amino acids were all DTT adducts of PTH-dehydro-α-aminobutyric acid.

From these results, phosphorylation of the peptide represented by the amino acid sequence of SEQ ID NO: 46 by tau protein kinase I requires phosphorylation by tau protein kinase II and tau protein kinase II. The site of phosphorylation by I was found to be ninth serine. Example 5 A peptide represented by the amino acid sequence of SEQ ID NO: 47 in the sequence listing was synthesized, and then a peptide obtained by phosphorylating the peptide with tau protein kinase II was synthesized. When phosphorylation of each peptide was attempted with tau protein kinase I in the presence of [γ- ³² P] ATP, incorporation of ³² P was recognized only in the case of the phosphorylated peptide.

[0060]

Industrial Applicability The phosphorylation method of the present invention acts specifically on tau protein, and elucidates the etiology of Alzheimer's disease and Alzheimer's senile dementia, and further seeks a drug to prevent or treat it. The application of is expected.

[0061]

[Sequence list] SEQ ID NO: 1 Sequence length: 1932 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to genomic RNA Origin Organism: Rat Sequence GGCCAAGAGA ACGAAGTCTT TTTTTTTTTT TTCTTGCGGG AGAACTTAAT GCTGCATTTA 60 TTATTAACCT AGTACCCTAA CATAAAACAA AAGGAAGAAA AGGATTAAGG AAGGAAAAGG 120 TGAATCGAGA AGAGCCATC ATG TCG GGG CGA CCG AGA ACC ACC TCC TTT GCG 172 Met Ser Gly Arg Pro Arg Thr Thr Ser Phe TGA TCA GAG CAG CTAG AGC ATG AAA 220 Glu Ser Cys Lys Pro Val Gln Gln Pro Ser Ala Phe Gly Ser Met Lys 15 20 25 GTT AGC AGA GAT AAA GAT GGC AGC AAG GTA ACC ACA GTG GTG GCA ACT 268 Val Ser Arg Asp Lys Asp Gly Ser Lys Val Thr Thr Val Val Ala Thr 30 35 40 CCT GGA CAG GGT CCT GAC AGG CCA CAG GAA GTC AGT TAC ACA GAC ACT 316 Pro Gly Gln Gly Pro Asp Arg Pro Gln Glu Val Ser Tyr Thr Asp Thr 45 50 55 AAA GTC ATT GGA AAT GGG TCA TTT GGT GTG GTA TAT CAA GCC AAA CTT 364 Lys Val Ile Gly Asn Gly Ser Phe Gly Val Val Tyr Gln Ala Lys Leu 60 65 70 75 TGT GAC TCA GGA GAA CTG GTG GCC ATC AAG AAA GTT CTT CAG GAC AAG 412 Cys Asp Ser Gly Glu Leu Val Ala Ile Lys Lys Val Leu Gln Asp Lys 80 85 90 CGA TTT AAG AAC CGA GAG CTC CAG ATC ATG AGA AAG CTA GAT CAC TGT 460 Arg Phe Lys Asn Arg Glu Leu Gln Ile Met Arg Lys Leu Asp His Cys 95 100 105 AAC ATA GTC CGA TTG CGG TAT TTC TTC TAC TCG AGT GGC GAG AAG AAA 508 Asn Ile Val Arg Leu Arg Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys 110 115 120 GAT GAG GTC TAC CTT AAC CTG GTG CTG GAC TAT GTT CCG GAA ACA GTG 556 Asp Glu Val Tyr Leu Asn Leu Val Leu Asp Tyr Val Pro Glu Thr Val 125 130 135 TAC AGA GTC GCC AGA CAC TAT AGT CGA GCC AAG CAG ACA CTC CCT GTG 604 Tyr Arg Val Ala Arg His Tyr Ser Arg Ala Lys Gln Thr Leu Pro Val 140 145 150 155 ATC TAT GTC AAG TTG TAT ATG TAC CAG CTG TTC AGA AGT CTA GCC TAT 652 Ile Tyr Val Lys Leu Tyr Met Tyr Gln Leu Phe Arg Ser Leu Ala Tyr 160 165 170 ATC CAT TCC TTT GGG ATC TGC CAT CGA GAC ATT AAA CCA CAG AAC CTC 70 0 Ile His Ser Phe Gly Ile Cys His Arg Asp Ile Lys Pro Gln Asn Leu 175 180 185 TTG CTG GAT CCT GAT ACA GCT GTA TTA AAA CTC TGC GAC TTT GGA AGT 748 Leu Leu Asp Pro Asp Thr Ala Val Leu Lys Leu Cys Asp Phe Gly Ser 190 195 200 GCA AAG CAG CTG GTC CGA GGA GAG CCC AAT GTT TCA TAT ATC TGT TCT 796 Ala Lys Gln Leu Val Arg Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser 205 210 215 CGG TAC TAC AGG GCA CCA GAG CTG ATC TTT GGA GCC ACC GAT TAC ACG 844 Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr 220 225 230 235 TCT AGT ATA GAT GTA TGG TCT GCA GGC TGT GTG TTG GCT GAA TTG TTG 892 Ser Ser Ile Asp Val Trp Ser Ala Gly Cys Val Leu Ala Glu Leu Leu 240 245 250 CTA GGA CAA CCA ATA TTT CCT GGG GAC AGT GGT GTG GAT CAG TTG GTG 940 Leu Gly Gln Pro Ile Phe Pro Gly Asp Ser Gly Val Asp Gln Leu Val 255 260 265 GAA ATA ATA AAG GTC CTA GGA ACA CCA ACA AGG GAG CAA ATT AGA GAA 988 Glu Ile Ile Lys Val Leu Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu 270 275 280 ATG AAC CCA AAT TAT ACA GAA TTC AAA TTC CCC CAA ATC AA G GCA CAT 1036 Met Asn Pro Asn Tyr Thr Glu Phe Lys Phe Pro Gln Ile Lys Ala His 285 290 295 CCT TGG ACG AAG GTC TTT CGG CCC CGA ACT CCA CCA GAG GCA ATC GCA 1084 Pro Trp Thr Lys Val Phe Arg Pro Arg Thr Pro Pro Glu Ala Ile Ala 300 305 310 315 CTG TGT AGC CGT CTC CTG GAG TAC ACG CCG ACC GCC CGG CTA ACA CCA 1132 Leu Cys Ser Arg Leu Leu Glu Tyr Thr Pro Thr Ala Arg Leu Thr Pro 320 325 330 CTG GAA GCT TGT GCA CAT TCA TTT TTT GAT GAA TTA CGG GAC CCA AAT 1180 Leu Glu Ala Cys Ala His Ser Phe Phe Asp Glu Leu Arg Asp Pro Asn 335 340 345 GTC AAA CTA CCA AAT GGG CGA GAC ACA CCT GCC CTC TTC AAC TTT ACC 1228 Val Lys Leu Pro Asn Gly Arg Asp Thr Pro Ala Leu Phe Asn Phe Thr 350 355 360 ACT CAA GAA CTG TCA AGT AAC CCA CCT CTG GCC ACC ATC CTT ATC CCT 1276 Thr Gln Glu Leu Ser Ser Asn Pro Pro Leu Ala Thr Ile Leu Ile Pro 365 370 375 CCT CAC GCT CGG ATT CAG GCA GCT GCT TCA CCG CCT GCA AAC GCC ACA 1324 Pro His Ala Arg Ile Gln Ala Ala Ala Ser Pro Pro Ala Asn Ala Thr 380 385 390 390 395 GCA GCC TCA GAT ACT AAT GCT GG A GAC CGT GGA CAG ACC AAT AAC GCC 1372 Ala Ala Ser Asp Thr Asn Ala Gly Asp Arg Gly Gln Thr Asn Asn Ala 400 405 410 GCT TCT GCA TCA GCC TCC AAC TCT ACC TGA ACAG CCCCAAGTAG CCAGCTGCGC1426 Ala Ser Ala Ser Ala Ser Asn thr Stop 415 420 AGGGAAGACC AGCACTTACT TGAGTGCCAC TCAGCAACAC TGGTCACGTT TGGAAAGAAA 1486 ATTAAAAAGA GGAAAACAAA AACAAAAACA AAAAACCCCG GCTTTGGTTT GTTTCTTCTT 1546 TCTTCTTTTC CTCTATTTTC TTTTTTAAAA ATCTGTTTCT CCTTTTAAAA AAATTAAGAT 1606 GAAGTCAAGT CTGATGTCAT GGGTAACCCC ACCTACTTGG AAGGCTGAGT CTAGAGGTTT 1666 ACAGCTCAAG CCCATGCTGG ACTACAGTGG GAGTCCAAGG CCAGCNTGGG CAACTTAAAA 1726 AGAACTTGTT TCAAAAACGA CAAAGTTGGC TGATAATATG GCTCTCCAAG AGCCACAATA 1786 AATAAATATG TAAATAAACT CAAATAAGTC TTGTAATTTA AATTACACTA AACTAGGTTA 1846 ACTTTTAAAC TCTCATCTTT AAGAACTACA GGTTTAAAAA CCCAACGGTT GTTTTATGTA 1906 TTAGGGAAAA ATGAAAAATC TAATATAAAA AGAAGCAGCA ACAGCAGCAG GAGCCAACCA 1966 AAGGAT 1972 N: Unidentified

SEQ ID NO: 2 Sequence length: 352 Sequence type: amino acid Topology: linear Sequence type: protein Origin Organism name: Human sequence Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Ala Glu Glu Ala 35 40 45 Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val 50 55 60 Thr Gln Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp 65 70 75 80 Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro 85 90 95 Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg 100 105 110 Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly 115 120 125 Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser 130 135 140 Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro 145 150 155 160 Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro L ys 165 170 175 Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 180 185 190 Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asp Leu 195 200 205 Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val 210 215 220 Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His 225 230 235 240 His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp 245 250 255 Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr 260 265 270 His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr 275 280 285 Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val 290 295 300 Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser 305 310 315 320 Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu 325 330 335 Ala Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 340 345 350

SEQ ID NO: 3 Sequence length: 184 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser 1510 15 Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg 20 25 30 Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala 35 40 45 Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser 50 55 60 Thr Glu Asp Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val 65 70 75 80 Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu 85 90 95 Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser 100 105 110 Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu 115 120 125 Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr 130 135 140 His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly 145 150 155 160 Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro 165 170 175 Arg His Leu Se r Asn Val Ser Ser 180

SEQ ID NO: 4 Sequence length: 176 Sequence type: amino acid Topology: linear Sequence type: Peptide sequence Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser 1510 15 Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg 20 25 30 Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala 35 40 45 Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser 50 55 60 Thr Glu Asp Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val 65 70 75 80 Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu 85 90 95 Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser 100 105 110 Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu 115 120 125 Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr 130 135 140 His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly 145 150 155 160 Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro 165 170 175

SEQ ID NO: 5 Sequence length: 167 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser 1510 15 Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg 20 25 30 Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala 35 40 45 Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser 50 55 60 Thr Glu Asp Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val 65 70 75 80 Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu 85 90 95 Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser 100 105 110 Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu 115 120 125 Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr 130 135 140 His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly 145 150 155 160 Ala Glu Ile Val Tyr Lys Ser 165

SEQ ID NO: 6 Sequence length: 38 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser 1510 15 Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg 20 25 30 Thr Pro Pro Lys Ser Pro 35

SEQ ID NO: 7 Sequence length: 33 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser 1510 15 Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg 20 25 30 Thr

SEQ ID NO: 8 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 9 Sequence length: 5 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 10 Sequence length: 181 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr 1510 15 Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro 20 25 30 Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro 35 40 45 Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asp 50 55 60 Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro 65 70 75 80 Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile 85 90 95 His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu 100 105 110 Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile 115 120 125 Thr His Val Pro Gly Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu 130 135 140 Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile 145 150 155 160 Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu 165 170 175 Ser Asn Val Ser Ser 180

SEQ ID NO: 11 Sequence Length: 173 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr 1510 15 Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro 20 25 30 Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro 35 40 45 Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asp 50 55 60 Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro 65 70 75 80 Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile 85 90 95 His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu 100 105 110 Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile 115 120 125 Thr His Val Pro Gly Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu 130 135 140 Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile 145 150 155 160 Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro 165 170

SEQ ID NO: 12 Sequence length: 164 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr 1510 15 Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro 20 25 30 Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro 35 40 45 Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asp 50 55 60 Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro 65 70 75 80 Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile 85 90 95 His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu 100 105 110 Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile 115 120 125 Thr His Val Pro Gly Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu 130 135 140 Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile 145 150 155 160 Val Tyr Lys Ser

SEQ ID NO: 13 Sequence length: 35 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr 1510 15 Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro 20 25 30 Lys Ser Pro 35

SEQ ID NO: 14 Sequence length: 30 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr 1510 15 Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr 20 25 30

SEQ ID NO: 15 Sequence length: 178 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr 1510 15 Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro 20 25 30 Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp 35 40 45 Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asp Leu Lys His 50 55 60 Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val Asp Leu 65 70 75 80 Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys 85 90 95 Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys 100 105 110 Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val 115 120 125 Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg 130 135 140 Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys 145 150 155 160 Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val 165 170 175 Ser Ser

SEQ ID NO: 16 Sequence length: 170 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr 1510 15 Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro 20 25 30 Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp 35 40 45 Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asp Leu Lys His 50 55 60 Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val Asp Leu 65 70 75 80 Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys 85 90 95 Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys 100 105 110 Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val 115 120 125 Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg 130 135 140 Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys 145 150 155 160 Ser Pro Val Val Ser Gly Asp Thr Ser Pro 165 170

SEQ ID NO: 17 Sequence Length: 161 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr 1510 15 Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro 20 25 30 Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp 35 40 45 Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asp Leu Lys His 50 55 60 Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val Asp Leu 65 70 75 80 Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys 85 90 95 Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys 100 105 110 Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val 115 120 125 Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg 130 135 140 Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys 145 150 155 160 Ser

SEQ ID NO: 18 Sequence length: 32 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr 1510 15 Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro 20 25 30

SEQ ID NO: 19 Sequence Length: 27 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr 1510 15 Arg Glu Pro Lys Lys Val Ala Val Val Arg Thr 20 25

SEQ ID NO: 20 Sequence length: 152 Sequence type: amino acid Topology: linear Sequence type: Peptide sequence Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala 1510 15 Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser 20 25 30 Thr Glu Asp Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val 35 40 45 Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu 50 55 60 Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser 65 70 75 80 Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu 85 90 95 Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr 100 105 110 His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly 115 120 125 Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro 130 135 140 Arg His Leu Ser Asn Val Ser Ser 145 150

SEQ ID NO: 21 Sequence Length: 144 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala 1510 15 Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser 20 25 30 Thr Glu Asp Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val 35 40 45 Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu 50 55 60 Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser 65 70 75 80 Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu 85 90 95 Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr 100 105 110 His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly 115 120 125 Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro 130 135 140

SEQ ID NO: 22 Sequence length: 135 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala 1510 15 Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser 20 25 30 Thr Glu Asp Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val 35 40 45 Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu 50 55 60 Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser 65 70 75 80 Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu 85 90 95 Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr 100 105 110 His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly 115 120 125 Ala Glu Ile Val Tyr Lys Ser 130 135

SEQ ID NO: 23 Sequence length: 6 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 24 Sequence length: 148 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 1510 15 Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asp Leu 20 25 30 Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val 35 40 45 Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His 50 55 60 His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp 65 70 75 80 Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr 85 90 95 His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr 100 105 110 Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val 115 120 125 Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser 130 135 140 Asn Val Ser Ser 145

SEQ ID NO: 25 Sequence length: 140 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 1 5 10 15 Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asp Leu 20 25 30 Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val 35 40 45 Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His 50 55 60 His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp 65 70 75 80 Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr 85 90 95 His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr 100 105 110 Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val 115 120 125 Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro 130 135 140

SEQ ID NO: 26 Sequence length: 131 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Pro Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 1 5 10 15 Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asp Leu 20 25 30 Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Val Tyr Lys Pro Val 35 40 45 Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His 50 55 60 His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp 65 70 75 80 Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr 85 90 95 His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr His Lys Leu Thr 100 105 110 Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val 115 120 125 Tyr Lys Ser 130

SEQ ID NO: 27 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val 1 5 10 15 Ser Ser

SEQ ID NO: 28 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 29 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 30 Sequence length: 6 Sequence type: amino acid Topology: linear Sequence type: peptide Origin Organism name: bovine

SEQ ID NO: 31 Sequence length: 25 Sequence type: amino acid Topology: Linear Sequence type: Peptide Origin Organism name: Bovine Sequence Val Thr Thr Val Val Ala Thr Pro Gly Gln Gly Pro Asp Arg Pro Gln 1 5 10 15 Glu Val Ser Tyr Thr Asp Xaa Xaa Xaa 20 25 Xaa: Unidentified amino acid

SEQ ID NO: 32 Sequence length: 14 Sequence type: amino acid Topology: linear Sequence type: peptide Origin Organism name: bovine sequence Val Ile Gly Asn Gly Ser Phe Gly Val Val Tyr Gln Ala Lys 15 Ten

SEQ ID NO: 33 Sequence length: 21 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin Organism name: Bovine Sequence Xaa Thr Leu Pro Val Ile Tyr Val Xaa Xaa Xaa Tyr Gln Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 Xaa: Unidentified amino acid

SEQ ID NO: 34 Sequence length: 25 Sequence type: amino acid Topology: Linear Sequence type: Peptide Origin Organism name: Bovine Sequence Leu Pro Asn Gly Arg Asp Thr Pro Ala Leu Phe Asn Xaa Thr Thr Gln 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa 20 25 Xaa: Unidentified amino acid

SEQ ID NO: 35 Sequence length: 29 Sequence type: amino acid Topology: linear Sequence type: peptide Origin Organism name: Bovine Sequence Val Phe Arg Pro Arg Thr Pro Pro Glu Ala Ile Ala Leu Leu Xaa Arg 1 5 10 15 Xaa Leu Glu Tyr Thr Xaa Xaa Ala Xaa Xaa Thr Pro Xaa 20 25 Xaa: Unidentified amino acid

SEQ ID NO: 36 Sequence length: 30 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Origin Organism name: Bovine Sequence Val Phe Arg Pro Arg Thr Pro Pro Glu Ala Ile Ala Leu Xaa Xaa Xaa 1 5 10 15 Leu Leu Glu Tyr Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa: Unidentified amino acid

SEQ ID NO: 37 Sequence length: 23 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid (sense primer) Sequence ACN CCN CCN GAG GCN AT H GCN YT 23 N: Inosine H : A or C or TY: C or T

SEQ ID NO: 38 Sequence length: 20 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (antisense primer) Sequence CKN GCN GGN GGN GTR TAY TC 20 N: Inosine Y: T or CR: A or GK: G or T

SEQ ID NO: 39 Sequence length: 56 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid sequence ACG CCG CCG GAG GCA GTC GCG CTT TGT AGC CGT CTG CTG GAG TAT ACC 48 CCC CCG GC 56

SEQ ID NO: 40 Sequence length: 41 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: other nucleic acid (antisense primer) Sequence CTY CGN TAG CGN GAA ACA TCG GCA GAC GAC CTC ATA TGN GG 41 N: Inosine Y: T or C

SEQ ID NO: 41 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics Symbol indicating characteristics: modified-site (phosphorylation) Location: 5 Characteristic determination Method: E sequence Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg 1 5 10

SEQ ID NO: 42 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics Symbol indicating characteristics: modified-site (phosphorylation) Location: 8 Characteristic determination Method: E sequence Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg 1 5 10

SEQ ID NO: 43 Sequence length: 12 Sequence type: amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Symbol indicating characteristics: modified-site (phosphorylation) Location: 5, 8 Characteristics Determined method: E sequence Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg 1 5 10

SEQ ID NO: 44 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics Symbol indicating characteristics: modified-site (phosphorylation) Location: 10 Characteristic determination Method: E sequence Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg 1 5 10

SEQ ID NO: 45 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 1 5 10 15

SEQ ID NO: 46 Sequence length: 34 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro 1510 15 Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu 20 25 30 Pro Lys

SEQ ID NO: 47 Sequence Length: 55 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val 1 5 10 15 Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly 20 25 30 Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu 35 40 45 Val Ser Ala Ser Leu Ala Lys 50 55

[Brief description of the drawings]

FIG. 1 is a drawing showing a restriction map of tau protein kinase I.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Kazutomo Imabori 11-916 Minamiotani, Machida-shi, Tokyo 2 Mitsubishi Chemical Life Science Research Institute, Inc. (56) References FEBS Lett. , Vol. 289, no. 1 (1991), p. 37-43 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/48 C12N 9/12 BIOSIS (DIALOG)

Claims (1)

(57) [Claims] 1. A protein or a peptide which can be phosphorylated by a phosphorylase II having the following physicochemical properties is phosphorylated by the phosphorylase II, and further activated by a phosphorylase I having the following physicochemical properties. A method for phosphorylating a protein, which comprises phosphorylating. Phosphorylase II (i) action: using ATP as a phosphate donor, phosphorylates serine and threonine with proline next to the C-terminal side in tau protein, and phosphorylates 4 residues per tau protein molecule. Introduce a group. (J) Substrate specificity: specifically phosphorylates tau protein and MAP2, which are proteins associated with microtubules, in proteins of brain extract. Histone H1 and the neurofilament H subunit also phosphorylate. Slightly phosphorylates β-casein. (K) Optimum pH: The optimum pH in the absence of tubulin is 6.5, and the optimum pH in the presence of tubulin is 6.5.
0. (L) Suitable temperature for action: 37 ° C (m) Molecular weight: The measured value obtained by gel filtration in the presence of 0.5 M sodium chloride is about 50,000, and a complex with tau protein is formed in the absence of sodium chloride. Its measured value by gel filtration is about 100,000. The measured value obtained by SDS-polyacrylamide gel electrophoresis is about 30,000, and there are 23,000 other proteins, and the two proteins are combined to be about 50,000 by gel filtration. (N) Stability: 25% glycerol and 0.02% polyoxyethylene sorbitan monolaurate, pH 6.0.
5. Stable at -20 ° C for at least one year. (O) Activation: cA, a known kinase activator
It is not activated by MP, cGMP, Ca 2+, calmodulin and phospholipids. Under tubulin polymerization conditions,
The phosphorylation of tau protein is activated. (P) Inhibition: Activity is halved with 40 mM NaCl or KCl. Phosphorylase I (a) action: Using ATP as a phosphate donor, phosphorylates serine and threonine in tau protein and introduces a phosphate group of 4 residues per one molecule of tau protein. (B) Substrate specificity: specifically phosphorylates tau protein and MAP2, which are microtubule-associated proteins, in proteins of brain extract. Does not phosphorylate histones. Slightly phosphorylates α-casein. (C) Optimum pH: The optimum pH in the absence of tubulin is 6.5, and the optimum pH in the presence of tubulin is 6.5.
0. (D) Appropriate temperature for action: 37 ° C. (e) Molecular weight: The measured value obtained by gel filtration in the presence of 0.5 M sodium chloride is about 50,000, and a complex with tau protein is formed in the absence of sodium chloride. Its measured value by gel filtration is about 100,000. The measured value obtained by SDS-polyacrylamide gel electrophoresis was about 45,000.
Thousands. (F) Stability: pH 6.5 in 25% glycerol and 0.02% polyoxyethylene sorbitan monolaurate.
5. Stable at -20 ° C for at least one year. (G) Activation: cA, a known kinase activator
It is not activated by MP, cGMP, Ca 2+, calmodulin and phospholipids. Under tubulin polymerization conditions,
The phosphorylation of tau protein is activated. (H) Inhibition: 90 mM NaCl or KCl halves the activity.