U.S. patent application number 09/866712 was filed with the patent office on 2002-05-16 for methods of inhibiting tau-protein kinase i activity, inhibiting neuronal cell death and treating alzheimer's disease and tau-protein kinase i.
Invention is credited to Hoshino, Toshimitsu, Imahori, Kazutomo, Saito, Ken-Ichi, Sato, Showbu, Shiratsuchi, Akiko, Takashima, Akihiko.
Application Number | 20020058637 09/866712 |
Document ID | / |
Family ID | 46251800 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058637 |
Kind Code |
A1 |
Takashima, Akihiko ; et
al. |
May 16, 2002 |
Methods of inhibiting tau-protein kinase I activity, inhibiting
neuronal cell death and treating alzheimer's disease and
tau-protein kinase I
Abstract
A preventive or therapeutic agent for Arzheimer's disease which
comprises a substance exhibiting an inhibitory action to
tau-protein kinase I as an effective component is provided. A
pharmaceutical composition comprising said agent and a method of
inhibiting neuronal cell death in the brain are also provided.
Inventors: |
Takashima, Akihiko;
(Machida-shi, JP) ; Hoshino, Toshimitsu;
(Machida-shi, JP) ; Imahori, Kazutomo; (Meguro-ku,
JP) ; Saito, Ken-Ichi; (Asao-ku, JP) ;
Shiratsuchi, Akiko; (Sagamihara-shi, JP) ; Sato,
Showbu; (Machida-shi, JP) |
Correspondence
Address: |
WENDEROTH, LIND & PONACK, L.L.P.
2033 K STREET N. W.
SUITE 800
WASHINGTON
DC
20006-1021
US
|
Family ID: |
46251800 |
Appl. No.: |
09/866712 |
Filed: |
May 30, 2001 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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09866712 |
May 30, 2001 |
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09216958 |
Dec 21, 1998 |
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6248559 |
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09216958 |
Dec 21, 1998 |
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08461018 |
Jun 5, 1995 |
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6071694 |
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08461018 |
Jun 5, 1995 |
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08204091 |
Mar 2, 1994 |
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Current U.S.
Class: |
514/44A ;
435/194 |
Current CPC
Class: |
A61K 31/70 20130101;
C12N 9/12 20130101; C12N 2310/315 20130101; C12N 15/1137 20130101;
C12Y 207/11026 20130101; A61K 31/00 20130101 |
Class at
Publication: |
514/44 ;
435/194 |
International
Class: |
A61K 048/00; C12N
009/12 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 2, 1993 |
JP |
041160/1993 |
Mar 22, 1993 |
JP |
085143/1993 |
Aug 2, 1993 |
JP |
191246/1993 |
Claims
We claim:
1. A preventive or therapeutic agent for Arzheimer's disease which
comprises a substance exhibiting an inhibitory action to
tau-protein kinase I as an effective component.
2. The preventive or therpeutic agent for Arzheimer's disease
according to claim 1 in which the substance exhibiting an
inhibitory action to tau-protein kinase I is the one which, when
said substance is incubated together with neurons and amymoid
beta-protein, has an action of inhibiting the death of said
neurons.
3. A preventive or therapeutic agent for Arizheimer's disease which
comprises antisense oligonucleotide capable of hybridizing with
mRNA or DNA of tau-protein kinase I.
4. The preventive or therapeutic agnet for Arzheimer's disease
according to claim 3 in which the primary structure of the
tau-protein kinase I is expressed by the amino acid sequence which
is described in the Seq. ID No. 1 or No. 2 of the attached Sequence
Listing.
5. A pharmaceutical composition for prevention or therapy of
Arzheimer's disease, which comprises a substance exhibiting an
inhibitory action of tau-protein kinase and a
pharmaceutically-acceptable carrier.
6. The pharmaceutical composition according to claim 5 in which the
substance exhibiting an inhibitory action to tau-protein kinase I
is the one which, when said substance is incubated together with
neurons and amyloid beta-protein, has an action of inhibiting the
death of said neurons.
7. A pharmaceutical composition for preventinon or therapy of
Arzheimer's disease, which comprises antisense oligonucleotide
capable of hybridizing with mRNA or DNA of tau-protein kinase
I.
8. The pharmaceutical composition according to claim 7 in which the
primary structure of tau-protein kinase I is represented by the
amino acid sequence which is described in the Seq. ID No. 1 or 2 of
the attached Sequence Listing.
9. A method for screening an agent useful for a prevention or
therapy of Arzheimer's disease, characterized in that amyloid
beta-protein, nerve cells and an agent presumed to be effective as
a preventive or therapeutic agent for Arzheimer's disease are
incubated and, when death of said nerve cells is inhibited, then
said agent is judged to be effective as a preventive or therapeutic
agent for Arzheimer's disease.
10. A method of inhibiting the neuronal cell death in the brain,
characterized in that a substance exhibiting an inhibitory action
to tau-protein kinase I is applied to the neurons in the brain.
11. A method of inhibiting the neuronal cell death in the brain,
characterized in that an antisense oligonucleotide capable of
hybridizing with mRNA or DNA of tau-protein kinase I is applied to
the neurons in the brain.
12. The method according to claim 11 in which the primary structure
of tau-protein kinase I is expressed by the amino acid sequence
described in the Seq. ID No. 1 or No. 2 of the attached Sequence
Listing.
13. Tau-Protein kinase I originated from human being, which is
characterized by being represented by the amino acid sequence
described in the Seq. ID No. 1 of the attached Sequence Listing or
the partial sequence thereof.
14. Gene which encodes the human-originated tau-protein kinase I
described in claim 13.
15. The gene according to claim 14 in which said gene is
represented by the base sequence descrbed in the Seq. ID No. 1 of
the attached Sequence Listing.
16. Recombinant human-originated tau-protein kinase I.
17. Recombinant vector which is capable of expressing recombinant
human-originated tau-protein kinase. I.
18. Transformant which is obtained by subjecting the host cells to
a transformation using the recombinant vector of claim 17.
19. A method of producing recombinant human-originated tau-protein
kinase I, characterized in that, the transformant of claim 18 is
incubated and the recombinant human-originated protein kinase I is
collected from its culture.
Description
[0001] The present invention relates to a preventive or a
therapeutic agent for Arzheimer's disease, a method of screesing
Arzheimer's disease and tau-protein kinase I which is originated
from human being. More particularly, it relates to a preventive or
a therapeutic agent for Arzheimer's disease usig a tau-protein
kinase I inhibitor; a method of screening a preventive or a
therapeutic agent for Arzheimer's disease utilizing an amyloid
beta-protein; a human-originated tau-protein kinase I which
phosphorylates tau-protein, partial peptides thereof or peptides
similar thereto; a gene which encodes the kinase; and a method of
producing the same.
[0002] Arzheimer's disease is a progressive dementia which develops
in late middle ages (45 to 65 years old) and its etiological
changes are shrinkage of cerebral cortex due to a-neuronal cell
loss and degeneration of the neurons while, from the pathological
view, many senile plaques and neurofibrillary tangles are noted in
the brain. There is no pathologically substantial difference
between the disease and senile dementia caused by the so-called
natural aging which develops in the senile period of 65 years and
older ages and, therefore, this is called senile dementia of
Arzheimer type.
[0003] Numbers of the patients of this disease are increasing with
an increase of population of aged people and the disease is
becoming serious in the society. There are various theories on the
cause of this disease but, the cause has been still ambiguous and,
accordingly, there has been a demand for prompt clarification.
[0004] It has been known that the quantities which appear in the
two pathological changes which are characteristic to Arizheimer's
disease and to senile dementia of Arzheimer type are well
correlated with the degree of cognitive impairement. Accordingly,
studies for clarifying the cause of those diseases by clarifying,
in a molecular level, the accumulated insoluble substances
resulting in those two pathological changes have been carried out
since the first half of the 1980's.
[0005] It has been clarified already that a main component of the
senile plaques which is one of those pathological changes is
amyloid beta-protein (hereinafter, it may be abbreviated as
"A.beta.P") [Annu., Rev. Neurosci., 12, 463-490 (1989)]. A
neurofibrillary tangle which is another pathological change is due
to an accumulation of a double-stranded fibrous substance called
PHF (paired helical filament) in the neurons and, recently, the
components thereof have been identified as ubiquitin and
tau-protein which is one of the microtubule-associated proteins
characteristic to brain [J. Biochem., 99, 1807-1810 (1986); Proc.
Natl. Acad. Sci. USA, 83, 4913-4917 (1986)].
[0006] It is believed now that, in Arzheimer's disease, the amyloid
beta-protein is extremely accumulated in the neurons and that, as a
result of its correlation with the formation of PHF, death of the
neurons is resulted.
[0007] It has been known that the tau-protein (hereinafter, the
protein may be abbreviated as a "tau") is usually a series of
related proteins forming several bands at the molecular weights of
48-65 kd on SDS polyacrylamide gel electrophoresis and that it
promotes the formation of microtubules.
[0008] It has been proved already by the use of polyclonal antibody
to PHF [anti-ptau: J. Biolchem. 99, 1807-1810(1986)1 and also of
monoclonal antibody [tau-1 antibody; Proc. Natl. Acad. Asic. USA,
83, 4913-4917(1986)] that the tau which is incorporated in the PHF
of the brain of Arzheimer's disease is extremely phosphorylated as
compared with the normal one.
[0009] The present inventors have isolated an enzyme which
catalyzes such an abnormal phosphorylation, named it "tau-protein
kinase I" (hereinafter, it may be abbreviated as "TPK-I") and
clarified its biochemical properties [Seikagaku, vol. 64, no. 5,
page 308 (1992)]. The inventors have further cloned the cDNA of rat
TPK-I from the cDNA library of cerabral cortex of rats based upon
the partial amino acid sequence of TPK-I, whereby the base sequence
has been determined and the amino acid sequence has been proposed
(Seq. ID No. 2 in the Sequence Listing; Japanese Patent Application
177241/92, FEBS Lett., 325, 167-172 (1993)).
[0010] As a result thereof, it has been confirmed that the primary
structure of the rat TPK-I is identical with that of the enzyme
which is known as a rat GSK-3.beta. (glycogen syntase kinase
3.beta.) [EMBO J., 9, 2431-2438 (1990)]. However, in finding the
drugs which are effective for the prevention or the therapy of
human diseases, the primary struc ture which are targets for the
drug usually vary depending upon the animal species. Therefore,
there are many cases that the interaction between the drug and the
protein (in other words, sensitivity and effectiveness of the drug)
greatly differs depending upon the animal species (e.g. Nature,
360, 161 (1992)]. Thus, in order to find drugs which are really
effective to human being, it is desired that the investigation is
carried out using proteins which are originated from human being.
Particularly in the case of finding the drugs effective for the
diseases which have not been found in animals other than human
being such as Arzheimer's disease, it is believed to be essential
to use proteins originated from human being. However, there has
been no report on the separation and purification of TPK-I (or
GSK-3.beta.) from human tissues and, moreover, there has been no
report on gene (cDNA) which encodes human TPK-I (or
GSK-3.beta.).
[0011] An object of the present invention is to clarify the
correlation between the death of neurons and accumulation of PHF
and amyloid beta-protein characteristically found in the brains of
Arzneimer's disease and also to apply it to the clarification of
the cause of Arzheimer's disease and further to the investigation
to the preventive or therapeutic agents therefor.
[0012] Another object of the present invention is to clarify the
structure of the human-originated TPK-I (which is essential for the
progress of investigations for such drugs) on the molecular
biological basis and to offer a method of producing it by means of
gene technology.
[0013] The present inventors have carried out the investigations
for achieving the above-given objects and confirmed that, when
amyloid beta-protein acted to the neurons in the brain, activity of
TPK-I significantly increases whereupon the extremely
phosphorylated tau-protein found in PHF of the brains of
Arznheimer's disease is resulted and, moreover, the neurons are
killed, and that the above-mentioned increase in the TPK-I activity
and neuronal cell death in the brain is inhibited by the treatment
with the antisense oligonucleotide of the TPK-I.
[0014] In addition, with a view that the accumulation of PHF
results in the degeneration of neurons in the brains of Arzheimer's
disease and successively induces the death, the present inventors
have for the first time cloned the gene (which encodes the
human-originated TPK-I which is thought to be a key enzyme for the
PHF formation) from the cDNA library of human fetus brain,
whereupon its primary struture is determined and a method for
constant supply (or production) of the human-originated TPK-I has
been established.
[0015] The present invention has been achieved as a result of the
above-given findings followed by further investigations, and its
characteristic features are as follows:
[0016] (1) a preventive or a therapeutic agent for Arzheimer's
disease, which comprises a substance exhibiting an inhibitory
action to tau-protein kinase I as an effective component;
[0017] (2) a preventive or a therapeutic agent for Arzheimer's
disease, which comprises an antisense oligonucleotide capable of
hybridizing with mRNA or DNA of tau-protein kinase I as an
effective component;
[0018] (3) a pharmaceutical composition for prevention or therapy
of Arzheimer's disease, which comprises a substance exhibiting an
inhibitory action to tau-protein kinase I and a pharmaceutically
acceptable carrier;
[0019] (4) a pharmaceutical composition for prevention or therapy
of Arzheimer's disease, which comprises an antisense
oligonucleotide capable of hybridizing with mRNA and DNA of
tau-protein kinase I;
[0020] (5) a method of screening a preventive or a therapeutic
agent for Arzheimer's disease in which, when amyloid beta-pro tein,
nerve cells and a drug which is presumed to be effective as a
preventive or a therapeutic agent to Arzheimer's disease are
incubated and the death of said nerve cells is inhibited, then said
drug is judged to be effective as a preventive or a therapeutic
agent for Arzheimer's disease;
[0021] (6) a method of inhibiting the death of neurons in the
brain, chracterized in that a substance which exhibits an
inhibitory action to tau-protein kinase I to the neuron in the
brain is applied;
[0022] (7) a method of inhibiting the death of neurons in the
brain, characterized in that an antisense oligonucleotide which is
capable of hybridizing with mRNA or DNA of tau-protein kinase I is
applied to the cranial nerve cells;
[0023] (8) human-originated tau-protein kinase I characterized in
being represented by an amino acid sequence given in the Seq. ID
No. 1 of the attached Sequence Listing or a partial sequence
thereof;
[0024] (9) gene which encodes the human-originated tau-protein
kinase I which is represented by the amino acid sequence given in
the Seq. ID No. 1 of the attached Sequence Listing or a partial
sequence thereof;
[0025] (10) recombinant human-originated tau-protein kinase I;
[0026] (11) recombinant vector which is capable of expressing the
recombinant human-originated tau-protein kinase I;
[0027] (12) transformant which is obtained by a transformation of
the host cells by a recombinant vector which is capable of
expressing the recombinant human-originated tau-protein kinase I;
and
[0028] (13) a method of producing a recombinant human-originated
tau-protein kinase I, characterized in that a transformant obtained
by a transformation of host cells by a recombinant vector which is
capable of expressing the recombinant human-originated tau-protein
kinase I is incubated and then the recombinant human-originated
tau-protein kinase I is collected from said culture.
[0029] The present invention will be further illustrated as
hereunder.
[0030] With regard to the substance which exhibits an inhibitory
action to tau-protein kinase I in the present invention, any
substance will do provided that, when said substance is incubated
together with nerve cells and amyloid beta-protein, death of said
nerve cells is inhibited. For example, it is chemically-synthesized
substance, a substance which is extracted from living cells of
microorganisms, etc.
[0031] Further, in accordance with the present invention, an
antisense oligonucleotide (hereinafter, it may be abbreviated as
"TPK-I antisense oligonucleotide") which is capable of hybridizing
with mRNA or DNA of TPK-I is used for prevention or therapy of
Arzheimer's disease.
[0032] Antisense oligonucleotide is capable of inhibiting the
protein synthesis in a level of gene and, therefore, it has been
receiving attention in the medical field as a synthetic inhibitor
for the proteins causing the disease. The principle is that, when
the antisense RNA or the antisense DNA forms a base pair with mRNA
in a sense sequence, spread of the gene information is interrupted
and synthesis of protein which is the final product is inhibited
[Igaku no Ayumi, vol.162, no.13, 909-911(1992)].
[0033] With regard to the TPK-I antisense oligonucleotide applied
in the present invention, anything will do provided that it is
capable of hybridizing with mRNA or DNA of TPK-I and that it has a
sequence for inhibiting the synthesis of TPK-I by, for example,
inhibition of transcription, inhibition of splicing of pre-mRNA,
inhibition of mRNA septum transmission, inhibition of translation,
etc. Usually, that comprising about 15 to 30 nucleotides is
used.
[0034] Furthermore, the antisense oligonucleotides applicable are a
phosphorothioate type in which an oxygen atom which is bonded by
means of a double bond with a phosphorus atom at the phosphodiester
bond connecting deoxyribonucleosides is substituted with a sulfur
atom; a methyl phosphate type in which methyl group is introduced
instead of the sulfur atom; a phosphonate type without
substitution; and an alpha-oligonucleotide type [Anticancer Drug
Des. 6 (66), 606-646 (1991); Anticancer Research, 10, 1169-1182
(1990)]. In addition, in the present invention, it is not always
necessary to use a nucleotide type in which a nucleoside derivative
is bonded provided that the substance can form a hybrid with the
aimed sequence. For example, the antisense compounds which are
described in Antisense Research and Development, 1, 65-113 (1991),
etc. may be used as well.
[0035] Specific examples of the TPK-I antisense oligonucleotides
used in the present invention are TPK-I antisense oligonucleotide
chain: 5'-TCTCGGTCGCCCCGACAT-3' (Seq. ID No. 5 of the Sequence
Listing) which is complementary to TPK-I sense oligonucleotide
chain: 5'-ATGTCGGGGCGACCGAGA-3' (Seq. ID No. 4 of the Sequence
Listing) corresponding to the first six amino acid residues: Met
Ser Gly Arg Pro Arg in the translation initiating domain of TPK-I
in the primary structure of the rat GSK-3.beta. [same as the
primary structure of the rat TPK-I (Seq. ID No. 2 of the Sequence
Listing) described in the above-referenced EMBO J., 9,
2431-2438(1990)]; the TPK-I antisense oligonucleotide chain:
5'-TCTGGGCCGCCCTGACAT-3' (Seq. ID No. 7 of the Sequence Listing)
which is complementary to the TPK-I sense oligonucleotide chain:
5'-ATGTCAGGGCGGGCCCAGA-3' (Seq. ID No. 6 of the Sequence Listing)
corresponding to the first six amino acid residues: Met Ser Gly Arg
Pro Arg in the translation initiating domain of TPK-I in the
primary structure of human TPK-I (Seq. ID No. 1 of the Sequence
Listing; refer to the examples which will given later); and the
like.
[0036] The above-mentioned TPK-I sense oligonucleotide and TPK-I
antisense oligonucleotide can be easily synthesized by means of
commercially-available automatic DNA synthesizers such as a DNA
synthesizer manufactured by Applied Biosystems, that manufactured
by MilliGen, etc. As mentioned already, the TPK-I antisense
oligonucleotides of the present invention are not particularly
limited to those having the above-given sequences provided that
they are capable of hybridizing with mRNA or DNA of TPK-I and, so
far as the hybrid-forming ability is not deteriorated, a part of
the sequence may be substituted with any base. In addition, the
antisense oligonucleotides which are changed or modified for
passing through a blood-brain barrier as described in Science, 259,
373-377 (1993). are included in the coverage of the present
invention as well.
[0037] When the TPK-I antisense oligonucleotides or the substances
having an inhibitory action to TPK-I as mentioned above are used as
preventive or therapeutic agents for Arzheimer's disease, they may
be made into preparations meeting with the particular administering
route together with usual carriers. For example, in the case of
oral administration, preparations in the form of tablets, capsules,
granules, diluted powder, liquid, etc. are prepared.
[0038] In preparing solid preparations for oral use, commonly-used
fillers, binders and lubricants as well as colorants,
disintegrating agents, etc. may be used. Examples of the fillers
are lactose, starch, talc, magnesium stearate, crystalline
cellulose, methyl cellulose, carboxymethyl cellulose, glycerol,
sodium alginate, gum arabic, etc. Examples of the binders are
polyvinylalcohol, polyvinyl ether, ethyl cellulose, gum arabic,
shellac, white sugar, etc. Examples of the lubricants are magnesium
stearate, talc, etc. Besides those, commonly-used ones may be used
for the colorants, disintegrating agents, etc. as well. Tablets may
be coated by known methods. Liquid preparations may be aqueous or
oily suspensions, solutions, syrups, elixiers and the like and may
be manufactured by commonly-used methods. In preparing injections,
pH-adjusting agents, buffers, stabilizers, isotonic agents, local
anesthetics, etc. may be added to TPK-I antisense oligonucleotides
or the substances having an inhibitory action to TPK-I and
subcutaneous, intramuscular or intravenous injections may be
prepared by common methods. With regard to the bases for the
manufacture of suppositories, oily ones such as cacao butter,
polyethylene glycol, Witepsol (registered trade mark of Dynamite
Nobel) may be used.
[0039] Doses of the preparations manufactured as such are not
always the same but vary depending upon the symptoms, body weights,
ages, etc. of the patients. Usually, however, the amount
corresponding to about 1 to 1,000 mg/kg of said drug per day for
adults will do and it is preferred to administer by dividing that
for 1 to 4 times daily. In some instances, the administration may
be carried out once daily to every several or more days.
[0040] Examples of the nerve cells used in the present invention
are the neuron in the brain collected from mammals and the neuronal
cell lines in which the nerve projections are extended by the
induction of growth factors such as NGF (nerve growth factor;
neurotrophic factor), IGF (insulin-like growth factor), etc. An
example of the former is a culture prepared by incubation of
tissues of hippocampus of mammals (such as rat) in a complete
culture medium. Examples of the latter are PC 12 cells induced by
NGF, FGF (fibroblast growth factor), EGF (epidermal growth factor),
interleukin 6, etc. [Ann. Rev. Pharma col. Toxicol., 31, 205-228
(1991)]; SH-SY5Y cells induced by IGF [The Journal of Cell Biology,
102, 1949-1954 (1986)]; and those which are disclosed in Cell
Culture in the Neurosciences, New York: Plenum Press, pages 95-123
(1955) such as NGF-induced MJB cells, NMB cells, NGP cells,
SK-N-SH-SY5Y cells, LAN-1 cells, KA-9 cells, IMR-32 cells and
5-bromodeoxyuridine-induced IMR-32 cells, NMB cells, NGP cells,
etc.
[0041] Amyloid beta-protein is a main component of senile plaques
of Arzheimer's disease and it has been known that said substance is
composed of a peptide comprising the following 43 amino acid
residues [Science, 250, 279-282 (1990) and Proc. Natl. Acad. Sci.
USA, 87, 9020-9023 (1990)].
[0042] Amino Acid Sequence of Amyloid beta-Protein (Seq. ID No. 3
of the Sequence Listing): Asp Ala Glu Phe Arg His Asp Aer Gly Tyr
Alu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn
Lys Gloy Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr
The present invention will be further illustrated as hereinafter by
an example on the behavior of hippocampus cells of rats and the
phosphorylation activity of TPK-I when the hippocampus cells were
treated with a certain amount of A.beta.P and also with TPK-I sense
oligonucleotide (hereinafter, referred to as "ITPKI-sense") and
TPK-I antisense oligonucleotide (hereinafter, referred to as
"TPKI-antisense") as controls under certain conditions. When the
present invention is carried out as a method of screening the
preventive and the therapeutic agent of Arzheimer's disease,
hippocampus cells of rat are used as the neurons and, as the agent
presumed to be the preventive or the therapeutic agent, TPKI-sense
or TPKI-antisense is used.
[0043] Certain amount of TPKI-antisense was added to the culture of
the hippocampus cells at certain temperature, then certain aount of
A.beta.P was added thereto, the mixture was kept at certain
temperature and the living cell numbers with an elapse of time were
measured by a method described in the examples which will be given
later. For comparison, the living cell numbers were measured for
the case in which only A.beta.P was added followed by the same
treatments and the case in which TPKI-antisense and A.beta.P were
added followed by the same treatments. The result showed that, as
given in the examples later, the living cell numbers when
TPKI-antisense and A.beta.P were added were significantly more than
those when only A.beta.P was added and when TPKI-sense and A.beta.P
were added and that the TPKI-antisense has an action of inhibiting
the death of the cells by ASP.
[0044] The results of the observations of the samples using a phase
contrast microscope (magnifying power: 400) when TPKI-antisense and
A.beta.P were added to the cell culture followed by allowing to
stand for 24 hours, when only A.beta.P was added followed by
allowing to stand for the same time and when TPKI-sense and
A.beta.P were added followed by allowing to stand for the same time
showed that the cell toxicity by A.beta.P was little being similar
to the controls only when TPKI-antisense was acted.
[0045] Further, the phosphorylation activities of tau-protein by
TPKI after 24 hours were measured by the method given in the
examples when only A.beta.P was added and allowed to stand and when
TPKI-antisense and A.beta.P were added and allowed to stand same as
above. The result was that, as shown in the examples given later,
the phosphorylation activity of TPK-I when TPKI-antisense and
A.beta.P were added was about one half of that when only A.beta.P
was added and that TPKI-antisense exhibits an activity of
inhibiting the phosphorylation activity of TPK-I.
[0046] Out of the above results, it may be concluded that, when the
present invention is carried out as a method of screening the
preventive and the therapeutic agent for Arzheimer's disease,
TPKI-anti'sense is effective as said preventive and therapeutic
agent. Incidentally, the effectiveness of the agents other than the
TPK-I antisense oligonucleotide can be evaluated similarly.
[0047] Now, the method of obtaining the human-originated TPK-I and
the method of production thereof will be illustrated as
hereunder.
[0048] The TPK-I originated from human being of the present
invention may, for example, be manufactured as follows. Thus,
microtubule fractions were obtained from an extract of human brain
immediately after death by means of temperature-depending
polymerization and depolymerization and then, operations such as
phosphocellulose column chromatography, gel filtration,
hydroxyapatite column chromatography, S-Sepharose column
chromatography, heparin column chromatography, etc. are combined
according to a method by Uchida, et al. [Seikagaku, vol.64, no.5,
page 308 (1992)] whereby pure protein is obtained. The (partial)
primary structure of such a pure protein may be determined by
conducting a conventional amino acid analysis. It is not easy to
obtain the human brain tissues in large quantities and it is
difficult to purify the human TPK-I and, therefore, it is also
possible that, by a method which will be given later, gene is
previously cloned and the amino acid sequence is deduced therefrom
whereby the primary structure is determined.
[0049] The human TPK-I of the present invention prepared as such is
a protein in which the primary structure is represented by the
amino acids described in the Seq. ID No. 1 of the Sequence Listing
(420 amino acid residues; molecular weight: 46,719; isoelectric
point: 9.21) and alterations such as removal, substitution,
modification or addition of some amino acids may be carried out,
within such a range that the functions (action, substrate
specificity, etc.) will not be deteriorated.
[0050] The gene (cDNA) which encodes the above TPK-I may be cloned
by such a method that in which the corresponding protein is
purified from natural material, its partial amino acid sequence is
determined and the DNA probe corresponding thereto is utilized;
that in which homology with the protein of the same species or the
corresponding protein of the different animal species is utilized;
that in which an antibody which is specific to the corresponding
protein is utilized; that in which a detection of the specific
function of the protein is utilized; etc. The present inventors
have previously purified TPK-I from an extract of brain of rat or
bovine and, depending upon the information of the partial amino
acid sequence thereof, they cloned the rat TPK-IcDNA from the rat
brain cDNA library (Seq. ID No. 2 of the Sequence Listing; Japanese
Patent Application No. 177241/92, FEBS Lett., 325, 167-172
(1993)).
[0051] Usually, however, the homology of the primary structures of
rats with human beings in the same protein is, in most cases,
around 90% or more and, therefore, it is possible to clone human
TPK-IcDNA from rat TPK-IcDNA by utilizing said homology. Thus,
lamda-phage is infected to Escherichia coli by a method of
Tomizawa, et al. ["Experiments in Bacteriophage" (Iwanami Shoten),
pages 99-174 (1970)] from cDNA library containing the gene which
encodes human TPK-I such as human fetus brain cDNA library followed
by culturing. The plaques formed thereby were selected by a plaque
hybridization method ["Molecular Cloning" Cold Spring Harbor
Laboratory, pages 320-328 (1982)] using a rat TPK-IcDNA or DNA
fragments having a partial structure thereof as a probe. The phage
is promulgated from positive plaques by a method of Tomiza wa, et
al., then DNA is prepared by a method of T. Maniatis, et al.
["Molecular Cloning", Cold Spring Harbor Laboratory, page 85
(1982)] or after subjecting to a subcloning if necessary, cleaved
by a suitable restriction enzyme such as EcoRI and cloned to a
plasmid such as pUC18 or pUC19. As such, cDNA of human TPK-I is
prepared and its base sequence can be determined, for example, by a
dideoxy method of Sanger, et al. [Proc. Natl. Acad. Sci. USA, 74,
5463 (1977)].
[0052] An example of the above-mentioned gene (cDNA) encoding the
human-originated TPK-I is that which has a base sequence given in
the Seq. ID No. 1 of the Sequence Listing.
[0053] In the human TPK-I prepared by the present invention, 5
amino acid residues were different out of the 420 amino acid
residues in the amino acid sequence of the rat TPK-I and the
homology in the bases in the translation domain was 92.5%.
[0054] The above-prepared human TPK-I or its partial peptides can
be expressed and generated by means of gene recombination. Thus,
the 5'-terminal of the human TPK-IcDNA or its fragment is modified
or added with precursor gene, inserted into the downstream of the
promoter of the expressed vector and then the vector is induced
into host cells such as bacteria, yeasts, insect cells, animal
cells, etc. The transformed host cells as such is cultured under a
suitable condition whereby the recombinant human TPK-I is produced
in or outside the cells.
[0055] Examples of the transformable host cells are bacteria
(procaryotic cells) such as Escherichia coli (K-12 strain),
Bacillus subtitlis, etc.; yeasts such as Saccharomyces cerevisiae;
insect cells such as ovary-originated cells (Sf9 cell strain) of
Spodoptera spp.; and (mammalian) animal cells such as
ovary-originated cells (CHO cells) of Chinese hamster, mouse C127
cells, kidney-originated cells (COS cells) of African green monkey,
mouse L cells, mouse FM3A cells, kidney-originated cells (HEK
cells, 293 cells) of human fetus, etc.
[0056] The expression vector which is suitably used is that which
contains a promoter at the position where a gene (cDNA) coding the
human TPK-I or the DNA fragment threreof can be transcribed. For
example, when cells are used as a host, it is preferred that the
expression vector is composed of promoter, ribosome binding (SD)
sequence, human TPK-I-encoding gene or fragment thereof,
transcription terminating factor and promoter-controling gene. Even
when eucaryonic cells such as (mammalian) animal cells, insect
cells, yeasts, etc. are used as host cells, the fundamental unit
comprising the expression vector is the same as that in the case of
the above-mentioned bacteria.
[0057] Examples of the promoter when bacteria are used as host
cells are those originated from Escherichia coli, phage, etc. such
as lactose operon (lac), tryptophan-synthesizing enzyme (trp),
lamda-phage P.sub.L' E. coli T7 polymerase promoter and tac (hybrid
promoter originated from trp and lac UV5). The examples in the case
of yeasts are promoters of gene of enzymes such as phosphoglyceric
acid kinase (PGK), glyceraldehyde-3-phosphoric acid dehydrogenase
(GPD), repressible acidic phosphatase (PHOS) and alcohol
dehydrogenase 1 (ADH1). The examples in the case of insect cells
are used as the host are promoter of polyhedron gene of
baculovirus, etc. The examples in the case of (mammalian) animal
cells are SV40 initial promoter, SV40 late promoter, apolipoprotein
E gene promoter, etc.
[0058] Examples of the ribosome binding sequence are those which
are originated from E. coli, phage, etc. and those which are
partially complementary to the base sequence of the 3'-terminal
domain of 16S ribosome RNA.
[0059] Though the transcription terminating factor is not always
necessary, it is preferred to have that which is
rho.rho.-independent such as lipoprotein terminator, trp operon
terminator, etc.
[0060] With regard to the sequence of those factors necessary for
the expression on the expression plasmid (vector), it is desired
that they are placed in the order of promoter, SD sequence, human
TPK-I-encoding gene or fragment thereof and transcription
terminating factor from the 5'-upstream side.
[0061] Specific examples of the expression vectors satisfying those
requirements in case that bacteria are used as the host are
pKK233-2 (manufactured by Farmacia) and pET3C [Gene, 56, 125
(1987)]. Expression vector pGEX series (Farmacia) which are
expressed as fused protein may be used as well in the same manner.
When yeasts are used as a host, the vector in which the
above-mentioned promoter (and, further, gene which complements the
auxotropic mutant as a selected marker such as trp1 and leu2) is
incorporated into YEp vector having a replication origin of 2
micron DNA is suitably used. In the case of insect cells, an
example is Maxbac (trademark) which is a baculovirus expression
system manual version 1.4 of Invitrogen. In the case of animal
cells, those having the above-mentioned promoter and selective
marker gene such as neomycin-resisting gene (Neo) and dihydrofolic
acid reductase gene (DHFR) are suitably used. When eucaryonic cells
are used as a host, a shuttle vector to E. coli may be used as
well.
[0062] Transformation of host cells may be carried out by
conventional manner. Culture of the transformant may be carried out
by a method depending upon a method of T. Maniatis, et al.
described in "Molecular Cloning" Cold Spring Harbor Laboratory,
1982. Though the culture temperature is not always the same
depending upon the conditons such as the host cells, a temperature
of about 25 to 40.degree. C. is suitable.
[0063] The human TPK-I produced by such host-vector systems can be
purified by a series of purifying steps corresponding to hosts and
culturing conditions such as extraction from the host, salting-out
and chromatography using various columns. In the case of column
chromatography, the suitably-used ones are phosphocellulose column
chromatography, hydroxyapatite column chromatography, S-Sepharose
column chromatography, heparin column chromatography, blue
Sepharose column chromatographny, etc.
[0064] The recombinant human TPK-I prepared as such is capable of
phosphorylating proteins such as tau-protein, glycogen-synthesizing
enzyme, protooncogene product c-jun, etc. and partial peptides
thereof and the progress of the phosphorylation can be confirmed
by, for example, the conditions as given below. Thus, the
recombinant human TPK-I of the present invention is added, together
with a suitable amount of the substrate protein, to a buffer of pH
5.0-8.0 containing 0.2-4.0 mM magnesium acetate and 0.2-4.0 mM
adenosine triphosphate, the mixture is incubated at the room
temperature to 40.degree. C. and the phosphorylation of the
substrate protein is checked and determined by radiochemical,
proteinochemical or immunochemical means. Consequently, when an
agent is added to this reaction system and the resulting promotion
or inhibition of the phosphorylation reaction is checked, it is
possible to find the agent having a physiologically important
meaning whereby the investigation on the agent which is effective
for the prevention or the therapy of human disease is now
possible.
EXAMPLES
[0065] The present invention will be illustrated by way of the
following examples though the present invention is not limited to
those examples so far as they are not out of the characteristic
feature of the present invention. Incidentally, judgement of the
cytotoxity, measurement of the phosphorylation of tau-proten and
immunohistochemistry by Alz-50 antibody were carried out in
accordance with the following methods. Further, in each of the
following examples, at least three independent experiments were
carried out and the data were given by their average values.
[0066] Judgement of Cytotoxity:
[0067] Numbers of many normal and healthy cells were counted by a
phase contrast microscope as an index of the living cells after the
treatment. Normal cells mean those which have morphologically flat
circumference and many nerve cell projections while the degenerated
cells were judged by checking the irregular shape, degeneration of
the neural projections, etc. Numbers of the living cells were
counted in a well. In the standard culture liquid, the cell numbers
were not less than 400 per well. The result was confirmed by an
immunohistochemical means.
[0068] Measurement of Degree of Phosphorylation of tau-Protein:
[0069] Hippocampus cells were collected from the culture medium by
washing with an ice-cooled phosphate buffer for three times. The
cells were suspended in a buffer A (pH: 6.8) which contained 1 mM
EGTA, 0.5 mM magnesium acetate and 20 mM
2-(N-morpholino)-ethanesulfonic acid containing a phosphatase
inhibitor (1 mM okadaic acid; manufactured by Seikagaku Kogyo) and
a protease inhibitor (1 mM phenylmethylsulfonyl fluoride and each 1
micro gram/ml of leupeptin, pepstatin and aprotinin), homogenized
and centrifuged at 14,000 rpm for one hour and the supernatant
liquid was used for checking the phosphorylation.
[0070] The rat tau-protein expressed in E. coli BL21 by a gene
recombination was purified by a method described in J. Biol. Chem.,
267, 10897-10901 (1992).
[0071] The hippocampus extract (1 microliter) was added to a
solution of the rat tau-protein (400 micrograms/ml) dissolved in a
buffer A containing 1 mM [.tau.-.sup.32P]ATP (10-20 Ci/mmole) and
then 10 micromoles of okadaic acid was added to make the final
volume 10 microliters. This was incubated at 37.degree. C. for
three hours and the reaction was stopped by adding a buffer for
electrophoresis. After subjecting to a 10% polyacrylamide gel
electrophoresis, the .sup.32p in the tau-protein was observed by a
laser image analyzer (Fuji BAS 2000).
[0072] Immunohistochemistry by an Alz-50 Antibody:
[0073] The cultured medium of the hippocampus cells was fixed in a
phosphate buffer for ten minutes using 4% paraformaldehyde. The
fixed culture liquid was incubated for 30 minutes in a Tris buffer
containing 0.2% Triton X-100 so that the cells were made
permeable.
[0074] Then this culture medium was subjected to an immunolabelling
using a 1:5 diluted Alz-50 mouse monoclonal antibody [Science, 232,
648-650(1986)], Vectastain ABC avidin-biotin-enzyme peroxide
detector kit (manufactured by Vector Laboratory) and
diaminobenzidine tetrahydrochloride as a dye.
Example 1.
[0075] Preparation of Culture Medium of Cells:
[0076] The primary culture medium of hippocampus of rats was
prepared in accordance with a method described in Brain Res., 126,
397-425 (1977). Thus, the hippocampus tissues were collected from
embryo of the rats of 18 days after fertilization and digested in
papain (protease) (10 U/ml) at 37.degree. C. for 20 minutes. The
resulting cells were added to a Dulbecco's modified Eagle's medium
supplied with 5% bovine fetus serum, 5% horse serum, 10
micrograms/ml insulin, 0.1 mg/ml transferrin, 1 microgram/ml
aprotinin, 1 mM sodium pyruvate and 84 micrograms/ml gentamycin.
This was planted to a well for tissue culture covered with
poly-L-lysine at the density of 2.times.10.sup.5 cells/cm.sup.2,
cultured for three days and treated with 1 micromole of
cytosine-beta-arabinofuran- oside for 24 hours and the cells of the
fifth day of the culture were used.
[0077] Preparation of A.beta.P:
[0078] A.beta.P peptide (Seq. ID No. 3 of the Sequence Listing)
comprising the already-mentioned 43 amino acid residues was
synthesized by a method which was described in Science, 250,
279-282(1990) and Proc. Natl. Acad. Sci. USA, 87, 9020-9023(1990)
and, after being purified, it was dissolved in 35% acetonitrile to
prepare a stock solution of 2M.
[0079] Preparation of TPKI-Sense and TPKI-Antisense:
[0080] Rat GSK-3.beta. [EMBO J., 9, 2431-2438(1990)], i.e. the
TPKI-sense comprising the followng 18 bases corresponding to the
translation initiating domain of the primary structure of rat TPK-I
(FEBS Lett., 325, 167-172 (1993)) and the TPKI-antisense which is
complementary thereto were synthesized using an automatic DNA
synthesizer (MilliGen), recovered from 20% acrylamide-urea gel and
purified by means of an ethanol precipitating method and the
precipitate was dissolved in water to adjust to a concentration of
1 micromole.
[0081] TPKI-Sense: 5'-ATGTCGGGGCGACCGAGA-3' (Seq. ID No. 4 of the
Sequence Listing) TPKI-Antisense: 5'-TCTCGGTCGCCCCGACAT-3' (Seq. ID
No. 5 of the Sequence Listing)
[0082] Inhibiting Action for the Death of Cranial Nerve Cells:
[0083] The culture medium of the hippocampus prepared by the
above-mentioned method was subjected to the following treatments
(b) to (d), numbers of the living cells with an elapse of time were
counted and the result is given in Table
[0084] (a) Nontreated culture medium (control):
[0085] (b) TPKI-antisense (1 micromole) was added to 1 ml of the
cell culture medium and, after five minutes, 20 micromoles of
A.beta.P was added followed by keeping at 37.degree. C. for 24
hours.
[0086] (c) A.beta.P (20 micromoles) was added to 1 ml of the cell
cuture medium followed by keeping at 37.degree. C. for 24
hours.
[0087] (d) TPKI-sense (1 micromole) was added to 1 ml of the cell
culture medium and, after five minutes, 20 micromoles of A.mu.P was
added followed by keeping at 37.degree. C. for 24 hours.
1TABLE 1 Numbers of Living Cells (%) After Treating Agents 6 hours
21 hours (Control) 100 100 A.beta.P + TPKI-Antisense 83.0 72.6
A.beta.P 41.3 25.4 A.beta.P + TPKI-Sense 49.5 17.1
[0088] Table 1 shows the numbers of the living cells with an elapse
of time after the above-mentioned tretments (b), (c) and (d) and
the numbers are given in terms of percentages to the control.
[0089] As shown in Table 1, the numbers of the living cells after 6
and 21 hours of the treatment of the hippocampus cells with
TPKI-antisense and A.beta.B (b) were significantly more than those
of the case treated only with A.beta.B (c) and of the case treated
with TPKI-sense and A.beta.P (d). This fact clearly shows that the
TPKI-antisense significantly inhibits the death of the cells by
A.beta.P.
[0090] Further, it was clarified by the observations of the
above-mentioned cases of (b) to (d) after 24 hours using a phase
contrast microscope (magnifying power: 400) that, only in the case
of (b) where TPKI-antisense and A.beta.P were acted to the
hippocampus cells, the cytotoxity by A.beta.P was little and
similar to the case of the control.
[0091] Phosphorylation of tau-Protein:
[0092] Phosphorylating activity of the TPK-I was measured by the
above-mentioned method for the samples of (1) untreated cell
culture medium (control); (2) a sample in which 1 micromole of
TPKI-antisense was added to 1 ml of the cell culture medium
followed by adding 20 micromoles of A.beta.P after 5 hours; and (3)
a sample in which 20 micromoles of A.beta.P was added to 1 ml of
the cell culture medium and the result is given in Table 2. The
phosphorylating activity of TPK-I in Table 2 shows that (units/mg
protein) per mg of the protein in the supernatant liquid wherein
one unit is equivalent to the intensity of the radioactivity
measured by a laser image analyzer (BAS 2000; Fuji).
2 TABLE 2 Phosphorylating Activity of TPKI Treating Agent (unit/mg
protein) (Control) 39.6 A.beta.P + TPKI-Antisense 31.6 A.beta.P
66.2
[0093] As shown in Table 2, the phosphorylating activity of the
case (2) in which TPKI-antisense and A.beta.P were acted on the
cell culture medium was only about one-half of that of the case (2)
in which only A.beta.P was acted. Thus, it is clear that the
TPKI-antisense significantly inhibits the phosphorylating activity
of TPK-I by A.beta.P.
Example 2
[0094] Cloning of Human TPK-IcDNA.
[0095] Commercially-available human fetus brain cDNA library
(prepared by inserting a 1:1 mixture of cDNA synthesized from mRNA
of human fetus brain using oligo dT and ramdom primer to
lamda-ZAPII; manufactured by Strategen) was infected to a host
which was E. coli XL1-blue [W. O. Bullock, et al: Biotechnique, 5,
376-379(1987)] to form plaques. The plaques (numbers: 450,000) were
screened using a probe which was prepared by a part of the
translation domain (170 base pair from the 1137th HindIII site to
the 1306th A; Seq. ID No. 8 of the Sequence Listing) of the rat
TPK-IcDNA (Seq. ID No. 2 of the Sequence Listing) whereby 19
positive clones were obtained. Among those, two clones were
subcloned to Bluescript SK (Strategen) and then EcoRI fragments
which hybridize with the above probe were subcloned to vector pUC19
[C. Yanisch-Perrou, et al: Gene, 33, 103 (1985)]. The restriction
enzyme map of the EcoRI fragments of the two clones prepared as
such is given in FIG. 1.
[0096] Base sequence was determined for entire domains of the clone
#1 by a dideoxy method and it lacked the N-terminal moiety of human
TPK-I protein. Total length of clone #2 was 2.2 kilobases and, out
of a comparison with the restriction enzyme map, it was presumed to
probably contain clone #1. Therefore, the base sequences
corresponding to 5'-untranslated domain and N-terminal domain
mostly comprising human TPK-I protein were determined by a dideoxy
method of Sanger, et al. The base sequence of cDNA out of the both
results and the amino acid sequence of TPK-I supposed therefrom are
given in Seq. ID No. 1 of the Sequence Listing.
[0097] Further, comparison with the amino acid sequence of the rat
TPK-I is given in FIG. 2.
Example 3
[0098] Expression of Human TPK-I by Insect Cells.
[0099] NruI-EcoRI fragments containing entire length of translation
domain of human TPK-IcDNA were inserted to a SmaI-EcoRI part of
transfer vector PVL1392 [Invitrogen; N. R. Webb and M. D. Summers:
Technique, 173-188(1990)] prepared by insertion of virus-originated
DNA fragments containing baculovirus (nuclear polyhedrosis virus)
polyhedron gene and promoter thereof into vector PUC8 (E.
coli-hosted plasmid vector) whereupon an expression vector PVL-TPKI
was prepared.
[0100] Cell strain Sf9 originated from ovalium cells of Spodoptera
spp. was cultured in a medium for insect cells FNM-FH [This was
prepared as follows; thus, 0.35 mg/lit of sodium bicarbonate
(manufactured by Wako Pure Chemical), 3.3 mg/ml of TC lactalbumin
hydrolysate (manufactured by Difco) and 3.3 mg/ml of TC yeast late
were added to a Grace's insect medium (Sigma), adjusted to pH 6.2,
sterilized and then a heat-processed 10t bovine fetus serum, 50
micrograms/ml of gentamycin sulfate and 2.5 micrograms/ml of
amphotericin B were added thereto.] and co-infected with wild
baculovirus DNA and vector DNA whereupon a homogeneous
recombination between them took place to some extent and, as a
result, recombinant virus having a TPK-I expressing system was
prepared.
[0101] Selection of the wild virus-infected cells and the
recombinant virus-infected cells was conducted visually and, as a
result of repeated selections for three times, the recombinant
virus-infected cells were separated. The virus-containing liquid
with high infectivity obtained from the supernatant of the cells
was further infected to Sf9 cells and cultured for 72 hours to
recover 5.times.108 cells. They were suspended in 30 ml of a buffer
A [comprising 10 mM sodium phosphate (pH: 7.05), 1 mM
ethylenediaminetetraacetic acid, 5 mM ethyleneglycol bis
(2-amino-ethyl ether) tetraacetate, 2 mM dithiothreitol, 10 mM
magnesium chloride, 0.1 mM sodium orthovanadate, 40 micrograms/ml
phenyl methanesulfonyl fluoride, 1 microgram/ml leupeptin, 1
microgram/ml pepstatin and 1 microgram/ml antipain], homogenized
and centrifuged at 105 G to recover the supernatant liquid. Then
the supernatant was subjected to a phosphocellulose column
chromatography (filled with P-11; Whatman) and fractionated with a
buffer B [comprising 25 mM tris(hydroxymethyl)aminomethane
hydrochloride (pH: 7.5), 1 mM ethylenediaminetetraacetic acid, lmM
dithiothreitol, 0.1% beta-mercaptoethanol, 5 glycerol and 50 mM
sodium chloride] with a gradient of sodium chloride concentrations
of 50 mM to 250 mM. Each fraction of the eluate was analyzed by
means of an electrophoresis and an immunoblot technique and an
anti-TPK-I antibody positive fraction which cross-reacts with an
anti-rat TPK-I amino terminal antibody [rabbit antiserum obtained
as an antigen by expressing rat TPK-I amino terminal 36 residues
(Seq. ID No. 9 of the Sequence Listing) as an E. coli-hosted vector
system as its fused protein with beta-galactosidase] was pooled.
This was concentrated by means of an ultrafiltration, subjected to
a blue sepharose column chromatography (filled with Blue-Sepharose
CL-6B of Farmacia) and fractionated by a buffer C [comprising 20 mM
N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (pH: 7.5), 1
mM ethylenediaminetetraacetic acid, 0.1 mM dithiothreitol and 5%
glycerol] with a sodium chloride concentration gradient of 0 to 1M.
Anti-TPK-I antibody positive fraction was pooled by analysis of
immunoblotting and electrophoresis for each fraction, subjected to
an ultrafiltration and dialyzed against a buffer D [comprising 100
mM 2-(N-morpholino)-ethanesul- fonic acid (pH: 6.5), 0.5 mM
magnesium acetate, 1 mM ethyleneglycol bis(2-aminoethyl ether)
tetraacetate, 10% glycerol, 0.02% polyoxyethylenesorbitan
monolaurate (Tween 20), 0.1 mM phenylmethanesulfonyl fluoride, 1
microgram/ml pepstatin, 1 microgram/ml antipain, 1 microgram/ml
leupeptin and 5 mM beta-mercaptoethanol] to give 1 ml of enzyme
liquid. Total protein obtained was 0.4 mg.
[0102] Progress of phosphorylation was checked using this enzyme
solution by the following two methods.
[0103] (Phosphorylating Method I)
[0104] tau-Protein extracted from bovine brain followed by
purification (2 microliters; 1.5 mg/ml concentration) and 1
microliters of the above partially-purified enzyme solution were
mixed. To the mixture was added a solution containing 2 mM
adenosinetriphosphate and 2 mM magnesium acetate and
[gamma-32P]adenosinetriphosphate so that the phosphorylation of
tau-protein was conducted at room temperature for 20 hours whereby
the amount of phosphoric acid incorporated in tau-protein was
evaluated.
[0105] (Phosphorylating Method II)
[0106] Phosphorylation reaction which was the same as in the method
I was conducted with an exception that no
[gamma-32P]adenosinetriphosphate was contained followed by
subjecting to an SDS electrophoresis to blot to nitrocellulose. The
blotted tau-protein was subjected to an immunodyeing with anti-tau
antibody (rabbit antiserum to chicken fetus brain-originated
tau-protein) and anti-p-tau antibody [Ihara, et al: J. Biochem.,
99, 1807-1910(1986)].
[0107] As a result of the method I, incorporation of tau-protein
into phosphoric acid was confirmed while the result of the method
II was that:
[0108] 1) mobility of tau-protein after the reaction was less than
that of tau-protein which was not phosphorylated; and
[0109] 2) tau-protein which was not phosphorylated did not react
with anti-p-tau antibody while tau-protein after the reaction
reacted with anti-p-tau antibody.
[0110] Those results indicate that the outcome was the same as that
in the phosphorylation of tau-protein using the TPK-I purified from
animal brain.
Example 4
[0111] Phosphorylation of Peptide by Recombinant Human TPK-I.
[0112] Peptide (hereinafter, abbreviated as "K2") represented by
the amino acid sequence described in the Seq. ID No. 10 of the
Sequence Listing was synthesized. This peptide was phosphorylated
by the same manner as in the phosphorylating method II in Example 3
with an exception that tau-protein kinase II (TPK-II) purified from
bovine brain microtubule was used instead of TPK-I whereupon
phosphorylated peptide (hereinafter, abbreviated as "p-K2") was
obtained.
[0113] Phosphorylations of K2 and p-K2 were conducted according to
a phosphorylating method I of Example 3 using human-originated
partially purified TPK-I prepared in Example 3 whereupon the
progress of phosphorylation of p-K2 was clearly noted while
phosphorylation of K2 was slow and its initial speed was about
one-tenth of that of p-K2.
[0114] The result shows that the outcome was the same as the
phosphorylation of K2 and p-K2 using TPK-I purified from animal
brain.
Example 5
[0115] Expression of Recombination of Human TPK-I by Escherichia
coli.
[0116] SacI-EcoRI fragment of human TPK-IcDNA clone #2 obtained in
Example 2 was introduced into an SacI-EcoRI part of vector PUC19
[C. Yanisch-Perrou, et al: Gene, 33, 103 (1985)] to prepare pUSE2.
In the meanwhile, in order to prepare an NdeI part in an
oligonucleotide from 598th to 629th members of cDNA represented by
the base sequence described in the Seq. ID No. 1 of the Sequence
Listing, a plus strand oligonucleotide (Seq. ID No. 11 of the
Sequence Listing) wherein CAT was inserted between 613th and 615th
member and a minus strand oligonucleotide (Seq. ID No. 12 of the
Sequence Listing) from 1076th to 1047th members were syn thesized
and a cDNA fragment (Seq. ID No. 13 of the Sequence Listing)
ranging from 598th to 1076th members and having NdeI part
duplicating with the initiation codon was obtained by a PCR method
[Saiki, et al: Nature, 324, 126 (1986)].
[0117] Fragments ranging from a 5'-terminal of the cDNA fragment
obtained by a PCR method to a SacI part were inserted to SmaI-SacI
part of pUSE2. The NdeI-EcoRI fragment of the plasmid vector was
introduced into NdeI-BamHI part of pET3C [A. H. Rosen berg, et al:
Gene, 56, 125(1987)] which is one of the vectors having E. coli T7
polymerase promoter to construct pET3C/TPKI.
[0118] pET3C/TPKI was transformed by conventional means using E.
coli BL21 (DE3) [F. W. Studier and B. A. Moffatt: J. Mol. Biol.
189, 113 (1986)] as a host to prepare a recombinant. The resulting
recombinant E. coli was cultured at 37.degree. C. until the middle
stage of the logarithmic growth phase, kept at 21.degree. C., 0.3
mM (final concentration) of IPTG
(isopropyl-beta-D(-)-thiogalactopyranoside) was added and cultured
for four hours more. The living cells (5 g) were suspended in 50 ml
of a buffer E [comprising 20 mM of 2-(N-morpholino)-ethanesulfonic
acid (pH: 6.5), 1 mM of ethylenediaminetetraacetic acid, SmM of
beta-mercaptoethanol and 50 mM of sodium chloride], disintegrated
with ultrasonic wave and centrifuged at 100,000 g for one hour. The
supernatant was subjected to a phosphocellulose column
chromatography (filled with P-11; Whatman) which was balanced with
a buffer E and then subjected to a gradient elution with sodium
chloride concentrations of 50 to 500 mM whereby the fraction which
was positive to anti-TPK-I antibody was pooled and concentrated.
This was dialyzed against a buffer F [comprising 20 mM of
N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (pH: 7.2), 1
mM of ethylene-diaminetetraacetic acid and 5 mM of
beta-mercaptoethanol], subjected to a blue sepharose column
chromatography (filled with Blue-Sepharose CL-6B of Farmacia) and
eluted with a concentration gradient of 0 to 1M of sodium chloride.
Anti-TPK-I antibody positive fractions were collected and dialyzed
against a buffer D.
[0119] Phosphorylation of tau-protein was conducted by the
phosphorylating methods I and II by the same manner as in Example 3
using the resulting partially-purified TPK-I. It was found that, as
a result of the phosphorylating method I, 1.2 moles of phosphoric
acid was incorporated into one molecule of tau-protein while, as a
result of the phosphorylating method II, the mobility of
electrophoresis of tau-protein after the reaction became small and
the reaction with anti-ptau antibody became positive.
[0120] When the partially-purified TPK-I was used for
phosphorylation of the peptides K2 and p-K2 by the same manner as
in Example 4, the phosphorylation of p-Ka proceeded while that of
K2 hardly proceeded.
[0121] Those results show that the recombinant TPK-I prepared in
this example had the same property as that of TPK-I purified from
animal brain and of recombinant TPK-I prepared in Example 3.
[0122] (Merit of the Invention)
[0123] In accordance with the preventive and the therapeutic agent
of Arzheimer's disease of the present invention, the
phosphorylating activation of tau-protein kinase I by amyloid
beta-protein was inhibited whereby the death of the neuron in the
brain can be inhibited. Further, it is possible to conduct a
screening of the preventive or the therapeutic agent of Arzheimer's
disease utilizing the above mechanism.
[0124] Moreover, the human-originated TPK-I of the present
invention is an enzyme which specifically acts to tau-protein which
is suggested to be related to Arzheimer's disease and also to
senile dementia of Arzheimer's disease type and, therefore, its
application to clarification of cause of those diseases and to the
investigations for the agents for the prevention and the therapy
thereof can be expected.
BRIEF EXPLANATION OF THE DRAWINGS
[0125] FIG. 1 is a drawing which shows the restriction enzyme map
of the human TPK-I.
[0126] FIG. 2 is a drawing which shows the comparison of amino acid
sequences of human TPK-I and rat TPK-I. In the drawing, each amino
acid is represented by a single letter.
Sequence CWU 1
1
* * * * *