Title: Ti-3 protein derived from triatoma infestans exhibiting activity to inhibit platelet aggregation
Abstract: The present invention provides Ti-3 protein, which is a protein obtained from salivary gland of Triatoma infestans, and the present invention provides Ti-3 gene encoding the protein. As the Ti-3 protein exhibits inhibitory activity on platelet aggregation, a medicine comprising the Ti-3 protein as an active ingredient will serve as a platelet aggregation inhibitor possibly effective for prevention and treatment of myocardial infarction, pulmonary infarction and cerebral infarction. Moreover, the Ti-3 protein will also serve as a highly prospective lead compound in the development of novel platelet aggregation inhibitors.
Patent Number: 6,956,106 Issued on 10/18/2005 to Morita, et al.
| Inventors:
|
Morita; Akihiro (Tsu, JP);
Isawa; Haruhiko (Tsu, JP);
Yuda; Masao (Tsu, JP);
Orito; Yuki (Suzuka, JP);
Chinzei; Yasuo (Tsu, JP)
|
| Assignee:
|
Mie University (Tsu City, JP)
|
| Appl. No.:
|
401038 |
| Filed:
|
March 28, 2003 |
Foreign Application Priority Data
| Oct 02, 2002[JP] | 2002-289683 |
| Current U.S. Class: |
530/350 |
| Intern'l Class: |
C07K 014/70.5 |
| Field of Search: |
530/350
435/71
514/12
|
References Cited [Referenced By]
| Foreign Patent Documents |
| 7-506959 | Aug., 1995 | JP.
| |
| 93/05150 | Mar., 1993 | WO.
| |
Primary Examiner: Weber; Jon
Assistant Examiner: Mondesi; Robert B.
Attorney, Agent or Firm: Burns, Doane, Swecker & Mathis, LLP
Claims
1. An isolated and purified protein derived from
Triatoma infestans consisting
of the amino acid sequence of following (a), or (b)
(a) an amino acid sequence represented by amino acids Nos. -(18)-164 of SEQ ID
NO:1;
(b) an amino acid sequence exhibiting 95% homology or more with amino acid sequence
(a), wherein amino acid sequence (b) exhibits activity to inhibit platelet aggregation.
2. An isolated protein derived from
Triatoma infestans consisting of the
amino acid sequence of following (a), or (b):
(a) an amino acid sequence represented by amino acids Nos. 1-164 of SEQ ID NO:1;
(b) an amino acid sequence exhibiting 95% homology or more with amino acid sequence
(a), wherein amino acid sequence (b) exhibits activity to inhibit platelet aggregation.
3. A platelet aggregation inhibitor containing the protein according to claim
2 as an active ingredient.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to Ti-3 protein, which is a protein derived from
salivary gland of
Triatoma infestans (an assassin bug) exhibiting activity
to inhibit platelet aggregation, and to a gene encoding the Ti-3 protein.
2. Description of the Related Art
With progression of aging of society, treatment of adult diseases as a social
problem is becoming more important. What is the most important for the welfare
of such aging society is treatment and prevention of symptoms related to adult
diseases, particularly cardiovascular disorders resulting from vascular sclerosis
such as hypertension, pulmonary hypertension, myocardial infarction, cerebral infarction,
pulmonary infarction and vascular spasms followed by subarachnoid hemorrhage. Such
vascular disorders can be prevented and treated by administration of vasodepressors,
blood coagulation inhibitors and platelet aggregation inhibitors to the patients.
The known peptide having an anticoagulant activity which may be used as a medicine
for treating such vascular disorders includes hirudine, a peptide isolated from
salivary gland of leech. Hirudine is an anticoagulant peptide isolated from salivary
gland of assassin bugs and it exhibits anti-thrombic activity. Ti-3 protein of
this invention and a gene encoding the protein are novel. However, general knowledge
on physiologically active substances isolated from salivary glands of assassin
bugs are described as reviews in the following scientific journals:
(1) Ribeiro, J. M., "Role of saliva in blood-feeding by arthropods", Annu.
Rev. Entomol., 1987, vol.32, p463-478
(2) Basanova, A. V., Baskova, I. P., and Zavalova, L. L., "Vascular-platelet
and plasma hemostatis regulators from bloodsucking animals" Biochemistry, 2002,
vol67, p143-150
However, hirudine has some problems when applied as a medicine, those are,
synthesis of hirudine is difficult, and it has some adverse side-effects. To achieve
large amount of production of the compound to be used as a medicine in safely,
it is necessary to solve the above problems. Thus, isolation of a protein whose
synthesis is easy and capable of inhibiting the aggregation of platelets without
causing any notable side-effects has been needed. If such a protein were produced,
it would be a useful lead compound in the production of an anticoagulant, and be
highly promising in the development of new medicines.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a protein exhibiting activity
to inhibit platelet aggregation isolated from salivary gland of
Triatoma infestans,
a blood-sucking insect. Another object of the present invention is to provide a
method to achieve production of the protein in large amount, utilizing the system
of baculovirus.
To achieve the above objects, the present application provides following inventions.
This invention provides Ti-3 protein derived from
Triatoma infestans, consisting
of an amino acid sequence represented by amino acids Nos. (-18)-164 shown in SEQ
ID No. 1 of the sequence listing. Other proteins consisting of an amino acid sequence
in which a part of above-mentioned amino acid sequence is deleted, substituted,
or another amino acid sequence is added to the above-mentioned amino acid sequence
are also included in this invention, so far as exhibiting the activity to inhibit
platelet aggregation.
This invention further provides Ti-3 protein derived from
Triatoma infestans,
consisting of an amino acid sequence represented by amino acids Nos. 1-164 shown
in the SEQ ID No. 1 of the sequence listing. Other proteins consisting of an amino
acid sequence in which a part of above-mentioned amino acid sequence is deleted,
substituted, or another amino acid sequence is added to the above-mentioned amino
acid sequence are also included in this invention, so far as the exhibiting the
activity to inhibit platelet aggregation.
This invention further provides Ti-3 gene derived from
Triatoma infestans,
consisting of a base sequence represented by bases Nos. 1-695 shown in the SEQ
ID No. 2 of the sequence listing. Other genes consisting of a base sequence in
which a part of above-mentioned base sequence is deleted, substituted, or another
base sequence is added to the above-mentioned base sequence are also included in
this invention, so far as encoding proteins exhibiting the activity to inhibit
platelet aggregation.
Furthermore, this invention provides a platelet aggregation inhibitor
containing the above-mentioned protein as an effective ingredient.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a figure showing the amino acid sequence of the Ti-3 protein, and
the base sequence of the gene encoding the protein.
FIG. 2 is a graph illustrating the effect of Ti-3 protein on collagen-induced
platelet aggregation.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The salivary glands derived from blood-sucking insects and mites contain substances
having specific activities toward blood or blood vessels. Therefore, the present
inventors identified an active substance having inhibitory effect to platelet aggregation,
which is derived from salivary gland of such an insect. Then the inventors achieved
isolation and purification of the substance, and further analyzed on the properties
of the active substance. In a subsequent study, the inventors achieved cDNA cloning
of the gene. Moreover, the inventors developed a method for production of the protein
having the activity to inhibit platelet coagulation in a large scale, utilizing
the system of baculovirus.
In concrete, the inventors paid attention to
Triatoma infestans (Ti),
which
is a blood-sucking insect, and tried to isolate a protein exhibiting inhibitory
activity on platelet aggregation derived from the insect. Salivary glands were
removed from approximately 40 individuals of
Triatoma infestans, total mRNA
was extracted from the salivary glands, dsDNA was synthesized using poly(A)
+
mRNA as a template by reverse transcriptase, then cDNA library of the salivary
gland was constructed by inserting the dsDNA into a transfer vector. From cDNA
library of the Ti salivary gland, colonies were picked up at random and their base
sequences were determined. The inventors sequenced on 550 colonies, they picked
up cDNAs containing secretion signal and excluded overlapping cDNAs. As a consequence
44 cDNAs were obtained and total base sequences of them were determined. FIG. 1
shows base sequence of the Ti-3 gene thus obtained and amino acid sequence of the
Ti-3 protein encoded by the gene.
Out of them, transfer vector constructs were prepared on 16 cDNAs to achieve
protein expression in an expression system using baculovirus (AcNPV). The viruses
were transfected with the construct, and viral clones failing to form apocytes,
namely clones expressing proteins encoded by the inserts, were isolated. Expression
of the protein was confirmed by SDS-PAGE, and the protein was isolated and purified
by HPLC based on gel filtration chromatography and ion exchange chromatography,
then Ti-3 protein according to this invention was obtained. Then, investigation
of the protein according to this invention thus obtained was performed on its effect
toward platelet aggregation.
The substance of the target was added to washed human platelets, and collagen
was added to induce platelet aggregation. Ten minutes later, the light transmission
of the sample was measured, and then inhibitory effect of the substance on the
platelet aggregation was investigated. As a consequence, the Ti-3 protein inhibited
collagen-induced platelet aggregation in dose-dependent manner. From this result,
it was concluded that the Ti-3 protein according to this invention exhibited the
effect to inhibit platelets aggregation, that is, the protein would be effective
as a platelet aggregation inhibitor. Therefore, the Ti-3 protein will be effective
as an active ingredient in medicines for treatment or prevention of myocardial
infarction, pulmonary infarction and cerebral infarction.
The Ti-3 protein is defined by the amino acid sequence consisting of amino acids
Nos. (-18)-164 shown in the SEQ ID No. 1 of the sequence listing. In this specification,
the protein consisting of an amino acid sequence in which a part of said amino
acid sequence represented SEQ ID NO: 1 in the sequence listing is deleted, substituted
or added with another amino acid sequence means a protein consisting of an amino
acid sequence in which 20 or less, preferably 10 or less, and more preferably 5
or less amino acids of the sequence is deleted, substituted or added to the amino
acid sequence represented by SEQ ID NO: 1 in the sequence listing. Moreover, such
protein exhibits homology 95% or more, preferably 97% or more and still preferably
99% or more with the amino acid sequence represented by SEQ ID NO: 1 in the sequence
listing. Such polypeptide is also within the range of this invention so far as
it exhibits function as Ti-3 protein of inhibiting platelet aggregation. Meanwhile,
in the SEQ ID NO: 1 in the sequence listing, the region corresponding to amino
acids Nos. -18 to -1 represent a signal peptide. The peptide received processing
to yield a mature protein consisting of 164 represented by amino acids Nos. 1-164.
Moreover, the Ti-3 gene codes for the Ti-3 protein described above, and
it consists of the base sequence represented by bases Nos. 1-695 shown in the SEQ
ID No. 2 of the sequence listing. The region corresponding to bases Nos. 21-566
represents an open reading frame, and encodes the above protein. According to technique
of gene recombination, artificial modification can be achieved at a specific site
of basic DNA, without alteration or with improvement of basic characteristic of
said DNA. Concerning a gene having native sequence provided according to this invention
or modified sequence different from said native sequence, it is also possible to
perform artificial modification such as insertion, deletion or substitution to
obtain gene of equivalent or improved characteristic compared with said native
gene. Moreover, a gene with such mutation is also included in the range of this invention.
That is, the gene consisting of a base sequence in which a part of said gene
represented by base sequence represented by SEQ ID NO: 2 in the sequence listing
is deleted, substituted or added with another base sequence means a gene consisting
of a base sequence in which 20 or less, preferably 10 or less, and more preferably
5 or less bases of the sequence is deleted, substituted or added to the base sequence
represented by SEQ ID NO: 2 in the sequence listing. Moreover, such gene exhibits
homology 95% or more, preferably 97% or more and still preferably 99% or more with
the base sequence represented by SEQ ID NO: 2 in the sequence listing. Such gene
is also within the range of this invention so far as it encodes a protein exhibiting
function as Ti-3 protein of inhibiting platelets aggregation. In addition, such
gene hybridizes with the base sequence represented by SEQ ID No.2 in the sequence
listing under a stringent condition.
The condition for hybridization can be selected by a skilled artisan ad libitum.
For example, hybridization can be performed by the following procedure. DNA molecules
or RNA molecules to be tested are transferred onto a membrane, then the membrane
is hybridized with a labeled probe in a proper hybridization buffer. The hybridization
buffer may comprise, for example, 5×SSC, 0.1 (weight) % N-lauroylsarcosine,
0.02 (weight) % SDS, 2 (weight) % of blocking reagent for nucleic acid hybridization,
and 50% formamide. The blocking reagent for nucleic acid hybridization may comprise,
for example, a buffer (pH 7.5) containing 0.1M maleic acid and 0.15M sodium chloride
and commercially available blocking reagent for hybridization dissolved into the
buffer at the concentration of 10%. The 20×SSC solution may comprise 3M sodium
chrolide and 0.3M citrate, and the SSC solution may be preferably utilized at the
concentration of 3 to 6×SSC, more preferably at the concentration of 4 to 5×SSC.
The temperature for hybridization may preferably be 40 to 80° C., more preferably
be 50 to 70° C., further more preferably be 55 to 65° C. Incubation may
be performed from several hours to overnight, then washed by a washing buffer.
The temperature for washing may preferably be room temperature, more preferably
it may be the temperature used for hybridization. The formulation for the washing
buffer may preferably comprise 6×SSC and 0.1% (weight %) SDS, more preferably
may comprise 4×SSC and 0.1% (weight %) SDS, further preferably may comprise
2×SSC and 0.1% (weight %) SDS, more further preferably may comprise 1×SSC
and 0.1% (weight %) SDS, most preferably may comprise 0.1×SSC and 0.1% (weight
%) SDS. The membrane may be washed by such washing buffer, then DNA molecule or
RNA molecule may be distinguished by the hybridization with the labeled probe.
It is possible to produce Ti-3 protein according to this invention in a large
scale, utilizing baculovirus expression system in which cDNA encoding the Ti-3
protein is inserted. An exemplary production method will be mentioned below. The
Ti-3 protein is allowed to express in BmN4 culture cells (silkworm cell) or in
silkworm larvae, utilizing
Bombyx mori nuclear polyhedral virus (BmNPV).
The extract derived from the culture medium or from the body fluid of the silkworm
larvae is subjected to chromatography to isolate the target protein. Alternatively,
cDNA encoding the Ti-3 protein can be inserted into
Autographa californica nuclear
polyhedral virus (AcNPV), and the Ti-3 protein is allowed to express in Sf9 cells
derived from
Spodoptera frugiperda or in Tn5 cells derived from
Trichoplusia
ni. Supernatant of the culture medium is subjected to chromatography in the
same manner to isolate the target protein.
The Ti-3 protein according to this invention can be produced in a large scale,
utilizing an
E. coli expression system in which cDNA encoding the Ti-3 protein
is inserted. For such a purpose, cDNA encoding the Ti-3 protein can be amplified;
and the cDNA can be inserted into a plasmid such as pMAL-c2g to construct a vector
for expression of a fusion protein with maltose binding protein (MBP).
E. coli
is transformed with the expression vector, cultivated in a medium containing
IPTG to induce expression of the Ti-3 protein fused with MBP in the
E. coli.
The
E. coli strains preferred for such a purpose include BL21 strain, for
example. The MBP fused Ti-3 protein induced in
E. coli bodies can be recovered
by disrupting the cell bodies. The MBP fused Ti-3 protein thus recovered can be
purified by affinity chromatography utilizing an amylose resin.
The Ti-3 protein of this invention having inhibitory effect on platelet aggregation
can be produced in a large scale by; identification, isolation and purification
of the active substance exhibiting inhibitory effect on platelet aggregation derived
from salivary gland of a blood-sucking insect, cloning of the cDNA encoding the
active substance, and expressing the protein in a baculovirus expression system.
If the active site responsible for the inhibitory effect on platelet aggregation
will be determined on the protein and progression on structural analysis of the
protein will be achieved, the active substance will be produced through conventional
synthetic procedure using technique of molecular designing.
Moreover, the Ti-3 protein according to this invention will be quite valuable
as a prospective lead compound in the development of novel medicines having an
inhibitory effect on platelet aggregation. Furthermore, the structure of the Ti-3
protein can be modified in various manners to develop novel compounds exhibiting
higher inhibitory potency on platelet aggregation. Thus, the Ti-3 protein according
to this invention provides a physiologically active substance that becomes the
basis for such investigation. Furthermore, it is quite useful as a lead compound
useful for further development of novel platelet aggregation inhibitors.
EXAMPLES
The present invention will be illustrated below by means of examples. However,
the present invention is not limited in any way to those examples.
Procedure for Gene Isolation
Salivary glands were removed from thorax of
Triastoma infestans.
The mRNA was extracted from the salivary glands and purified using Microprep mRNA
purification kit (Amersham Pharmacia). Using the mRNA as a template, cDNA library
of the salivary glands was constructed using Superscript Plasmid System (Life Technology).
The clones (approximately 550 clones in total) were randomly picked up from this
library and plasmids were extracted and purified using QiAPrep Spin Miniprep kit
(Qiagen). The base sequence of the cDNA inserted in the plasmid was determined
with ABI PRISM 310 genetic analyzer (PE Biosystems). Then the inventors analyzed
on the base sequence of the cDNA thus determined, using a Genetyx ver. 8.5 (Software
Development). As a result, it was revealed that four cDNA clones were possesing
an identical base sequence, and it was designated to be Ti-3 gene.
Massive Expression of the Recombinant Protein and Purification of the Protein
The full length cDNA of Ti-3 gene was amplified by PCR and it was inserted into
BamHI restriction site of plasmid pAcYM1 to produce a transfer vector, and then
it was purified using Plasmid Mini kit (Qiagen). The plasmid vector was introduced
into insect culture cells (Sf-9) using Baculogold Linearized Baculovirus DNA (Phaemingen)
to produce recombinant baculoviruses. The recombinant viruses were allowed to infect
another insect culture cells (Tn-5) and massive expression of the recombinant Ti-3
was achieved. The recombinant Ti-3 protein was secreted into the culture medium
and the recombinant protein was purified via two steps of purification, that is,
cation ion exchange chromatography with MONO S (Amersham Pharmacia) and gel-filtration
chromatography with TSK2000SW (Toso). The purified product was used in the subsequent experiment.
Aggregation of Blood Platelets
Platelet aggregation was determined using washed human platelets by turbidimetric
assay method. In other word, 50 μl of washed human platelets (6.0×10
5/ml)
was added into 40 μl of solution comprising the purified Ti-3 protein dissolved
into a buffer solution containing 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl, and
the mixture was incubated at 37° C. After 10 minutes of incubation, 10 μl
of collagen (Chronolog) solution (20 μg/ml) was added to it, and the mixture
was further incubated for ten minutes. The light transmission of the solution was
measured at 600 nm using Micro Plate reader MPR-A4i (Toso).
FIG. 2 shows the effect of Ti-3 protein on collagen-induced platelet aggregation.
FIG. 2 shows that the Ti-3 protein inhibits platelet aggregation in a dose dependent
manner. This demonstrates that the Ti-3 protein isolated from salivary gland of
Triatoma infestans can inhibit collagen-induced platelet aggregation.
The present invention provides Ti-3 protein, a novel protein derived from
Triatoma
infestans, and a gene coding for the Ti-3 protein. The Ti-3 protein has an
inhibitory activity on platelet aggregation, therefore, a medicine comprising the
Ti-3 protein as an active ingredient will serve as a platelet aggregation inhibitor
possibly effective for prevention and treatment of myocardial infarction, pulmonary
infarction and cerebral infarction. Moreover, the Ti-3 protein will also serve
as a highly prospective lead compound in the development of new platelet aggregation inhibitors.
SEQUENCE LISTING
<100> GENERAL INFORMATION:
<160> NUMBER OF SEQ ID NOS: 2
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO: 1
<211> LENGTH: 182
<212> TYPE: PRT
<213> ORGANISM: Triatoma infestans
<220> FEATURE:
<221> NAME/KEY: SIGNAL
<222> LOCATION:(1)...(18)
<400> SEQUENCE: 1
Met Lys Met Ile Ile Ala Val Thr Phe Leu Gly Ile Val Thr Ile Ala
-15 -10 -5
Phe Ala Glu Glu Cys Arg Leu Met Gln Pro Ala Ala Asn Phe Asp Ala
1 5 10
Ala Thr Tyr Phe Ser Ile Pro His Val Tyr Val Thr His Ser Lys Asn
15 20 25 30
Glu Pro Lys Thr Asp Val Cys Arg Glu Tyr Asp Thr Ser Lys Thr Asp
35 40 45
Gly Gly Ser Thr Thr Val Ile Thr Ser Asn Tyr Lys Ile Lys Gly Gln
50 55 60
Ala Val Asn Asn Lys Val Thr Cys Thr Ser Thr Gly Leu Lys Asn Gly
65 70 75
Gln Thr Gly Gln Phe Ser Val Val Cys Gln Pro Pro Thr Gly Ala Ala
80 85 90
Val Thr Leu Thr Thr Ser Val Leu Ala Thr Asp Asn Gln Asn Tyr Ala
95 100 105 110
Ile Leu Gln Arg Cys Pro Thr Ser Gly Gln Gly Asn Ile Leu Val Leu
115 120 125
Gln Thr Ala Lys Glu Gly Val Asn Pro Gly Val Lys Asp Phe Phe Gln
130 135 140
Lys Lys Gly Trp Asn Ile Asp Ser Trp Phe Ser Arg Thr Asn Val Asn
145 150 155
Cys Glu Asn Ile Gln Ser
160
<200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO: 2
<211> LENGTH: 695
<212> TYPE: DNA
<213> ORGANISM: Triatoma infestans
<400> SEQUENCE: 2
gtaaatttca ctttgacaac atgaagatga tcattgcagt gacatttctt gggattgtga 60
cgatcgcatt tgctgaagaa tgccgactca tgcaacctgc ggcaaacttt gatgctgcaa 120
cttatttcag cattcctcat gtatatgtga ctcattcaaa gaatgaacca aaaacagatg 180
tatgtcgaga atatgatact tcaaaaactg atggtggcag cactacagta attacctcaa 240
attacaaaat caaaggacag gcagttaaca ataaagttac atgtactagt accgggctaa 300
aaaatgggca gacgggccaa ttttctgtag tttgccaacc accaactggc gccgctgtca 360
ctttaactac gtcagttctt gccacggata atcaaaacta tgctatactt caaagatgtc 420
ctacgagtgg acaaggcaat attttggtat tacaaacagc taaagaaggc gtaaatccag 480
gagttaaaga cttttttcaa aaaaaaggtt ggaacataga ctcatggttt tctaggacaa 540
atgttaattg tgaaaacatc cagagttaaa catgtaaaaa aaaaaaaaat aaataaataa 600
agatttatta ttttgaataa actgacatta aaacaaaaaa aaaaaaaaaa aaaaaaaaaa 660
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 695
*
