CA 02479908 2004-09-20
SPECIFICATION
Calcineurin activator
TECHNICAL FIELD
The present invention relates to the use of a killer protein (KLKP)
produced by killer yeast, Kluyveromyces lactis. The present invention
specifically relates to a calcineurin activator comprising a killer protein
(KLKP) produced by Kluyveromyces lactis killer yeast as an active ingredient
and having the action of increasing intracellular calcium ion concentration
through the influx of calcium ions into eukaryotic cells. Furthermore, the
present invention relates to an agent for inhibiting eukaryotic cell
proliferation and an agent for inhibiting the eukaryotic cell cycle,
comprising
the calcineurin activator.
BACKGROUND ART
Recently, various studies have been conducted on the yeast (S.
cerevisiae) killer system. A Kl killer protein activates TOKI excessively,
and destroys cell membrane functions (Ahmed, A. et al., Cell 99, 283-291
(1999)). Furthermore, destruction and delocalization of chitin synthase III
confer resistance against K. lactis killer protein (KLKP) on yeast
(Jablonowski, D., et al., Yeast 18 p. 1285 (2001 )). It has been reported that
KLKP is encoded by a gene on a pGKLI plasmid and consists of 3 subunits:
the a subunit, the (3 subunit, and the y subunit (Stark MJ et al., EMBO J.,
Aug,
5(8), p.1995- (1986)).
Furthermore, we have previously reported that the action of KLKP is
activated by Ca2+. However, the mechanism of the killer activity has not yet
been elucidated. Moreover, such yeast killer protein has been thought to
have killer activity only against yeast, and the industrial applications
thereof
1
CA 02479908 2004-09-20
have been limited to the field of fermentation and the like.
Calcineurin (CaN) is the only dephosphorylation enzyme of
eukaryotes, which is controlled by a Ca2+/calmodulin complex and plays a
central role in the signaling system mediated by Ca2+, such as concerning cell
proliferation, cell differentiation, and regulation mechanisms for
transcriptional control. The CaN action mechanism that has been elucidated
in yeast represents important information for analyzing CaN functions in
higher eukaryotes. A CaN inhibitor has been put to practical use as an
immunosuppressant. CaN possesses various functions and is expected to be
applied in medicine or agriculture. CaN functions that have already been
elucidated or industrially applied are shown in Table 1.
Table 1. CaN actions and industrial applications thereof
Action revealed usin east Action on hi her eukaryotes
Site of action of immunosuppresantImmunosuppression
=
CaN inhibition (practical use of immunosuppressant
--~ medical traps lantation)
Ion homeostasis* 1 CaN inhibition ~ Side effects
of
immunosuppressant
Renal disorder
Hyperlipidemia
Elevated blood ressure
Cell cycle suppression *2 CaN activation
Inositol phospholipid metabolismT cell activation
Cell wall synthesis Induction of cancer cell
differentiation
Apoptosis
Reconstruction of myofibrils
Plants
Tem erature~salt stress resistance
*Reported by collaborator (1. EMBO Journal, 12, p. 4063 (1993); 2. Nature,
392, p.303 (1998))
It has been reported that CaN activated with Caz+ using budding yeast
activates a negative regulation factor of the cell cycle engine, so as to
cause a
2
CA 02479908 2004-09-20
delay in the cell cycle (NATURE, 392, p.303 (1998)).
As described above, a substance having the action of activating or
inhibiting calcineurin has been expected to be industrially applied widely as
an agent for inhibiting eukaryotic cell proliferation, in medicines, and the
like. However, conventionally the number of easily available calcineurin
activators has been very small.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a calcineurin
activator comprising a killer protein (KLKP) produced by Kluyveromyces
lactis killer yeast as an active ingredient and having the action of
increasing
intracellular calcium ion concentration through the influx of calcium ions
into eukaryotic cells. Furthermore, another object of the present invention
is to provide an agent for inhibiting eukaryotic cell proliferation and an
agent
for inhibiting eukaryotic cell cycle, comprising the calcineurin activator. A
composition containing the calcineurin activator can be broadly applied as an
agent for inhibiting eukaryotic cell proliferation, an anticancer agent, and
the
like. Thus, another object of the present invention is to provide such an
agent for inhibiting eukaryotic cell proliferation, an anticancer agent, an
immunosuppressant, an immunostimulant, a therapeutic agent against
diseases relating to memory, and a therapeutic agent against cardiovascular
diseases.
We have discovered that a killer protein (KLKP) produced by
Kluyveromyces lactis killer yeast increases intracellular calcium ion
concentration through the influx of calcium (Ca2+) into budding yeast cells,
and then activates calcineurin (CaN) so as to induce a delay at the G2 phase
of the cell cycle. We have also discovered that KLKP induces suppressed
proliferation of plant culture cells, thereby completing the present
invention.
Specifically, the present invention relates to a calcineurin activator
3
CA 02479908 2004-09-20
comprising Kluyveromyces lactis killer protein (KLKP) as an active
ingredient and having the action of increasing intracellular calcium ion
concentration through the influx of calcium ions into eukaryotic cells.
Furthermore, the present invention relates to the above calcineurin
activator comprising one of the following proteins as an active ingredient and
having the action of increasing intracellular calcium ion concentration
through the influx of calcium ions into eukaryotic cells:
(a) a Kluyveromyces lactis killer protein (KLKP), consisting of 3 subunits: a
subunit consisting of an amino acid sequence represented by SEQ ID NO: 2, a
subunit consisting of an amino acid sequence represented by SEQ ID NO: 3,
and a subunit consisting of an amino acid sequence represented by SEQ ID
NO: 4; or
(b) a protein, being the same as protein (a) except for differing from protein
(a) in that at least one of the 3 subunits consists of an amino acid sequence
derived from the amino acid sequence represented by SEQ ID NO: 2, 3, or 4
by deletion, substitution, or addition of 1 or several amino acids, and having
Kluyveromyces lactis killer protein (KLKP) activity.
Furthermore, the present invention relates to an agent for inhibiting
eukaryotic cell proliferation comprising calcium ions and the above
calcineurin activator.
Furthermore, the present invention relates to an agent for inhibiting
eukaryotic cell cycle comprising calcium ions and the above calcineurin
activator.
Furthermore, the present invention relates to an agent for inhibiting
eukaryotic cell cycle comprising a prepared product comprising cheese whey
or cheese-whey-derived calcium at a high concentration and the above
calcineurin activator.
Furthermore, the present invention relates to a method for preventing
aerobic deterioration of silage, comprising a step of simultaneously or
4
CA 02479908 2004-09-20
separately adding calcium ions and the above calcineurin activator to silage.
Furthermore, the present invention relates to the above method,
wherein calcium ions are added by adding a prepared product comprising
cheese whey or cheese-whey-derived calcium at a high concentration.
Furthermore, the present invention relates to a pharmaceutical
composition comprising the above calcineurin activator, which is selected
from the group consisting of an anticancer agent, an immunosuppressant, an
immunostimulant, a therapeutic agent against disorders relating to memory,
and a therapeutic agent against cardiovascular diseases.
Furthermore, the present invention relates to a method for screening
for a compound involved in calcineurin inhibition, comprising causing a
calcineurin activator comprising Kluyveromyces lactis killer protein (KLKP)
as an active ingredient and having the action of increasing intracellular
calcium ion concentration through the influx of calcium ions into eukaryotic
cells and a test substance to act on a KLKP-sensitive yeast strain, and using
the ability of the test substance to cancel the sensitivity of the
KLKP-sensitive cell as an index.
Furthermore, the present invention relates to a method for screening
for a remedy against diseases in which calcineurin is involved, comprising
causing a calcineurin activator comprising Kluyveromyces lactis killer protein
(KLKP) as an active ingredient and having the action of increasing
intracellular calcium ion concentration through the influx of calcium ions
into eukaryotic cells and a test substance to act on a KLKP-sensitive yeast
strain, and using the ability of the test substance to cancel the sensitivity
of
the KLKP-sensitive yeast as an index.
Furthermore, the present invention relates to the above method,
wherein the remedy against diseases in which calcineurin is involved is
selected from the group consisting of an anti-cancer agent, an
immunosuppressant, an immunostimulant, a therapeutic agent against
CA 02479908 2004-09-20
disorders relating to memory, and a therapeutic agent against cardiovascular
diseases.
The present invention is explained in detail as follows.
1. Obtainment of killer protein (KLKP) produced by Kluyveromyces lactic
killer yeast
Killer yeast means yeast that produces a killer toxin selectively
suppressing the growth of other types of yeast. A killer protein KLKP
produced by the killer yeast used in the present invention can be obtained
from Kluyveromyces lactic (Kitamoto, H. K. et al., (1995), 7th European
Congress on Biotechnology, Abstract Book, 62). The killer protein KLKP
produced by Kluyveromyces lactic consists of 3 subunits: a., ~3, and y, and
DNAs encoding these subunits are present on a linear plasmid, pGKLl. An
example of Kluyveromyces lactic is the IF01267 strain. The Kluyveromyces
lactic IF01267 strain can be obtained from the Institute for Fermentation,
Osaka (IFO) (2-17-85, Jusohhommachi, Yodogawa-ku, Osaka-shi). The
above killer yeast is cultured, and then KLKP can be isolated and purified
from the culture product. Here, the culture product means a culture
supernatant and cultured microbial bodies. Yeast can be cultured by known
methods. Moreover, KLKP can be isolated and purified from the culture
product using known biochemical techniques using, for example, a
hydroxyapatite column. Whether or not a purified protein is KLKP can be
determined by measuring if the protein has yeast killer activity.
Moreover, a gene encoding KLKP is isolated from Kluyveromyces
lactic, and then a recombinant KLKP can be obtained by genetic engineering
techniques.
Experiments required for obtaining recombinant KLKP, such as
mRNA preparation, cDNA production, the RT-PCR method, the RACE
method, determination of the nucleotide sequence of DNA, and examination
of expression by Northern blot can be conducted by methods described in
6
CA 02479908 2004-09-20
general experimental manuals such as the one edited by Sambrook et al
(Molecular Cloning, A laboratory manual, 2001, Eds., Sambrook, J. & Russell,
DW. Cold Spring Harbor Laboratory Press).
The DNA sequence of the pGKLl linear plasmid wherein KLKP is
present is shown in SEQ ID NO: 1. The DNA has been registered with
GenBank under accession number X00762. Of these, a sequence between
positions 3229 and 6669 encodes the large subunit of the killer protein.
Among the amino acid sequences of the large subunit, an amino acid
sequence between positions 30 and 892 corresponds to the a subunit, and an
amino acid sequence between positions 895 and 1146 corresponds to the (3
subunit. A sequence between positions 7939 and 8688 of SEQ ID NO: 1
encodes the small subunit of KLKP, that is, the y subunit. The amino acid
sequences of the a, (3, and y subunits of KLKP are shown in SEQ ID NOS: 2,
3, and 4, respectively.
A protein that is also included in examples of the protein of the
present invention is a protein consisting of the a, ~3, and y subunits
represented by SEQ ID NOS: 2, 3, and 4, respectively, wherein: at least 1 or
preferably 1 or several (e.g., 1 to 10 and further preferably 1 to S) amino
acids may be deleted from the amino acid sequence of at least one of the 3
subunits; at least 1 or preferably 1 or several (e.g., 1 to 10 and further
preferably 1 to 5) amino acids may be added to the amino acid sequence
represented by SEQ ID NO: 2; or at least 1 or preferably 1 or several (e.g., 1
to 10 and further preferably 1 to 5) amino acids may be substituted with other
amino acids, and having killer yeast activity or the action of activating
calcineurin.
An example of such an amino acid sequence derived from the amino
acid sequence represented by SEQ ID NO: 2, 3, or 4 by deletion, substitution,
or addition of 1 or several amino acids is an amino acid sequence having at
least 80% or more, preferably 90% or more, more preferably 95% or more,
7
CA 02479908 2004-09-20
and particularly preferably 97% or more homology with the amino acid
sequence of SEQ ID NO: 2, 3, or 4 when calculation is conducted using
BLAST or the like (for example, when calculation is conducted with default,
that is, initial setting conditions). A protein consisting of 3 subunits
having
such homologies and having killer yeast activity or the action of activating
calcineurin is also included among the examples of the protein of the present
invention.
Here, killer yeast activity means activity suppressing the proliferation
of wild yeasts and can be assayed using as an index the suppression of the
proliferation of yeast when yeast is cultured with the protein of the present
invention added to the yeast. For example, killer yeast activity can be
assayed by a method that involves culturing yeast in a medium on which a
paper disk containing the protein of the present invention is placed, and
measuring the area of the inhibition zone.
Furthermore, the action of activating calcineurin means the action of
increasing the intracellular calcium ion level so as to activate calcineurin,
the
calcium-signaling pathway. This activation action can be measured by the
method described below.
The gene is incorporated into an appropriate vector, and then an
appropriate host is transformed with the vector, so that a recombinant KLKP
can be expressed and obtained.
The recombinant vector of the present invention can be obtained by
ligating (inserting) the gene of the present invention into an appropriate
vector. Examples of a vector to be used for the insertion of the gene of the
present invention are not specifically limited, as long as such vectors can be
replicated in a host, and include a plasmid DNA and a phage DNA.
An example of a method that is employed for inserting the gene of the
present invention into a vector involves, first, cleaving a purified DNA with
an appropriate restriction enzyme, and then inserting the cleaved product at a
8
CA 02479908 2004-09-20
restriction enzyme site or a multicloning site of an appropriate vector DNA,
so as to ligate the product to the vector.
It is necessary for the gene of the present invention to be incorporated
into a vector so that the gene functions can be exerted. Hence, in addition
to a promoter and the gene of the present invention, if desired, a sequence
containing a cis element such as an enhancer, a splicing signal consisting of
a
splice donor site located on the 5' terminal side of an intron and a splice
receptor site located on the 3' terminal side of the intron, a polyA addition
signal, a selection marker, a ribosome binding sequence (SD sequence), and
the like can be ligated to the vector of the present invention.
A transformant of the present invention can be obtained by
introducing the recombinant vector of the present invention into a host so
that
a target gene can be expressed. Here, an example of a host is not
specifically limited, as long as it can express the DNA of the present
invention. Examples of such a host include: bacteria belonging to the genus
Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus
subtilis, and the genus Pseudomonas such as Pseudomonas putida; yeast such
as Kluyveromyces lactic, Saccharomyces cerevisiae, and
Schizosaccharomyces pombe; animal cells such as COS cells and CHO cells;
and insect cells such as S 121.
Examples of a method for introducing a recombinant vector into
bacteria are not specifically limited, as long as such methods are methods for
introducing DNA into bacteria. Examples thereof include a method using
calcium ions [Cohen, S. N. et al., Proc. Natl. Acad. Sci., U.S.A., 69, 2110
( 1972)] and electroporation.
The protein of the present invention can be obtained by culturing the
above transformant and then collecting the protein from the culture product.
The "culture product of the transformant" means any of a culture supernatant,
cultured cells, cultured microbial bodies, and disrupted products of cells or
9
CA 02479908 2004-09-20
microbial bodies.
After culture, when the protein of the present invention is produced
within microbial bodies or cells, KLKP is extracted by disrupting the
microbial bodies or cells. In addition, when the protein of the present
invention is produced outside of microbial bodies or cells, the broth is
directly used or the broth is subjected to centrifugation or the like to
remove
the cells. Subsequently, through the use of one of or an appropriate
combination of general biochemical methods employed for isolation and
purification of proteins, such as ammonium sulfate precipitation, gel
chromatography, ion exchange chromatography, and affinity chromatography,
the protein of the present invention can be isolated and purified from the
above culture product. In a manner similar to purification from
Kluyveromyces lactis killer yeast, it is preferable to conduct purification by
chromatography using hydroxyapatite gel.
2. Use of killer protein (KLKP) produced by Kluyveromyces lactis killer
yeast as calcineurin activator
KLKP purified from the above culture product of Kluyveromyces
lactis killer yeast or KLKP obtained by genetic engineering techniques can be
used as the calcineurin (CaN) activator of the present invention.
The killer protein that is produced by Kluyveromyces lactis killer
yeast increases the calcium concentration within eukaryotic cells through the
influx of calcium ions into the cells so as to activate calcineurin, thereby
inducing a delay at the G2 phase of the cell cycle.
Here, the influx of calcium ions into cells includes not only the influx
of calcium ions existing extracellularly into cells, but also the influx of
calcium ions existing in the organelles within cells into cytoplasms. Hence
an increase in the intracellular calcium ion concentration means an increase
in the intracytoplasmic calcium ion concentration.
CA 02479908 2004-09-20
The calcineurin activator of the present invention comprising KLKP
as an active ingredient can be used as a reagent for studying the signaling
system mediated by Ca2+, such as cell proliferation, cell differentiation, and
regulatory mechanisms for transcriptional control in eukaryotes. Moreover,
the activation of calcineurin induces a delay at the G2 phase of the cell
cycle.
Thus, the calcineurin activator of the present invention can be used as an
agent for inhibiting the cell cycle of eukaryotic cells such as yeast, plant,
and
animal cells. Furthermore, the calcineurin activator of the present invention
inhibits the cell cycle, so that it can be used as an agent for inhibiting
cell
proliferation. Furthermore, the activation of calcineurin results in T-cell
proliferation, induction of cancer cell differentiation, apoptosis, and
reconstruction of myofibrils. Thus, the calcineurin activator can also be
used as an agent for activating the immune system, an anti-cancer agent, or
the like.
The action of KLKP to activate calcineurin can be confirmed as
described below.
Based on the fact that the calcium-signaling pathway activates Swel,
the action of KLKP can be confirmed using changes in killer sensitivity when
the calcium-signaling pathway is blocked.
Moreover, the activation of calcineurin, the calcium-signaling
pathway, can also be confirmed using the fact that when KLKP is caused to
coexist with calcium in Ozdsl (Zdsl-disrupted strain), the cell cycle is
delayed at the G2 phase, as an index. Delay at the GZ phase of the cell
cycle can be confirmed by FACS analysis.
3. Use of the killer protein (KLKP) produced by Kluyveromyces lactis killer
yeast as agent for inhibiting cell proliferation
The Killer protein (KLKP) produced by Kluyveromyces lactis killer
yeast of the present invention can be used as an agent for inhibiting cell
11
CA 02479908 2004-09-20
proliferation.
KLKP can be used as an agent for inhibiting cell proliferation by
causing KLKP to come into contact with cells, or to be expressed within
cells.
For example, KLKP can be used as an antifungal agent or the like for
suppressing mycetes proliferation. In addition, KLKP inhibits cell
proliferation, so that it can also be used as an agent for inhibiting the
proliferation of an organism body itself, such as plant bodies. The type of
KLKP to be caused to come into contact with cells or organism bodies can be
appropriately determined depending on the species and quantities of
organisms whose proliferation is to be inhibited by KLKP. For example, in
the case of cultured tobacco cells, KLKP is added at a final concentration
between 1/100 and 1/1000 to a broth supplemented with cells where the ratio
of cells to solution by live weight is 1/40.
4. Prevention of deterioration of silage (feed of cattle) using killer protein
(KLKP) produced by Kluyveromyces lactis killer yeast
Silage is feed of ruminant domestic animals having an improved
preservative quality, which is produced by lactate fermentation of grass.
When silage is exposed to an aerobic condition, aerobic deterioration
proceeds due to the lactate metabolism of yeast. To prevent such aerobic
deterioration, it is necessary to suppress the proliferation of
lactic-acid-assimilating wild yeasts that cause deterioration.
The killer protein (KLKP) produced by Kluyveromyces lactis killer
yeast suppresses the proliferation of fungal cells such as yeast in the
presence
of calcium ions. Thus, KLKP can be used for preventing silage
deterioration. Per kg of silage, 0.1 g to 0.5 g of calcium ions and 0.1 g to
0.5 g of KLKP may be added. In addition, as a supply source of calcium
ions, for example, cheese whey can be used. Calcium ions and KLKP may
12
CA 02479908 2004-09-20
be added simultaneously or separately to silage.
A composition comprising calcium ions or a supply source of calcium
ions and KLKP for preventing silage deterioration is also encompassed within
the scope of the present invention.
5. Use of killer protein (KLKP) produced by Kluyveromyces lactic killer
yeast as medicine
A calcineurin activator comprising KLKP as an active ingredient can
also be used as a medicine. Since calcineurin has various functions,
activation of calcineurin can be used for preventing and treating various
diseases.
The calcineurin activator of the present invention comprising KLKP
as an active ingredient can be utilized as, for example, an anti-cancer agent.
Furthermore, the calcineurin activator of the present invention comprising
KLKP as an active ingredient can also be utilized as an immunosuppresant,
an immunostimulant, a therapeutic agent against disorders relating to memory,
and a therapeutic agent against cardiovascular diseases. A pharmaceutical
composition containing the calcineurin activator comprising KLKP as an
active ingredient is also encompassed within the scope of the present
invention. Here, examples of disorders relating to memory include
hypomnesia, amnesia, anterograde amnesia, and emotional amnesia.
Examples of cardiovascular diseases include ischemic neuronopathy.
A method for treating disorders relating to memory, a method for
treating cardiovascular diseases, and a method for suppressing or activating
immunity by administering the calcineurin activator of the present invention
comprising KLKP as an active ingredient to patients are also encompassed
within the scope of the present invention. Moreover, the use of the
calcineurin activator of the present invention comprising KLKP as an active
ingredient in production of an anti-cancer agent, an immunosuppresant, an
13
CA 02479908 2004-09-20
immunostimulant, a therapeutic agent against disorders relating to memory,
or a therapeutic agent against cardiovascular diseases is also encompassed
within the scope of the present invention.
A medicine of the present invention can be administered in various
forms. Examples of such forms for administration include tablets, capsules,
granules, powder, syrup, and the like used for oral administration, and
injections, drops, suppositories, and the like for parenteral administration.
Such a composition is produced by known methods, and contains a carrier, a
diluent, and an excipient that are generally used in the field of
pharmaceutical preparations. For example, as a carrier and an excipient for
a tablet, lactose, magnesium stearate, or the like is used. An injection is
prepared by dissolving, suspending, or emulsifying KLKP or a salt thereof in
a sterile aqueous or oil solution that is generally used for an injection. As
an aqueous solution for injection, physiological saline, an isotonic solution
containing glucose and other adjuvants, or the like is employed. The
aqueous solution for injection can be used together with an appropriate
solubilizer, for example, alcohol, polyalcohol such as propylene glycol, or a
nonionic surface active agent. As an oil solution, sesame oil, soybean oil,
or the like is used. As a solubilizer, benzyl benzoate, benzyl alcohol, or the
like may be used together.
The dose differs depending on symptom, age, body weight, and the
like, and ranges from approximately 0.001 mg to 100 mg per day in general
oral administration. Administration is conducted once or several separate
times per day. In the case of parenteral administration, a dose ranging
from 0.001 mg to 100 mg per administration is conducted by subcutaneous
injection, intramuscular injection, or intravenous injection.
Furthermore, the calcineurin activator of the present invention
comprising Kluyveromyces lactis killer protein (KLKP) as an active
ingredient and having the action of increasing intracellular calcium ion
14
CA 02479908 2004-09-20
concentration through the influx of calcium ions into eukaryotic cells can be
used for screening for a remedy against diseases in which calcineurin is
involved. For example, screening can be conducted as described below.
When a KLKP-sensitive strain such as Ozds 1 (Zds 1-disrupted strain)
is placed in a microplate together with a medium and calcium, and then
KLKP is added to the microplate, or recombinant KLKP is expressed,
proliferation is generally inhibited. If proliferation is not inhibited when
various drugs are added, the drugs can be determined to be calcineurin
inhibitors.
This screening can be conducted using not only yeast, but also animal
cells.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. I shows the results of searching killer proteins that are activated
by calcium.
Fig. 2 shows the killer effect of KLKP against the W303 strain. (a)
shows the results when Ca2+ coexisted with KLKP and (b) shows the results
when other ions coexisted with KLKP.
Fig. 3 shows the killer effect against Ca2+ signaling system variants.
Fig. 3A shows the results for (a) the parent W303 strain and (b) the zdsl
gene-disrupted strain (~zdsl) thereof. Fig. 3B shows the results for double
gene (including tlzdsl)-disrupted strains. (c) shows the results for
~zds 1 Oswe 1, (d) shows the results for ~zds 1 ~cnb 1, and (e) shows the
results
for 4zds 1 ~mpk 1.
Fig. 4 shows the G2/M-phase cell cycle model participating in a
budding yeast Ca2+ signaling system.
Fig. 5 shows the results of FACS analyses conducted for the Ozdsl
strain (Zdsl-destrupted strain) on which KLKP was caused to act.
Fig. 6 shows the results of FACS analyses conducted for double
CA 02479908 2004-09-20
(Ozdsl and Ca2+ signaling system) variants on which KLKP was caused to act.
(a) shows the results for Ozds 1 Ocnb 1, (b) shows the results for Azds 1 Oswe
1,
and (c) shows the results for Ozds 1 ~mpk 1.
Fig. 7 shows the effect of KLKP on viable count. (a) shows the
results for the parent W303 strain, and (b) shows the results for 4zdsl.
Fig. 8 shows photographs showing the appearance of (a) chitin (9
hours later) and (b) actin (6 hours later) of cells on which KLKP was caused
to act.
Fig. 9 shows emission levels in wild-type cells and cells caused to
express aequorin (Fig. 9 (a)) and an increase in intracellular calcium level
by
KLKP (reporter assay method) (Fig. 9 (b)).
Fig. 10 shows the result of analyzing the action of KLKP using 45Ca.
Fig. 11 shows photographs showing the growth of cultured tobacco
cells on which KLKP was caused to act.
BEST MODE OF CARRYING OUT THE INVENTION
The present invention will be further described specifically by
referring to examples, but the technical scope of the present invention is not
limited by these examples.
Example 1 Purification of killer protein (KLKP) produced by
Kluyveromyces lactis killer yeast.
100 ml of YPD medium (yeast extract 1%, peptone 2%, and glucose
2%) in which the Kluyveromyces lactis yeast IF01267 strain had been
inoculated was put in a 500 ml flask, and then the yeast was subjected to
rotation and shake culture overnight at 28°C and 220 rpm. 1.3 L of the
broth was centrifuged. The resulting supernatant was filtered with a 0.2 pm
filter, and then concentrated to 10 ml using a ultrafiltration system (Asahi
Kasei Corporation, ACP-1010 and SLP-0053). After substitution with a 10
mM potassium phosphate buffer (pH 6.8), the resultant was adsorbed to a
16
CA 02479908 2004-09-20
hydroxyapatite column (Nacalai Tesque, Inc., 187-37, 100-200 mesh) that had
been equilibrated with the same buffer. After washing with a 10 mM
potassium phosphate buffer (pH 6.8), the killer protein was eluted using a
400 mM potassium phosphate buffer (pH 6.8). Fractions having killer
activity were collected, dialyzed using a 50 mM citrate-phosphate buffer (pH
6.0), and then filtered and sterilized using a 0.2 ~m filter. Glycerol was
added at a final concentration of 10% to 10 ml, and then the resultant was
stored at -80°C.
Example 2 Increase in intracellular Caz+ level and activation of calcineurin
by killer protein (KLKP) produced by Kluyveromyces lactic killer yeast
9 types of killer yeast culture filtrates were caused to act on budding
yeast in the presence of Ca2+, and then growth inhibition was examined.
The 9 types of killer yeasts were the Saccharomyces cerevisiae NCYC235
strain (hereinafter, the name of a killer type is K1), the Saccharomyces
cerevisiae NCYC738 strain (K2), the Saccharomyces cerevisiae NCYC761
strain (K3), the Candida glabrata NCYC388 strain (K4), the Pichia anomala
NCYC434 strain (KS), the Kluyveromyces marxianus NCYC587 strain (K6),
the Candida valida NCYC327 strain (K7), the Kluyveromyces lactic
NCYC575 strain (K10), and the Williopsis saturnus var. saturnus IF00117
strain. Furthermore, proliferation of cell cycle-related gene variants in the
presence of various metal salts was examined on a 96-well microplate using
KLKP that had been purified using the hydroxyapatite column from the killer
yeast culture filtrate. Similarly, after shake culture within a flask in the
presence of 50 mM Ca2+ and 1/100 KLKP, viable count was examined with
time. Furthermore, the cell cycles of immobilized cells were examined
using a flow cytometry system (FACS), thereby analyzing the morphology,
nuclei, actin, and chitin by fluorescence microscope observation. As a
result, the following matters were revealed.
17
CA 02479908 2004-09-20
Of the killer yeast strains tested, 4 strains (the Saccharomyces
cerevisiae NCYC738 strain (K2), the Saccharomyces cerevisiae NCYC761
strain (K3), the Candida valida NCYC327 strain (K7) and the Kluyveromyces
lactis NCYC575 strain (K10)) inhibited the proliferation of budding yeast
while coexisting with Ca2+. However, the Kluyveromyces lactis broth used
in this study showed strong activity of inhibiting proliferation, even if it
had
been diluted 10-fold, a dilution at which no other killer yeasts showed such
activity (Fig. 1). In Fig. 1, white bars indicate cases where calcium was
present, and black bars indicate cases where no calcium was present. The
horizontal axis indicates the degree of proliferation.
Furthermore, the proliferation of the budding yeast W303 strain was
inhibited by KLKP at high Ca2+ concentrations, but was not affected by Zn2+,
Mn2+, Na+, K+, or Mg2+ (Fig. 2 (a) and (b)). The ~zdsl strain
(Zds 1-desrupted strain) showing sensitivity to high concentrations of Ca2+,
which had been isolated from the W303 strain, showed sensitivity to KLKP at
concentrations lower than those to which the W303 strain showed sensitivity.
Furthermore, the growth inhibition of the Ozdsl strain by KLKP was
suppressed by the addition of EGTA, a CaZ+ chelating agent (Fig. 3(b)).
Zdsl suppresses the transcription of Swel, which is the negative regulation
factor of the cell cycle engine. In ~zdsl, Swel is transcribed and the
Cdc28-Clb cell cycle engine is phosphorylated, so that the Ozdsl strain cell
cycle is delayed at the G2 phase. In addition, Ca2+ signaling system
calcineurin and Mpkl MAP kinase transcibe and activate Swel, respectively
in the presence of Ca2+, thereby negatively controlling the cell cycle engine
(Fig. 4). The mechanism shown in Fig. 4 has been reported by Mizunuma et
al (NATURE, 392, p.303 (1998)). Hence, in 0 zdsl, the effect of Ca2+ on
cell cycle can be observed. The double gene (Ozdsl and Ca2+ signaling
system)-disrupted strains Ozds 1 ~swe 1, 4zds 1 ~cnb 1 (calcineurin subunit),
and Ozdsl0mpkl showed KLKP sensitivity lower than that of Ozdsl at 0.1
18
CA 02479908 2004-09-20
mM to 300 mM Ca2++ (Fig. 3(c), (d), and (e)).
As a result of FACS analyses, G2-phase cells increased in the Ozdsl
strain by the addition of KLKP (Fig. 5). In the meantime, in ~zdslOcnbl
and 4zds 14swe 1, G 1-phase cells increased by the addition of KLKP, and in
OzdslOmpkl, G2-phase cells did not increase as in the case where no KLKP
had been added (Fig. 6). Regarding the 2 peaks in the results of FACS
analyses in Figs. 5 and 6, the ls' peak shows an increase in cells at the Gl
phase, and the 2°d peak shows an increase in cells at the G2 phase.
These results revealed that the addition of KLKP results in a delay at
the G2 phase of the cell cycle of budding yeast, and this action is activated
by Ca2+. In this mechanism, activation of Swel kinase by a Caz+ signaling
pathway is involved. Although KLKP is known to arrest the cell cycle of
budding yeast at the G1 phase, it has been reported that a high concentration
of KLKP often arrests the cell cycle at a different phase. Hence, it was
concluded that suppression of the cell cycle by KLKP takes place at two
phases (the Gl phase and the G2 phase), and the mechanism activated by
Ca2+ causes a delay at the G2 phase. When the Ca2+ signaling pathway is
blocked, a suppressed cell cycle is observed at the other G1 phase as
observed in Fig. 6.
Under the culture conditions, the viable count of the W303 strain was
hardly ever affected. However, the ~zdsl strain rapidly died due to the
addition of KLKP, showing hypersensitivity to KLKP (Fig. 7 (a) and (b)).
The gray lines in Fig. 7 show the total number of microbial bodies. In
addition, Fig. 7 (a) shows the result for the parent W303 strain, and Fig. 7
(b)
shows the result for the ~zds strain.
Under the culture conditions, the viable count of the W303 strain was
hardly ever affected. However, the ~zds 1 strain rapidly died by the addition
of KLKP, showing hypersensitivity to KLKP (Fig. 7).
KLKP is known to recognize chitin. Chitin is generally localized at
19
CA 02479908 2004-09-20
bud scars. However, in both the cases of the W303 strain and the ~zdsl
strain, chitin masses were scattered on the cell cortex due to the addition of
Ca2+ and KLKP (Fig. 8(a)). Moreover, in the case of actin, it is known that
cables generally run in the direction of budding, that patches are localized
at
budding positions, and that Ca2+ has an effect on actin localization. In both
the cases of the W303 strain and Ozdsl strain, actin localization became
deficient due to the addition of KLKP. Deficiency in chitin and actin
localization due to KLKP was enhanced by Caz+ coexisting with KLKP, and
this was more serious in the dzdsl strain than in the W303 strain (Fig. 8
(b)).
As described above, KLKP generally exerts a bacteriostatic (fungistatic)
effect, inducing deficiency in actin localization. Furthermore, KLKP also
induces deficiency in chitin localization of KLKP-hypersensitive strains due
to the activation of KLKP by Caz+. In addition, it was observed that KLKP
kills cells.
Furthermore, intracellular calcium concentrations increased by KLKP
were measured.
Yeast that had been caused to express a luminescent protein aequorin
precursor was cultured in a medium containing no calcium ions. The yeast
was caused to absorb a substrate (coelenterazine), CaZ+ and KLKP were
caused to act thereon, and then emission levels were measured using a photon
counter. In addition, a calcium ionophore (A23187) was used as a control of
Ca2+ influx.
Furthermore, KLKP was caused to act on yeast that had been
transformed with a plasmid having a (3-galactosidase gene ligated
downstream of the promoter of a protein to be induced by Ca2+. 5 minutes
later, the cells were washed with water. Subsequently, a liquid medium was
added to the cells. The resultant was shaken for 4 hours, and then
(3-galactosidase was expressed corresponding to intracellular Ca2+
concentrations. The cells were treated with ether, a substrate (ONPG) was
CA 02479908 2004-09-20
added to the cells, and then the ~3-galactosidase activity of the cells was
examined. The results are shown in Fig. 9 (a)a to f (aequorin measurement
method), and the (3-galactosidase activity is shown in Fig. 9 (b) (reporter
assay method). In Fig. 9(a), (a)a shows the results for the wild type, and
(a)b to (a)f show the results for the cells expressing the aequorin precursor.
As a result, the cells (Fig. 9 (a)b to f) that had been caused to express
the aequorin precursor slightly emitted light due to the addition of Ca2+
(Fig.
9(a)c) and the emission levels increased 2.4-fold due to the addition of Ca2+
and a calcium ionophore (Fig. 9 (a)d). However, similar to the case of the
calcium ionophore, KLKP alone did not induce light emission (Fig. 9(a)e).
Nevertheless, the addition of Ca2+ and KLKP induced light emission 2.4-fold
more intense than that resulting from the addition of Ca2+ (Fig. 9(a)f). Thus,
it was confirmed that Ca2+ in an amount equivalent to that of the calcium
ionophore increased within the cells immediately after the addition of KLKP.
Subsequently, reporter assay was conducted. In the case of cells
subjected to KLKP treatment, it was considered that intracellular Ca2+
concentration increased because of increased (3-galactosidase activity (Fig. 9
(
By the above two methods, it was confirmed that intracellular Ca2+
concentration was increased by KLKP treatment.
Furthermore, the action of KLKP was analyzed using 45Ca. 45Ca was
added to yeast cells that had been cultured in a medium lacking calcium. 9
minutes later, when the calcium absorption level reached a flat level, KLKP
was added. Cells were sampled every 2 minutes after the addition of 45Ca,
so that the intracellular 45Ca level was measured. As a result, the
intracellular calcium level after the addition of KLKP increased to a level
1.5-fold to 2-fold greater than the case where no KLKP had been added (Fig.
11 ).
As shown in Fig. 9 and Fig. 11, the influx of calcium ions from
21
CA 02479908 2004-09-20
outside of cells due to KLKP was confirmed.
The activation of calcineurin due to KLKP was confirmed by the
following methods.
Based on the fact that the calcium-signaling pathway activates Swel
(Fig. 4), the 4zdsl strain was used to enable to observe Swel activation due
to the Caz+ signaling.
When the calcium-signaling pathway was blocked, killer sensitivity
decreased. In particular, in the case of double disruption (where both the
calcium-signaling pathway and Cnbl were disrupted), the double disruption
strain came to have enhanced resistance compared with the parent strain, and
did not become killer-sensitive without calcineurin.
It is known that the ~zdsl strain is delayed at the G2 phase when
calcium is added. As a result of FACS analyses, when KLKP was caused to
co-exist with calcium, the strain is further delayed at the G2 phase (Fig. 5).
Thus, it was confirmed that the calcium-signaling system in Fig. 4 was
activated.
The above results revealed the presence of a mechanism wherein
intracellular calcium concentration was increased by KLKP, resulting in an
activated calcium-signaling pathway, calcineurin, and a delay at the G2 phase
of the cell cycle.
Example 3 Suppression of cultured plant cell proliferation by killer protein
(KLKP) produced by Kluyveromyces lactis killer yeast
1 ml of cultured tobacco cells (BY cells) that had been grown at 28°C
for 7 days was added to 20 ml of a Murashige and Skoog medium containing
3% sucrose. KLKP was added to the medium at a final concentration
between 1/100 and 1/100000, and then cultured for 5 days at 28°C and
115
rpm. The broth containing cells was transferred into a calibrated test tube,
and then allowed to stand. The amount of the precipitated cells was
22
CA 02479908 2004-09-20
measured. Furthermore, cells to which no KLKP had been added and cells
to which KLKP had been added at a concentration of 1/100 were stained with
the DAPI fluorescence reagent, so that the morphology of the cells and the
nuclei could be observed.
As a result, in the presence of KLKP at a concentration of 1/100, the
proliferation of the culture cells was approximately half of that of the cells
to
which no KLKP had been added. Even in the presence of KLKP at a
concentration of 1/1000, the amount of the culture cells was approximately
80% of that of the cells to which no KLKP had been added, showing that cell
proliferation was inhibited by KLKP. Furthermore, the cell size became
greater than the general cell size through the addition of KLKP, showing that
cell division was inhibited by KLKP (Fig. 11 ). In Fig. 11, Fig. 11 (a) shows
increases in cells, and Fig. 11 (b) shows changes in cell morphology. In
addition, it was revealed that KLKP acts also on eukaryotes other than yeast.
Industrial Applicability
As shown in Example 2, the killer protein (KLKP) produced by
Kluyveromyces lactis killer yeast can be utilized as a calcineurin activator
in
eukaryotic cells, because the protein causes an increase in intracellular
calcium ion concentration and activates calcineurin in eukaryotic cells.
Furthermore, KLKP can arrest the cell cycle of eukaryotic cells at the G2
phase in the presence of calcium ions, so that it can be utilized as an agent
for inhibiting the cell cycle. In particular, as shown in Example 3, KLKP
can suppress proliferation of eukaryotic cells other than yeast, so that it
can
be used as an agent for inhibiting the proliferation of a variety of cells. In
particular, deterioration of silage can be prevented by adding the calcineurin
activator of the present invention comprising KLKP as an active ingredient to
silage together with a calcium ion supply source.
Furthermore, the calcineurin activator of the present invention
23
CA 02479908 2004-09-20
comprising KLKP as an active ingredient can be used as a medicine such as
an anti-cancer agent.
All publications cited herein are incorporated herein by reference in
their entirety. It is easily understood by those skilled in the art that
various
changes and modifications may be made in the present invention without
departing from the technical idea and the scope of the present invention
described in the attached claims. It is intended that the present invention
encompasses such modifications and changes.
24
CA 02479908 2004-09-20
SEQUENCE LISTING
<110> National Institute of Agrobiological Science
<120> Calcineurin activator
<130> PH-1537PCT
<140>
<141>
<150> JP2002/081415
<151> 2002-03-22
<160> 4
<170> PatentIn Ver. 2. 1
<210> 1
<211> 8874
<212> DNA
<213> Kluyveromyces lactis
<400> 1
acacataaca taggggagag tactaaaagt gagattattg gaagattagt acgtctccat 60
ttttttctgt ttttttgttt ttatatatta ggttattttt tttcagtttt atatcaactc 120
tgtataacaa gtctattttt ttatatttta agtctatttt acacttttga cctataagtc 180
attttattat acacattttc caactataat atatgaatta cattattaat ttaaaaatgg 240
attacaaaga taaggcttta aatgatctaa gaaatgtata tgccgacttt gattcacttc 300
ctttagattt tagacaaata ttaataaaag atagagccac acttcttcaa aaagaagatg 360
I/16
CA 02479908 2004-09-20
tagaaaagaa aatattggaa agacaagaag atgcaaagaa atatgcagaa tatttaaaac 420
aatcagaaat accagaacga atatctttgc ctaacattaa aagacataaa ggtgtttcta 480
tatcttttga agaaacatca gaagatatgg ttttggaacc aagacctttt atttttgatg 540
gattaaatat tagatgtttt agacgagaga caattttctc tctcaaaaat aaaatattaa 600
acatggtaaa agaaagttct tcttttaaaa atgtttctag acaatcagtt tctttcatgt 660
attttaaaat ttttaataaa gggaaagtta tagcttctac aaaaagtgta aatatttatg 720
aagataaaat agatgagaga ttagaagatt tgtgtaataa ttttgacgat gtattaaaga 780
aaattataga tgtaacttat ggttatgaaa gtttatttgt ttcagaaaca tattcttatg 840
ttatatttta tgctaaatct atatatttcc ctcaacctag atgtgtgaat aattggggta 900
ataatattcc taatattctt actttcgata gttttaagct tttcacagct aataaaaata 960
atgtttcttg tattaaacag tgctctcgtt ttctgtggca aaaagatttt aatacattag 1020
aagaaatgat agaatataaa aatggtaata tttgtatagt tactcctcaa ttacatataa 1080
atgatgtaag agacataaaa tcatttaacg acatacgttt atattcagaa agtcctatta 1140
aaacattcag tgttatagat aatactataa catatttgtt ttattttaaa gaacatttag 1200
gagttatatt taatattact aaatccagac atgatagaag agtcactaaa tttagtcctt 1260
tgtcaaaatt ttctgatgtt aaaaatataa cagtatgttt tgatatagaa tcttattttg 1320
atccagaaaa agaatctaat caagttaata taccctttat atgttgtgca tctataatat 1380
ataataaagt cataggaaat attgtagatt ttgaaggaag agattgtgta gctcaaatga 1440
tagaatatgt tgtagatata tgtggagagc ttaatatatc ttcagtggaa ctaattgcac 1500
ataatggtgg aggttatgat tttcattata ttttaagtag tatgtataat cctgcagcta 1560
ttaaaaatat attaattaga aataactcat ttataagttt taattttgct cacgatggag 1620
tcaaattttc tgtaaaagat tcctatagtt tcttgttatg tagtttagca aatgcttcaa 1680
aagcattttt aaacgaagaa acctttaaga aaacagattt tccccatcat gatttaaaaa 1740
cagcagatga tttatataaa gtatataaag aatggtcatc tgtaaacact gaaataaatc 1800
atgtagtgga aaaagaaaaa cttcttataa catcagaaca tatagttaat ttcactaaaa 1860
atgataaatc taaaactcta atagaatggt ctaaagatta ttgtagaaat gatgttttgg 1920
ttttatctaa ggtatggtta gaatttaaaa atgctgtaga agatattttt aattgtgaat 1980
tagtagatca aactatgaca ttagcaggac taagttataa attatttcaa gcaaatatgc 2040
cttttgatgt tgaattaaga catccaaata aagaagatta ttttaacatg agagaggctt 2100
taataggagg gagatgtatt agtgtcaatg gaatatataa agatgtttta tgtttagatg 2160
2/16
CA 02479908 2004-09-20
taaaatcatt atatccagca tctatggcat tttatgacca gccatatgga tctttcaaaa 2220
gagtatctag tagacctaaa gatgaattag gtatttatta tgtcagagta actcctaata 2280
gaaataataa atccaacttt tttcctataa gaagtcacaa taaaattact tataataatt 2340
ttgaagaaag tacatatata gcatggtata caaatgtaga tatagatata ggtttgtctg 2400
aaggtcataa tatagaatat atcccctttg attcttatgg aaatataggt tattcttggt 2460
ctaaaaaagg taaaatattc gaaaaatata taaaagacgt gctgtacaaa ttaaaaataa 2520
agtatgaaaa acaaaacaat aaagttaaaa gaaatgttat caaaattatt atgaacagtt 2580
tatggggcaa attcgcacaa aaatgggtaa attttgagta ttttataaaa tcagaagatg 2640
atatagattt tgagtcagaa gaggcatata agatatggga cactgatttt atgctgataa 2700
agaaaattaa agaatctact tattcatcta aacctataca aaatggagta tttacattaa 2760
gttgggcaag ataccacatg aaaagtatat gggatgcagg ggctaaagaa ggagcagaat 2820
gtatctattc ggacacagat agtatttttg tacataaaga acattttaaa aagaatgcta 2880
aatttatgtt aaatggttta aaagttccta ttataggatc agaagtagga caattagaat 2940
tagaatgtga gtttgataaa ttgttatgtg caggtaaaaa gcaatacatg ggattttata 3000
cttattttca agatggaaaa ccatgtataa aagaaaagaa aagatttaag ggtattccta 3060
gtaattatat aatacctgaa ttatatgctc atttactttc aggtgcagac aaagaagcta 3120
aaatacaatt tttgaaattt agaagagaat ggggatcagt taaaggatat atagaaaata 3180
agaccgtgaa agctacttaa tatatgaaag tttttataat aattataaaa tgaatatatt 3240
ttacatattt ttgtttttgc tgtcattcgt tcaaggtttg gagcatactc atcgaagagg 3300
ctccttagtc aaaagagcag tatgttatga cactgatcaa gttccactta atattttctt 3360
tggttataat agagcagata agactgattc taataagaat atggctctaa acatctttaa 3420
tgtttttaga ggttttctag ctggagaagg tggagagtct ttttacaatt ctaatggtaa 3480
tgtttatgga tttatgtggg taggtagtat ggttcataat agaggtttta aagataatat 3540
tttacctata atggaaaatg aagttaagaa ttatggtatt cctaaaacct tgtatttaga 3600
atatgacgga ggtggagatc ctatgaaatc ttttggtatt attttagata caacaagtag 3660
agatactgta gttaaagctg caaaattatg gagtcaaggt aaaaaattaa atagttatga 3720
aggatctaaa aattatcaag ctactgcatg ctatttatct tatgcatata gaaagcccat 3'780
tgttaatgat aattttgtag gaacttgcga ctatttcact ttagaaagtg gtaaaacacc 3840
agcagaccaa tctggtatta atggagagtc tctacaaggt tataatccta atttagattt 3900
ctctaaatta tcagcaggac aacctatttg taaaaccata ggtaatcctc ctaattttaa 3960
3/16
CA 02479908 2004-09-20
accttctaag aattcagacg gttcttgtaa aacatacaag gtatcatctg gagagtcttg 4020
ttcttctata gcagttaaat attatccatt aagtttaaat gatatagaaa attataataa 4080
aggtaattat ggatggaaag gatgttctag tcttcaaaaa gattataact tatgtgtgag 4140
tgatggtagt gctcctagac cagtttcaaa tcctatagca gaatgtggtc cattagctcc 4200
aggagagaaa tataatgcta aatgtccttt aaatgcttgt tgtagtgaat ttggtttctg 4260
tggtttaact aaagattatt gtgacaaaaa gagtagtact actggtgctc ctggtacaga 4320
tggctgtttt tctaattgtg gttatggttc tacttctaat gtaaaatcat ctacttttaa 4380
aaagattgct tattggttag atgctaaaga taaattagct atggatccga agaatattcc 4440
taatggtcct tatgatattt tacattatgc ttttgttaat ataaattcag actttagtat 4500
tgatgattct gcattttcaa aatctgcctt tttaaaagtt acttcttcca aaaagatacc 4560
tagttttggt ggttgggatt ttagtacatc tcctagtact tacactatat ttagaaatgc 4620
tgttaaaaca gatcaaaata gaaatacgtt tgctaacaat ttaatcaatt ttatgaataa 4680
atataatctt gatggtatag atttagattg ggaatatcca ggtgctcctg atattccaga 4740
tattcctgct gatgattcaa gtagtggatc taattatcta actttcctta agttattaaa 4800
gggtaaaatg ccttctggta aaaccttatc tatagccatt ccttcttcct attggtattt 4860
aaaaaatttc cctatttctg atattcaaaa cactgtagat tatatggttt acatgacgta 4920
tgatatacat ggtatatggg aatacggtaa agccaatagt tatataaact gccatactcc 4980
tcgtaaagaa attgaagatg ctataaaaat gttagataaa gctggagtta aatttaataa 5040
agtatttgga ggtgtggcaa attacggtag atcctacaaa atggttaata caaattgtta 5100
taattatgga tgcggttttc aaagagaggg aggaaattct agagatatga ctaatacacc 5160
aggtgttctt tctgattcag aaattattga tattgatagt tcagataaaa agaatgatag 5220
atgggtagat actaacacag attgtatttt tatgaaatat gacggaaatt ctgttgtttc 5280
atggcctaaa agtagatacg atttagaaga tatgtttaaa aattatggat ttgctggtac 5340
ttctttatgg gccgctaatt atttcaaaca tgatgaatgg aagaacgatg aagatgataa 5400
taatgatgat acagaagatc ctttcgatga agagaatgta tatttcgatg tttatgattg 5460
caaaaacaaa gctggttatg atctggacaa tccagtttat gggtgtagat tagaaacagc 5520
tataaatatt attatatgga atggtacaga atctgttaat acagttttaa atatattaaa 5580
tgattacgat aattatatta aatattatga agctctaact agagcacatt atgattcagt 5640
catggaaaaa tacgaaaaat ggctgtttga agaagatgga tattacacat attatactga 5700
tgtagacgga gatgatataa ttataactcc tccagataag aagaaaagag attacataca 5760
4/16
CA 02479908 2004-09-20
agagaaatat tcttttgaaa aagaatttat gatgtctcaa aatatgacag aattaacaga 5820
aattaaagtt aataaaacta ttaattttat gttaaatgga acatctctag ctgtaaaaga 5880
atataacaac gaaaaagttt tatataaaag aggagatata cctcctcctg gttctaataa 5940
tagattaatt agaaacagta ttattttaga taaagataaa gaagcagcta ttgcgtcttt 6000
caaacaatat tctggaatag aattatctaa agattctttt gtacaaagag ataaagataa 6060
aaagtttgat ctaaatggta aacattatac atttatgcat agtactattc tgaatgctat 6120
tgttttattc cctaatgttt taacaaatat tgattctgac tatattcatc atatttcaga 6180
tttaattgaa caagctcata acagtttagg taatgaaagt cctgataata tttatgaggt 6240
cttagaaagt gtggttgttt ttatgtctgt atcagaaata gctgattata catatacaga 6300
aggtaaaaag ataaaagaaa aatacgataa gatgaagaaa actatgattg ttggtattat 6360
attgggtatc ataggtggtt tgtctctatt tttaggacct ataggtatag ctacatctgt 6420
tcttgcagat tttgctctat taggagcaga tgccgctata aacggagagt taaatccatc 6480
agacctagca ttcgctttag caggtttatt cttaccagta tttgcttctt taggaaaaac 6540
atttaaattt gctgaagctt tacaaaaaat taatattaat aaatctaaaa actttgataa 6600
tttaaatgaa tttgagaaaa taagattttt cagatctaaa ttagggaaag ttaagatgtg 6660
tggctcttaa aagtaatgga tgaccattat tcttgtgtaa attgtcaaaa tctacatctt 6720
catatttatg atatttaaat atatattttt cgttttcaaa atctaaatgt tgacacatac 6780
ctccttcttt ttttgcttta ttcatcataa tattataaaa ttcaatacta ccagaagcat 6840
aagctattct tattaaatct atatctggac tataattttc taaatcttca gttatattca 6900
taatagcata atttactaat attgcatatc tttggcgtgg aaaatcgata agtagttttt 6960
gaaccatata tttatttaaa gttttataag tgtaaaaata aaaaggccta taaagagaca 7020
caaagtttga atcataaata tcattcacta ataaatttaa tactgctttt ttacacaaat 7080
catctggata ttctttatga tgtttaagta cataagctga atttaaaaaa ttaaattcaa 7140
ctgtatttat atttatatct aaataaggtt tataagagac catattatag tacacacttt 7200
tatctacaga aacacaatcc ataggaccaa attctgtatt ttgactataa tctatatatg 7260
tatataacat atcatctata atttgttcta tattactttg tttagaagta taattatatt 7320
taaaaaatat ttctaaagtt gtgtctttat tcctgagtat agtttcagga agtaaatatt 7380
tgtctttttc tactttttct aaaatatttt tattttcatg tattttataa ttatatatag 7440
tatcttcttc gcaaaaagat cttctattaa aaattataga taatctaaaa caaacttctg 7500
tacatatttt atcacattta tcacaaacat catcccaacc taataaaaca catattgttt 7560
5/16
CA 02479908 2004-09-20
taataactaa actatattta ggatcttttt ctaataaata tatacaagtt tctttagtag 7620
gaacattagg ataccaaatt cctgaaggca aatatttaaa attaaaatca caaaccttgt 7680
tactcataat atatctagca gatatactgg aaagtattcc agatgttaat tttaaacctg 7740
aattttccat tttaactgca aaattataac tatttcttat tcctatacat aaatgaaagt 7800
ttaaatcatc ttcatcaaaa agctcttcat tatcataata tttaataaaa ttttcataat 7860
ctgaataata agcataagta catgctttaa aataatctga aagattatta tctaattcta 7920
aacacatttt taattaaaat gaagatatat catatattta gtgtttgtta tctaataaca 7980
ttatgtgctg ctgcagctac tactgcgaga gaggagtttt tcttatgtta tgatttaatt 8040
agatatttaa aacaatatga aaaaacagga gagagtaaat tagtagaaca aacatttttt 8100
aatagtatta aaaacttaga cataaactct agagagtata tggaacttgt atataacaaa 8160
atagcaggta tttccaatga aagaaataaa tttgaaaata tatataaaga tggagattct 8220
ataagtcaag ttgtagaaag agctgtaagc gaaaagaaac ttacatttgg attaaacggt 8280
aaaggattat atgttccaga aaacggagaa ccccgactaa aaggttatgc ttctattata 8340
gaaagaataa ctctggattt aatggaaata tattctatta aaggacttaa tgatatacct 8400
agagatataa aatttaatat ggaaaaaata agacaagaaa gatacaacca aatgaaagaa 8460
gctctaaata gtgttgaagg ttataaagga aaaattgtag cctcagactc agattggtgt 8520
ttcaaagatc ctcaaggcaa tagaataaca gattttgata gtattaataa agaattaggt 8580
cttggtagaa gagatgtaaa attagataaa ggtcatgatg atttaattaa attatgtact 8640
gaaaaaatag atagtatgaa taatctacag aatggaaaat gtgtataata aaatgactta 8700
taggtcaaaa gtgtaaaata gacttaaaat ataaaaaaat agacttgtta tacagagttg 8760
atataaaact gaaaaaaaat aacctaatat ataaaaacaa aaaaacagaa aaaaatggag 8820
acgtactaat cttccaataa tctcactttt agtactctcc cctatgttat gtgt 8874
<210> 2
<2I1> 863
<212> PRT
<213> ICluyveromyces lactis
<400> 2
6/ 16
CA 02479908 2004-09-20
Ala Val Cys Tyr Asp Thr Asp Gln Val Pro Leu Asn Ile Phe Phe Gly
1 5 10 15
Tyr Asn Arg Ala Asp Lys Thr Asp Ser Asn Lys Asn Met Ala Leu Asn
20 25 30
Ile Ph a Asn Val Phe Arg Gly Phe Leu Ala Gly Glu Gly Gly Glu Ser
35 40 45
Phe Tyr Asn Ser Asn Gly Asn Val Tyr Gly Phe Met Trp Val Gly Ser
50 55 60
Met Val His Asn Arg Gly Phe Lys Asp Asn Ile Leu Pro Ile Met Glu
65 70 75 80
Asn Glu Val Lys Asn Tyr Gly Ile Pro Lys Thr Leu Tyr Leu Glu Tyr
85 90 95
Asp Gly Gly Gly Asp Pro Met Lys Ser Phe Gly Ile Ile Leu Asp Thr
100 105 110
Thr Ser Arg Asp Thr Val Val Lys Ala Ala Lys Leu Trp Ser Gln Gly
115 120 125
Lys Lys Leu Asn Ser Tyr Glu Gly Ser Lys Asn Tyr Gln Ala Thr Ala
130 135 140
Cys Tyr Leu Ser Tyr Ala Tyr Arg Lys Pro Ile Val Asn Asp Asn Phe
145 150 155 160
7/16
CA 02479908 2004-09-20
Val Gly Thr Cys Asp Tyr Phe Thr Leu Glu Ser Gly Lys Thr Pro Ala
165 170 175
Asp Gln Ser Gly Ile Asn Gly Glu Ser Leu Gln Gly Tyr Asn Pro Asn
180 185 190
Leu Asp Phe Ser Lys Leu Ser Ala Gly Gln Pro Ile Cys Lys Thr Ile
195 200 205
Gly Asn Pro Pro Asn Phe Lys Pro Ser Lys Asn Ser Asp Gly Ser Cys
210 215 220
Lys Thr Tyr Lys Val Ser Ser Gly Glu Ser Cys Ser Ser Ile Ala Val
225 230 235 240
Lys Tyr Tyr Pro Leu Ser Leu Asn Asp Ile Glu Asn Tyr Asn Lys Gly
245 250 255
Asn Tyr Gly Trp Lys Gly Cys Ser Ser Leu Gln Lys Asp Tyr Asn Leu
260 265 270
Cys Val Ser Asp Gly Ser Ala Pro Arg Pro Val Ser Asn Pro Ile Ala
275 280 285
Glu Cys Gly Pro Leu Ala Pro Gly Glu Lys Tyr Asn Ala Lys Cys Pro
290 295 300
Leu Asn Ala Cys Cys Ser Glu Phe Gly Phe Cys Gly Leu Thr Lys Asp
305 310 315 320
8/16
CA 02479908 2004-09-20
Tyr Cys Asp Lys Lys Ser Ser Thr Thr Gly Ala Pro Gly Thr Asp Gly
325 330 335
Cys Phe Ser Asn Cys Gly Tyr Gly Ser Thr Ser Asn Val Lys Ser Ser
340 345 350
Thr Phe Lys Lys Ile Ala Tyr Trp Leu Asp Ala Lys Asp Lys Leu Ala
355 360 365
Met Asp Pro Lys Asn Ile Pro Asn Gly Pro Tyr Asp Ile Leu His Tyr
370 375 380
Ala Phe Val Asn Ile Asn Ser Asp Phe Ser Ile Asp Asp Ser Ala Phe
385 390 395 400
Ser Lys Ser Ala Phe Leu Lys Val Thr Ser Ser Lys Lys Ile Pro Ser
405 410 415
Phe Gly Gly Trp Asp Phe Ser Thr Ser Pro Ser Thr Tyr Thr Ile Phe
420 425 430
Arg Asn Ala Val Lys Thr Asp Gln Asn Arg Asn Thr Phe Ala Asn Asn
435 440 445
Leu Ile Asn Phe Met Asn Lys Tyr Asn Leu Asp Gly Ile Asp Leu Asp
450 455 460
Trp Glu Tyr Pro Gly Ala Pro Asp Ile Pro Asp Ile Pro Ala Asp Asp
465 470 475 480
9/ 16
CA 02479908 2004-09-20
Ser Ser Ser Gly Ser Asn Tyr Leu Thr Phe Leu Lys Leu Leu Lys Gly
485 490 495
Lys Met Pro Ser Gly Lys Thr Leu Ser Ile Ala Ile Pro Ser Ser Tyr
500 505 510
Trp Tyr Leu Lys Asn Ph a Pro Ile Ser Asp Ile Gln Asn Thr Val Asp
515 520 525
Tyr Met Val Tyr Met Thr Tyr Asp Ile His Gly Ile Trp Glu Tyr Gly
530 535 540
Lys Ala Asn Ser Tyr Ile Asn Cys His Thr Pro Arg Lys Glu Ile Glu
545 550 555 560
Asp Ala Ile Lys Met Leu Asp Lys Ala Gly Val Lys Phe Asn Lys Val
565 570 575
Phe Gly Gly Val Ala Asn Tyr Gly Arg Ser Tyr Lys Met Val Asn Thr
580 585 590
Asn Cys Tyr Asn~Tyr Gly Cys Gly Phe Gln Arg Glu Gly Gly Asn Ser
595 600 605
Arg Asp Met Thr Asn Thr Pro Gly Val Leu Ser Asp Ser Glu Ile Ile
610 615 620
Asp Ile Asp Ser Ser Asp Lys Lys Asn Asp Arg Trp Val Asp Thr Asn
625 630 635 640
10/16
CA 02479908 2004-09-20
Thr Asp Cys Ile Phe Met Lys Tyr Asp Gly Asn Ser Val Val Ser Trp
645 650 655
Pro Lys Ser Arg Tyr Asp Leu Glu Asp Met Phe Lys Asn Tyr Gly Ph a
&60 665 670
Ala Gly Thr Ser Leu Trp Ala Ala Asn Tyr Phe Lys His Asp Glu Trp
675 680 685
Lys Asn Asp Glu Asp Asp Asn Asn Asp Asp Thr Glu Asp Pro Phe Asp
690 695 700
Glu Glu Asn Val Tyr Phe Asp Val Tyr Asp Cys Lys Asn Lys Ala Gly
705 710 715 720
Tyr Asp Leu Asp Asn Pro Val Tyr Gly Cys Arg Leu Glu Thr Ala Ile
725 730 735
Asn Ile Ile Ile Trp Asn Gly Thr Glu Ser Val Asn Thr Va1 Leu Asn
740 745 750
Ile Leu Asn Asp Tyr Asp Asn Tyr Ile Lys Tyr Tyr Glu Ala Leu Thr
755 760 765
Arg Ala His Tyr Asp Ser Val Met Glu Lys Tyr Glu Lys Trp Leu Phe
770 775 780
Glu Glu Asp Gly Tyr Tyr Thr Tyr Tyr Thr Asp Val Asp Gly Asp Asp
785 790 795 800
1 I/16
CA 02479908 2004-09-20
Ile Ile Ile Thr Pro Pro Asp Lys Lys Lys Arg Asp Tyr Ile Gln Glu
805 810 815
Lys Tyr Ser Phe Glu Lys Glu Phe Met Met Ser Gln Asn Met Thr Glu
820 825 830
Leu Thr Glu Ile Lys Val Asn Lys Thr Ile Asn Phe Met Leu Asn Gly
835 840 845
Thr Ser Leu Ala Val Lys Glu Tyr Asn Asn Glu Lys Val Leu Tyr
850 855 860
<210> 3
<211> 252
<212> PRT
<213> Kluyveromyces lactis
<400> 3
Gly Asp Ile Pro Pro Pro Gly Ser Asn Asn Arg Leu Ile Arg Asn Ser
1 5 10 15
Ile Ile Leu Asp Lys Asp Lys Glu Ala Ala Ile Ala Ser Phe Lys Gln
20 25 30
Tyr Ser Gly Ile Glu Leu Ser Lys Asp Ser Phe Val Gln Arg Asp Lys
35 40 45
Asp Lys Lys Phe Asp Leu Asn Gly Lys His Tyr Thr Phe Met His Ser
50 55 60
12/16
CA 02479908 2004-09-20
Thr Ile Leu Asn Ala Ile Val Leu Phe Pro Asn Val Leu Thr Asn Ile
65 70 75 80
Asp Ser Asp Tyr Ile His His Ile Ser Asp Leu Ile Glu Gln Ala His
85 90 95
Asn Ser Leu Gly Asn Glu Ser Pro Asp Asn Ile Tyr Glu Val Leu Glu
100 105 110
Ser Val Val Val Phe Met Ser Val Ser Glu Ile Ala Asp Tyr Thr Tyr
115 120 125
Thr Glu Gly Lys Lys Ile Lys Glu Lys Tyr Asp Lys Met Lys Lys Thr
130 135 140
Met Ile Val Gly Ile Ile Leu Gly Ile Ile Gly Gly Leu Ser Leu Phe
145 150 155 160
Leu Gly Pro Ile Gly Ile Ala Thr Ser Val Leu Ala Asp Phe Ala Leu
165 170 175
Leu Gly Ala Asp Ala Ala Ile Asn Gly Glu Leu Asn Pro Ser Asp Leu
180 185 190
Ala Phe Ala Leu Ala Gly Leu Phe Leu Pro Val Phe Ala Ser Leu Gly
195 200 205
Lys Thr Ph a Lys Phe Ala Glu Ala Leu Gln Lys Ile Asn Ile Asn Lys
210 215 220
13/16
CA 02479908 2004-09-20
Ser Lys Asn Phe Asp Asn Leu Asn GIu Phe Glu Lys Ile Arg Phe Phe
225 230 235 240
Arg Ser Lys Leu Gly Lys Val Lys Met Cys Gly Ser
245 250
<210> 4
<211> 249
<212> PRT
<213> Kluyveromyces lactis
<400> 4
Met Lys Ile Tyr His Ile Phe Ser Val Cys Tyr Leu Ile Thr Leu Cys
1 5 10 15
Ala Ala Ala Ala Thr Thr Ala Arg Glu Glu Phe Phe Leu Cys Tyr Asp
20 25 30
Leu Ile Arg Tyr Leu Lys Gln Tyr Glu Lys Thr Gly Glu Ser Lys Leu
35 40 45
Val Glu Gln Thr Phe Phe Asn Ser Ile Lys Asn Leu Asp Ile Asn Ser
50 55 60
Arg Glu Tyr Met Glu Leu Val Tyr Asn Lys Ile Ala Gly Ile Ser Asn
65 70 75 80
Glu Arg Asn Lys Phe Glu ASII Ile Tyr Lys Asp Gly Asp Ser Ile Ser
14/16
CA 02479908 2004-09-20
85 90 95
Gln Val Val Glu Arg Ala Val Ser Glu Lys Lys Leu Thr Phe Gly Leu
100 105 110
Asn Gly Lys Gly Leu Tyr Val Pro Glu Asll Gly Glu Pro Arg Leu Lys
115 120 125
Gly Tyr Ala Ser Ile Ile Glu Arg Ile Thr Leu Asp Leu Met Glu Ile
130 135 140
Tyr Ser Ile Lys Gly Leu Asn Asp Ile Pro Arg Asp Ile Lys Phe Asn
145 150 155 160
Met Glu Lys Ile Arg Gln Glu Arg Tyr Asn Gln Met Lys Glu Ala Leu
165 170 175
Asn Ser Val Glu Gly Tyr Lys Gly Lys Ile Val Ala Ser Asp Ser Asp
180 185 190
Trp Cys Phe Lys Asp Pro Gln Gly Asn Arg Ile Thr Asp Phe Asp Ser
195 200 205
Ile Asn Lys Glu Leu Gly Leu Gly Arg Arg Asp Val Lys Leu Asp Lys
210 215 220
Gly His Asp Asp Leu Ile Lys Leu Cys Thr Glu Lys Ile Asp Ser Met
225 230 235 240
Asn Asn Leu Gln Asn Gly Lys Cys Val
15/16
<IMG>
