U.S. patent application number 12/650211 was filed with the patent office on 2010-07-08 for novel notch-origin polypeptides and biomarkers and reagents using the same.
Invention is credited to MASAYASU OKOCHI, MASATOSHI TAKEDA.
Application Number | 20100173331 12/650211 |
Document ID | / |
Family ID | 30767709 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100173331 |
Kind Code |
A1 |
OKOCHI; MASAYASU ; et
al. |
July 8, 2010 |
NOVEL NOTCH-ORIGIN POLYPEPTIDES AND BIOMARKERS AND REAGENTS USING
THE SAME
Abstract
It is intended to provide extracellular markers whereby Notch
signal transduction can be detected. Polypeptides (N.beta.), which
are novel peptides originating in Notch protein and released form
cells in the step of the nuclear migration of NICH (Notch
intracellular cytoplasmic domain) due to the extracellular
digestion and the subsequent protein digestion in the membrane
during a series of the Notch protein digestion, are referred to as
markers. These peptides (N.beta.) are released from the cells in
proportion to the Notch signal depending on presenilin. By
detecting these peptides, the Notch signal transduction, cell
differentiation, cell tumorigenesis, apoptosis, Alzheimer's
disease, etc. can be monitored.
Inventors: |
OKOCHI; MASAYASU;
(SUITA-SHI, JP) ; TAKEDA; MASATOSHI; (SUITA-SHI,
JP) |
Correspondence
Address: |
Hamre, Schumann, Mueller & Larson, P.C.
P.O. Box 2902-0902
Minneapolis
MN
55402
US
|
Family ID: |
30767709 |
Appl. No.: |
12/650211 |
Filed: |
December 30, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10521591 |
Oct 19, 2005 |
7378170 |
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PCT/JP2003/009059 |
Jul 17, 2003 |
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12650211 |
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10521691 |
Aug 31, 2005 |
7666982 |
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PCT/JP03/09059 |
Jul 17, 2003 |
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10521591 |
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Current U.S.
Class: |
435/7.9 ;
436/501 |
Current CPC
Class: |
G01N 33/6893 20130101;
C07K 14/705 20130101; G01N 33/6896 20130101 |
Class at
Publication: |
435/7.9 ;
436/501 |
International
Class: |
G01N 33/542 20060101
G01N033/542; G01N 33/566 20060101 G01N033/566 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 18, 2002 |
JP |
2002-210040 |
Claims
1-21. (canceled)
22. A method for detecting a condition that exhibits abnormal Notch
signal transduction, the method comprising the steps of: detecting
a level of a polypeptide having an amino acid sequence selected
from a group consisting of SEQ ID NOS: 10 to 18 expressed in a
sample obtained from an object; comparing the level detected in the
detecting step with a level of a corresponding polypeptide detected
from a normal object; and identifying abnormality of a Notch signal
transduction when the level of the polypeptide in the sample
obtained from the object is either significantly higher or lower
than the level of the corresponding polypeptide of the normal
object.
23. The method for detecting a condition that exhibits abnormal
Notch signal transduction according claim 22, wherein the detecting
step of the level of the polypeptide is conducted by using a
reagent comprising an antibody that recognizes the polypeptide.
24. The method for detecting a condition that exhibits abnormal
Notch signal transduction according claim 23, wherein the antibody
is at least one selected from the group consisting of a monoclonal
antibody and a polyclonal antibody.
25. The method for detecting a condition that exhibits abnormal
Notch signal transduction according claim 23, wherein the reagent
further comprises a labeled antibody that recognizes the
polypeptide or a labeled antibody that recognizes as an antigen
said antibody that recognizes the polypeptide.
26. The method for detecting a condition that exhibits abnormal
Notch signal transduction according claim 25, wherein the labeled
antibody is labeled by using a fluorescent substance, an enzyme, or
a radioactive substance.
27. The method for detecting a condition that exhibits abnormal
Notch signal transduction according claim 22, wherein the method
detects a functional disorder of presenilin.
28. The method for detecting a condition that exhibits abnormal
Notch signal transduction according claim 27, wherein the
functional disorder of presenilin is at least one selected from the
group consisting of abnormal cell differentiation, tumor,
apoptosis, and Alzheimer's disease.
29. The method for detecting a condition that exhibits abnormal
Notch signal transduction according claim 22, wherein the object is
a mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of application Ser. No.
10/521,691, filed Aug. 31, 2005, which is a U.S. National Stage
application of PCT/JP03/09059, filed Jul. 17, 2003, which
applications are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to novel polypeptides derived
from novel intramembranous endoproteolysis of Notch proteins
(hereinafter also referred to collectively as "Notch") and to
biomarkers and reagents using the same. In the description of the
present invention, the following abbreviations are used for
cleavage sites of Notch: S1 for Site-1, S2 for Site-2, S3 for
Site-3, and S4 for Site-4. As will be described later, Site-4 (S4)
is a novel intramembranous cleavage site discovered by the
inventors of the present invention.
BACKGROUND ART
[0003] Notch is a type I transmembrane protein present on a cell
surface. It contains a repeated EGF-like domain in its
extracellular domain and NICD (Notch Intracellular Cytoplasmic
Domain), which is a transcription factor containing an ankyrin
repeated domain, in its intracellular domain. It has been known
that Notch plays a role in intracellular signaling relating to cell
differentiation. For example, in the developmental process of a
cranial nerve system, some of the cells derived from ectoderm
differentiate into neuronal precursor cells (stem cells) and
further into nerve cells or glial cells, during which intracellular
signaling via Notch is important. The mechanism of the
intracellular signaling via Notch is as follows. First, Notch is
expressed as a receptor on a Notch signal-receiving cell. During
the transport to the cell surface, the Notch undergoes the cleavage
at the extracellular domain (S1) by a protease such as furin, and
the two Notch fragments resulting from the S1 cleavage are held
together through an S--S bond on the cell surface. Next, when a
Notch signal-sending cell is present near the Notch
signal-receiving cell, a Notch ligand (e.g., Delta, Serrate, or
Lag-2, belonging to a DSL family) is expressed on the surface of
the Notch signal-sending cell. Under these two conditions, the
Notch ligand interacts with the Notch receptor on the cell surface,
whereby sequential proteolytic events are induced to trigger signal
transduction. More specifically, the Notch is cleaved at a site
(S2) close to the cell surface, which triggers the cleavage at a
site (S3) that is either inside the cell membrane or in close
proximity to the cell membrane inside the cell. NICD, which is the
intracellular domain of the Notch resulting from the S3 cleavage,
is released to an intracellular space and translocates to the
nucleus, where it binds to a CSL family (CPB, SuH, or Lag-1;
transcription factor) to regulate the transcription of target
genes. Presenilin, which is associated with Alzheimer's disease, is
involved in the S3 cleavage.
[0004] As described above, Notch plays an extremely important role
in intracellular signaling for cell differentiation. Moreover,
recent studies have revealed that Notch is involved not only in the
differentiation of a cranial nerve system as described above but
also in cell tumorigenesis, apoptosis, Alzheimer's disease, etc.,
which causes Notch to become a focus of attention (see Okochi et
al., "Biology of Alzheimer's disease and presenilin", Bunshi
Seishin Igaku, Vol. 1, No. 3, 2001; Kageyama et al., "Notch pathway
in neural development", Tanpakushitsu Kakusan Koso, Vol. 45, No. 3,
2000; and Brian et al., "A carboxy-terminal deletion mutant of
Notch 1 accelerates lymphoid oncogenesis in E2A-PBX1 transgenic
mice", Blood, Vol. 96, No. 5, 2000 Sep. 1, pp 1906-1913).
Therefore, the detection of Notch signal transduction is extremely
important for research and diagnosis of cell differentiation, cell
tumorigensis, apoptosis, Alzheimer's disease, etc., and the earlier
possible establishment of the technology for detecting Notch signal
transduction is being demanded.
DISCLOSURE OF INVENTION
[0005] Therefore, with the foregoing in mind, it is an object of
the present invention to provide a substance that can serve as an
extracellular secreted marker for detecting Notch signal
transduction.
[0006] The inventors of the present invention hypothesized that,
during a series of proteolytic events of Notch, a polypeptide
remaining in a cell membrane is released to an extracellular space
as a result of the cleavage occurring at S3, and decided to examine
this hypothesis. This is because, if the polypeptide remaining in
the cell membrane is released to an extracellular space, it can
serve as a marker for Notch signal transduction. Through a series
of studies on Notch signal transduction, the inventors of the
present invention found out that a fourth cleavage occurs at a site
(in the transmembrane domain) different from the S3 cleavage site
and a polypeptide resulting from this fourth cleavage is released
to an extracellular space. Based on this finding, the inventors
arrived at the present invention.
[0007] That is, the novel polypeptide according to the present
invention is a polypeptide derived from a Notch protein. In a
series of proteolytic events of the Notch protein, the polypeptide
is released to an extracellular space when NICD (Notch
intracellular cytoplasmic domain) translocates to a nucleus as a
result of the intramembranous endoproteolysis that occurs
subsequent to the extracellular proteolysis. This polypeptide can
be detected by using an antibody or the like, and thus can be used
as a marker for detecting Notch signal transduction. Furthermore,
since Notch signal transduction is involved in cell
differentiation, cell tumorigensis, Alzheimer's disease, apoptosis,
etc., the novel polypeptide according to the present invention also
can be used as a marker for detecting them. Moreover, as will be
described later, there are several types of novel polypeptide
according to the present invention with their C-termini being
different from each other. Hereinafter, the novel polypeptide
according to the present invention is referred to also as
"Notch-.beta. (N.beta.)". Also, the above-described intramembranous
endoproteolysis is not limited to that occurring in a cell membrane
but includes that occurring in an organelle membrane.
BRIEF DESCRIPTION OF DRAWINGS
[0008] FIG. 1A is a schematic illustration of structures of
N.DELTA.E, FLAG-NEXT (F-NEXT), and NICD. FIGS. 1B and 1C are
electrophoretograms showing an example of the production of
FLAG-tagged novel polypeptides (N.beta.s) according to the present
invention.
[0009] FIGS. 2A and 2B are electrophoretograms showing an example
of the production of novel polypeptides (N.beta.s) according to the
present invention.
[0010] FIG. 3A is a chart showing the result of mass spectroscopy
with regard to a group of novel polypeptides according to the
present invention.
[0011] FIG. 3B shows a major site of a novel cleavage (S4 cleavage)
of a Notch protein and major cleavage sites of an Alzheimer's
disease (3-amyloid precursor protein (h.beta.APP).
[0012] FIG. 4A shows an example of amino acid sequences of the
novel polypeptides as a principle part of the present invention.
FIG. 4B is a view showing the comparison between intramembranous
amino acid sequences of Notch-1 to Notch-4 and that of
h.beta.APP.
[0013] FIGS. 5A and 5B are electrophoretograms showing an example
of the effect of inhibition of presenilin (PS) function upon
extracellular release of novel polypeptides (N.beta.s) according to
the present invention.
[0014] FIG. 6A is a chart showing the result of mass spectroscopy,
which shows an example of the effect of Alzheimer's disease
pathogenic presenilin mutants upon N.beta. release. FIG. 6B shows
N.beta. species whose secretion is relatively increased by the
effect of Alzheimer's disease pathogenic presenilin mutants. FIG.
6C shows the result of a semiquantitative analysis of the relative
increase of their secretion.
[0015] FIG. 7 is a schematic illustration of an example of
extracellular release of novel polypeptides (N.beta.s) according to
the present invention and illustrates the C-terminus of the
released peptide is changed by Alzheimer's disease pathogenic
presenilin mutants.
[0016] FIG. 8A illustrates how cleavages occur in transmembrane
domains of Notch-1 and .beta.APP. FIG. 8B is a schema specifically
illustrating F-NEXT V1744G and F-NEXT V1744L mutants. FIG. 8C is an
electrophoretogram showing an example of inhibition of NICD
production caused by mutating V1744. FIG. 8D is an
electrophoretogram showing an example of F-N.beta. secretion in the
corresponding cell culture media. FIG. 8E shows the result of the
measurement of S3 and S4 cleavage efficiencies in the cells.
[0017] FIGS. 9A, 9B, and 9C are charts showing the result of mass
spectroscopy with regard to F-N.beta. peptides released from
wild-type F-NEXT, F-NEXT V1744G mutant, and F-NEXT V1744L mutant,
respectively.
[0018] FIG. 10A is a schema specifically illustrating a S4 cleavage
site mutant prepared in an example of the present invention. FIGS.
10B and 10C are examples of electrophretograms showing molecular
weights of F-N.beta.s released from wild-type F-NEXT, F-NEXT
G1730-1733 mutant, and F-NEXT L1730-1733 mutant, respectively. FIG.
10D shows the result of the measurement of S3 and S4 cleavage
efficiencies in the cells.
[0019] FIGS. 11A and 11B are charts showing the result of mass
spectroscopy with regard to F-N.beta. peptides released from F-NEXT
G1730-1733 mutant and F-NEXT L1730-1733 mutant, respectively.
BEST MODE FOR CARRYING OUT THE INVENTION
[0020] Hereinafter, the present invention will be described further
in detail.
[0021] A polypeptide according to the present invention is released
to an extracellular space in proportion to Notch signal
transduction. Besides, novel proteolysis that occurs immediately
before the release of the polypeptide to the extracellular space is
presenilin dependent, and inhibition of the presenilin function
causes a decrease in the release of the polypeptide of the present
invention.
[0022] The novel polypeptide according to the present invention is
produced and released as a result of the proteolysis (S4 cleavage)
of a Notch protein that occurs simultaneously with or either before
or after the proteolysis of the Notch protein at a S3 cleavage
site. The proteolysis (S4 cleavage) occurs on a N-terminal side
with respect to the S3 cleavage site in a transmembrane domain of
the Notch protein.
[0023] The novel polypeptide (N.beta.) according to the present
invention is a polypeptide including an amino acid sequence
selected from SEQ ID NOS: 1 to 18. In these SEQ ID NOS: 1 to 18,
SEQ ID NOS: 1 to 9 represent murine amino acid sequences, while SEQ
ID NOS: 10 to 18 represent human amino acid sequences. In the amino
acid sequences represented by the SEQ ID NOS: 1 to 18, one or
several of the amino acids may be deleted, substituted, or
inserted. Polypeptides represented by such amino acid sequences
also are derived from Notch proteins, and are released to an
extracellular space when NICD translocates to a nucleus as a result
of intramembranous endoproteolysis that occurs subsequent to
extracellular proteolysis in a series of proteolytic events of the
Notch proteins. These polypeptides also are released to an
extracellular space in proportion to a Notch signal in a
presenilin-dependent manner. It is to be noted that the novel
polypeptide according to the present invention may be derived from
a living organism or may be synthesized artificially. The living
organism is not limited to a particular type, and may be, for
instance, a human, a mouse, a rat, a rabbit, a goat, a swine, a
bovine, a drosophila, or a nematode. Also, the type of tissue or
cell from which the novel polypeptide of the present invention is
derived is not particularly limited. More specifically, somatic
cells and tissues, such as nerve, marrow, and cancer cells and
tissues, may be the source of the polypeptide of the present
invention, regardless of whether undifferentiated or
differentiated.
[0024] A biomarker according to the present invention contains the
above-described polypeptide of the present invention. The biomarker
of the present invention can be used for detecting Notch signal
transduction, cell differentiation, tumor, apoptosis, Alzheimer's
disease, or the like. The biomarker of the present invention
further may contain other components, or alternatively, it may be
the novel polypeptide itself (i.e., the biomarker may contain the
novel polypeptide alone). This biomarker can be detected using a
reagent containing an antibody that can recognize the novel
polypeptide. The antibody that can recognize the novel polypeptide
can be prepared by an ordinary method, and may be a monoclonal
antibody or a polyclonal antibody. In addition to the antibody that
can recognize the novel polypeptide, the reagent further may
contain a labeled antibody against this antibody or a labeled
antibody that can recognize the novel polypeptide. The labeling can
be achieved, for example, by using a fluorescent substance, an
enzyme (e.g., an enzyme that acts on a substrate that develops
color when reacting with the enzyme), a radioactive substance, or a
carrier such as agarose.
[0025] A gene according to the present invention is a gene encoding
the novel polypeptide of the present invention, and may be DNA or
RNA. A vector according to the present invention is a vector
containing the above-described gene, and a transformant according
to the present invention is a transformant transformed with the
above-described vector.
[0026] Next, an example of the extracellular release of the novel
polypeptide according to the present invention will be described
with reference to the left region of FIG. 7. It is to be noted that
the right region of FIG. 7 shows an example of the extracellular
release of amyloid-.beta. (A.beta.) in Alzheimer's disease. As
shown in the left region of FIG. 7, the amino terminus of NEXT
(Notch Extracellular Truncation) is produced as a result of
extracellular cleavage by TACE (TNF.beta.-Converting Enzyme). The
NEXT resulting from the S2 cleavage then undergoes S3 cleavage, and
NICD resulting from the S3 cleavage translocates to the nucleus.
Cleavage at S4 (the fourth cleavage site of Notch newly discovered
by the inventors of the present invention) occurs simultaneously
with or either before or after the S3 cleavage, so that N.beta. (a
novel polypeptide according to the present invention) is released
to an extracellular space.
[0027] Next, an example of C-terminus amino acid sequences of novel
polypeptides of the present invention will be described with
reference to FIG. 4B. FIG. 4B shows sequences near the C-termini of
N.beta.s or fragments released to an extracellular space with
regard to 4 types of murine Notch (mNotch-1 to mNotch-4), 4 types
of human Notch (hNotch-1 to hNotch-4), and h.beta.APP. As shown in
FIG. 4B, the major S4 cleavage site resides a few amino acid
residues closer to the N-terminus with respect to the center of
putative transmembrane domain (TM) (indicated by the triangular
arrowhead on the left in the drawing). Furthermore, as shown in
FIG. 4B, amino acid sequences around the major cleavage site are
not conserved in mNotch-1 to mNotch-4, though valine 1743 as the S3
cleavage site is conserved (indicated by the triangular arrowhead
on the right in the drawing). Thus, the S4 cleavage site is
characterized by its diversity, unlike the S3 cleavage. It is
speculated that this diversity might reflect the peculiarity of the
mechanism by which S4 secretase recognizes the sequence of the
cleavage site.
EXAMPLES
[0028] Hereinafter, the present invention will be described by way
of examples. Reagents, materials, and experimental procedures used
in the respective examples are as follows.
[0029] (Reagent)
[0030] A .gamma.-Secretase inhibitor,
[(2R,4R,5S)-2-Benzyl-5-(Boc-amino)-4-hydroxy-6-phenyl-hexanoyl]-Leu-Phe-N-
H2, was purchased from Bachem.
[0031] (Plasmids)
[0032] cDNAs encoding Notch .DELTA.E-M1727V (N.DELTA.E) and NICD
with C-terminal 6.times. c-myc tag inserted in pcDNA3 hygro were
prepared in the manner described in Schroeter et al. (Schroeter, E.
H., Kisslinger, J. A., Kopan, R. (1998), "Notch-1 signalling
requires ligand-induced proteolytic release of intracellular
domain", Nature, 393, 382-386). The cDNAs were gift from Dr. R.
Kopan. N-terminally FLAG-tagged NEXT, i.e., FLAG-NEXT (F-NEXT), was
prepared by 2-step site-directed mutagenesis. In the first step,
F-NEXT M1727V was produced using the ExSite PCR-Based Site-Directed
Mutagenesis Kit (Stratagene). N.DELTA.E was used as a template, and
the following two primers 1 and 2 (SEQ ID NO: 19 and SEQ ID NO: 20)
were prepared.
TABLE-US-00001 Primer 1:
5-P-ATCGTCGTCCTTGTAGTCTCTCAAGCCTCTTGCGCCGAGCGCGGGC AGCAGCGTTAG-3'
Primer 2: 5-P-GACAAGATGGTGATGAAGAGTGAGCCGGTGGAGCCTCCGCTGCCCT
CGCAGCTG-3'
[0033] In the second step, F-NEXT was prepared by site-directed
mutagenesis using Quick Change Site-Directed Mutagenesis Kit
(Stratagene). The F-NEXT M1727V was used as a template, and the
following two primers 3 and 4 (SEQ ID NO: 21 and SEQ ID NO: 22)
were prepared.
TABLE-US-00002 Primer 3: 5-CCTCGCAGCTGCACCTCATGTACGTGGCAGCG-3'
Primer 4: 5-CGCTGCCACGTACATGAGGTGCAGCTGCGAGG-3'
[0034] Each mutant was sequenced to verify successful
mutagenesis.
[0035] (Antibodies)
[0036] The polyclonal antibody (L652) is an antibody against a
polypeptide with the amino acid sequence from V 1722 to G 1743 of
human Notch-1 (i.e., the sequence between S2 and S3). The antibody
(L652) was produced in the following manner. First, the
above-described polypeptide serving as an antigen was provided.
This polypeptide is characterized in that it contains a lot of
hydrophobic amino acids. On this account, the antibody was produced
in the same manner as that used for producing an antibody against
the Alzheimer's disease amyloid .beta.-protein. More specifically,
the antibody was produced in the following manner. The polypeptide
was dissolved in water directly without being conjugated with any
carrier protein. After addition of the same volume of 2.times.
phosphate buffer, the polypeptide was emulsified with adjuvant and
injected into rabbits (Wild-Bode, C., Yamazaki, T., Capell, A.,
Leimer, U., Steiner, H., Ihara, Y., Haass, C. (1997),
"Intracellular generation and accumulation of amyloid beta-peptide
terminating at amino acid 42", J Biol Chem 272, 16085-16088). A
monoclonal antibody (9E10) against c-myc and a reagent (M2-agarose)
in which a monoclonal antibody against FLAG is covalently bound to
agarose were obtained commercially.
[0037] (Cell Cultures and Cell Lines)
[0038] Human embryonic kidney 293 (K293), N2a and COS cells were
cultured in DMEM supplemented with 10% fetal bovine serum, 1%
penicillin/streptomycin, and 200 .mu.g/ml zeocin (to select for PS1
expression), and/or 100 .mu.g/ml hygromycin (to select for
N.DELTA.E and F-NEXT expression). The K293 can stably express
wild-type PS1, PS1 L286V, or PS1 D385N (Okochi et al, 2000, Kulic
et al, 2000, Wolfe et al., 1999). The transfection with N.DELTA.E
or F-NEXT was performed by means of a product named Lipofectamine
2000 (Invitrogen).
[0039] (Pulse-Chase)
[0040] To determine N.DELTA.E N-terminal fragment (NTF: N.beta.)
release from N.DELTA.E expressing cells, K293 cells stably
transfected with N.DELTA.E or NICD were grown to confluence in a 10
cm dish. The cells were then metabolically pulse-labeled for 2
hours with 300 .mu.Ci [.sup.3H] amino acids (tritiated amino acid
mixture, Amersham) in Earle's Balanced Salt Solution supplemented
with MEM Vitamine Solution (Gibco) and several cold amino acids,
followed by a 6-hour chase by 10% FCS/DMEM. To examine N.beta.
release, cells expressing F-NEXT were, at first, starved of
methinine for 40 min with methionine-free media and then
pulse-labeled for 1 hour with 400 .mu.Ci [.sup.35S] amino acid
mixture (Redivue Promix, Amersham) in methionine-free DMEM,
followed by chasing for various time periods with the chase media
containing 10% FCS/DMEM supplemented with excess cold
methionine.
[0041] (Immunoprecipitation/SDS-PAGE)
[0042] At the End of the Respective Chase Periods, the Media were
Collected and put on ice immediately, followed by centrifugation at
3000.times.g to exclude cell debris. Next, a protease inhibitor
cocktail (1:1000; Sigma) and 0.025% of sodium azide were added. The
thus-obtained samples were immunoprecipitated with L652 or
M2-agarose (Sigma) overnight and then washed three times with RIPA
buffer containing 0.1% SDS, 0.5% deoxycholic acid, and 1%
TritonX-100, followed by SDS-PAGE using Tris-Tricine 10% to 20%
gradient gel (Invitrogen). The cells were scraped in ice-cold PBS,
and then harvested by means of 1500.times.g centrifugation,
followed by lysation with 100 .mu.l of 10.times.RIPA. 900 .mu.l of
PBS with a protease inhibitor mix (1:500; Sigma) was then added to
the lysed cells. The insoluble fraction was separated by
15000.times.g centrifugation and the resultant supernatant was used
for immunoprecipitation. The samples for immunoprecipitation were
pretreated by protein A sepharose (Sigma) and immunoprecipitated
with 9E10 or M2 agarose. Next, the washed protein samples were
separated by 8% or Tris-Tricine SDS-PAGE. After fixation, the gel
was shaken in Amplify Fluorographic Reagent (Amersham), dried, and
autoradiographed.
[0043] (Immunoprecipitation/MALDI-TOF MS Analysis)
[0044] After cells stably expressing the F-NEXT and their
derivatives were grown to confluence in a 20 cm dish, the culture
media were replaced with fresh 10% FCS/DMEM. After the cells with
the fresh conditioned media were cultured for 3 hours in a CO.sub.2
incubator, the culture media were collected and immediately put on
ice and centrifuged to eliminate cell debris. After supplementation
with a protease inhibitor mix (1:1000) and 0.025% sodium azide, the
media were immunoprecipitated with M2-agarose for 4 hours at
4.degree. C. The samples were then washed three times for 10 min at
4.degree. C. with an MS wash buffer containing 0.1%
n-octylglucoside, 140 mM NaCl, 10 mM Tris (pH 8.0), and 0.025%
sodium azide. The samples were then washed once again with 10 mM
Tris (pH 8.0) containing 0.025% sodium azide. Peptides bound to the
resultant precipitates were eluted with TFA/Acetonitrile/Water
(TFA:acetonitrile:water=1:20:20) saturated with .alpha.-cyano-4
hydroxy cinnamic acid. The solubilized samples were dried on a
stainless plate and subjected to a MALDI-TOF MS analysis. MS peaks
were calibrated using angiotensin (Sigma) and insulin .beta.-chain
(Sigma).
Example 1
Detection of N-Terminal Fragment (NTF; F-N.beta.) of FLAG-NEXT
(F-NEXT) in Culture Media
[0045] FIG. 1A is a schematic illustration of structures of
N.DELTA.E, NICD, and F-NEXT. As shown in FIG. 1A, in F-NEXT, a
signal peptide and also a FLAG sequence and two methionines
subsequent to the signal peptide are inserted into the N-terminus
of NEXT. The 1727th amino acid residue was not mutated in the
F-NEXT. However, in N.DELTA.E (murine Notch-1 (mNotch-1)),
methionine 1727 was artificially mutated to valine, as indicated by
the inverse triangle in FIG. 1A (Schroeter, E. H., Kisslinger, J.
A., Kopan, R. (1998), "Notch-1 signalling requires ligand-induced
proteolytic release of intracellular domain", Nature, 393,
382-386). The triangular arrowhead indicates a S3 cleavage
site.
[0046] Cells stably expressing N.DELTA.E or F-NEXT were
pulse-labeled for 1 hour with [.sup.35S] and chased for the time
period indicated in FIG. 1B. The resultant cell lysates were
immunoprecipitated with 9E10 and analyzed by 8% SDS-PAGE. As shown
in the upper panel of FIG. 1B, proteolysis of N.DELTA.E (the middle
region of the panel) and F-NEXT (the right region of the panel) was
observed after a 2-hour chase, which resulted in NICD bands
migrating faster than those of N.DELTA.E and F-NEXT. With regard to
the NICD production efficiency, there was no difference between the
cells expressing N.DELTA.E and the cells expressing F-NEXT.
[0047] Next, the culture media were immunoprecipitated with
M2-agarose and analyzed by 8% SDS-PAGE. As shown in the lower panel
of FIG. 1B, a band of F-N.beta.s (an aggregate of novel polypeptide
groups according to the present invention) of about 4 kDa was
identified only in the 2-hour chased media of the cells stably
expressing F-NEXT. The result indicates an entirely new finding
that, during the NICD production, an amino terminal fragment on the
side opposite to the NICD is secreted into an extracellular
space.
[0048] F-NEXT expressing cells were pulse-labeled with [.sup.35S]
for 1 hour and chased for the time periods indicated in FIG. 1C.
F-N.beta.s in the media and the lysates were examined by the
above-described experimental procedures. As shown FIG. 1C,
accumulation of F-N.beta.s (an aggregate of novel polypeptide
groups according to the present invention) in accordance with the
extension of chase period was observed in the media, but was hardly
detectable in the cell lysates. However, with longer exposure of a
film when taking a picture of electrophoresis gel, a F-N.beta. band
with the same molecular weight (hereinafter referred to as "MW") as
in the media was also detectable in the lysates (data not
shown).
[0049] The results shown in FIGS. 1B and 1C were reproduced when
F-NEXT M1727V mutant was used or when CHO, COS, and N2a were used
as the expressing cells (data not shown).
Example 2
Detection of N-Terminal Fragment (NTF: N.beta.) of N.DELTA.A in
Culture Media
[0050] K293 cells stably expressing N.DELTA.E or NICD were
pulse-labeled with [.sup.3H] for 2 hours and chased for 6 hours.
Chased media and cell lysates were immunoprecipitated with an
antibody L652 against N.DELTA.E, and the thus-obtained samples were
separated by Tris-Tricine SDS-PAGE. As shown in FIG. 2A, a
N.DELTA.E NTF band (indicated by the triangular arrowhead) of MW 3
to 4 kDa was detected in the culture media of the N.DELTA.E cells,
but not from the culture media or cell lysate of the NICD cells.
Thus, it was considered that the band shown in FIG. 2A was of
wild-type N.beta.s that were not FLAG-tagged.
[0051] The same media and lysates as in the above were
immunoprecipitated with an anti-c-myc antibody (9E10). As shown in
the lower panel of in FIG. 2B, about 100 kDa bands of N.DELTA.E and
NICD were detected in the lysates (indicated by the triangular
arrowhead), but not in the media. This result suggests that
N.DELTA.E and NICD were expressed in the respective cells at
substantially the same rate.
Example 3
Identification of C-Termini of N.beta.s Released to Culture
Media
[0052] FIG. 3B is a schematic illustration of intramembranous
cleavage of murine Notch-1 (mNotch-1) and human .beta.APP
(h.beta.APP). As a result of the intramembranous cleavage of
mNotch-1, NICD and N.beta. are produced. In the present example,
N.beta. secretion and a novel cleavage site at the C-terminus of
N.beta. were confirmed. On the other hand, as a result of the
intramembranous cleavage of h.beta.APP, an intracellular fragment
CTF.gamma.50 (Sastre, M., Steiner, H., Fuchs, K., Capell, A.,
Multhaup, G., Condron, M. M., Teplow, D. B., Haass, C. (2001),
"Presenilin-dependent gamma-secretase processing of beta-amyloid
precursor protein at a site corresponding to the S3 cleavage of
Notch", EMBO Rep. 2, 835-841.) and several types of A.beta.
fragments are produced.
[0053] Culture media of cells stably expressing F-NEXT were
immunoprecipitated with M2-agarose, and MW of N.beta.s were
analyzed by means of MALDI-TOF MS according to the above-described
experimental procedures. The result is shown in the large graph
shown in FIG. 3A. As shown in the graph, multiple peaks were
observed around MW 4000, but no significant peaks of MW more than
4500 were identified. The small graph shown in FIG. 3A shows the
details of the peaks from MW 3000 to 4500. The same major peaks
were identified when CHO, COS and N2a were used as host cells (data
not shown). These peaks also were identified when transfected with
F-NEXT M1727V mutant (data not shown).
[0054] FIG. 4A shows a list of amino acid sequences of N.beta.s
corresponding to the MALDI-TOF MS peaks shown in the small graph of
FIG. 3A. The C-terminus of the major N.beta. species was alanine
1731. Bold letters indicate an amino acid sequence of the major
peak. As shown in FIG. 4A, no peaks of MW around 5060,
corresponding to a S3 cleavage site, were identified. From these
results, it can be concluded that N.beta.s are released to an
extracellular space and the cleavage site of the proteolysis
occurring just before the N.beta. release is a novel fourth
cleavage site (S4) that is different from the conventionally
reported three cleavage sites (S1, S2, and S3).
[0055] FIG. 4B shows a list of amino acid sequences of
transmembrane domains of human (h) and murine (m) Notch-1 to
Notch-4. S1, S2, and S3 cleavages are phenomena common to Notch-1
to Notch-4, and they serve as a common signal transduction
mechanism through which Notch proteins, whatever their species,
achieve signal transduction. From these facts, it is speculated
that S4 cleavage also is a phenomenon common to Notch proteins of
all types. As shown in FIG. 4B, the S4 cleavage site is conserved
partially, similarly to the S3 cleavage site. From this fact, it is
speculated that S4 cleavage is a phenomenon common to Notch-1 to
Notch-4.
Example 4
Confirmation of Presenilin (PS) Function Dependence of
Extracellular Release of N.beta.
[0056] Cells expressing wild-type PS1 or PS1 D385N that is a PS1
dominant negative mutant obtained by artificially causing loss of
presenilin function were stably transfected with F-NEXT. An hour
pulse with [.sup.35S] and then a 2-hour chase were performed, and
the resulting culture media and lysates were analyzed to determine
an N.beta. release level from the cells expressing both the PS1
derivative and F-NEXT at the same time. First, the chased media
were immunoprecipitated with M2-agarose to detect N.beta. release.
As shown in the upper panel of FIG. 5A, N.beta. release from the
PS1 D385N expressing cells decreased drastically as compared with
the case of the wild-type PS1 expressing cells. That is, it was
confirmed that the S4 cleavage efficiency decreases drastically in
the cells expressing the mutant obtained by artificially causing
loss of presenilin function. Also, the lysates collected at the
same time with the culture media were immunoprecipitated with 9E10.
As shown in the lower panel of FIG. 5A, NICD band after the 2-hour
chase was hardly visible in the PS1 D385N expressing cells. That
is, the report that the S3 cleavage efficiency decreases
drastically in the cells expressing the mutant obtained by
artificially causing loss of presenilin function was reproduced at
the same time.
[0057] Next, cells stably expressing F-NEXT were pulse-labeled for
1 hour and chased for 2 hours with or without a .gamma.-secretase
inhibitor (L685,458) that is designed to bind the active center of
presenilin. More specifically, 1 .mu.M of L685,458 was added to the
culture media 2 hours before methionine starvation. During the
pulse-chase period, every medium used contained the same
concentration of L685,458. The chased media were immunoprecipitated
with M2-agarose to detect release. As shown in the upper panel of
FIG. 5B, N.beta. release from the cells treated with the
.gamma.-secretase inhibitor decreased drastically. Also, the
corresponding lysates were immunoprecipitated with 9E10. As shown
in the lower panel of FIG. 5B, the NICD band after the 2-hour chase
period was hardly visible due to inhibition of S3 cleavage. From
these results, it can be said that the N.beta. release to an
extracellular space is caused by presenilin-dependent proteolysis,
and hence, inhibition of the presenilin function results in the
inhibition of S4 cleavage and N.beta. release that occurs
subsequent to the S4 cleavage.
Example 5
Effect of Presenilin (PS) Mutant Associated with Familial
Alzheimer's Disease (FAD) Upon S4 Cleavage
[0058] Heretofore, various studies have been made on PS mutation
associated with FAD, and an increase in A.beta. secretion has been
confirmed in every type of FAD pathogenic PS mutant. In the present
example, it was confirmed that PS dependent S4 proteolysis also
relates to PS mutation associated with FAD.
[0059] K293 cells expressing wild-type (wt) PS1 or PS1 mutants
associated with FAD, namely, PS1 C92S, PS1 L166P, and PS1 L286V,
were stably transfected with F-NEXT. Then, the culture media of the
cells expressing PS1 derivatives and F-NEXT were analyzed by
MALDI-TOF MS, in order to examine the change in C-termini of
F-N.beta.s. As shown in FIG. 6A, in contrast to the cells
expressing wild-type PS1, characteristic change in a proteolysis
pattern of C-termini of N.beta.s was observed in the cells
expressing PS1 mutations associated with FAD. In particular, the
cells expressing the PS1 L166P mutation causing a significant
increase in A.beta.42 production demonstrated a tendency to
elongate F-N.beta. peptides, and an increase in the production of
F-N.beta. species (F-N.beta. 1733 and F-N.beta. 1735) that were
longer than F-N.beta.1731 by 2 and 4 amino acid residues,
respectively, was confirmed (see FIG. 6B). Furthermore, as shown in
FIG. 6A, an increase in F-N.beta. 1734 level was observed in the
PS1 C92S cells, whereas an increase in F-N.beta. 1735 level and a
decrease in F-N.beta. 1734 level were observed in the PS1 L286V
cells. These results demonstrate that FAD pathogenic mutations
affects a pattern of the S4 cleavage site so that the S4 cleavage
site tends to shift toward the C-terminal side, thereby causing
elongation of released peptides. Similarly to A.beta.42, the
aggressive PS1 L166P mutation affects the length of F-N.beta.s most
significantly. It has been known that PS1 L166P mutation causes FAD
during the young adult years. These effects were not specific to
K293 cells, and the same effects of the PS mutations associated
with FAD also were confirmed when using Neuro 2a cells (data not
shown). Therefore, it can be said that every type of FAD pathogenic
mutation affects the C-terminus of F-N.beta. (see FIG. 6C).
Example 6
Effect of Proteolysis at S3 Upon Efficiency of Proteolysis at
S4
[0060] In order to examine the correlation between two cleavages
occurring in a cell membrane, i.e., proteolysis at S4 that produces
a Notch-.beta. peptide and proteolysis at S3 that produces NICD
determining signal transduction level, a mutant in which
proteolysis at S3 is inhibited was prepared in the present example
and it was confirmed using this mutant that there is no change in a
S4 cleavage efficiency even in the case where a S3 cleavage
efficiency is decreased artificially.
[0061] It has been reported that partial inhibition of S3 cleavage
is caused by mutating V1744 of Notch-1 that resides on a C-terminal
side with respect to a S3 cleavage site (Schroeter et al., Nature,
1998). Thus, at first, the change in a S4 cleavage activity caused
by the inhibition of S3 cleavage was examined. In order to
efficiently detect the products resulting from intramembranous
endoproteolysis, NEXT analogues were FLAG tagged at their N-termini
and myc-tagged at their C-termini. Thereafter, valine 1744 of the
plasmid expressing the analogues (F-NEXT; Okochi, 2002) was mutated
into glycine or leucine (hereinafter these mutants are referred to
as F-NEXT V1744G and F-NEXT V1744L, respectively) (FIG. 8B). An
F-NEXT expressing construct with or without S3 cleavage site
mutation was stably transfected into K293 cells constantly
expressing excessive wild-type PS1 or PS1 D385N lacking a
.gamma.-secretase function. The cells then were metabolically
labeled with .sup.35S methionine. Thereafter, newly radiolabeled
F-N.beta.s and NICD present in the cell sediments and the
corresponding culture media were detected by a method
(IP-autoradiography) combining immunoprecipitation and radiation
dosimetry performed after the separation by electrophoresis.
[0062] The cell sediments were pulsed for 30 minutes, followed by
IP-autoradiography with an anti-c-myc antibody (9E10). As a result,
F-NEXT expression was observed. The cells were then chased for 2
hours. As a result, NICD production caused by the degradation of
F-NEXT was observed, whereas NICD production was inhibited
significantly in the V1744G and V1744L mutants (the upper panel of
FIG. 8C). These results were in conformity with the conventional
reports. Even in the case where the degradation of NICD was
inhibited by adding Lactacystin as a proteasome inhibitor, the
amount of radiolabeled NICD measured after a 2-hour chase was
significantly small in the cells expressing V1744G and V1744L
mutants. The intramembranous endoproteolysis and the NICD
production caused by this F-NEXT were not at all observed in the
PS1 D385N expressing cells (the lower panel of FIG. 8C). From these
results, it can be said that the proteolysis shown in the upper
panel of FIG. 8C was caused by PS/.gamma.-secretase.
[0063] Next, the culture media after a 2-hour chase were analyzed
using an anti-FLAG antibody (M2). F-N.beta.s secreted from the
F-NEXT V1744G mutant cells and the F-NEXT V1744L mutant cells were
approximately the same level as those secreted from the wild-type
F-NEXT cells (FIG. 8D). Furthermore, F-N.beta. production was not
observed in the cells expressing PS1 D385N mutant. From these
results, it can be said that PS/.gamma.-secretase affects this
cleavage.
[0064] To further support the above-described conclusions, the S3
cleavage efficiency and the S4 cleavage efficiency were calculated.
The ratio of NICD to F-NEXT analogues in the cell sediments and the
ratio of F-N.beta.s in the culture media to the F-NEXT analogues in
the corresponding cell sediments were determined. As a result, it
was confirmed that although the V1744G mutant and the V1744L mutant
both decrease the S3 cleavage activity in contrast to the wild-type
PS1, they do not affect the S4 cleavage activity (FIG. 8E).
Example 7
Correlation Between Decrease in S3 Cleavage Efficiency and Accuracy
of S4 Cleavage
[0065] In PS1 mutants that cause Alzheimer's disease, the change in
accuracy of S4 cleavage occurs as well as a decrease in S3 cleavage
activity. If the S3 cleavage is a precondition for the S4 cleavage,
a decrease in S3 cleavage efficiency caused by a PS1 mutant should
affect the accuracy of the S4 cleavage. Thus, in the present
example, a S3 cleavage site mutant was prepared, and it was
confirmed using this mutant that the accuracy of the S4 cleavage
does not change even in the case where the S3 cleavage efficiency
is decreased artificially.
[0066] The cause of a familial Alzheimer's disease (FAD) is
considered to be that FAD pathogenic PS mutants affect the accuracy
of proteolysis by PS/.gamma.-secretase and increase the production
of A.beta.42, which is elongated A.beta.. Similarly, the FAD
pathogenic PS mutants affect the accuracy of Notch cleavage by
PS/.gamma.-secretase and increase the production of elongated
F-N.beta.. Moreover, it has been reported that some of the PS
mutants cause a decrease in S3 cleavage efficiency. Thus, the
effect of S3 mutants that cause a decrease in S3 cleavage
efficiency upon the accuracy of S4 cleavage was examined.
F-N.beta.s contained in the culture media of the cells expressing
wild-type F-NEXT, F-NEXT V1744G mutant, or F-NEXT V1744L mutant
were immunoprecipitated with M2 agarose and then analyzed by
MALDI-TOF MS. As a result, as shown in FIGS. 9B and 9C, the major
cleavage site of the F-NEXT V1744G mutant and the F-NEXT V1744L
mutant was between alanine 1731 and alanine 1732 as in the case of
the wild type, and a pattern of several minor S4 cleavage sites
located apart from each other were not at all affected by the
mutations. In other words, it was confirmed that mutations that
cause a decrease in S3 cleavage efficiency do not affect the
accuracy of S4 cleavage at all. These data suggest that FAD
pathogenic PS mutations indirectly affect the accuracy of S4
cleavage.
Example 8
Effect of Decrease in S4 Cleavage Efficiency Upon S3 Cleavage
Efficiency
[0067] Based on the assumption that S4 cleavage site mutation may
exhibit a similar effect to that of the above-described
artificially prepared S3 point mutants, the effect of a decrease in
S4 cleavage efficiency upon a S3 cleavage efficiency was examined
using F-NEXT G1730-1733 mutant and F-NEXT L1730-1733 mutant
prepared by mutating four alanine residues around the S4 cleavage
site into glycine residues and leucine residues, respectively (FIG.
10A). As a result, out of these two S4 cleavage site mutants, the
F-NEXT L1730-1733 mutant with inhibited S4 cleavage activity
exhibited a decrease in S3 cleavage efficiency. This result
suggests that there is a proteolytic pathway through which NICD is
produced by the S4 cleavage-dependent S3 proteolysis during
intramembranous endoproteolysis of Notch-1.
[0068] Next, analysis also was made with regard to the assumption
that the S4 cleavage site mutants similarly may affect the S4
cleavage. As indicated by the triangular arrowhead in FIG. 10A, a
S4 cleavage site of Notch is in the center of four sequential
alanine residues. The F-NEXT G1730-1733 mutant and the F-NEXT
L1730-1733 mutant were prepared by mutating these four sequential
alanine residues into glycine residues and leucine residues,
respectively. These mutants then were subjected to the same
pulse-chase experiment as that performed with respect to the S3
mutants. After a 2-hour chase, radiolabeled F-N.beta.s in the
culture media were analyzed. As a result, F-N.beta. secretion was
observed in the wild-type F-NEXT cells and the S4 mutated F-NEXT
cells (FIG. 10B). However, although there was substantially no
difference in the amount of F-N.beta. production between the
wild-type F-NEXT and the F-NEXT G1730-1733 mutant, the amount of
F-N.beta. production seemed to be decreased in the F-NEXT
L1730-1733 mutant as compared with the wild-type F-NEXT (FIG.
10B).
[0069] Next, production of radiolabeled NICD from F-NEXT contained
in the corresponding cell sediments was analyzed. As a result, a
similar level of NICD production to that of the wild-type F-NEXT
cells was observed in the G1730-1733 mutant cells, whereas NICD
production was decreased in the L1730-1733 mutant cells as compared
with the cells expressing wild-type F-NEXT (the upper panel of FIG.
10C). These data suggest that S3 cleavage is inhibited in the
L1730-1733 mutant.
[0070] In order to establish this result clearly, the S4 cleavage
efficiency and the S3 cleavage efficiency were calculated in the
same manner as in FIG. 8E. As a result, out of the two S4 mutants,
the G1730-1733 mutant that hardly affected the S4 activity did not
affect the S3 cleavage activity at all (FIG. 10D). In contrast, it
was confirmed that the L1730-1733 mutant with inhibited S4 cleavage
activity exhibited a decrease in S3 cleavage efficiency (FIG. 10D).
Furthermore, from the facts that the PS/.gamma.-secretase mechanism
causes cleavage at both S3 and S4 and that no intermediate
proteolysis product resulting from the S3 cleavage in close
proximity to the cell membrane and the S4 cleavage at an
approximate center of the transmembrane domain was found, it is
considered the S3 cleavage and the S4 cleavage occur substantially
at the same time. These results suggest that there is a proteolytic
pathway through which NICD is produced by the S4 cleavage-dependent
S3 proteolysis during intramembranous endoproteolysis of
Notch-1.
Example 9
Correlation Between S4 Cleavage Site and Activity
[0071] Subsequently, C-termini of F-N.beta. G1730-1733 and
F-N.beta. L1730-1733 were determined. The amount of F-N.beta.s
released from F-NEXT G1730-1733 substantially was equal to that
released from the cell expressing wild-type F-NEXT (FIG. 1B).
However, the G1730-1733 mutant did not have a S4 cleavage site
between glycine 1731 and glycine 1732, as indicated by the inverse
triangles in FIG. 11A. The major S4 cleavage sites of this mutant
shifted toward the C-terminus of four sequential glycine residues
to reside between phenylalanine 1734 and valine 1735, between
valine 1735 and leucine 1736, and between phenylalanine 1738 and
valine 1739, respectively. That is, S4 cleavage did not occur
around the glycine residues, but minor cleavage sites were present
apart from each other on the N-terminal side of the four glycine
residues, so that MW of F-N.beta.s released from the F-NEXT
G1730-1733 increased (FIG. 10B). Furthermore, as indicated by the
inverse triangles in FIG. 11B, the F-NEXT L1730-1733 mutant had a
major S4 cleavage site in the center of four sequential alanine
residues, i.e., between leucine 1731 and leucine 1732, in the
similar topology to that of the wild-type F-NEXT, and a minor
cleavage site was hardly observed. Moreover, MW of F-N.beta.s
released from the F-NEXT L1730-1733 mutant decreased (FIG.
10B).
INDUSTRIAL APPLICABILITY
[0072] As specifically described above, a novel polypeptide
according to the present invention is derived from a Notch protein.
In a series of proteolytic events of the Notch protein, the
polypeptide is released to an extracellular space when NICD
translocates to a nucleus as a result of intramembranous
endoproteolysis that occurs subsequent to extracellular
proteolysis. By using the novel polypeptide as a marker, it is
possible to detect Notch signal transduction. Also, it is possible
to detect cell differentiation, cell tumorigensis, apoptosis,
Alzheimer's disease, etc., for example.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 57 <210> SEQ ID NO 1 <211> LENGTH: 21 <212>
TYPE: PRT <213> ORGANISM: mouse <400> SEQUENCE: 1 Val
Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu 1 5 10
15 Met Tyr Val Ala Ala 20 <210> SEQ ID NO 2 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: mouse
<400> SEQUENCE: 2 Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro
Ser Gln Leu His Leu 1 5 10 15 Met <210> SEQ ID NO 3
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
mouse <400> SEQUENCE: 3 Val Lys Ser Glu Pro Val Glu Pro Pro
Leu Pro Ser Gln Leu His Leu 1 5 10 15 Met Tyr <210> SEQ ID NO
4 <211> LENGTH: 20 <212> TYPE: PRT <213>
ORGANISM: mouse <400> SEQUENCE: 4 Val Lys Ser Glu Pro Val Glu
Pro Pro Leu Pro Ser Gln Leu His Leu 1 5 10 15 Met Tyr Val Ala 20
<210> SEQ ID NO 5 <211> LENGTH: 22 <212> TYPE:
PRT <213> ORGANISM: mouse <400> SEQUENCE: 5 Val Lys Ser
Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu 1 5 10 15 Met
Tyr Val Ala Ala Ala 20 <210> SEQ ID NO 6 <211> LENGTH:
23 <212> TYPE: PRT <213> ORGANISM: mouse <400>
SEQUENCE: 6 Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu
His Leu 1 5 10 15 Met Tyr Val Ala Ala Ala Ala 20 <210> SEQ ID
NO 7 <211> LENGTH: 24 <212> TYPE: PRT <213>
ORGANISM: mouse <400> SEQUENCE: 7 Val Lys Ser Glu Pro Val Glu
Pro Pro Leu Pro Ser Gln Leu His Leu 1 5 10 15 Met Tyr Val Ala Ala
Ala Ala Phe 20 <210> SEQ ID NO 8 <211> LENGTH: 25
<212> TYPE: PRT <213> ORGANISM: mouse <400>
SEQUENCE: 8 Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu
His Leu 1 5 10 15 Met Tyr Val Ala Ala Ala Ala Phe Val 20 25
<210> SEQ ID NO 9 <211> LENGTH: 26 <212> TYPE:
PRT <213> ORGANISM: mouse <400> SEQUENCE: 9 Val Lys Ser
Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu 1 5 10 15 Met
Tyr Val Ala Ala Ala Ala Phe Val Leu 20 25 <210> SEQ ID NO 10
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
human <400> SEQUENCE: 10 Val Gln Ser Glu Thr Val Glu Pro Pro
Pro Pro Ala Gln Leu His Phe 1 5 10 15 Met <210> SEQ ID NO 11
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
human <400> SEQUENCE: 11 Val Gln Ser Glu Thr Val Glu Pro Pro
Pro Pro Ala Gln Leu His Phe 1 5 10 15 Met Tyr <210> SEQ ID NO
12 <211> LENGTH: 20 <212> TYPE: PRT <213>
ORGANISM: human <400> SEQUENCE: 12 Val Gln Ser Glu Thr Val
Glu Pro Pro Pro Pro Ala Gln Leu His Phe 1 5 10 15 Met Tyr Val Ala
20 <210> SEQ ID NO 13 <211> LENGTH: 21 <212>
TYPE: PRT <213> ORGANISM: human <400> SEQUENCE: 13 Val
Gln Ser Glu Thr Val Glu Pro Pro Pro Pro Ala Gln Leu His Phe 1 5 10
15 Met Tyr Val Ala Ala 20 <210> SEQ ID NO 14 <211>
LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 14 Val Gln Ser Glu Thr Val Glu Pro Pro Pro
Pro Ala Gln Leu His Phe 1 5 10 15 Met Tyr Val Ala Ala Ala 20
<210> SEQ ID NO 15 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: human <400> SEQUENCE: 15 Val Gln
Ser Glu Thr Val Glu Pro Pro Pro Pro Ala Gln Leu His Phe 1 5 10 15
Met Tyr Val Ala Ala Ala Ala 20 <210> SEQ ID NO 16 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 16 Val Gln Ser Glu Thr Val Glu Pro Pro Pro
Pro Ala Gln Leu His Phe 1 5 10 15 Met Tyr Val Ala Ala Ala Ala Phe
20 <210> SEQ ID NO 17 <211> LENGTH: 25 <212>
TYPE: PRT <213> ORGANISM: human <400> SEQUENCE: 17 Val
Gln Ser Glu Thr Val Glu Pro Pro Pro Pro Ala Gln Leu His Phe 1 5 10
15 Met Tyr Val Ala Ala Ala Ala Phe Val 20 25 <210> SEQ ID NO
18 <211> LENGTH: 26 <212> TYPE: PRT <213>
ORGANISM: human <400> SEQUENCE: 18 Val Gln Ser Glu Thr Val
Glu Pro Pro Pro Pro Ala Gln Leu His Phe 1 5 10 15 Met Tyr Val Ala
Ala Ala Ala Phe Val Leu 20 25 <210> SEQ ID NO 19 <211>
LENGTH: 57 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer 1 which
is derived from mouse Notch-1 gene for use in site spesific
mutagenesis. <400> SEQUENCE: 19 atcgtcgtcc ttgtagtctc
tcaagcctct tgcgccgagc gcgggcagca gcgttag 57 <210> SEQ ID NO
20 <211> LENGTH: 54 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer 2 which is derived from mouse Notch-1 gene for
use in site spesific mutagenesis. <400> SEQUENCE: 20
gacaagatgg tgatgaagag tgagccggtg gagcctccgc tgccctcgca gctg 54
<210> SEQ ID NO 21 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer 3 which is derived from mouse
Notch-1 gene for use in site spesific mutagenesis. <400>
SEQUENCE: 21 cctcgcagct gcacctcatg tacgtggcag cg 32 <210> SEQ
ID NO 22 <211> LENGTH: 32 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer 4 which is derived from mouse Notch-1 gene for
use in site spesific mutagenesis. <400> SEQUENCE: 22
cgctgccacg tacatgaggt gcagctgcga gg 32 <210> SEQ ID NO 23
<211> LENGTH: 70 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of F-NEXT which is derived from mouse
Notch-1 peptide and has FLAG sequence at N-terminal region.
<400> SEQUENCE: 23 Met Pro Arg Leu Leu Thr Pro Leu Leu Cys
Leu Thr Leu Leu Pro Ala 1 5 10 15 Arg Ala Ala Arg Gly Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met 20 25 30 Val Met Lys Ser Glu Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His 35 40 45 Leu Met Tyr Val
Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly 50 55 60 Cys Gly
Val Leu Leu Ser 65 70 <210> SEQ ID NO 24 <211> LENGTH:
31 <212> TYPE: PRT <213> ORGANISM: mouse <400>
SEQUENCE: 24 Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala Ala
Ala Phe Val 1 5 10 15 Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu
Ser Arg Lys Arg 20 25 30 <210> SEQ ID NO 25 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 25 Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu
Met Val Gly Gly Val Val 1 5 10 15 Ile Ala Thr Val Ile Val Ile Thr
Leu Val Met Leu Lys Lys Lys 20 25 30 <210> SEQ ID NO 26
<211> LENGTH: 45 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of F-NEXT which is derived from mouse
Notch-1 peptide and has FLAG sequence at N-terminal region.
<400> SEQUENCE: 26 Leu Arg Asp Tyr Lys Asp Asp Asp Asp Lys
Met Val Met Lys Ser Glu 1 5 10 15 Pro Val Glu Pro Pro Leu Pro Ser
Gln Leu His Leu Met Tyr Val Ala 20 25 30 Ala Ala Ala Phe Val Leu
Leu Phe Phe Val Gly Cys Gly 35 40 45 <210> SEQ ID NO 27
<211> LENGTH: 38 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of F-NEXT which is derived from mouse
Notch-1 peptide and has FLAG sequence at N-terminal region.
<400> SEQUENCE: 27 Leu Arg Asp Tyr Lys Asp Asp Asp Asp Lys
Met Val Met Lys Ser Glu 1 5 10 15 Pro Val Glu Pro Pro Leu Pro Ser
Gln Leu His Leu Met Tyr Val Ala 20 25 30 Ala Ala Ala Phe Val Leu 35
<210> SEQ ID NO 28 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 28 Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu 1 5 10 15 Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala 20 25
30 Ala Ala Ala Phe Val 35 <210> SEQ ID NO 29 <211>
LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Partial amino
acid sequence of F-NEXT which is derived from mouse Notch-1 peptide
and has FLAG sequence at N-terminal region. <400> SEQUENCE:
29 Leu Arg Asp Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu
1 5 10 15 Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr
Val Ala 20 25 30 Ala Ala Ala Phe 35 <210> SEQ ID NO 30
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of F-NEXT which is derived from mouse
Notch-1 peptide and has FLAG sequence at N-terminal region.
<400> SEQUENCE: 30 Leu Arg Asp Tyr Lys Asp Asp Asp Asp Lys
Met Val Met Lys Ser Glu 1 5 10 15 Pro Val Glu Pro Pro Leu Pro Ser
Gln Leu His Leu Met Tyr Val Ala 20 25 30 Ala Ala Ala 35 <210>
SEQ ID NO 31 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Partial amino acid sequence of F-NEXT which is
derived from mouse Notch-1 peptide and has FLAG sequence at
N-terminal region. <400> SEQUENCE: 31 Arg Gly Leu Arg Asp Tyr
Lys Asp Asp Asp Asp Lys Met Val Met Lys 1 5 10 15 Ser Glu Pro Val
Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr 20 25 30 Val Ala
Ala 35 <210> SEQ ID NO 32 <211> LENGTH: 33 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 32 Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu 1 5 10 15 Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala 20 25
30 Ala <210> SEQ ID NO 33 <211> LENGTH: 31 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 33 Asp Tyr Lys
Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu Pro Val 1 5 10 15 Glu
Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala 20 25 30
<210> SEQ ID NO 34 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 34 Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu 1 5 10 15 Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala 20 25
30 <210> SEQ ID NO 35 <211> LENGTH: 30 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 35 Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu 1 5 10 15 Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr 20 25 30
<210> SEQ ID NO 36 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 36 Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu 1 5 10 15 Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met 20 25 <210>
SEQ ID NO 37 <211> LENGTH: 23 <212> TYPE: PRT
<213> ORGANISM: mouse <400> SEQUENCE: 37 Leu His Leu
Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe 1 5 10 15 Val
Gly Cys Gly Val Leu Leu 20 <210> SEQ ID NO 38 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 38 Leu His Phe Met Tyr Val Ala Ala Ala Ala
Phe Val Leu Leu Phe Phe 1 5 10 15 Val Gly Cys Gly Val Leu Leu 20
<210> SEQ ID NO 39 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: mouse <400> SEQUENCE: 39 Leu Leu
Tyr Leu Leu Ala Val Ala Val Val Ile Ile Leu Phe Phe Ile 1 5 10 15
Leu Leu Gly Val Ile Met Ala 20 <210> SEQ ID NO 40 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 40 Leu Leu Tyr Leu Leu Ala Val Ala Val Val
Ile Ile Leu Phe Ile Ile 1 5 10 15 Leu Leu Gly Val Ile Met Ala 20
<210> SEQ ID NO 41 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: mouse <400> SEQUENCE: 41 Leu Leu
Pro Leu Leu Val Ala Gly Ala Val Phe Leu Leu Ile Ile Phe 1 5 10 15
Ile Leu Gly Val Met Val Ala 20 <210> SEQ ID NO 42 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 42 Leu Leu Pro Leu Leu Val Ala Gly Ala Val
Leu Leu Leu Val Ile Leu 1 5 10 15 Val Leu Gly Val Met Val Ala 20
<210> SEQ ID NO 43 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: mouse <400> SEQUENCE: 43 Ile Leu
Cys Ser Pro Val Val Gly Val Leu Leu Leu Ala Leu Gly Ala 1 5 10 15
Leu Leu Val Leu Gln Leu Ile 20 <210> SEQ ID NO 44 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 44 Val Leu Cys Ser Pro Val Ala Gly Val Ile
Leu Leu Ala Leu Gly Ala 1 5 10 15 Leu Leu Val Leu Gln Leu Ile 20
<210> SEQ ID NO 45 <211> LENGTH: 24 <212> TYPE:
PRT <213> ORGANISM: human <400> SEQUENCE: 45 Gly Ala
Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 1 5 10 15
Ile Val Ile Thr Leu Val Met Leu 20 <210> SEQ ID NO 46
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of transmembrane region of F-NEXT which
is derived from mouse Notch-1 peptide. <400> SEQUENCE: 46 Leu
His Leu Met Tyr Val Ala Ala 1 5 <210> SEQ ID NO 47
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of transmembrane region of F-NEXT which
is derived from mouse Notch-1 peptide. <400> SEQUENCE: 47 Leu
His Leu Met Tyr Val Ala Ala Ala Ala 1 5 10 <210> SEQ ID NO 48
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of transmembrane region of F-NEXT which
is derived from mouse Notch-1 peptide. <400> SEQUENCE: 48 Leu
His Leu Met Tyr Val Ala Ala Ala Ala Phe 1 5 10 <210> SEQ ID
NO 49 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Partial amino acid sequence of transmembrane region of
F-NEXT which is derived from mouse Notch-1 peptide. <400>
SEQUENCE: 49 Leu His Leu Met Tyr Val Ala Ala Ala Ala Phe Val 1 5 10
<210> SEQ ID NO 50 <211> LENGTH: 28 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
transmembrane region of F-NEXT which is derived from mouse Notch-1
peptide. <400> SEQUENCE: 50 Leu His Leu Met Tyr Val Ala Ala
Ala Ala Phe Val Leu Leu Phe Phe 1 5 10 15 Val Gly Cys Gly Val Leu
Leu Ser Arg Lys Arg Arg 20 25 <210> SEQ ID NO 51 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Partial amino
acid sequence of transmembrane region of F-NEXT which is derived
from mouse Notch-1 peptide. <400> SEQUENCE: 51 Leu His Leu
Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe 1 5 10 15 Val
Gly Cys Gly Val Leu Leu Ser 20 <210> SEQ ID NO 52 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Partial amino
acid sequence of transmembrane region of F-NEXT which is derived
from mouse Notch-1 peptide. <400> SEQUENCE: 52 Leu His Leu
Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe 1 5 10 15 Val
Gly Cys Gly Val Leu Leu Ser 20 <210> SEQ ID NO 53 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Partial amino
acid sequence of transmembrane region of F-NEXT(V1744G) which is
derived from mouse Notch-1 peptide. <400> SEQUENCE: 53 Leu
His Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe 1 5 10
15 Val Gly Cys Gly Gly Leu Leu Ser 20 <210> SEQ ID NO 54
<211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of transmembrane region of
F-NEXT(V1744L) which is derived from mouse Notch-1 peptide.
<400> SEQUENCE: 54 Leu His Leu Met Tyr Val Ala Ala Ala Ala
Phe Val Leu Leu Phe Phe 1 5 10 15 Val Gly Cys Gly Leu Leu Leu Ser
20 <210> SEQ ID NO 55 <211> LENGTH: 24 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
transmembrane region of F-NEXT which is derived from mouse Notch-1
peptide. <400> SEQUENCE: 55 Leu His Leu Met Tyr Val Ala Ala
Ala Ala Phe Val Leu Leu Phe Phe 1 5 10 15 Val Gly Cys Gly Val Leu
Leu Ser 20 <210> SEQ ID NO 56 <211> LENGTH: 24
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Partial amino acid sequence
of transmembrane region of F-NEXT(mutant) which is derived from
mouse Notch-1 peptide. <400> SEQUENCE: 56 Leu His Leu Met Tyr
Val Gly Gly Gly Gly Phe Val Leu Leu Phe Phe 1 5 10 15 Val Gly Cys
Gly Val Leu Leu Ser 20 <210> SEQ ID NO 57 <211> LENGTH:
24 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Partial amino
acid sequence of transmembrane region of F-NEXT(mutant) which is
derived from mouse Notch-1 peptide. <400> SEQUENCE: 57 Leu
His Leu Met Tyr Val Leu Leu Leu Leu Phe Val Leu Leu Phe Phe 1 5 10
15 Val Gly Cys Gly Val Leu Leu Ser 20
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 57 <210>
SEQ ID NO 1 <211> LENGTH: 21 <212> TYPE: PRT
<213> ORGANISM: mouse <400> SEQUENCE: 1 Val Lys Ser Glu
Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu 1 5 10 15 Met Tyr
Val Ala Ala 20 <210> SEQ ID NO 2 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: mouse <400>
SEQUENCE: 2 Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu
His Leu 1 5 10 15 Met <210> SEQ ID NO 3 <211> LENGTH:
18 <212> TYPE: PRT <213> ORGANISM: mouse <400>
SEQUENCE: 3 Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu
His Leu 1 5 10 15 Met Tyr <210> SEQ ID NO 4 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: mouse
<400> SEQUENCE: 4 Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro
Ser Gln Leu His Leu 1 5 10 15 Met Tyr Val Ala 20 <210> SEQ ID
NO 5 <211> LENGTH: 22 <212> TYPE: PRT <213>
ORGANISM: mouse <400> SEQUENCE: 5 Val Lys Ser Glu Pro Val Glu
Pro Pro Leu Pro Ser Gln Leu His Leu 1 5 10 15 Met Tyr Val Ala Ala
Ala 20 <210> SEQ ID NO 6 <211> LENGTH: 23 <212>
TYPE: PRT <213> ORGANISM: mouse <400> SEQUENCE: 6 Val
Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu 1 5 10
15 Met Tyr Val Ala Ala Ala Ala 20 <210> SEQ ID NO 7
<211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM:
mouse <400> SEQUENCE: 7 Val Lys Ser Glu Pro Val Glu Pro Pro
Leu Pro Ser Gln Leu His Leu 1 5 10 15 Met Tyr Val Ala Ala Ala Ala
Phe 20 <210> SEQ ID NO 8 <211> LENGTH: 25 <212>
TYPE: PRT <213> ORGANISM: mouse <400> SEQUENCE: 8 Val
Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu 1 5 10
15 Met Tyr Val Ala Ala Ala Ala Phe Val 20 25 <210> SEQ ID NO
9 <211> LENGTH: 26 <212> TYPE: PRT <213>
ORGANISM: mouse <400> SEQUENCE: 9 Val Lys Ser Glu Pro Val Glu
Pro Pro Leu Pro Ser Gln Leu His Leu 1 5 10 15 Met Tyr Val Ala Ala
Ala Ala Phe Val Leu 20 25 <210> SEQ ID NO 10 <211>
LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 10 Val Gln Ser Glu Thr Val Glu Pro Pro Pro
Pro Ala Gln Leu His Phe 1 5 10 15 Met <210> SEQ ID NO 11
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
human <400> SEQUENCE: 11 Val Gln Ser Glu Thr Val Glu Pro Pro
Pro Pro Ala Gln Leu His Phe 1 5 10 15 Met Tyr <210> SEQ ID NO
12 <211> LENGTH: 20 <212> TYPE: PRT <213>
ORGANISM: human <400> SEQUENCE: 12 Val Gln Ser Glu Thr Val
Glu Pro Pro Pro Pro Ala Gln Leu His Phe 1 5 10 15 Met Tyr Val Ala
20 <210> SEQ ID NO 13 <211> LENGTH: 21 <212>
TYPE: PRT <213> ORGANISM: human <400> SEQUENCE: 13 Val
Gln Ser Glu Thr Val Glu Pro Pro Pro Pro Ala Gln Leu His Phe 1 5 10
15 Met Tyr Val Ala Ala 20 <210> SEQ ID NO 14 <211>
LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 14 Val Gln Ser Glu Thr Val Glu Pro Pro Pro
Pro Ala Gln Leu His Phe 1 5 10 15 Met Tyr Val Ala Ala Ala 20
<210> SEQ ID NO 15 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: human <400> SEQUENCE: 15 Val Gln
Ser Glu Thr Val Glu Pro Pro Pro Pro Ala Gln Leu His Phe 1 5 10 15
Met Tyr Val Ala Ala Ala Ala 20 <210> SEQ ID NO 16 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 16 Val Gln Ser Glu Thr Val Glu Pro Pro Pro
Pro Ala Gln Leu His Phe 1 5 10 15 Met Tyr Val Ala Ala Ala Ala Phe
20 <210> SEQ ID NO 17 <211> LENGTH: 25 <212>
TYPE: PRT <213> ORGANISM: human <400> SEQUENCE: 17 Val
Gln Ser Glu Thr Val Glu Pro Pro Pro Pro Ala Gln Leu His Phe 1 5 10
15 Met Tyr Val Ala Ala Ala Ala Phe Val 20 25 <210> SEQ ID NO
18 <211> LENGTH: 26 <212> TYPE: PRT <213>
ORGANISM: human <400> SEQUENCE: 18 Val Gln Ser Glu Thr Val
Glu Pro Pro Pro Pro Ala Gln Leu His Phe 1 5 10 15
Met Tyr Val Ala Ala Ala Ala Phe Val Leu 20 25 <210> SEQ ID NO
19 <211> LENGTH: 57 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer 1 which is derived from mouse Notch-1 gene for
use in site spesific mutagenesis. <400> SEQUENCE: 19
atcgtcgtcc ttgtagtctc tcaagcctct tgcgccgagc gcgggcagca gcgttag 57
<210> SEQ ID NO 20 <211> LENGTH: 54 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer 2 which is derived from mouse
Notch-1 gene for use in site spesific mutagenesis. <400>
SEQUENCE: 20 gacaagatgg tgatgaagag tgagccggtg gagcctccgc tgccctcgca
gctg 54 <210> SEQ ID NO 21 <211> LENGTH: 32 <212>
TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer 3 which is derived from mouse
Notch-1 gene for use in site spesific mutagenesis. <400>
SEQUENCE: 21 cctcgcagct gcacctcatg tacgtggcag cg 32 <210> SEQ
ID NO 22 <211> LENGTH: 32 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer 4 which is derived from mouse Notch-1 gene for
use in site spesific mutagenesis. <400> SEQUENCE: 22
cgctgccacg tacatgaggt gcagctgcga gg 32 <210> SEQ ID NO 23
<211> LENGTH: 70 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of F-NEXT which is derived from mouse
Notch-1 peptide and has FLAG sequence at N-terminal region.
<400> SEQUENCE: 23 Met Pro Arg Leu Leu Thr Pro Leu Leu Cys
Leu Thr Leu Leu Pro Ala 1 5 10 15 Arg Ala Ala Arg Gly Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met 20 25 30 Val Met Lys Ser Glu Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His 35 40 45 Leu Met Tyr Val
Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly 50 55 60 Cys Gly
Val Leu Leu Ser 65 70 <210> SEQ ID NO 24 <211> LENGTH:
31 <212> TYPE: PRT <213> ORGANISM: mouse <400>
SEQUENCE: 24 Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala Ala
Ala Phe Val 1 5 10 15 Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu
Ser Arg Lys Arg 20 25 30 <210> SEQ ID NO 25 <211>
LENGTH: 31 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 25 Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu
Met Val Gly Gly Val Val 1 5 10 15 Ile Ala Thr Val Ile Val Ile Thr
Leu Val Met Leu Lys Lys Lys 20 25 30 <210> SEQ ID NO 26
<211> LENGTH: 45 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of F-NEXT which is derived from mouse
Notch-1 peptide and has FLAG sequence at N-terminal region.
<400> SEQUENCE: 26 Leu Arg Asp Tyr Lys Asp Asp Asp Asp Lys
Met Val Met Lys Ser Glu 1 5 10 15 Pro Val Glu Pro Pro Leu Pro Ser
Gln Leu His Leu Met Tyr Val Ala 20 25 30 Ala Ala Ala Phe Val Leu
Leu Phe Phe Val Gly Cys Gly 35 40 45 <210> SEQ ID NO 27
<211> LENGTH: 38 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of F-NEXT which is derived from mouse
Notch-1 peptide and has FLAG sequence at N-terminal region.
<400> SEQUENCE: 27 Leu Arg Asp Tyr Lys Asp Asp Asp Asp Lys
Met Val Met Lys Ser Glu 1 5 10 15 Pro Val Glu Pro Pro Leu Pro Ser
Gln Leu His Leu Met Tyr Val Ala 20 25 30 Ala Ala Ala Phe Val Leu 35
<210> SEQ ID NO 28 <211> LENGTH: 37 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 28 Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu 1 5 10 15 Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala 20 25
30 Ala Ala Ala Phe Val 35 <210> SEQ ID NO 29 <211>
LENGTH: 36 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Partial amino
acid sequence of F-NEXT which is derived from mouse Notch-1 peptide
and has FLAG sequence at N-terminal region. <400> SEQUENCE:
29 Leu Arg Asp Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu
1 5 10 15 Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr
Val Ala 20 25 30 Ala Ala Ala Phe 35 <210> SEQ ID NO 30
<211> LENGTH: 35 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of F-NEXT which is derived from mouse
Notch-1 peptide and has FLAG sequence at N-terminal region.
<400> SEQUENCE: 30 Leu Arg Asp Tyr Lys Asp Asp Asp Asp Lys
Met Val Met Lys Ser Glu 1 5 10 15 Pro Val Glu Pro Pro Leu Pro Ser
Gln Leu His Leu Met Tyr Val Ala 20 25 30 Ala Ala Ala 35 <210>
SEQ ID NO 31 <211> LENGTH: 35 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Partial amino acid sequence of F-NEXT which is
derived from mouse Notch-1 peptide and has FLAG sequence at
N-terminal region. <400> SEQUENCE: 31 Arg Gly Leu Arg Asp Tyr
Lys Asp Asp Asp Asp Lys Met Val Met Lys 1 5 10 15 Ser Glu Pro Val
Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr 20 25 30 Val Ala
Ala 35 <210> SEQ ID NO 32 <211> LENGTH: 33 <212>
TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Partial amino acid sequence of F-NEXT which is
derived from mouse Notch-1 peptide and has FLAG sequence at
N-terminal region. <400> SEQUENCE: 32 Leu Arg Asp Tyr Lys Asp
Asp Asp Asp Lys Met Val Met Lys Ser Glu 1 5 10 15 Pro Val Glu Pro
Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala 20 25 30 Ala
<210> SEQ ID NO 33 <211> LENGTH: 31 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 33 Asp Tyr Lys
Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu Pro Val 1 5 10 15 Glu
Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala 20 25 30
<210> SEQ ID NO 34 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 34 Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu 1 5 10 15 Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala 20 25
30 <210> SEQ ID NO 35 <211> LENGTH: 30 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 35 Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu 1 5 10 15 Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr 20 25 30
<210> SEQ ID NO 36 <211> LENGTH: 29 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
F-NEXT which is derived from mouse Notch-1 peptide and has FLAG
sequence at N-terminal region. <400> SEQUENCE: 36 Leu Arg Asp
Tyr Lys Asp Asp Asp Asp Lys Met Val Met Lys Ser Glu 1 5 10 15 Pro
Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met 20 25 <210>
SEQ ID NO 37 <211> LENGTH: 23 <212> TYPE: PRT
<213> ORGANISM: mouse <400> SEQUENCE: 37 Leu His Leu
Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe 1 5 10 15 Val
Gly Cys Gly Val Leu Leu 20 <210> SEQ ID NO 38 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 38 Leu His Phe Met Tyr Val Ala Ala Ala Ala
Phe Val Leu Leu Phe Phe 1 5 10 15 Val Gly Cys Gly Val Leu Leu 20
<210> SEQ ID NO 39 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: mouse <400> SEQUENCE: 39 Leu Leu
Tyr Leu Leu Ala Val Ala Val Val Ile Ile Leu Phe Phe Ile 1 5 10 15
Leu Leu Gly Val Ile Met Ala 20 <210> SEQ ID NO 40 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 40 Leu Leu Tyr Leu Leu Ala Val Ala Val Val
Ile Ile Leu Phe Ile Ile 1 5 10 15 Leu Leu Gly Val Ile Met Ala 20
<210> SEQ ID NO 41 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: mouse <400> SEQUENCE: 41 Leu Leu
Pro Leu Leu Val Ala Gly Ala Val Phe Leu Leu Ile Ile Phe 1 5 10 15
Ile Leu Gly Val Met Val Ala 20 <210> SEQ ID NO 42 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 42 Leu Leu Pro Leu Leu Val Ala Gly Ala Val
Leu Leu Leu Val Ile Leu 1 5 10 15 Val Leu Gly Val Met Val Ala 20
<210> SEQ ID NO 43 <211> LENGTH: 23 <212> TYPE:
PRT <213> ORGANISM: mouse <400> SEQUENCE: 43 Ile Leu
Cys Ser Pro Val Val Gly Val Leu Leu Leu Ala Leu Gly Ala 1 5 10 15
Leu Leu Val Leu Gln Leu Ile 20 <210> SEQ ID NO 44 <211>
LENGTH: 23 <212> TYPE: PRT <213> ORGANISM: human
<400> SEQUENCE: 44 Val Leu Cys Ser Pro Val Ala Gly Val Ile
Leu Leu Ala Leu Gly Ala 1 5 10 15 Leu Leu Val Leu Gln Leu Ile 20
<210> SEQ ID NO 45 <211> LENGTH: 24 <212> TYPE:
PRT <213> ORGANISM: human <400> SEQUENCE: 45 Gly Ala
Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 1 5 10 15
Ile Val Ile Thr Leu Val Met Leu 20 <210> SEQ ID NO 46
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of transmembrane region of F-NEXT which
is derived from mouse Notch-1 peptide. <400> SEQUENCE: 46 Leu
His Leu Met Tyr Val Ala Ala 1 5 <210> SEQ ID NO 47
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of transmembrane region of F-NEXT which
is derived from mouse Notch-1 peptide. <400> SEQUENCE: 47 Leu
His Leu Met Tyr Val Ala Ala Ala Ala 1 5 10 <210> SEQ ID NO
48
<211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of transmembrane region of F-NEXT which
is derived from mouse Notch-1 peptide. <400> SEQUENCE: 48 Leu
His Leu Met Tyr Val Ala Ala Ala Ala Phe 1 5 10 <210> SEQ ID
NO 49 <211> LENGTH: 12 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Partial amino acid sequence of transmembrane region of
F-NEXT which is derived from mouse Notch-1 peptide. <400>
SEQUENCE: 49 Leu His Leu Met Tyr Val Ala Ala Ala Ala Phe Val 1 5 10
<210> SEQ ID NO 50 <211> LENGTH: 28 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
transmembrane region of F-NEXT which is derived from mouse Notch-1
peptide. <400> SEQUENCE: 50 Leu His Leu Met Tyr Val Ala Ala
Ala Ala Phe Val Leu Leu Phe Phe 1 5 10 15 Val Gly Cys Gly Val Leu
Leu Ser Arg Lys Arg Arg 20 25 <210> SEQ ID NO 51 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Partial amino
acid sequence of transmembrane region of F-NEXT which is derived
from mouse Notch-1 peptide. <400> SEQUENCE: 51 Leu His Leu
Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe 1 5 10 15 Val
Gly Cys Gly Val Leu Leu Ser 20 <210> SEQ ID NO 52 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Partial amino
acid sequence of transmembrane region of F-NEXT which is derived
from mouse Notch-1 peptide. <400> SEQUENCE: 52 Leu His Leu
Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe 1 5 10 15 Val
Gly Cys Gly Val Leu Leu Ser 20 <210> SEQ ID NO 53 <211>
LENGTH: 24 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Partial amino
acid sequence of transmembrane region of F-NEXT(V1744G) which is
derived from mouse Notch-1 peptide. <400> SEQUENCE: 53 Leu
His Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe 1 5 10
15 Val Gly Cys Gly Gly Leu Leu Ser 20 <210> SEQ ID NO 54
<211> LENGTH: 24 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Partial amino acid sequence of transmembrane region of
F-NEXT(V1744L) which is derived from mouse Notch-1 peptide.
<400> SEQUENCE: 54 Leu His Leu Met Tyr Val Ala Ala Ala Ala
Phe Val Leu Leu Phe Phe 1 5 10 15 Val Gly Cys Gly Leu Leu Leu Ser
20 <210> SEQ ID NO 55 <211> LENGTH: 24 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Partial amino acid sequence of
transmembrane region of F-NEXT which is derived from mouse Notch-1
peptide. <400> SEQUENCE: 55 Leu His Leu Met Tyr Val Ala Ala
Ala Ala Phe Val Leu Leu Phe Phe 1 5 10 15 Val Gly Cys Gly Val Leu
Leu Ser 20 <210> SEQ ID NO 56 <211> LENGTH: 24
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Partial amino acid sequence
of transmembrane region of F-NEXT(mutant) which is derived from
mouse Notch-1 peptide. <400> SEQUENCE: 56 Leu His Leu Met Tyr
Val Gly Gly Gly Gly Phe Val Leu Leu Phe Phe 1 5 10 15 Val Gly Cys
Gly Val Leu Leu Ser 20 <210> SEQ ID NO 57 <211> LENGTH:
24 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Partial amino
acid sequence of transmembrane region of F-NEXT(mutant) which is
derived from mouse Notch-1 peptide. <400> SEQUENCE: 57 Leu
His Leu Met Tyr Val Leu Leu Leu Leu Phe Val Leu Leu Phe Phe 1 5 10
15 Val Gly Cys Gly Val Leu Leu Ser 20
* * * * *