U.S. patent application number 12/191950 was filed with the patent office on 2009-06-04 for beta-secretase enzyme compositions and methods.
This patent application is currently assigned to Elan Pharmaceuticals, Inc.. Invention is credited to John P. Anderson, Guriqbal Basi, Minh Tam Doan, Normand Frigon, Varghese John, Lisa McConlogue, Michael Power, Sukanto Sinha, Gwen Tatsuno, Jay Tung, Shuwen Wang.
Application Number | 20090144840 12/191950 |
Document ID | / |
Family ID | 34799884 |
Filed Date | 2009-06-04 |
United States Patent
Application |
20090144840 |
Kind Code |
A1 |
Anderson; John P. ; et
al. |
June 4, 2009 |
Beta-secretase enzyme compositions and methods
Abstract
Disclosed are various forms of an active, isolated
.beta.-secretase enzyme in purified and recombinant form. This
enzyme is implicated in the production of amyloid plaque components
which accumulate in the brains of individuals afflicted with
Alzheimer's disease. Recombinant cells that produce this enzyme
either alone or in combination with some of its natural substrates
(.beta.-APPwt and .beta.-APPsw) are also disclosed, as are
antibodies directed to such proteins. These compositions are useful
for use in methods of selecting compounds that modulate
.beta.-secretase. Inhibitors of .beta.-secretase are implicated as
therapeutics in the treatment of neurodegenerative diseases, such
as Alzheimer's disease.
Inventors: |
Anderson; John P.; (San
Francisco, CA) ; Basi; Guriqbal; (Palo Alto, CA)
; Doan; Minh Tam; (Hayward, CA) ; Frigon;
Normand; (Milbrae, CA) ; John; Varghese; (San
Francisco, CA) ; Power; Michael; (Fremont, CA)
; Sinha; Sukanto; (San Francisco, CA) ; Tatsuno;
Gwen; (Oakland, CA) ; Tung; Jay; (Belmont,
CA) ; Wang; Shuwen; (Hershey, PA) ;
McConlogue; Lisa; (Burlingame, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Elan Pharmaceuticals, Inc.
South San Francisco
CA
|
Family ID: |
34799884 |
Appl. No.: |
12/191950 |
Filed: |
August 14, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11069377 |
Feb 28, 2005 |
7427478 |
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12191950 |
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09723722 |
Nov 28, 2000 |
7115410 |
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11069377 |
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09501708 |
Feb 10, 2000 |
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09723722 |
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09471669 |
Dec 24, 1999 |
7456007 |
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09501708 |
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60139172 |
Jun 15, 1999 |
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60119571 |
Feb 10, 1999 |
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60139172 |
Jun 15, 1999 |
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60119571 |
Feb 10, 1999 |
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60114408 |
Dec 31, 1998 |
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Current U.S.
Class: |
800/18 ;
435/7.4 |
Current CPC
Class: |
H01Q 3/44 20130101; H01Q
9/0442 20130101; H01Q 1/364 20130101; H01Q 15/02 20130101; A61K
38/00 20130101; H01Q 9/0407 20130101; H01Q 1/38 20130101; A01K
2217/075 20130101; C12N 9/6478 20130101 |
Class at
Publication: |
800/18 ;
435/7.4 |
International
Class: |
G01N 33/573 20060101
G01N033/573; A01K 67/027 20060101 A01K067/027 |
Claims
1-107. (canceled)
108. A knock-out mouse, characterized by inactivation or deletion
of an endogenous .beta.-secretase gene.
109. The knock-out mouse of claim 108, wherein said
.beta.-secretase gene encodes a protein having at least 90%
sequence identity to the sequence SEQ ID NO: 65.
110. The knock-out mouse of claim 108, wherein said deletion is
inducible
111. The knock-out mouse of claim 110, wherein said inducible
expression is effected by a Cre-lox expression system inserted into
the mouse genome.
112-131. (canceled)
132. A method for identifying a compound that decreases the
activity of a beta-secretase polypeptide comprising steps of: (a)
contacting a beta-secretase polypeptide comprising an amino acid
sequence at least 95% identical to a fragment of the beta-secretase
amino acid sequence of SEQ ID NO: 43, wherein said fragment is a
continuous fragment that includes active aspartic acid catalytic
sites and exhibits beta-secretase activity, which is an ability to
cleave a 695 isotype of APP between amino acids 596 and 597 with a
test compound and a substrate cleavable by the beta secretase
polypeptide; (b) measuring the beta-secretase activity of the
polypeptide on the substrate in the presence and absence of the
test compound, and (c) comparing the beta-secretase activity of the
polypeptide cell in the presence and absence of the test compound,
wherein the beta-secretase activity is determined using an antibody
specific for the C-terminus of the N-terminal fragment or the
N-terminus of the C-terminal fragment generated by the beta
secretase cleavage of the substrate, wherein decreased
beta-secretase activity in the presence of the test compound
identifies the test compound as a compound that decreases the
beta-secretase activity of the beta-secretase polypeptide.
133. A method according to claim 132, wherein the substrate is APP
or a fragment thereof cleavable by .beta.-secretase.
134. A method according to claim 132, wherein the substrate
comprises a Swedish mutation of APP.
135. A method according to claim 132, wherein the substrate is a
fragment of the amyloid precursor protein comprising SEQ ID NO: 54
or its Swedish mutation SEQ ID NO: 51.
136. The method of claim 132, wherein the substrate is selected
from the group consisting of MBP-C125st, MBP-C124sw, APPwt, APPsw
and beta-secretase cleavable fragments thereof.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/723,722, filed Nov. 28, 2000, which is a continuation of
U.S. application Ser. No. 09/501,708, filed Feb. 10, 2000, which
claims the benefit under 35 U.S.C. 119(e) of U.S. Application No.
60/119,571, filed Feb. 10, 1999, and which also claims the benefit
under 35 U.S.C. 119(e) of U.S. Application No. 60/139,172, filed
Jun. 15, 1999. Each of these applications is hereby incorporated
herein by reference in its entirety. This application is also a
continuation-in-part of U.S. application Ser. No. 09/471,669, filed
Dec. 24, 1999, which also claims the benefit under 35 U.S.C. 119(e)
of U.S. Application No. 60/119,571, filed Feb. 10, 1999, and claims
the benefit under 35 U.S.C. 119(e) of U.S. Application No.
60/139,172, filed Jun. 15, 1999.
FIELD OF THE INVENTION
[0002] The invention relates to the discovery of various active
forms of .beta.-secretase, an enzyme that cleaves .beta.-amyloid
precursor protein (APP) at one of the two cleavage sites necessary
to produce .beta.-amyloid peptide (A.beta.). The invention also
relates to inhibitors of this enzyme, which are considered
candidates for therapeutics in the treatment of amyloidogenic
diseases such as Alzheimer's disease. Further aspects of the
present invention include screening methods, assays, and kits for
discovering such therapeutic inhibitors, as well as diagnostic
methods for determining whether an individual carries a mutant form
of the enzyme.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease is characterized by the presence of
numerous amyloid plaques and neurofibrillatory tangles present in
the brain, particularly in those regions of the brain involved in
memory and cognition. .beta.-amyloid peptide (A.beta.) is a 39-43
amino acid peptide that is major component of amyloid plaques and
is produced by cleavage of a large protein known as the amyloid
precursor protein (APP) at a specific site(s) within the N-terminal
region of the protein. Normal processing of APP involves cleavage
of the protein at point 16-17 amino acids C-terminal to the
N-terminus of the .beta.-AP region, releasing a secreted
ectodomain, .alpha.-sAPP, thus precluding production of .beta.-AP.
Cleavage by .beta.-secretase enzyme of APP between Met.sup.671 and
Asp.sup.672 and subsequent processing at the C-terminal end of APP
produces A.beta. peptide, which is highly implicated in the
etiology of Alzheimer's pathology (Seubert, et al., in
Pharmacological Treatment of Alzheimer's disease, Wiley-Liss, Inc.,
pp. 345-366, 1997; Zhao, J., et al. J. Biol. Chem. 271:
31407-31411, 1996).
[0004] It is not clear whether .beta.-secretase enzyme levels
and/or activity is inherently higher than normal in Alzheimer's
patients; however, it is clear that its cleavage product, A.beta.
peptide, is abnormally concentrated in amyloid plaques present in
their brains. Therefore, it would be desirable to isolate, purify
and characterize the enzyme responsible for the pathogenic cleavage
of APP in order to help answer this and other questions surrounding
the etiology of the disease. In particular, it is also desirable to
utilize the isolated enzyme, or active fragments thereof, in
methods for screening candidate drugs for ability to inhibit the
activity of .beta.-secretase. Drugs exhibiting inhibitory effects
on .beta.-secretase activity are expected to be useful therapeutics
in the treatment of Alzheimer's disease and other amyloidogenic
disorders characterized by deposition of A.beta. peptide containing
fibrils.
[0005] U.S. Pat. No. 5,744,346 (Chrysler, et al.) describes the
initial isolation and partial purification of .beta.-secretase
enzyme characterized by its size (apparent molecular weight in the
range of 260 to 300 kilodaltons when measured by gel exclusion
chromatography) and enzymatic activity (ability to cleave the
695-amino acid isotype of .beta.-amyloid precursor protein between
amino acids 596 and 597). The present invention provides a
significant improvement in the purity of .beta.-secretase enzyme,
by providing a purified .beta.-secretase enzyme that is at least
200 fold purer than that previously described. Such a purified
protein has utility in a number of applications, including
crystallization for structure determination. The invention also
provides methods for producing recombinant forms of
.beta.-secretase enzymes that have the same size and enzymatic
profiles as the naturally occurring forms. It is a further
discovery of the present invention that human .beta.-secretase is a
so-called "aspartyl" (or "aspartic") protease.
SUMMARY OF THE INVENTION
[0006] This invention is directed to a .beta.-secretase protein
that has now been purified to apparent homogeneity, and in
particular to a purified protein characterized by a specific
activity of at least about 0.2.times.10.sup.5 and preferably at
least 1.0.times.10.sup.5 nM/h/.mu.g protein in a representative
.beta.-secretase assay, the MBP-C125sw substrate assay. The
resulting enzyme, which has a characteristic activity in cleaving
the 695-amino acid isotype of .beta.-amyloid precursor protein
(.beta.-APP) between amino acids 596 and 597 thereof, is at least
10,000-fold, preferably at least 20,000-fold and, more preferably
in excess of 200,000-fold higher specific activity than an activity
exhibited by a solubilized but unenriched membrane fraction from
human 293 cells, such as have been earlier characterized.
[0007] In one embodiment, the purified enzyme is fewer than 450
amino acids in length, comprising a polypeptide having the amino
acid sequence SEQ ID NO: 70 [63-452]. In preferred embodiments, the
purified protein exists in a variety of "truncated forms" relative
to the proenzyme referred to herein as SEQ ID NO:2 [1-501], such as
forms having amino acid sequences SEQ ID NO:70 [63-452], SEQ ID
NO:69 [63-501],: SEQ ID NO:67 [58-501], SEQ ID NO:68 [58-452], SEQ
ID NO:58 [46-452], SEQ ID NO:74 [22-452]. More generally, it has
been found that particularly useful forms of the enzyme,
particularly with regard to the crystallization studies described
herein, are characterized by an N-terminus at position 46 with
respect to SEQ ID NO: 2 and a C-terminus between positions 452 and
470 with respect to SEQ ID NO: 2, and more particularly, by an
N-terminus at position 22 with respect to SEQ ID NO: 2 and a
C-terminus between positions 452 and 470 with respect to SEQ ID NO:
2. These forms are considered to be cleaved in the transmembrane
"anchor" domain. Other particularly useful purified forms of the
enzyme include: SEQ ID NO: 43 [46-501], SEQ ID NO: 66 [22-501], and
SEQ ID NO: 2 [1-501]. More generally, it is appreciated that useful
forms of the enzyme have an N-terminal residue corresponding to a
residue selected from the group consisting of residues 22, 46, 58
and 63 with respect to SEQ ID NO: 2 and a C-terminus selected from
a residue between positions 452 and 501 with respect to SEQ ID NO:
2 or a C-terminus between residue positions 452 and 470 with
respect to SEQ ID NO: 2. Also described herein are forms of enzyme
isolated from a mouse, exemplified by SEQ ID NO: 65.
[0008] This invention is further directed to a crystalline protein
composition formed from a purified .beta.-secretase protein, such
as the various protein compositions described above. According to
one embodiment, the purified protein is characterized by an ability
to bind to the .beta.-secretase inhibitor substrate
P10-P4'staD.fwdarw.V which is at least equal to an ability
exhibited by a protein having the amino acid sequence SEQ ID NO:71
[46-419], when the proteins are tested for binding to said
substrate under the same conditions. According to another
embodiment, the purified protein forming the crystallization
composition is characterized by a binding affinity for the
.beta.-secretase inhibitor substrate SEQ ID NO: 72
(P10-P4'staD.fwdarw.V) which is at least 1/100 of an affinity
exhibited by a protein having the amino acid sequence SEQ ID NO: 43
[46-501], when said proteins are tested for binding to said
substrate under the same conditions. Proteins forming the
crystalline composition may be glycosylated or deglycosylated.
[0009] The invention also includes a crystalline protein
composition containing a .beta.-secretase substrate or inhibitor
molecule, examples of which are provided herein, particularly
exemplified by peptide-derived inhibitors such as SEQ ID NO: 78,
SEQ ID NO: 72, SEQ ID NO: 81, and derivatives thereof. Generally
useful inhibitors in this regard will have a K.sub.i of no more
than about 50 .mu.M to 0.5 mM.
[0010] Another aspect of the invention is directed to an isolated
protein, comprising a polypeptide that (i) is fewer than about 450
amino acid residues in length, (ii) includes an amino acid sequence
that is at least 90% identical to SEQ ID NO: 75 [63-423] including
conservative substitutions thereof, and (iii) exhibits
.beta.-secretase activity, as evidenced by an ability to cleave a
substrate selected from the group consisting of the 695 amino acid
isotype of beta amyloid precursor protein (.beta.APP) between amino
acids 596 and 597 thereof, MBP-C125wt and MBP-C125sw. Peptides
which fit these criteria include, but are not limited to
polypeptides which include the sequence SEQ ID NO: 75 [63-423],
such as SEQ ID NO: 58 [46-452], SEQ ID NO: 58 [46-452], SEQ ID NO:
58 [46-452], SEQ ID NO: 74 [22-452], and may also include
conservative substitutions within such sequences.
[0011] According to a further embodiment, the invention includes
isolated protein compositions, such as those described above, in
combination with a .beta.-secretase substrate or inhibitor
molecule, such as MBP-C125wt, MBP-C125sw, APP, APPsw, and
.beta.-secretase-cleavable fragments thereof. Additional
.beta.-secretase-cleavable fragments useful in this regard are
described in the specification hereof. Particularly useful
inhibitors include peptides derived from or including SEQ ID NO:
78, SEQ ID NO: 81 and SEQ ID NO: 72. Generally, such inhibitors
will have K.sub.1s of less than about 1 .mu.M. Such inhibitors may
be labeled with a detectable reporter molecule. Such labeled
molecules are particularly useful, for example, in ligand binding
assays.
[0012] In accordance with a further aspect, the invention includes
protein compositions, such as those described above, expressed by a
heterologous cell. In accordance with a further embodiment, such
cells may also co-express a .beta.-secretase substrate or inhibitor
protein or peptide. One or both of the expressed molecules may be
heterologous to the cell.
[0013] In a related embodiment, the invention includes antibodies
that bind specifically to a .beta.-secretase protein comprising a
polypeptide that includes an amino acid sequence that is at least
90% identical to SEQ ID NO: 75 [63-423] including conservative
substitutions thereof, but which lacks significant immunoreactivity
with a protein a sequence selected from the group consisting of SEQ
ID NO: 2 [1-501] and SEQ ID NO: 43 [46-501].
[0014] In a further related embodiment, the invention includes
isolated nucleic acids comprising a sequence of nucleotides that
encodes a .beta.-secretase protein that is at least 95% identical
to a protein selected from the group consisting of SEQ ID NO: 66
[22-501], SEQ ID NO: 43[46-501], SEQ ID NO: 57 [1-419], SEQ ID NO:
74 [22-452], SEQ ID NO: 58 [46-452], SEQ ID NO: 59 [1-452], SEQ ID
NO: 60 [1-420], SEQ ID NO: 67 [58-501], SEQ ID NO: 68 [58-452], SEQ
ID NO: 69 [63-501], SEQ ID NO: 70 [63-452], SEQ ID NO: 75 [63-423],
and SEQ ID NO: 71 [46-419], or a complementary sequence of any of
such nucleotides. Specifically excluded from this nucleotide is a
nucleic acid encoding a protein having the sequence SEQ ID NO: 2
[1-501].
[0015] Additionally, the invention includes an expression vector
comprising such isolated nucleic acids operably linked to the
nucleic acid with regulatory sequences effective for expression of
the nucleic acid in a selected host cell, for heterologous
expression. The host cells can be a eukaryotic cell, a bacterial
cell, an insect cell or a yeast cell. Such cells can be used, for
example, in a method of producing a recombinant .beta.-secretase
enzyme, where the method further includes subjecting an extract or
cultured medium from said cell to an affinity matrix, such as a
matrix formed from a .beta.-secretase inhibitor molecule or
antibody, as detailed herein.
[0016] The invention is also directed to a method of screening for
compounds that inhibit A.beta. production, comprising contacting a
.beta.-secretase polypeptide, such as those full-length or
truncated forms described above, with (i) a test compound and (ii)
a .beta.-secretase substrate, and selecting the test compound as
capable of inhibiting A.beta. production if the .beta.-secretase
polypeptide exhibits less .beta.-secretase activity in the presence
of than in the absence of the test compound. Such an assay may be
cell-based, with one or both of the enzyme and the substrate
produced by the cell, such as the co-expression cell referred to
above. Kits embodying such screening methods also form a part of
the invention.
[0017] The screening method may further include administering a
test compound to a mammalian subject having Alzheimer's disease or
Alzheimer's disease like pathology, and selecting the compound as a
therapeutic agent candidate if, following such administration, the
subject maintains or improves cognitive ability or the subject
shows reduced plaque burden. Preferably, such a subject is a
comprising a transgene for human .beta.-amyloid precursor protein
(.beta.-APP), such as a mouse bearing a transgene which encodes a
human .beta.-APP, including a mutant variants thereof, as
exemplified in the specification.
[0018] In a related embodiment, the invention includes
.beta.-secretase inhibitor compound selected according to the
methods described above. Such compounds may be is selected, for
example, from a phage display selection system ("library"), such as
are known in the art. According to another aspect, such libraries
may be "biased" for the sequence peptide SEQ ID NO: 97
[P10-P4'D.fwdarw.V]. Other inhibitors include, or may be derived
from peptide inhibitors herein identified, such as inhibitors SEQ
ID NO: 78, SEQ ID NO: 72, SEQ ID NO: 78 and SEQ ID NO: 81.
[0019] Also forming part of the invention are knock-out mice,
characterized by inactivation or deletion of an endogenous
.beta.-secretase gene, such as genes encodes a protein having at
least 90% sequence identity to the sequence SEQ ID NO: 65. The
deletion or inactivation may be inducible, such as by insertion of
a Cre-lox expression system into the mouse genome.
[0020] According to a further related aspect, the invention
includes a method of screening for drugs effective in the treatment
of Alzheimer's disease or other cerebrovascular amyloidosis
characterized by A.beta. deposition. According to this aspect of
the invention, a mammalian subject characterized by overexpression
of .beta.-APP and/or deposition of A.beta. is given a test compound
selected for its ability to inhibit .beta.-secretase activity a
.beta.-secretase protein according to claim 37. The compound is
selected as a potential therapeutic drug compound, if it reduces
the amount of A.beta. deposition in said subject or if it maintains
or improves cognitive ability in the subject. According to one
preferred embodiment, the mammalian subject is a transgenic mouse
bearing a transgene encoding a human .beta.-APP or a mutant
thereof.
[0021] The invention also includes a method of treating a patient
afflicted with or having a predilection for Alzheimer's disease or
other cerebrovascular amyloidosis. According to this aspect, the
enzymatic hydrolysis of APP to A.beta. is blocked by administering
to the patient a pharmaceutically effective dose of a compound
effective to inhibit one or more of the various forms of the enzyme
described herein. According to another feature, the therapeutic
compound is derived from a peptide selected from the group
consisting of SEQ ID NO: 72, SEQ ID NO: 78, SEQ ID NO: 81 and SEQ
ID NO: 97. Such derivation may be effected by the various phage
selection systems described herein, in conjunction with the
screening methods of the invention, or other such methods.
Alternatively, or in addition, derivation may be achieved via
rational chemistry approaches, including molecular modeling, known
in the medicinal chemistry art. Such compounds will preferably be
rather potent inhibitors of .beta.-secretase enzymatic activity,
evidenced by a K.sub.i of less than about 1-50 .mu.M in a
MBP-C125sw assay. Such compounds also form the basis for
therapeutic drug compositions in accordance with the present
invention, which may also include a pharmaceutically effective
excipient.
[0022] According to yet another related aspect, the invention
includes a method of diagnosing the presence of or a predilection
for Alzheimer's disease in a patient. This method includes
detecting the expression level of a gene comprising a nucleic acid
encoding .beta.-secretase in a cell sample from said patient, and
diagnosing the patient as having or having a predilection for
Alzheimer's disease, if said expression level is significantly
greater than a pre-determined control expression level. Detectable
nucleic acids, and primers useful in such detection, are described
in detail herein. Such nucleic acids may exclude a nucleic acid
encoding the preproenzyme [1-501]. The invention is further
directed to method of diagnosing the presence of or a predilection
for Alzheimer's disease in a patient, comprising measuring
.beta.-secretase enzymatic activity in a cell sample from said
patient, and diagnosing the patient as having or having a
predilection for Alzheimer's disease, if said level enzymatic
activity level is significantly greater than a pre-determined
control activity level.
[0023] The diagnostic methods may be carried out in a whole cell
assay and/or on a nucleic acid derived from a cell sample of said
patient.
[0024] The invention also includes a method of purifying a
.beta.-secretase protein enzyme molecule. According to this aspect,
an impure sample containing .beta.-secretase enzyme activity with
an affinity matrix which includes a .beta.-secretase inhibitor,
such as the various inhibitor molecules described herein.
[0025] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1A shows the sequence of a polynucleotide (SEQ ID NO:
1) which encodes human .beta.-secretase translation product shown
in FIG. 2A.
[0027] FIG. 1B shows the polynucleotide of FIG. 1A, including
putative 5'- and 3'-untranslated regions (SEQ ID NO: 44).
[0028] FIG. 2A shows the amino acid sequence (SEQ ID NO: 2) of the
predicted translation product of the open reading frame of the
polynucleotide sequence shown in FIGS. 1A and 1B.
[0029] FIG. 2B shows the amino acid sequence of an active fragment
of human .beta.-secretase (SEQ ID NO: 43).
[0030] FIG. 3A shows the translation product that encodes an active
fragment of human .beta.-secretase, 452stop, (amino acids 1-452
with reference to SEQ ID NO: 2; SEQ ID NO: 59) including a
FLAG-epitope tag (underlined; SEQ ID NO: 45) at the C-terminus.
[0031] FIG. 3B shows the amino acid sequence of a fragment of human
.beta.-secretase (amino acids 46-452 (SEQ ID NO: 58) with reference
to SEQ ID NO: 2; including a FLAG-epitope tag (underlined; SEQ ID
NO: 45) at the C-terminus.
[0032] FIG. 4 shows an elution profile of recombinant
.beta.-secretase eluted from a gel filtration column.
[0033] FIG. 5 shows the full length amino acid sequence of
.beta.-secretase 1-501 (SEQ ID NO: 2), including the ORF which
encodes it (SEQ ID NO: 1), with certain features indicated, such as
"active-D" sites indicating the aspartic acid active catalytic
sites, a transmembrane region commencing at position 453, as well
as leader ("Signal") sequence (residues 1-21; SEQ ID NO: 46) and
putative pro region (residues 22-45; SEQ ID NO: 47) and where the
polynucleotide region corresponding the proenzyme region
corresponding to amino acids 46-501 (SEQ ID NO: 43) (nt 135-1503)
is shown as SEQ ID NO: 44 and contains an internal peptide region
(SEQ ID NO:56) and a transmembrane region (SEQ ID NO:62).
[0034] FIGS. 6A and 6B show images of silver-stained SDS-PAGE gels
on which purified .beta.-secretase-containing fractions were run
under reducing (6A) and non-reducing (6B) conditions.
[0035] FIG. 7 shows a silver-stained SDS-PAGE of .beta.-secretase
purified from heterologous 293T cells expressing the recombinant
enzyme.
[0036] FIG. 8 shows a silver-stained SDS-PAGE of .beta.-secretase
purified from heterologous Cos A2 cells expressing the recombinant
enzyme.
[0037] FIG. 9 shows a scheme in which primers derived from the
polynucleotide (SEQ ID NO. 76 encoding N-terminus of purified
naturally occurring .beta.-secretase (SEQ ID NO. 77) were used to
PCR-clone additional portions of the molecule, such as fragment SEQ
ID NO. 79 encoding by nucleic acid SEQ ID NO. 98, as
illustrated.
[0038] FIG. 10 shows an alignment of the amino acid sequence of
human .beta.-secretase ("Human Imapain.seq," 1-501, SEQ ID NO: 2)
compared to ("pBS/mImpain H#3 cons") consensus mouse sequence: SEQ
ID NO: 65.
[0039] FIG. 11A shows the nucleotide sequence (SEQ ID NO: 80) of an
insert used in preparing vector pCF.
[0040] FIG. 11B shows a linear schematic of pCEK.
[0041] FIG. 12 shows a schematic of pCEK.clone 27 used to transfect
mammalian cells with .beta.-secretase.
[0042] FIG. 13(A-E) shows the nucleotide sequence of pCEK clone 27
(SEQ ID NO: 48), with the ORF indicated by the amino acid sequence
SEQ ID NO: 2.
[0043] FIG. 14A shows a nucleotide sequence inserted into parent
vector pCDNA3 (SEQ ID NO: 80).
[0044] FIG. 14B shows a plot of .beta.-secretase activity in cell
lysates from COS cells transfected with vectors derived from clones
encoding .beta.-secretase.
[0045] FIG. 15A shows an image of an SDS PAGE gel loaded with
triplicate samples of the lysates made from heterologous cells
transfected with mutant APP (751 wt) and .beta.-galactosidase as
control (lanes d) and from cells transfected with mutant APP (751
wt) and .beta.-secretase (lanes f) where lanes a, b, and c show
lysates from untreated cells, cells transfected with
.beta.-galactosidase alone and cells transfected with
.beta.-secretase alone, respectively, and lane e indicates
markers.
[0046] FIG. 15B shows an image an image of an SDS PAGE gel loaded
with triplicate samples of the lysates made from heterologous cells
transfected with mutant APP (Swedish mutation) and
.beta.-galactosidase as control (lanes c) and from cells
transfected with mutant APP (Swedish mutation) and .beta.-secretase
(lanes e) where lanes a and b show lysates from cells transfected
with .beta.-galactosidase alone and cells transfected with
.beta.-secretase alone, and lane d indicates markers.
[0047] FIGS. 16A and 16B show Western blots of cell supernatants
tested for presence or increase in soluble APP (sAPP).
[0048] FIGS. 17A and 17B show Western blots of .alpha.-cleaved APP
substrate in co-expression cells.
[0049] FIG. 18 shows A.beta. (x-40) production in 293T cells
cotransfected with APP and .beta.-secretase.
[0050] FIG. 19A shows a schematic of an APP substrate fragment, and
its use in conjunction with antibodies SW192 and 8E-192 in the
assay.
[0051] FIG. 19B shows the .beta.-secretase cleavage sites in the
wild-type APP sequence (SEQ ID NO: 103) and Swedish APP sequence
(SEQ ID NO: 104).
[0052] FIG. 20 shows a schematic of a second APP substrate fragment
derived from APP 638, and it use in conjunction with antibodies
SW192 and 8E-192 in the assay.
[0053] FIG. 21 shows a schematic of pohCK751 vector.
BRIEF DESCRIPTION OF THE SEQUENCES
[0054] This section briefly identifies the sequence identification
numbers referred to herein. Number ranges shown in brackets here
and throughout the specification are referenced to the amino acid
sequence SEQ ID NO: 2, using conventional N.fwdarw.C-terminus
order.
[0055] SEQ ID NO: 1 is a nucleic acid sequence that encodes human
.beta.-secretase, including an active fragment, as exemplified
herein.
[0056] SEQ ID NO: 2 is the predicted translation product of SEQ ID
NO: 1 [1-501].
[0057] SEQ ID NOS: 3-21 are degenerate oligonucleotide primers
described in Example 1 (Table 4), designed from regions of SEQ ID
NO: 2.
[0058] SEQ ID NOS: 22-41 are additional oligonucleotide primers
used in PCR cloning methods described herein, shown in Table 5.
[0059] SEQ ID NO: 42 is a polynucleotide sequence that encodes the
active enzyme .beta.-secretase shown as SEQ ID NO: 43.
[0060] SEQ ID NO: 43 is the sequence of an active enzyme portion of
human .beta.-secretase, the N-terminus of which corresponds to the
N-terminus of the predominant form of the protein isolated from
natural sources [46-501].
[0061] SEQ ID NO: 44 is a polynucleotide which encodes SEQ ID NO:
2, including 5' and 3' untranslated regions.
[0062] SEQ ID NO: 45 is the FLAG sequence used in conjunction with
certain polynucleotides.
[0063] SEQ ID NO: 46 is the putative leader region of
.beta.-secretase [1-22].
[0064] SEQ ID NO: 47 is the putative pre-pro region of
.beta.-secretase [23-45].
[0065] SEQ ID NO: 48 is the sequence of the clone pCEK C1.27 (FIG.
13A-E).
[0066] SEQ ID NO: 49 is a nucleotide sequence of a fragment of the
gene which encodes human .beta.-secretase.
[0067] SEQ ID NO: 50 is the predicted translation product of SEQ ID
NO: 49.
[0068] SEQ ID NO: 51 is a peptide sequence cleavage site of APP
(Swedish mutation).
[0069] SEQ ID NOS: 52 and 53 are peptide substrates suitable for
use in .beta.-secretase assays used in the present invention.
[0070] SEQ ID NO: 54 is a peptide sequence cleavage site of APP
(wild type) recognized by human .beta.-secretase.
[0071] SEQ ID NO: 55 is amino acids 46-69 of SEQ ID NO: 2.
[0072] SEQ ID NO: 56 is an internal peptide just N-terminal to the
transmembrane domain of .beta.-secretase.
[0073] SEQ ID NO: 57 is .beta.-secretase [1-419].
[0074] SEQ ID NO: 58 is .beta.-secretase [46-452].
[0075] SEQ ID NO: 59 is .beta.-secretase [1-452].
[0076] SEQ ID NO: 60 is .beta.-secretase [1-420].
[0077] SEQ ID NO: 61 is EVM[hydroxyethylene]AEF.
[0078] SEQ ID NO: 62 is the amino acid sequence of the
transmembrane domain of .beta.-secretase shown in (FIG. 5).
[0079] SEQ ID NO: 63 is P26-P4' of APPwt.
[0080] SEQ ID NO: 64 is P26-P1' of APPwt.
[0081] SEQ ID NO: 65 is mouse .beta.-secretase (FIG. 10, lower
sequence).
[0082] SEQ ID NO: 66 is .beta.-secretase [22-501].
[0083] SEQ ID NO: 67 is .beta.-secretase [58-501].
[0084] SEQ ID NO: 68 is .beta.-secretase [58-452].
[0085] SEQ ID NO: 69 is .beta.-secretase [63-501].
[0086] SEQ ID NO: 70 is .beta.-secretase [63-452].
[0087] SEQ ID NO: 71 is .beta.-secretase [46-419].
[0088] SEQ ID NO: 72 is P1-P4'staD.fwdarw.V.
[0089] SEQ ID NO: 73 is P4-P4'staD.fwdarw.V.
[0090] SEQ ID NO: 74 is .beta.-secretase [22-452].
[0091] SEQ ID NO: 75 is .beta.-secretase [63-423].
[0092] SEQ ID NO: 76 is nucleic acid encoding the N-terminus of
naturally occurring .beta.-secretase.
[0093] SEQ ID NO: 77 is a peptide fragment at the N-terminus of
naturally occurring .beta.-secretase.
[0094] SEQ ID NO: 78 is a P3-P4'XD.fwdarw.V (VMXVAEF, where X is
hydroxyethylene or statine).
[0095] SEQ ID NO: 79 is a peptide fragment of naturally occurring
.beta.-secretase.
[0096] SEQ ID NO: 80 is a nucleotide insert in vector pCF used
herein.
[0097] SEQ ID NO: 81 is P4-P4'XD.fwdarw.V (EVMXVAEF, where X is
hydroxyethylene or statine).
[0098] SEQ ID NO: 82 is APP fragment SEVKMDAEF (P 5-P4'wt).
[0099] SEQ ID NO: 83 is APP fragment SEVNLDAEF (P5-P4'sw).
[0100] SEQ ID NO: 84 is APP fragment SEVKLDAEF.
[0101] SEQ ID NO: 85 is APP fragment SEVKFDAEF.
[0102] SEQ ID NO: 86 is APP fragment SEVNFDAEF.
[0103] SEQ ID NO: 87 is APP fragment SEVKMAAEF.
[0104] SEQ ID NO: 88 is APP fragment SEVNLAAEF.
[0105] SEQ ID NO: 89 is APP fragment SEVKLAAEF.
[0106] SEQ ID NO: 90 is APP fragment SEVKMLAEF.
[0107] SEQ ID NO: 91 is APP fragment SEVNLLAEF.
[0108] SEQ ID NO: 92 is APP fragment SEVKLLAEF.
[0109] SEQ ID NO: 93 is APP fragment SEVKFAAEF.
[0110] SEQ ID NO: 94 is APP fragment SEVNFAAEF.
[0111] SEQ ID NO: 95 is APP fragment SEVKFLAEF.
[0112] SEQ ID NO: 96 is APP fragment SEVNFLAEF.
[0113] SEQ ID NO: 97 is APP-derived fragment P10-P4'(D.fwdarw.V):
KTEEISEVNLVAEF.
[0114] SEQ ID NO: 98 is a nucleic acid fragment (FIG. 9).
[0115] SEQ ID NO: 99 is the N terminal peptide sequence of
.beta.-secretase isolated from human brain, recombinant 293T cells
and recombinant Cos A2 cells (Table 3).
[0116] SEQ ID NO: 100 is the N terminal peptide sequence of a form
of .beta.-secretase isolated from recombinant 293T cells.
[0117] SEQ ID NO: 101 is the N terminal peptide sequence of a form
of .beta.-secretase isolated from recombinant 293T cells.
[0118] SEQ ID NO: 102 is the N terminal peptide sequence of a form
of .beta.-secretase isolated from recombinant CosA2 cells.
[0119] SEQ ID NO: 103 is the .beta.-secretase cleavage sites in the
wild-type APP sequence.
[0120] SEQ ID NO: 104 is the .beta.-secretase cleavage sites in the
Swedish APP sequence.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0121] Unless otherwise indicated, all terms used herein have the
same meaning as they would to one skilled in the art of the present
invention. Practitioners are particularly directed to Sambrook, et
al. (1989) Molecular Cloning: A Laboratory Manual (Second Edition),
Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M., et
al. (1998) Current Protocols in Molecular Biology, John Wiley &
Sons, New York, N.Y., for definitions, terms of art and standard
methods known in the art of molecular biology, particularly as it
relates to the cloning protocols described herein. It is understood
that this invention is not limited to the particular methodology,
protocols, and reagents described, as these may be varied to
produce the same result.
[0122] The terms "polynucleotide" and "nucleic acid" are used
interchangeably herein and refer to a polymeric molecule having a
backbone that supports bases capable of hydrogen bonding to typical
polynucleotides, where the polymer backbone presents the bases in a
manner to permit such hydrogen bonding in a sequence specific
fashion between the polymeric molecule and a typical polynucleotide
(e.g., single-stranded DNA). Such bases are typically inosine,
adenosine, guanosine, cytosine, uracil and thymidine. Polymeric
molecules include double and single stranded RNA and DNA, and
backbone modifications thereof, for example, methylphosphonate
linkages.
[0123] The term "vector" refers to a polynucleotide having a
nucleotide sequence that can assimilate new nucleic acids, and
propagate those new sequences in an appropriate host. Vectors
include, but are not limited to recombinant plasmids and viruses.
The vector (e.g., plasmid or recombinant virus) comprising the
nucleic acid of the invention can be in a carrier, for example, a
plasmid complexed to protein, a plasmid complexed with lipid-based
nucleic acid transduction systems, or other non-viral carrier
systems.
[0124] The term "polypeptide" as used herein refers to a compound
made up of a single chain of amino acid residues linked by peptide
bonds. The term "protein" may be synonymous with the term
"polypeptide" or may refer to a complex of two or more
polypeptides.
[0125] The term "modified", when referring to a polypeptide of the
invention, means a polypeptide which is modified either by natural
processes, such as processing or other post-translational
modifications, or by chemical modification techniques which are
well known in the art. Among the numerous known modifications which
may be present include, but are not limited to, acetylation,
acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor
formation, covalent attachment of a lipid or lipid derivative,
methylation, myristlyation, pegylation, prenylation,
phosphorylation, ubiquitination, or any similar process.
[0126] The term ".beta.-secretase" is defined in Section III,
herein.
[0127] The term "biologically active" used in conjunction with the
term .beta.-secretase refers to possession of a .beta.-secretase
enzyme activity, such as the ability to cleave .beta.-amyloid
precursor protein (APP) to produce .beta.-amyloid peptide
(A.beta.).
[0128] The term "fragment," when referring to .beta.-secretase of
the invention, means a polypeptide which has an amino acid sequence
which is the same as part of but not all of the amino acid sequence
of full-length .beta.-secretase polypeptide. In the context of the
present invention, the full length .beta.-secretase is generally
identified as SEQ ID NO: 2, the ORF of the full-length nucleotide;
however, according to a discovery of the invention, the naturally
occurring active form is probably one or more N-terminal truncated
versions, such as amino acids 46-501 (SEQ ID NO:43), 22-501 (SEQ ID
NO:66), 58-501 (SEQ ID NO:67) or 63-501 (SEQ ID NO:69); other
active forms are C-terminal truncated forms ending between about
amino acids 450 and 452. The numbering system used throughout is
based on the numbering of the sequence SEQ ID NO: 2.
[0129] An "active fragment" is a .beta.-secretase fragment that
retains at least one of the functions or activities of
.beta.-secretase, including but not limited to the .beta.-secretase
enzyme activity discussed above and/or ability to bind to the
inhibitor substrate described herein as P10-P4'staD->V (SEQ ID
NO:72). Fragments contemplated include, but are not limited to, a
.beta.-secretase fragment which retains the ability to cleave
.beta.-amyloid precursor protein to produce .beta.-amyloid peptide.
Such a fragment preferably includes at least 350, and more
preferably at least 400, contiguous amino acids or conservative
substitutions thereof of .beta.-secretase, as described herein.
More preferably, the fragment includes active aspartyl acid
residues in the structural proximities identified and defined by
the primary polypeptide structure shown as SEQ ID NO: 2 and also
denoted as "Active-D" sites herein.
[0130] A "conservative substitution" refers to the substitution of
an amino acid in one class by an amino acid in the same class,
where a class is defined by common physicochemical amino acid
sidechain properties and high substitution frequencies in
homologous proteins found in nature (as determined, e.g., by a
standard Dayhoff frequency exchange matrix or BLOSUM matrix). Six
general classes of amino acid sidechains, categorized as described
above, include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly);
Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V
(Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example,
substitution of an Asp for another class III residue such as Asn,
Gln, or Glu, is considered to be a conservative substitution.
[0131] "Optimal alignment" is defined as an alignment giving the
highest percent identity score. Such alignment can be performed
using a variety of commercially available sequence analysis
programs, such as the local alignment program LALIGN using a ktup
of 1, default parameters and the default PAM. A preferred alignment
is the pairwise alignment using the CLUSTAL-W program in MacVector,
operated with default parameters, including an open gap penalty of
10.0, an extended gap penalty of 0.1, and a BLOSUM30 similarity
matrix.
[0132] "Percent sequence identity," with respect to two amino acid
or polynucleotide sequences, refers to the percentage of residues
that are identical in the two sequences when the sequences are
optimally aligned. Thus, 80% amino acid sequence identity means
that 80% of the amino acids in two or more optimally aligned
polypeptide sequences are identical. If a gap needs to be inserted
into a first sequence to optimally align it with a second sequence,
the percent identity is calculated using only the residues that are
paired with a corresponding amino acid residue (i.e., the
calculation does not consider residues in the second sequences that
are in the "gap" of the first sequence.
[0133] A first polypeptide region is said to "correspond" to a
second polypeptide region when the regions are essentially
co-extensive when the sequences containing the regions are aligned
using a sequence alignment program, as above. Corresponding
polypeptide regions typically contain a similar, if not identical,
number of residues. It will be understood, however, that
corresponding regions may contain insertions or deletions of
residues with respect to one another, as well as some differences
in their sequences.
[0134] A first polynucleotide region is said to "correspond" to a
second polynucleotide region when the regions are essentially
co-extensive when the sequences containing the regions are aligned
using a sequence alignment program, as above. Corresponding
polynucleotide regions typically contain a similar, if not
identical, number of residues. It will be understood, however, that
corresponding regions may contain insertions or deletions of bases
with respect to one another, as well as some differences in their
sequences.
[0135] The term "sequence identity" means nucleic acid or amino
acid sequence identity in two or more aligned sequences, aligned as
defined above.
[0136] "Sequence similarity" between two polypeptides is determined
by comparing the amino acid sequence and its conserved amino acid
substitutes of one polypeptide to the sequence of a second
polypeptide. Thus, 80% protein sequence similarity means that 80%
of the amino acid residues in two or more aligned protein sequences
are conserved amino acid residues, i.e., are conservative
substitutions.
[0137] "Hybridization" includes any process by which a strand of a
nucleic acid joins with a complementary nucleic acid strand through
base pairing. Thus, strictly speaking, the term refers to the
ability of the complement of the target sequence to bind to the
test sequence, or vice-versa.
[0138] "Hybridization conditions" are based in part on the melting
temperature (Tm) of the nucleic acid binding complex or probe and
are typically classified by degree of "stringency" of the
conditions under which hybridization is measured. The specific
conditions that define various degrees of stringency (i.e., high,
medium, low) depend on the nature of the polynucleotide to which
hybridization is desired, particularly its percent GC content, and
can be determined empirically according to methods known in the
art. Functionally, maximum stringency conditions may be used to
identify nucleic acid sequences having strict identity or
near-strict identity with the hybridization probe; while high
stringency conditions are used to identify nucleic acid sequences
having about 80% or more sequence identity with the probe.
[0139] The term "gene" as used herein means the segment of DNA
involved in producing a polypeptide chain; it may include regions
preceding and following the coding region, e.g., 5' untranslated
(5' UTR) or "leader" sequences and 3' UTR or "trailer" sequences,
as well as intervening sequences (introns) between individual
coding segments (exons).
[0140] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Such isolated polynucleotides may be part of a vector
and/or such polynucleotides or polypeptides may be part of a
composition, such as a recombinantly produced cell (heterologous
cell) expressing the polypeptide, and still be isolated in that
such vector or composition is not part of its natural
environment.
[0141] An "isolated polynucleotide having a sequence which encodes
.beta.-secretase" is a polynucleotide that contains the coding
sequence of .beta.-secretase, or an active fragment thereof, (i)
alone, (ii) in combination with additional coding sequences, such
as fusion protein or signal peptide, in which the .beta.-secretase
coding sequence is the dominant coding sequence, (iii) in
combination with non-coding sequences, such as introns and control
elements, such as promoter and terminator elements or 5' and/or 3'
untranslated regions, effective for expression of the coding
sequence in a suitable host, and/or (iv) in a vector or host
environment in which the .beta.-secretase coding sequence is a
heterologous gene.
[0142] The terms "heterologous DNA," "heterologous RNA,"
"heterologous nucleic acid," "heterologous gene," and "heterologous
polynucleotide" refer to nucleotides that are not endogenous to the
cell or part of the genome in which they are present; generally
such nucleotides have been added to the cell, by transfection,
microinjection, electroporation, or the like. Such nucleotides
generally include at least one coding sequence, but this coding
sequence need not be expressed.
[0143] The term "heterologous cell" refers to a recombinantly
produced cell that contains at least one heterologous DNA
molecule.
[0144] A "recombinant protein" is a protein isolated, purified, or
identified by virtue of expression in a heterologous cell, said
cell having been transduced or transfected, either transiently or
stably, with a recombinant expression vector engineered to drive
expression of the protein in the host cell.
[0145] The term "expression" means that a protein is produced by a
cell, usually as a result of transfection of the cell with a
heterologous nucleic acid.
[0146] "Co-expression" is a process by which two or more proteins
or RNA species of interest are expressed in a single cell.
Co-expression of the two or more proteins is typically achieved by
transfection of the cell with one or more recombinant expression
vectors(s) that carry coding sequences for the proteins. In the
context of the present invention, for example, a cell can be said
to "co-express" two proteins, if one or both of the proteins is
heterologous to the cell.
[0147] The term "expression vector" refers to vectors that have the
ability to incorporate and express heterologous DNA fragments in a
foreign cell. Many prokaryotic and eukaryotic expression vectors
are commercially available. Selection of appropriate expression
vectors is within the knowledge of those having skill in the
art.
[0148] The terms "purified" or "substantially purified" refer to
molecules, either polynucleotides or polypeptides, that are removed
from their natural environment, isolated or separated, and are at
least 90% and more preferably at least 95-99% free from other
components with which they are naturally associated. The foregoing
notwithstanding, such a descriptor does not preclude the presence
in the same sample of splice- or other protein variants
(glycosylation variants) in the same, otherwise homogeneous,
sample.
[0149] A protein or polypeptide is generally considered to be
"purified to apparent homogeneity" if a sample containing it shows
a single protein band on a silver-stained polyacrylamide
electrophoretic gel.
[0150] The term "crystallized protein" means a protein that has
co-precipitated out of solution in pure crystals consisting only of
the crystal, but possibly including other components that are
tightly bound to the protein.
[0151] A "variant" polynucleotide sequence may encode a "variant"
amino acid sequence that is altered by one or more amino acids from
the reference polypeptide sequence. The variant polynucleotide
sequence may encode a variant amino acid sequence, which contains
"conservative" substitutions, wherein the substituted amino acid
has structural or chemical properties similar to the amino acid
which it replaces. In addition, or alternatively, the variant
polynucleotide sequence may encode a variant amino acid sequence,
which contains "non-conservative" substitutions, wherein the
substituted amino acid has dissimilar structural or chemical
properties to the amino acid which it replaces. Variant
polynucleotides may also encode variant amino acid sequences, which
contain amino acid insertions or deletions, or both. Furthermore, a
variant polynucleotide may encode the same polypeptide as the
reference polynucleotide sequence but, due to the degeneracy of the
genetic code, has a polynucleotide sequence that is altered by one
or more bases from the reference polynucleotide sequence.
[0152] An "allelic variant" is an alternate form of a
polynucleotide sequence, which may have a substitution, deletion or
addition of one or more nucleotides that does not substantially
alter the function of the encoded polypeptide.
[0153] "Alternative splicing" is a process whereby multiple
polypeptide isoforms are generated from a single gene, and involves
the splicing together of nonconsecutive exons during the processing
of some, but not all, transcripts of the gene. Thus, a particular
exon may be connected to any one of several alternative exons to
form messenger RNAs. The alternatively-spliced mRNAs produce
polypeptides ("splice variants") in which some parts are common
while other parts are different.
[0154] "Splice variants" of .beta.-secretase, when referred to in
the context of an mRNA transcript, are mRNAs produced by
alternative splicing of coding regions, i.e., exons, from the
.beta.-secretase gene.
[0155] "Splice variants" of .beta.-secretase, when referred to in
the context of the protein itself, are .beta.-secretase translation
products that are encoded by alternatively-spliced .beta.-secretase
mRNA transcripts.
[0156] A "mutant" amino acid or polynucleotide sequence is a
variant amino acid sequence, or a variant polynucleotide sequence,
which encodes a variant amino acid sequence that has significantly
altered biological activity or function from that of the naturally
occurring protein.
[0157] A "substitution" results from the replacement of one or more
nucleotides or amino acids by different nucleotides or amino acids,
respectively.
[0158] The term "modulate" as used herein refers to the change in
activity of the polypeptide of the invention. Modulation may relate
to an increase or a decrease in biological activity, binding
characteristics, or any other biological, functional, or
immunological property of the molecule.
[0159] The terms "antagonist" and "inhibitor" are used
interchangeably herein and refer to a molecule which, when bound to
the polypeptide of the present invention, modulates the activity of
enzyme by blocking, decreasing, or shortening the duration of the
biological activity. An antagonist as used herein may also be
referred to as a ".beta.-secretase inhibitor" or ".beta.-secretase
blocker." Antagonists may themselves be polypeptides, nucleic
acids, carbohydrates, lipids, small molecules (usually less than
1000 kD), or derivatives thereof, or any other ligand which binds
to and modulates the activity of the enzyme.
.beta.-Secretase Compositions
[0160] The present invention provides an isolated, active human
.beta.-secretase enzyme, which is further characterized as an
aspartyl (aspartic) protease or proteinase, optionally, in purified
form. As defined more fully in the sections that follow,
.beta.-secretase exhibits a proteolytic activity that is involved
in the generation of .beta.-amyloid peptide from .beta.-amyloid
precursor protein (APP), such as is described in U.S. Pat. No.
5,744,346, incorporated herein by reference. Alternatively, or in
addition, the .beta.-secretase is characterized by its ability to
bind, with moderately high affinity, to an inhibitor substrate
described herein as P10-P4' staD.fwdarw.V (SEQ ID NO.: 72).
According to an important feature of the present invention, a human
form of .beta.-secretase has been isolated, and its naturally
occurring form has been characterized purified and sequenced.
[0161] According to another aspect of the invention, nucleotide
sequences encoding the enzyme have been identified. In addition,
the enzyme has been further modified for expression in altered
forms, such as truncated forms, which have similar protease
activity to the naturally occurring or full length recombinant
enzyme. Using the information provided herein, practitioners can
isolate DNA encoding various active forms of the protein from
available sources and can express the protein recombinantly in a
convenient expression system. Alternatively and in addition,
practitioners can purify the enzyme from natural or recombinant
sources and use it in purified form to further characterize its
structure and function. According to a further feature of the
invention, polynucleotides and proteins of the invention are
particularly useful in a variety of screening assay formats,
including cell-based screening for drugs that inhibit the enzyme.
Examples of uses of such assays, as well as additional utilities
for the compositions are provided in Section IV, below.
[0162] .beta.-secretase is of particular interest due to its
activity and involvement in generating fibril peptide components
that are the major components of amyloid plaques in the central
nervous system (CNS), such as are seen in Alzheimer's disease,
Down's syndrome and other CNS disorders. Accordingly, a useful
feature of the present invention includes an isolated form of the
enzyme that can be used, for example, to screen for inhibitory
substances which are candidates for therapeutics for such
disorders.
A. Isolation of Polynucleotides Encoding Human .beta.-Secretase
[0163] Polynucleotides encoding human .beta.-secretase were
obtained by PCR cloning and hybridization techniques as detailed in
Examples 1-3 and described below. FIG. 1A shows the sequence of a
polynucleotide (SEQ ID NO: 1) which encodes a form of human
.beta.-secretase (SEQ ID NO.: 2 [1-501]. Polynucleotides encoding
human .beta.-secretase are conveniently isolated from any of a
number of human tissues, preferably tissues of neuronal origin,
including but not limited to neuronal cell lines such as the
commercially available human neuroblastoma cell line IMR-32
available from the American Type Culture Collection (Manassas, Va.;
ATTC CCL 127) and human fetal brain, such as a human fetal brain
cDNA library available from OriGene Technologies, Inc. (Rockville,
Md.).
[0164] Briefly, human .beta.-secretase coding regions were isolated
by methods well known in the art, using hybridization probes
derived from the coding sequence provided as SEQ ID NO: 1. Such
probes can be designed and made by methods well known in the art.
Exemplary probes, including degenerate probes, are described in
Example 1. Alternatively, a cDNA library is screened by PCR, using,
for example, the primers and conditions described in Example 2
herein. Such methods are discussed in more detail in Part B,
below.
[0165] cDNA libraries were also screened using a 3'-RACE (Rapid
Amplification of cDNA Ends) protocol according to methods well
known in the art (White, B. A., ed., PCR Cloning Protocols; Humana
Press, Totowa, N.J., 1997; shown schematically in FIG. 9). Here
primers derived from the 5' portion of SEQ ID NO: 1 are added to
partial cDNA substrate clone found by screening a fetal brain cDNA
library as described above. A representative 3'RACE reaction used
in determining the longer sequence is detailed in Example 3 and is
described in more detail in Part B, below.
[0166] Human .beta.-secretase, as well as additional members of the
neuronal aspartyl protease family described herein may be
identified by the use of random degenerate primers designed in
accordance with any portion of the polypeptide sequence shown as
SEQ ID NO: 2. For example, in experiments carried out in support of
the present invention, and detailed in Example 1 herein, eight
degenerate primer pools, each 8-fold degenerate, were designed
based on a unique 22 amino acid peptide region selected from SEQ
ID: 2. Such techniques can be used to identify further similar
sequences from other species and/or representing other members of
this protease family.
Preparation of Polynucleotides
[0167] The polynucleotides described herein may be obtained by
screening cDNA libraries using oligonucleotide probes, which can
hybridize to and/or PCR-amplify polynucleotides that encode human
.beta.-secretase, as disclosed above. cDNA libraries prepared from
a variety of tissues are commercially available, and procedures for
screening and isolating cDNA clones are well known to those of
skill in the art. Genomic libraries can likewise be screened to
obtain genomic sequences including regulatory regions and introns.
Such techniques are described in, for example, Sambrook et al.
(1989) Molecular Cloning: A Laboratory Manual (2nd Edition), Cold
Spring Harbor Press, Plainview, N.Y. and Ausubel, F M et al. (1998)
Current Protocols in Molecular Biology, John Wiley & Sons, New
York, N.Y.
[0168] The polynucleotides may be extended to obtain upstream and
downstream sequences such as promoters, regulatory elements, and 5'
and 3' untranslated regions (UTRs). Extension of the available
transcript sequence may be performed by numerous methods known to
those of skill in the art, such as PCR or primer extension
(Sambrook et al., supra), or by the RACE method using, for example,
the MARATHON RACE kit (Cat. # K1802-1; Clontech, Palo Alto,
Calif.).
[0169] Alternatively, the technique of "restriction-site" PCR
(Gobinda et al. (1993) PCR Methods Applic. 2:318-22), which uses
universal primers to retrieve flanking sequence adjacent a known
locus, may be employed to generate additional coding regions.
First, genomic DNA is amplified in the presence of primer to a
linker sequence and a primer specific to the known region. The
amplified sequences are subjected to a second round of PCR with the
same linker primer and another specific primer internal to the
first one. Products of each round of PCR are transcribed with an
appropriate RNA polymerase and sequenced using reverse
transcriptase.
[0170] Inverse PCR can be used to amplify or extend sequences using
divergent primers based on a known region (Triglia T et al. (1988)
Nucleic Acids Res 16:8186). The primers may be designed using
OLIGO(R) 4.06 Primer Analysis Software (1992; National Biosciences
Inc, Plymouth, Minn.), or another appropriate program, to be 22-30
nucleotides in length, to have a GC content of 50% or more, and to
anneal to the target sequence at temperatures about 68-72.degree.
C. The method uses several restriction enzymes to generate a
suitable fragment in the known region of a gene. The fragment is
then circularized by intramolecular ligation and used as a PCR
template.
[0171] Capture PCR (Lagerstrom M et al. (1991) PCR Methods Applic
1:111-19) is a method for PCR amplification of DNA fragments
adjacent to a known sequence in human and yeast artificial
chromosome DNA. Capture PCR also requires multiple restriction
enzyme digestions and ligations to place an engineered
double-stranded sequence into a flanking part of the DNA molecule
before PCR.
[0172] Another method which may be used to retrieve flanking
sequences is that of Parker, J D et al. (1991; Nucleic Acids Res
19:3055-60). Additionally, one can use PCR, nested primers and
PromoterFinder.TM. libraries to "walk in" genomic DNA (Clontech,
Palo Alto, Calif.). This process avoids the need to screen
libraries and is useful in finding intron/exon junctions. Preferred
libraries for screening for full length cDNAs are ones that have
been size-selected to include larger cDNAs. Also, random primed
libraries are preferred in that they will contain more sequences
which contain the 5' and upstream regions of genes. A randomly
primed library may be particularly useful if an oligo d(T) library
does not yield a full-length cDNA. Genomic libraries are useful for
extension into the 5' nontranslated regulatory region.
[0173] The polynucleotides and oligonucleotides of the invention
can also be prepared by solid-phase methods, according to known
synthetic methods. Typically, fragments of up to about 100 bases
are individually synthesized, then joined to form continuous
sequences up to several hundred bases.
B. Isolation of .beta.-Secretase
[0174] The amino acid sequence for a full-length human
.beta.-secretase translation product is shown as SEQ ID NO: 2 in
FIG. 2A. According to the discovery of the present invention, this
sequence represents a "pre pro" form of the enzyme that was deduced
from the nucleotide sequence information described in the previous
section in conjunction with the methods described below. Comparison
of this sequence with sequences determined from the biologically
active form of the enzyme purified from natural sources, as
described in Part 4, below, indicate that it is likely that an
active and predominant form of the enzyme is represented by
sequence shown in FIG. 2B (SEQ ID NO: 43), in which the first 45
amino acids of the open-reading frame deduced sequence have been
removed. This suggests that the enzyme may be post-translationally
modified by proteolytic activity, which may be autocatalytic in
nature. Further analysis, illustrated by the schematics shown in
FIG. 5 herein, indicates that the enzyme contains a hydrophobic,
putative transmembrane region near its C-terminus. As described
below, a further discovery of the present invention is that the
enzyme can be truncated prior to this transmembrane region and
still retain .beta.-secretase activity.
1. Purification of .beta.-Secretase from Natural and Recombinant
Sources
[0175] According to an important feature of the present invention,
.beta.-secretase has now been purified from natural and recombinant
sources. U.S. Pat. No. 5,744,346, incorporated herein by reference,
describes isolation of .beta.-secretase in a single peak having an
apparent molecular weight of 260-300,000 (Daltons) by gel exclusion
chromatography. It is a discovery of the present invention that the
native enzyme can be purified to apparent homogeneity by affinity
column chromatography. The methods revealed herein have been used
on preparations from brain tissue as well as on preparations from
293T and recombinant cells; accordingly, these methods are believed
to be generally applicable over a variety of tissue sources. The
practitioner will realize that certain of the preparation steps,
particularly the initial steps, may require modification to
accommodate a particular tissue source and will adapt such
procedures according to methods known in the art. Methods for
purifying .beta.-secretase from human brain as well as from cells
are detailed in Example 5. Briefly, cell membranes or brain tissue
are homogenized, fractionated, and subjected to various types of
column chromatographic matrices, including wheat germ
agglutinin-agarose (WGA), anion exchange chromatography and size
exclusion. Activity of fractions can be measured using any
appropriate assay for .beta.-secretase activity, such as the
MBP-C125 cleavage assay detailed in Example 4. Fractions containing
.beta.-secretase activity elute from this column in a peak elution
volume corresponding to a size of about 260-300 kilodaltons.
[0176] The foregoing purification scheme, which yields
approximately 1,500-fold purification, is similar to that described
in detail in U.S. Pat. No. 5,744,346, incorporated herein by
reference. In accordance with the present invention, further
purification can be achieved by applying the cation exchange
flow-through material to an affinity column that employs as its
affinity matrix a specific inhibitor of .beta.-secretase, termed
"P1-P4'staD->V" (NH.sub.2-KTEEISEVN[sta]VAEF-CO.sub.2H; SEQ ID
NO.: 72). This inhibitor, and methods for making a Sepharose
affinity column which incorporates it, are described in Example 7.
After washing the column, .beta.-secretase and a limited number of
contaminating proteins were eluted with pH 9.5 borate buffer. The
eluate was then fractionated by anion exchange HPLC, using a Mini-Q
column. Fractions containing the activity peak were pooled to give
the final .beta.-secretase preparation. Results of an exemplary run
using this purification scheme are summarized in Table 1. FIG. 6A
shows a picture of a silver-stained SDS PAGE gel run under reducing
conditions, in which .beta.-secretase runs as a 70 kilodalton band.
The same fractions run under non-reducing conditions (FIG. 6B)
provide evidence for disulfide cross-linked oligomers. When the
anion exchange pool fractions 18-21 (see FIG. 6B) were treated with
dithiothreitol (DTT) and re-chromatographed on a Mini Q column,
then subjected to SDS-PAGE under non-reducing conditions, a single
band running at about 70 kilodaltons was observed. Surprisingly,
the purity of this preparation is at least about 200 fold higher
than the previously purified material, described in U.S. Pat. No.
5,744,346. By way of comparison, the most pure fraction described
therein exhibited a specific activity of about 253 nM/h/.mu.g
protein, taking into consideration the MW of substrate MBP-C26sw
(45 kilodaltons). The present method therefore provides a
preparation that is at least about 1000-fold higher purity
(affinity eluate) and as high as about 6000-fold higher purity than
that preparation, which represented at least 5 to 100-fold higher
purity than the enzyme present in a solubilized but unenriched
membrane fraction from human 293 cells.
TABLE-US-00001 TABLE 1 Preparation of .beta.-secretase from Human
Brain Total Specific Activity.sup.a Activity.sup.b % Purification
nM/h nM/h/.mu.g prot. Yield (fold) Brain Extract 19,311,150 4.7 100
1 WGA Eluate 21,189,600 81.4 110 17 Affinity Eluate 11,175,000
257,500 53 54,837 Anion Exchange 3,267,685 1,485,312 17 316,309
Pool .sup.aActivity in MBP-C125sw assay b Specific Activity = (
Product conc . nM ) ( Dilution factor ) ( Enzyme sol . vol ) (
Incub . time h ) ( Enzyme conc . .mu.g / vol ) ##EQU00001##
[0177] Example 5 also describes purification schemes used for
purifying recombinant materials from heterologous cells transfected
with the .beta.-secretase coding sequence. Results from these
purifications are illustrated in FIGS. 7 and 8. Further experiments
carried out in support of the present invention, showed that the
recombinant material has an apparent molecular weight in the range
from 260,000 to 300,000 Daltons when measured by gel exclusion
chromatography. FIG. 4 shows an activity profile of this
preparation run on a gel exclusion chromatography column, such as a
Superdex 200 (26/60) column, according to the methods described in
U.S. Pat. No. 5,744,346, incorporated herein by reference.
1. Sequencing of .beta.-Secretase Protein
[0178] A schematic overview summarizing methods and results for
determining the cDNA sequence encoding the N-terminal peptide
sequence determined from purified .beta.-secretase is shown in FIG.
9. N-terminal sequencing of purified .beta.-secretase protein
isolated from natural sources yielded a 21-residue peptide
sequence, SEQ ID NO. 77, as described above. This peptide sequence,
and its reverse translated fully degenerate nucleotide sequence,
SEQ ID NO. 76, is shown in the top portion of FIG. 4. Two partially
degenerate primer sets used for RT-PCR amplification of a cDNA
fragment encoding this peptide are also summarized in FIG. 4.
Primer set 1 consisted of DNA nucleotide primers #3427-3434, shown
in Table 3 (Example 3). Matrix RT-PCR using combinations of primers
from this set with cDNA reverse transcribed from primary human
neuronal cultures as template yielded the predicted 54 bp cDNA
product with primers #3428-3433, also described in Table 3.
[0179] In further experiments carried out in support of the present
invention, it was found that oligonucleotides from primer sets 1
and 2 could also be used to amplify cDNA fragments of the predicted
size from mouse brain mRNA. DNA sequence demonstrated that such
primers could also be used to clone the murine homolog(s) and other
species homologs of human .beta.-secretase and/or additional
members of the aspartyl protease family described herein by
standard RACE-PCR technology. The sequence of a murine homolog is
presented in FIG. 10 (lower sequence; "pBS/MuImPain H#3 cons"); SEQ
ID NO. 65. The murine polypeptide sequence is about 95% identical
to the human polypeptide sequence.
2. 5' and 3' RACE-PCR for Additional Sequence, Cloning, and mRNA
Analysis
[0180] The unambiguous internal nucleotide sequence from the
amplified fragment provided information which facilitated the
design of internal primers matching the upper (coding) strand for
3' RACE, and lower (non-coding) strand for 5' RACE (Frohman, M. A.,
M. K. Dush and G. R. Martin (1988). "Rapid production of
full-length cDNAs from rare transcripts amplification using a
single gene specific oligo-nucleotide primer." Proc. Natl. Acad.
Sci. U.S.A. 85 (23): 8998-9002). The DNA primers used for this
experiment (#3459 & #3460) are illustrated schematically in
FIG. 9, and the exact sequence of these primers is presented in
Table 4 of Example 3.
[0181] Primers #3459 and #3476 (Table 5) were used for initial 3'
RACE amplification of downstream sequences from the IMR-32 cDNA
library in the vector pLPCXlox. The library had previously been
sub-divided into 100 pools of 5,000 clones per pool, and plasmid
DNA was isolated from each pool. A survey of the 100 pools with the
primers described in Part 2, above, identified individual pools
containing .beta.-secretase clones from the library. Such clones
can be used for RACE-PCR analysis.
[0182] An approximately 1.8 Kb PCR fragment was observed by agarose
gel fractionation of the reaction products. The PCR product was
purified from the gel and subjected to DNA sequence analysis using
primer #3459 (Table 5). The resulting clone sequence, designated
23A, was determined. Six of the first seven deduced amino-acids
from one of the reading frames of 23A were an exact match with the
last 7 amino-acids of the N-terminal sequence (SEQ ID NO. 77)
determined from the purified protein isolated from natural sources
in other experiments carried out in support of this invention. This
observation provided internal validation of the sequences, and
defined the proper reading frame downstream. Furthermore, this DNA
sequence facilitated design of additional primers for extending the
sequence further downstream, verifying the sequence by sequencing
the opposite strand in the upstream direction, and further
facilitated isolating the cDNA clone.
[0183] A DNA sequence of human .beta.-secretase is illustrated as
SEQ ID NO: 42 corresponding to SEQ ID NO: 1 including 5'- and
3'-untranslated regions. This sequence was determined from a
partial cDNA clone (9C7e.35) isolated from a commercially available
human fetal brain cDNA library purchased from OriGene.TM., the 3'
RACE product 23A, and additional clones--a total of 12 independent
cDNA clones were used to determine the composite sequence. The
composite sequence was assembled by sequencing overlapping
stretches of DNA from both strands of the clone or PCR fragment.
The predicted full length translation product is shown as SEQ ID
NO: 2 in FIG. 1B.
3. Tissue Distribution of .beta.-Secretase and Related
Transcripts
[0184] Oligonucleotide primer #3460 (SEQ ID NO. 39, Table 5) was
employed as an end-labeled probe on Northern blots to determine the
size of the transcript encoding .beta.-secretase and to examine its
expression in IMR-32 cells. Additional primers were used to isolate
the mouse cDNA and to characterize mouse tissues, using Marathon
RACE ready cDNA preparations (Clontech, Palo Alto, Calif.). TABLE 2
summarizes the results of experiments in which various human and
murine tissues were tested for the presence of
.beta.-secretase-encoding transcripts by PCR or Northern
blotting.
[0185] For example, the oligo-nucleotide probe 3460 (SEQ ID NO: 39)
hybridized to a 2 Kb transcript in IMR-32 cells, indicating that
the mRNA encoding the .beta.-secretase enzyme is 2 Kb in size in
this tissue. Northern blot analysis of total RNA isolated from the
human T-cell line Jurkat, and human myelomonocyte line Thp1 with
the 3460 oligo-nucleotide probe 3460 also revealed the presence of
a 2 kb transcript in these cells.
[0186] The oligonucleotide probe #3460 also hybridizes to a
.about.2 kb transcript in Northern blots containing RNA from all
human organs examined to date, from both adult and fetal tissue.
The organs surveyed include heart, brain, liver, pancreas,
placenta, lung, muscle, uterus, bladder, kidney, spleen, skin, and
small intestine. In addition, certain tissues, e.g., pancreas,
liver, brain, muscle, uterus, bladder, kidney, spleen and lung,
show expression of larger transcripts of .about.4.5 kb, 5 kb, and
6.5 kb which hybridize with oligonucleotide probe #3460.
[0187] In further experiments carried out in support of the present
invention, Northern blot results were obtained with oligonucleotide
probe #3460 by employing a riboprobe derived from SEQ ID NO: 1,
encompassing nucleotides #155-1014. This clone provides an 860 bp
riboprobe, encompassing the catalytic domain-encoding portion of
.beta.-secretase, for high stringency hybridization. This probe
hybridized with high specificity to the exact match mRNA expressed
in the samples being examined. Northern blots of mRNA isolated from
IMR-32 and 1.degree.HNC probed with this riboprobe revealed the
presence of the 2 kb transcript previously detected with
oligonucleotide #3460, as well as a novel, higher MW transcript of
.about.5 kb. Hybridization of RNA from adult and fetal human
tissues with this 860 nt riboprobe also confirmed the result
obtained with the oligonucleotide probe #3460. The mRNA encoding
.beta.-secretase is expressed in all tissues examined,
predominantly as an .about.5 kb transcript. In adult, its
expression appeared lowest in brain, placenta, and lung,
intermediate in uterus, and bladder, and highest in heart, liver,
pancreas, muscle, kidney, spleen, and lung. In fetal tissue, the
message is expressed uniformly in all tissues examined.
TABLE-US-00002 TABLE 2 Tissue distribution of human and murine
.beta.-secretase transcripts Size Messages Found (Kb): Tissue/Organ
Human Mouse Heart 2.sup.a 3.5, 3.8, 5 & 7 Brain 2, 3, 4, and 7
3.5, 3.8, 5 & 7 Liver 2, 3, 4, and 7 3.5, 3.8, 5 & 7
Pancreas 2, 3, 4, and 7 nd.sup.d Placenta 2.sup.a, 4 and 7.sup.b nd
Lung 2.sup.a, 4 and 7.sup.b 3.5, 3.8, 5 & 7 Muscle 2.sup.a and
7.sup.b 3.5, 3.8, 5 & 7 Uterus 2.sup.a, 4, and 7 nd Bladder
2.sup.a, 3, 4, and 7 nd Kidney 2.sup.a, 3, 4, and 7 3.5, 3.8, 5
& 7 Spleen 2.sup.a, 3, 4, and 7 nd Testis nd 4.5 Kb, 2 Kb
Stomach nd 5.sup.a Sm. Intestine nd 3.5, 3.8, 5 & 7 f
Brain.sup.c 2.sup.a, 3, 4, and 7 nd f Liver 2.sup.a, 3, 4, and 7 nd
f Lung 2.sup.a, 3, 4, and 7 nd f Muscle 2.sup.a, 3, 4, and 7 nd f
Heart 2.sup.a, 3, 4, and 7 nd f Kidney 2.sup.a, 3, 4, and 7 nd f
Skin 2.sup.a, 3, 4, and 7 nd f Sm. Intestine 2.sup.a, 3, 4, and 7
nd Cell Line Human Mouse IMR32 2.sup.a, 5 &7 U937 2.sup.a THP1
2.sup.a Jurkat 2.sup.a HL60 none A293 5 & 7 NALM6 5 & 7
A549 5 & 7 Hela 2, 4, 5, & 7 PC12 2 & 5 J774 5 Kb, 2 Kb
P388D1 ccl46 5 Kb (very little), 2 Kb P19 5 Kb, 2 Kb RBL 5 Kb, 2 Kb
EL4 5 Kb, 2 Kb Clontech Human Brain region Tissue/Organ Human
Cerebellum 2 Kb, 4 Kb, 6 Kb Cerebral Cx 2 Kb, 4 Kb, 6 Kb Medulla 2
Kb, 4 Kb, 6 Kb Spinal Cord 2 Kb, 4 Kb, 6 Kb Occipital Pole 2 Kb, 4
Kb, 6 Kb Frontal Lobe 2 Kb, 4 Kb, 6 Kb Amygdala 2 Kb, 4 Kb, 6 Kb
Caudate N. 2 Kb, 4 Kb, 6 Kb Corpus Callosum 2 Kb, 4 Kb, 6 Kb
Hippocampus 2 Kb, 4 Kb, 6 Kb Substantia Nigra 2 Kb, 4 Kb, 6 Kb
Thalamus 2 Kb, 4 Kb, 6 Kb .sup.aby oligo 3460 probe only
.sup.bfaint .sup.cf = fetal .sup.dnd = not determined
4. Active Forms of .beta.-Secretase
[0188] a. N-Terminus
[0189] The full-length open reading frame (ORF) of human
.beta.-secretase is described above, and its sequence is shown in
FIG. 2A as SEQ ID NO: 2. However, as mentioned above, a further
discovery of the present invention indicates that the predominant
form of the active, naturally occurring molecule is truncated at
the N-terminus by about 45 amino acids. That is, the protein
purified from natural sources was N-terminal sequenced according to
methods known in the art (Argo Bioanalytica, Morris Plains, N.J.).
The N-terminus yielded the following sequence:
EGDEEPEEPGRRGSFVEMVDNLRG . . . (SEQ ID NO: 55). This corresponds to
amino acids 46-69 of the ORF-derived putative sequence. Based on
this observation and others described below, the N-terminus of an
active, naturally occurring, predominant human brain form of the
enzyme is amino acid 46, with respect to SEQ ID NO: 2. Further
processing of the purified protein provided the sequence of an
internal peptide: IGFAVSACHVHDEFR (SEQ ID NO: 56), which is amino
terminal to the putative transmembrane domain, as defined by the
ORF. These peptides were used to validate and provide reading frame
information for the isolated clones described elsewhere in this
application.
[0190] In additional studies carried out in support of the present
invention, N-terminal sequencing of .beta.-secretase isolated from
additional cell types revealed that the N-terminus may be amino
acid numbers 46, 22, 58, or 63 with respect to the ORF sequence
shown in FIG. 2A, depending on the tissue from which the protein is
isolated, with the form having as its N-terminus amino acid 46
predominating in the tissues tested. That is, in experiments
carried out in support of the present invention, the full-length
.beta.-secretase construct (i.e., encoding SEQ ID NO: 2) was
transfected into 293T cells and COS A2 cells, using the Fugene
technique described in Example 6. .beta.-secretase was isolated
from the cells by preparing a crude particulate fraction from the
cell pellet, as described in Example 5, followed by extraction with
buffer containing 0.2% Triton X-100. The Triton extract was diluted
with pH 5.0 buffer and passed through a SP Sepharose column,
essentially according to the methods described in Example 5A. This
step removed the majority of contaminating proteins. After
adjusting the pH to 4.5, .beta.-secretase was further purified and
concentrated on P10-P4'staD.fwdarw.V Sepharose, as described in
Examples 5 and 7. Fractions were analyzed for N-terminal sequence,
according to standard methods known in the art. Results are
summarized in Table 3, below.
[0191] The primary N-terminal sequence of the 293T cell-derived
protein was the same as that obtained from brain. In addition,
minor amounts of protein starting just after the signal sequence
(at Thr-22) and at the start of the aspartyl protease homology
domain (Met-63) were also observed. An additional major form found
in Cos A2 cells resulted from a Gly-58 cleavage.
TABLE-US-00003 TABLE 3 N-terminal Sequences and Amounts of
.beta.-secretase Forms in Various Cell Types Est. Amount N-terminus
Source (pmoles) (Ref.: SEQ ID NO: 2) Sequence Human brain 1-2 46
ETDEEPEEPGR . . . (SEQ ID NO: 99) Recombinant, 293T ~35 46
ETDEEPEEPGR . . . ~7 22 (SEQ ID NO: 99) ~5 63 TQHGIRL(P)LR . . .
(SEQ ID NO: 100) MVDNLRGKS . . . (SEQ ID NO: 101) Recombinant,
CosA2 ~4 46 ETDEEPEEPGR . . . ~3 58 (SEQ ID NO: 99) GSFVEMVDNL . .
. (SEQ ID NO: 102)
b. C-Terminus
[0192] Further experiments carried out in support of the present
invention revealed that the C-terminus of the full-length amino
acid sequence presented as SEQ ID NO: 2 can also be truncated,
while still retaining .beta.-secretase activity of the molecule.
More specifically, as described in more detail in Part D below,
C-terminal truncated forms of the enzyme ending just before the
putative transmembrane region, i.e., at or about 10 amino acids C
terminal to amino acid 452 with respect to SEQ ID NO: 2, exhibit
.beta.-secretase activity, as evidenced by an ability to cleave APP
at the appropriate cleavage site and/or ability to bind SEQ ID NO.
72.
[0193] Thus, using the reference amino acid positions provided by
SEQ ID NO: 2, one form of .beta.-secretase extends from position 46
to position 501 (.beta.-secretase 46-501; SEQ ID NO: 43). Another
form extends from position 46 to any position including and beyond
position 452, (.beta.-secretase 4-452+), with a preferred form
being .beta.-secretase 46-452 (SEQ ID NO: 58). More generally,
another preferred form extends from position 1 to any position
including and beyond position 452, but not including position 501.
Other active forms of the .beta.-secretase protein begin at amino
acid 22, 58, or 63 and may extend to any point including and beyond
the cysteine at position 420, and more preferably, including and
beyond position 452, while still retaining enzymatic activity
(i.e., .beta.-secretase 22-452+; .beta.-secretase 58-452+;
.beta.-secretase 63-452+). As described in Part D, below, those
forms which are truncated at a C-terminal position at or before
about position 452, or even several amino acids thereafter, are
particularly useful in crystallization studies, since they lack all
or a significant portion of the transmembrane region, which may
interfere with protein crystallization. The recombinant protein
extending from position 1 to 452 has been affinity purified using
the procedures described herein.
c. Crystallization of .beta.-secretase
[0194] According to a further aspect, the present invention also
includes purified .beta.-secretase in crystallized form, in the
absence or presence of binding substrates, such as peptide,
modified peptide, or small molecule inhibitors. This section
describes methods and utilities of such compositions.
1. Crystallization of the Protein
[0195] .beta.-secretase purified as described above can be used as
starting material to determine a crystallographic structure and
coordinates for the enzyme. Such structural determinations are
particularly useful in defining the conformation and size of the
substrate binding site. This information can be used in the design
and modeling of substrate inhibitors of the enzyme. As discussed
herein, such inhibitors are candidate molecules for therapeutics
for treatment of Alzheimer's disease and other amyloid diseases
characterized by A.beta. peptide amyloid deposits.
[0196] The crystallographic structure of .beta.-secretase is
determined by first crystallizing the purified protein. Methods for
crystallizing proteins, and particularly proteases, are now well
known in the art. The practitioner is referred to Principles of
Protein X-ray Crystallography (J. Drenth, Springer Verlag, NY,
1999) for general principles of crystallography. Additionally, kits
for generating protein crystals are generally available from
commercial providers, such as Hampton Research (Laguna Niguel,
Calif.). Additional guidance can be obtained from numerous research
articles that have been written in the area of crystallography of
protease inhibitors, especially with respect to HIV-1 and HIV-2
proteases, which are aspartic acid proteases.
[0197] Although any of the various forms of .beta.-secretase
described herein can be used for crystallization studies,
particularly preferred forms lack the first 45 amino acids of the
full length sequence shown as SEQ ID NO: 2, since this appears to
be the predominant form which occurs naturally in human brain. It
is thought that some form of post-translational modification,
possibly autocatalysis, serves to remove the first 45 amino acids
in fairly rapid order, since, to date, virtually no naturally
occurring enzyme has been isolated with all of the first 45 amino
acids intact. In addition, it is considered preferable to remove
the putative transmembrane region from the molecule prior to
crystallization, since this region is not necessary for catalysis
and potentially could render the molecule more difficult to
crystallize.
[0198] Thus, a good candidate for crystallization is
.beta.-secretase 46-452 (SEQ ID NO: 58), since this is a form of
the enzyme that (a) provides the predominant naturally occurring
N-terminus, and (b) lacks the "sticky" transmembrane region, while
(c) retaining .beta.-secretase activity. Alternatively, forms of
the enzyme having extensions that extend part of the way
(approximately 10-15 amino acids) into the transmembrane domain may
also be used. In general, for determining X-ray crystallographic
coordinates of the ligand binding site, any form of the enzyme can
be used that either (i) exhibits .beta.-secretase activity, and/or
(ii) binds to a known inhibitor, such as the inhibitor ligand
P10-P4'staD.fwdarw.V, with a binding affinity that is at least
1/100 the binding affinity of .beta.-secretase [46-501] (SEQ ID
NO:43) to P10-P4'staD.fwdarw.V (SEQ ID NO:72). Therefore, a number
of additional truncated forms of the enzyme can be used in these
studies. Suitability of any particular form can be assessed by
contacting it with the P10-P4'staD->V affinity matrix described
above. Truncated forms of the enzyme that bind to the matrix are
suitable for such further analysis. Thus, in addition to 46-452,
discussed above, experiments in support of the present invention
have revealed that a truncated form ending in residue 419, most
likely 46-419 (SEQ ID NO:71), also binds to the affinity matrix and
is therefore an alternative candidate protein composition for X-ray
crystallographic analysis of .beta.-secretase. More generally, any
form of the enzyme that ends before the transmembrane domain,
particularly those ending between about residue 419 and 452 are
suitable in this regard.
[0199] At the N-terminus, as described above, generally the first
45 amino acids will be removed during cellular processing. Other
suitable naturally occurring or expressed forms are listed in Table
3 above. These include, for example, a protein commencing at
residue 22, one commencing at residue 58 and one commencing at
residue 63. However, analysis of the entire enzyme, starting at
residue 1, can also provide information about the enzyme. Other
forms, such as 1-420 (SEQ ID NO 60) to 1-452 (SEQ ID NO: 59),
including intermediate forms, for example 1-440, can be useful in
this regard. In general, it will also be useful to obtain structure
on any subdomain of the active enzyme.
[0200] Methods for purifying the protein, including active forms,
are described above. In addition, since the protein is apparently
glycosylated in its naturally occurring (and mammalian-expressed
recombinant) forms, it may be desirable to express the protein and
purify it from bacterial sources, which do not glycosylate
mammalian proteins, or express it in sources, such as insect cells,
that provide uniform glycosylation patterns, in order to obtain a
homogeneous composition. Appropriate vectors and codon optimization
procedures for accomplishing this are known in the art.
[0201] Following expression and purification, the protein is
adjusted to a concentration of about 1-20 mg/ml. In accordance with
methods that have worked for other crystallized proteins, the
buffer and salt concentrations present in the initial protein
solution are reduced to as low a level as possible. This can be
accomplished by dialyzing the sample against the starting buffer,
using microdialysis techniques known in the art. Buffers and
crystallization conditions will vary from protein to protein, and
possibly from fragment to fragment of the active .beta.-secretase
molecule, but can be determined empirically using, for example,
matrix methods for determining optimal crystallization conditions.
(Drentz, J., supra; Ducruix, A., et al., eds. Crystallization of
Nucleic Acids and Proteins: A Practical Approach, Oxford University
Press, New York, 1992).
[0202] Following dialysis, conditions are optimized for
crystallization of the protein. Generally, methods for optimization
may include making a "grid" of 1 .mu.l drops of the protein
solution, mixed with 1 .mu.l well solution, which is a buffer of
varying pH and ionic strength. These drops are placed in individual
sealed wells, typically in a "hanging drop" configuration, for
example in commercially available containers (Hampton Research,
Laguna Niguel, Calif.). Precipitation/crystallization typically
occurs between 2 days and 2 weeks. Wells are checked for evidence
of precipitation or crystallization, and conditions are optimized
to form crystals. Optimized crystals are not judged by size or
morphology, but rather by the diffraction quality of crystals,
which should provide better than 3 .ANG. resolution. Typical
precipitating agents include ammonium sulfate (NH.sub.4SO.sub.4),
polyethylene glycol (PEG) and methyl pentane diol (MPD). All
chemicals used should be the highest grade possible (e.g., ACS) and
may also be re-purified by standard methods known in the art, prior
to use.
[0203] Exemplary buffers and precipitants forming an empirical grid
for determining crystallization conditions are commercially
available. For example, the "Crystal Screen" kit (Hampton Research)
provides a sparse matrix method of trial conditions that is biased
and selected from known crystallization conditions for
macromolecules. This provides a "grid" for quickly testing wide
ranges of pH, salts, and precipitants using a very small sample (50
to 100 microliters) of macromolecule. In such studies, 1 .mu.l of
buffer/precipitant(s) solution is added to an equal volume of
dialyzed protein solution, and the mixtures are allowed to sit for
at least two days to two weeks, with careful monitoring of
crystallization. Chemicals can be obtained from common commercial
suppliers; however, it is preferable to use purity grades suitable
for crystallization studies, such as are supplied by Hampton
Research (Laguna Niguel, Calif.). Common buffers include Citrate,
TEA, CHES, Acetate, ADA and the like (to provide a range of pH
optima), typically at a concentration of about 100 mM. Typical
precipitants include (NH.sub.4).sub.2SO.sub.4, MgSO.sub.4, NaCl,
MPD, Ethanol, polyethylene glycol of various sizes, isopropanol,
KCl; and the like (Ducruix).
[0204] Various additives can be used to aid in improving the
character of the crystals, including substrate analogs, ligands, or
inhibitors, as discussed in Part 2, below, as well as certain
additives, including, but not limited to: [0205] 5% Jeffamine
[0206] 5% Polypropyleneglycol P400 [0207] 5% Polyethyleneglycol 400
[0208] 5% ethyleneglycol [0209] 5% 2-methyl-2,4-pentanediol [0210]
5% Glycerol [0211] 5% Dioxane [0212] 5% dimethyl sulfoxide [0213]
5% n-Octanol [0214] 100 mM (NH4)2SO4 [0215] 100 mM CsCl [0216] 100
mM CoSO4 [0217] 100 mM MnCl2 [0218] 100 mM KCl [0219] 100 mM ZnSO4
[0220] 100 mM LiCl2 [0221] 100 mM MgCl2 [0222] 100 mM Glucose
[0223] 100 mM 1,6-Hexanediol 100 mM Dextran sulfate [0224] 100 mM
6-amino caproic acid [0225] 100 mM 1,6 hexane diamine [0226] 100 mM
1,8 diamino octane [0227] 100 mM Spermidine [0228] 100 mM Spermine
[0229] 0.17 mM n-dodecyl-.beta.-D-maltoside NP 40 [0230] 20 mM
n-octyl-.beta.-D-glucopyranoside
[0231] According to one discovery of the present invention, the
full-length .beta.-secretase enzyme contains at least one
transmembrane domain, and its purification is aided by the use of a
detergent (Triton X-100). Membrane proteins can be crystallized
intact, but may require specialized conditions, such as the
addition of a non-ionic detergent, such as C.sub.8G
(8-alkyl-.beta.-glucoside) or an n-alkyl-maltoside (C.sub.nM).
Selection of such a detergent is somewhat empirical, but certain
detergents are commonly employed. A number of membrane proteins
have been successfully "salted out" by addition of high salt
concentrations to the mixture. PEG has also been used successfully
to precipitate a number of membrane proteins (Ducruix, et al.,
supra). Alternatively, as discussed above, a C-terminal truncated
form of the protein that binds inhibitor but which lacks the
transmembrane domain, such as .beta.-secretase 46-452 (SEQ ID
NO:58), is crystallized.
[0232] After crystallization conditions are determined,
crystallization of a larger amount of the protein can be achieved
by methods known in the art, such as vapor diffusion or equilibrium
dialysis. In vapor diffusion, a drop of protein solution is
equilibrated against a larger reservoir of solution containing
precipitant or another dehydrating agent. After sealing, the
solution equilibrates to achieve supersaturating concentrations of
proteins and thereby induce crystallization in the drop.
[0233] Equilibrium dialysis can be used for crystallization of
proteins at low ionic strength. Under these conditions, a
phenomenon known as "salting in" occurs, whereby the protein
molecules achieve balance of electrostatic charges through
interactions with other protein molecules. This method is
particularly effective when the solubility of the protein is low at
the lower ionic strength. Various apparatuses and methods are used,
including microdiffusion cells in which a dialysis membrane is
attached to the bottom of a capillary tube, which may be bent at
its lower portion. The final crystallization condition is achieved
by slowly changing the composition of the outer solution. A
variation of these methods utilizes a concentration gradient
equilibrium dialysis set up. Microdiffusion cells are available
from commercial suppliers such as Hampton Research (Laguna Niguel,
Calif.).
[0234] Once crystallization is achieved, crystals characterized for
purity (e.g., SDS-PAGE) and biological activity. Larger crystals
(>0.2 mm) are preferred to increase the resolution of the X-ray
diffraction, which is preferably on the order of 10-1.5 Angstroms.
The selected crystals are subjected to X-ray diffraction, using a
strong, monochromatic X-ray source, such as a Synchrotron source or
rotating anode generator, and the resulting X-ray diffraction
patterns are analyzed, using methods known in the art.
[0235] In one application, .beta.-secretase amino acid sequence
and/or X-ray diffraction data is recorded on computer readable
medium, by which is meant any medium that can be read and directly
accessed by a computer. These data may be used to model the enzyme,
a subdomain thereof, or a ligand thereof. Computer algorithms
useful for this application are publicly and commercially
available.
2. Crystallization of Protein Plus Inhibitor
[0236] As mentioned above, it is advantageous to co-crystallize the
protein in the presence of a binding ligand, such as inhibitor.
Generally, the process for optimizing crystallization of the
protein is followed, with addition of greater than 1 mM
concentration of the inhibitor ligand during the precipitation
phase. These crystals are also compared to crystals formed in the
absence of ligand, so that measurements of the ligand binding site
can be made. Alternatively, 1-2 .mu.l of 0.1-25 mM inhibitor
compound is added to the drop containing crystals grown in the
absence of inhibitor in a process known as "soaking." Based on the
coordinates of the binding site, further inhibitor optimization is
achieved. Such methods have been used advantageously in finding
new, more potent inhibitors for HIV proteases (See, e.g.,
Viswanadhan, V. N., et al. J. Med. Chem. 39: 705-712, 1996; Muegge,
I., et al. J. Med. Chem. 42: 791-804, 1999).
[0237] One inhibitor ligand which is used in these
co-crystallization and soaking experiments is P10-P4'staD->V
(SEQ ID NO: 72), a statin peptide inhibitor described above.
Methods for making the molecule are described herein. The inhibitor
is mixed with .beta.-secretase, and the mixture is subjected to the
same optimization tests described above, concentrating on those
conditions worked out for the enzyme alone. Coordinates are
determined and comparisons are made between the free and ligand
bound enzyme, according to methods well known in the art. Further
comparisons can be made by comparing the inhibitory concentrations
of the enzyme to such coordinates, such as described by
Viswanadhan, et al, supra. Analysis of such comparisons provides
guidance for design of further inhibitors, using this method.
D. Biological Activity of .beta.-Secretase
[0238] 1. Naturally Occurring .beta.-Secretase
[0239] In studies carried out in support of the present invention,
isolated, purified forms of .beta.-secretase were tested for
enzymatic activity using one or more native or synthetic
substrates. For example, as discussed above, when .beta.-secretase
was prepared from human brain and purified to homogeneity using the
methods described in Example 5A, a single band was observed by
silver stain after electrophoresis of sample fractions from the
anion exchange chromatography (last step) on an SDS-polyacrylamide
gel under reducing (+.beta.-mercaptoethanol) conditions. As
summarized in Table 1, above, this fraction yielded a specific
activity of approximately 1.5.times.10.sup.9 nM/h/mg protein, where
activity was measured by hydrolysis of MBP-C125SW.
[0240] 2. Isolated Recombinant .beta.-Secretase
[0241] Various recombinant forms of the enzyme were produced and
purified from transfected cells. Since these cells were made to
overproduce the enzyme, it was found that the purification scheme
described with respect naturally occurring forms of the enzyme
(e.g., Example 5A) could be shortened, with positive results. For
example, as detailed in Example 6, 293T cells were transfected with
pCEKclone 27 (FIG. 12 and FIG. 13A-E) (SEQ ID NO:48) and Cos A2
cells were transfected with pCF.beta.A2 using "FUGENE" 6
Transfection Reagent (Roche Molecular Biochemicals Research,
Indianapolis, Ind.). The vector pCF was constructed from the parent
vector pcDNA3, commercially available from Invitrogen, by inserting
SEQ ID NO: 80 (FIG. 11A) between the HindIII and EcoRI sites. This
sequence encompasses the adenovirus major late promoter tripartite
leader sequence, and a hybrid splice created from adenovirus major
late region first exon and intron and a synthetically generated IgG
variable region splice acceptor.
[0242] pcDNA3 was cut with restriction endonucleases HindIII and
EcoRI, then blunted by filling in the ends with Klenow fragment of
DNA polymerase I. The cut and blunted vector was gel purified, and
ligated with isolated fragment from pED.GI. The pED fragment was
prepared by digesting with PvuII and SmaI, followed by gel
purification of the resulting 419 base-pair fragment, which was
further screened for orientation, and confirmed by sequencing.
[0243] To create the pCEK expression vector, the expression
cassette from pCF was transferred into the EBV expression vector
pCEP4 (Invitrogen, Carlsbad, Calif.). pCEP 4 was cut with BglII and
XbaI, filled in, and the large 9.15 kb fragment containing pBR,
hygromycin, and EBV sequences) ligated to the 1.9 kb NruI to XmnI
fragment of pCF containing the expression cassette (CMV,
TPL/MLP/IGg splice, Sp6, SVpolyA, M13 flanking region). pCF.beta.A2
(clone A2) contains full length .beta.-secretase in the vector pCF.
pCF vector replicates in COS and 293T cells. In each case, cells
were pelleted and a crude particulate fraction was prepared from
the pellet. This fraction was extracted with buffer containing 0.2%
Triton X-100. The Triton extract was diluted with pH 5.0 buffer and
passed through a SP Sepharose column. After the pH was adjusted to
4.5, .beta.-secretase activity containing fractions were
concentrated, with some additional purification on
P10-P4'(statine)D->V Sepharose, as described for the brain
enzyme. Silver staining of fractions revealed co-purified bands on
the gel. Fractions corresponding to these bands were subjected to
N-terminal amino acid determination. Results from these experiments
revealed some heterogeneity of .beta.-secretase species within the
fractions. These species represent various forms of the enzyme; for
example, from the 293T cells, the primary N-terminus is the same as
that found in the brain, where (with respect to SEQ ID NO: 2) amino
acid 46 is at the N-terminus. Minor amounts of protein starting
just after the signal sequence (at residue 23) and at the start of
the aspartyl protease homology domain (Met-63) were also observed.
An additional major form of protein was found in Cos A2 cells,
resulting from cleavage at Gly-58. These results are summarized in
Table 3, above.
2. Comparison of Isolated, Naturally Occurring .beta.-Secretase
with Recombinant .beta.-Secretase
[0244] As described above, naturally occurring .beta.-secretase
derived from human brain as well as recombinant forms of the enzyme
exhibit activity in cleaving APP, particularly as evidenced by
activity in the MBP-C125 assay. Further, key peptide sequences from
the naturally occurring form of the enzyme match portions of the
deduced sequence derived from cloning the enzyme. Further
confirmation that the two enzymes act identically can be taken from
additional experiments in which various inhibitors were found to
have very similar affinities for each enzyme, as estimated by a
comparison of IC.sub.50 values measured for each enzyme under
similar assay conditions. These inhibitors were discovered in
accordance with a further aspect of the invention, which is
described below. Significantly, the inhibitors produce near
identical IC.sub.50 values and rank orders of potency in
brain-derived and recombinant enzyme preparations, when compared in
the same assay.
[0245] In further studies, comparisons were made between the full
length recombinant enzyme having a C-terminal flag sequence
"FLp501" (SEQ ID NO: 2, +SEQ ID NO: 45) and a recombinant enzyme
truncated at position 452 "452Stop" (SEQ ID NO: 58 or SEQ ID NO:
59). Both enzymes exhibited activity in cleaving .beta.-secretase
substrates such as MBP-C125, as described above. The C-terminal
truncated form of the enzyme exhibited activity in cleaving the
MBP-C125sw substrate as well as the P26-P4' substrate, with similar
rank order of potency for the various inhibitor drugs tested. In
addition, the absolute IC.sub.50s were comparable for the two
enzymes tested with the same inhibitor. All IC.sub.50s were less
than 10 .mu.M.
1. Cellular .beta.-Secretase
[0246] Further experiments carried out in support of the present
invention have revealed that the isolated .beta.-secretase
polynucleotide sequences described herein encode .beta.-secretase
or .beta.-secretase fragments that are active in cells. This
section describes experiments carried out in support of the present
invention, cells were transfected with DNA encoding
.beta.-secretase alone, or were co-transfected with DNA
encoding-secretase and DNA encoding wild-type APP as detailed in
Example 8.
a. Transfection with .beta.-Secretase
[0247] In experiments carried out in support of the present
invention, clones containing genes expressing the full-length
polypeptide (SEQ ID NO: 2) were transfected into COS cells (Fugene
and Effectene methods). Whole cell lysates were prepared and
various amounts of lysate were tested for .beta.-secretase activity
according to standard methods known in the art or described in
Example 4 herein. FIG. 14B shows the results of these experiments.
As shown, lysates prepared from transfected cells, but not from
mock- or control cells, exhibited considerable enzymatic activity
in the MPB-C125sw assay, indicating "overexpression" of
.beta.-secretase by these cells.
b. Co-Transfection of Cells with .beta.-Secretase and APP
[0248] In further experiments, 293T cells were co-transfected with
pCEK clone 27, FIGS. 12 and 13 or poCK vector containing the full
length .beta.-secretase molecule (1-501; SEQ ID NO: 2) and with a
plasmid containing either the wild-type or Swedish APP construct
pohCK751, as described in Example 8. .beta.-specific cleavage was
analyzed by ELISA and Western analyses to confirm that the correct
site of cleavage occurs.
[0249] Briefly, 293T cells were co-transfected with equivalent
amounts of plasmids encoding .beta.APPsw or wt and .beta.-secretase
or control .beta.-galactosidase (.beta.-gal) cDNA according to
standard methods. .beta.APP and .beta.-secretase cDNAs were
delivered via vectors, pohCK or pCEK, which do not replicate in
293T cells (pCEK-clone 27, FIGS. 12 and 13; pohCK751 expressing
.beta.APP 751, FIG. 21). Conditioned media and cell lysates were
collected 48 hours after transfection. Western assays were carried
out on conditioned media and cell lysates. ELISAs for detection of
A.beta. peptide were carried out on the conditioned media to
analyze various APP cleavage products.
Western Blot Results
[0250] It is known that .beta.-secretase specifically cleaves at
the Met-Asp in APPwt and the Leu-Asp in APPsw to produce the
A.beta. peptide, starting at position 1 and releasing soluble APP
(sAPP.beta.). Immunological reagents, specifically antibody 92 and
92sw (or 192sw), respectively, have been developed that
specifically detect cleavage at this position in the APPwt and
APPsw substrates, as described in U.S. Pat. No. 5,721,130,
incorporated herein by reference. Western blot assays were carried
out on gels on which cell lysates were separated. These assays were
performed using methods well known in the art, using as primary
antibody reagents Ab 92 or Ab92S, which are specific for the C
terminus of the N-terminal fragment of APP derived from APPwt and
APPsw, respectively. In addition, ELISA format assays were
performed using antibodies specific to the N terminal amino acid of
the C terminal fragment.
[0251] Monoclonal antibody 13G8 (specific for C-terminus of
APP-epitope at positions 675-695 of APP695) was used in a Western
blot format to determine whether the transfected cells express APP.
FIG. 15A shows that reproducible transfection was obtained with
expression levels of APP in vast excess over endogenous levels
(triplicate wells are indicated as 1, 2, 3 in FIG. 15A). Three
forms of APP--mature, immature and endogenous--can be seen in cells
transfected with APPwt or APPSw. When .beta.-secretase was
co-transfected with APP, smaller C-terminal fragments appeared in
triplicate well lanes from co-transfected cells (Western blot FIG.
15A, right-most set of lanes). In parallel experiments, where cells
were co-transfected with .beta.-secretase and APPsw substrate,
literally all of the mature APP was cleaved (right-most set of
lanes labeled "1, 2, 3" of FIG. 15B). This suggests that there is
extensive cleavage by .beta.-secretase of the mature APP (upper
band), which results in C-terminal fragments of expected size in
the lysate for cleavage at the .beta.-secretase site.
Co-transfection with Swedish substrate also resulted in an increase
in two different sized CTF fragments (indicated by star). In
conjunction with the additional Western and ELISA results described
below, these results are consistent with a second cleavage
occurring on the APPsw substrate after the initial cleavage at the
.beta.-secretase site.
[0252] Conditioned medium from the cells was analyzed for
reactivity with the 192sw antibody, which is specific for
.beta.-s-APPsw. Analysis using this antibody indicated a dramatic
increase in .beta.-secretase cleaved soluble APP. This is observed
in the gel illustrated in FIG. 16B by comparing the dark bands
present in the "APPsw .beta.sec" samples to the bands present in
the "APPsw .beta.gal" samples. Antibody specific for .beta.-s-APPwt
also indicates an increase in .beta.-secretase cleaved material, as
illustrated in FIG. 16A.
[0253] Since the antibodies used in these experiments are specific
for the .beta.-secretase cleavage site, the foregoing results show
that p501 .beta.-secretase cleaves APP at this site, and the
overexpression of this recombinant clone leads to a dramatic
enhancement of .beta.-secretase processing at the correct
.beta.-secretase site in whole cells. This processing works on the
wildtype APP substrate and is enhanced substantially on the Swedish
APP substrate. Since approximately 20% of secreted APP in 293T
cells is .beta.-sAPP, with the increase observed below for APPsw,
it is probable that almost all of the sAPP is .beta.-sAPP. This
observation was further confirmed by independent Western assays in
which alpha and total sAPP were measured.
[0254] Monoclonal antibody 1736 is specific for the exposed
.alpha.-secretase cleaved .beta.-APP (Selkoe, et al.). When Western
blots were performed using this antibody as primary antibody, a
slight but reproducible decrease in .alpha.-cleaved APPwt was
observed (FIG. 17A), and a dramatic decrease in .alpha.-cleaved
APPsw material was also observed (note near absence of reactivity
in FIG. 17B in the lanes labeled "APPsw .beta.sec"). These results
suggest that the overexpressed recombinant p501 .beta.-secretase
cleaves APPsw so efficiently or extensively that there is little or
no substrate remaining for .alpha.-secretase to cleave. This
further indicates that all the sAPP in APPsw .beta.sec samples
(illustrated in FIG. 16B) is .beta.-sAPP.
A.beta. ELISA Results
[0255] Conditioned media from the recombinant cells was collected,
diluted as necessary and tested for A.beta. peptide production by
ELISA on microtiter plates coated with monoclonal antibody 2G3,
which is specific for recognizing the C-terminus of A.beta.(1-40),
with the detector reagent biotinylated mAb 3D6, which measures
A.beta.(x-40) (i.e., all N-terminus-truncated forms of the A.beta.
peptide). Overexpression of .beta.-secretase with APPwt resulted in
an approximately 8-fold increase in A.beta.(x-40) production, with
1-40 representing a small percentage of the total. There was also a
substantial increase in the production of A.beta.1-40 (FIG. 18).
With APPsw there was an approximate 2-fold increase in
A.beta.(x-40). Without adhering to any particular underlying
theory, it is thought that the less dramatic increase of
A.beta.(x-40) .beta.-sec/APPsw cells in comparison to the
.beta.-sec/APPwt cells is due in part to the fact that processing
of the APPsw substrate is much more efficient than that of the
APPwt substrate. That is, a significant amount of APPsw is
processed by endogenous .beta.-secretase, so further increases upon
transfection of .beta.-secretase are therefore limited. These data
indicate that the expression of recombinant .beta.-secretase
increases A.beta. production and that .beta.-secretase is rate
limiting for production of A.beta. in cells. This means that
.beta.-secretase enzymatic activity is rate limiting for production
of A.beta.3 in cells, and therefore provides a good therapeutic
target.
IV. Utility
A. Expression Vectors and Cells Expressing .beta.-Secretase
[0256] The invention includes further cloning and expression of
members of the aspartyl protease family described above, for
example, by inserting polynucleotides encoding the proteins into
standard expression vectors and transfecting appropriate host cells
according to standard methods discussed below. Such expression
vectors and cells expressing, for example, the human
.beta.-secretase enzyme described herein, have utility, for
example, in producing components (purified enzyme or transfected
cells) for the screening assays discussed in Part B, below. Such
purified enzyme also has utility in providing starting materials
for crystallization of the enzyme, as described in Section III,
above. In particular, truncated form(s) of the enzyme, such as
1-452 (SEQ ID NO: 59) and 46-452 (SEQ ID NO:58), and the
deglycosylated forms of the enzyme described herein are considered
to have utility in this regard, as are other forms truncated
partway into the transmembrane region, for example amino acid
residues 1-460 or 46-458 respectively in reference to SEQ ID
NO:2.
[0257] In accordance with the present invention, polynucleotide
sequences which encode human .beta.-secretase, splice variants,
fragments of the protein, fusion proteins, or functional
equivalents thereof, collectively referred to herein as
".beta.-secretase," may be used in recombinant DNA molecules that
direct the expression of .beta.-secretase in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
nucleic acid sequences that encode substantially the same or a
functionally equivalent amino acid sequence may be used to clone
and express .beta.-secretase. Such variations will be readily
ascertainable to persons skilled in the art.
[0258] The polynucleotide sequences of the present invention can be
engineered in order to alter a .beta.-secretase coding sequence for
a variety of reasons, including but not limited to, alterations
that modify the cloning, processing and/or expression of the gene
product. For example, alterations may be introduced using
techniques which are well known in the art, e.g., site-directed
mutagenesis, to insert new restriction sites, to alter
glycosylation patterns, to change codon preference, to produce
splice variants, etc. For example, it may be advantageous to
produce .beta.-secretase-encoding nucleotide sequences possessing
non-naturally occurring codons. Codons preferred by a particular
prokaryotic or eukaryotic host (Murray, E. et al. (1989) Nuc Acids
Res 17:477-508) can be selected, for example, to increase the rate
of .beta.-secretase polypeptide expression or to produce
recombinant RNA transcripts having desirable properties, such as a
longer half-life, than transcripts produced from naturally
occurring sequence. This may be particularly useful in producing
recombinant enzyme in non-mammalian cells, such as bacterial,
yeast, or insect cells. The present invention also includes
recombinant constructs comprising one or more of the sequences as
broadly described above. The constructs comprise a vector, such as
a plasmid or viral vector, into which a sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are also described in
Sambrook, et al., (supra).
[0259] The present invention also relates to host cells that are
genetically engineered with vectors of the invention, and the
production of proteins and polypeptides of the invention by
recombinant techniques. Host cells are genetically engineered
(i.e., transduced, transformed or transfected) with the vectors of
this invention which may be, for example, a cloning vector or an
expression vector. The vector may be, for example, in the form of a
plasmid, a viral particle, a phage, etc. The engineered host cells
can be cultured in conventional nutrient media modified as
appropriate for activating promoters, selecting transformants or
amplifying the .beta.-secretase gene. The culture conditions, such
as temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to those
skilled in the art. Exemplary methods for transfection of various
types of cells are provided in Example 6, herein.
[0260] As described above, according to a preferred embodiment of
the invention, host cells can be co-transfected with an enzyme
substrate, such as with APP (such as wild type or Swedish mutation
form), in order to measure activity in a cell environment. Such
host cells are of particular utility in the screening assays of the
present invention, particularly for screening for therapeutic
agents that are able to traverse cell membranes.
[0261] The polynucleotides of the present invention may be included
in any of a variety of expression vectors suitable for expressing a
polypeptide. Such vectors include chromosomal, nonchromosomal and
synthetic DNA sequences, e.g., derivatives of SV40; bacterial
plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived
from combinations of plasmids and phage DNA, viral DNA such as
vaccinia, adenovirus, fowl pox virus, and pseudorabies. However,
any other vector may be used as long as it is replicable and viable
in the host. The appropriate DNA sequence may be inserted into the
vector by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and related
sub-cloning procedures are deemed to be within the scope of those
skilled in the art.
[0262] The DNA sequence in the expression vector is operatively
linked to an appropriate transcription control sequence (promoter)
to direct mRNA synthesis. Examples of such promoters include: CMV,
LTR or SV40 promoter, the E. coli lac or trp promoter, the phage
lambda PL promoter, and other promoters known to control expression
of genes in prokaryotic or eukaryotic cells or their viruses. The
expression vector also contains a ribosome binding site for
translation initiation, and a transcription terminator. The vector
may also include appropriate sequences for amplifying expression.
In addition, the expression vectors preferably contain one or more
selectable marker genes to provide a phenotypic trait for selection
of transformed host cells such as dihydrofolate reductase or
neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0263] The vector containing the appropriate DNA sequence as
described above, as well as an appropriate promoter or control
sequence, may be employed to transform an appropriate host to
permit the host to express the protein. Examples of appropriate
expression hosts include: bacterial cells, such as E. coli,
Streptomyces, and Salmonella typhimurium; fungal cells, such as
yeast; insect cells such as Drosophila and Spodoptera Sf9;
mammalian cells such as CHO, COS, BHK, HEK 293 or Bowes melanoma;
adenoviruses; plant cells, etc. It is understood that not all cells
or cell lines will be capable of producing fully functional
.beta.-secretase; for example, it is probable that human
.beta.-secretase is highly glycosylated in native form, and such
glycosylation may be necessary for activity. In this event,
eukaryotic host cells may be preferred. The selection of an
appropriate host is deemed to be within the scope of those skilled
in the art from the teachings herein. The invention is not limited
by the host cells employed.
[0264] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for .beta.-secretase. For
example, when large quantities of .beta.-secretase or fragments
thereof are needed for the induction of antibodies, vectors, which
direct high level expression of fusion proteins that are readily
purified, may be desirable. Such vectors include, but are not
limited to, multifunctional E. coli cloning and expression vectors
such as Bluescript(R) (Stratagene, La Jolla, Calif.), in which the
.beta.-secretase coding sequence may be ligated into the vector
in-frame with sequences for the amino-terminal Met and the
subsequent 7 residues of beta-galactosidase so that a hybrid
protein is produced; pIN vectors (Van Heeke & Schuster (1989) J
Biol Chem 264:5503-5509); pET vectors (Novagen, Madison Wis.); and
the like.
[0265] In the yeast Saccharomyces cerevisiae a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase and PGH may be used. For reviews, see
Ausubel et al. (supra) and Grant et al. (1987; Methods in
Enzymology 153:516-544).
[0266] In cases where plant expression vectors are used, the
expression of a sequence encoding .beta.-secretase may be driven by
any of a number of promoters. For example, viral promoters such as
the 35S and 19S promoters of CaMV (Brisson et al. (1984) Nature
310:511-514) may be used alone or in combination with the omega
leader sequence from TMV (Takamatsu et al. (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO (Coruzzi et al (1984) EMBO J. 3:1671-1680;
Broglie et al. (1984) Science 224:838-843); or heat shock promoters
(Winter J and Sinibaldi R M (1991) Results. Probl. Cell Differ.
17:85-105) may be used. These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. For reviews of such techniques, see Hobbs S or Murry
L E (1992) in McGraw Hill Yearbook of Science and Technology,
McGraw Hill, New York, N.Y., pp 191-196; or Weissbach and Weissbach
(1988) Methods for Plant Molecular Biology, Academic Press, New
York, N.Y., pp 421-463.
[0267] .beta.-secretase may also be expressed in an insect system.
In one such system, Autographa californica nuclear polyhedrosis
virus (AcNPV) is used as a vector to express foreign genes in
Spodoptera frugiperda Sf9 cells or in Trichoplusia larvae. The
.beta.-secretase coding sequence is cloned into a nonessential
region of the virus, such as the polyhedrin gene, and placed under
control of the polyhedrin promoter. Successful insertion of Kv-SL
coding sequence will render the polyhedrin gene inactive and
produce recombinant virus lacking coat protein coat. The
recombinant viruses are then used to infect S. frugiperda cells or
Trichoplusia larvae in which .beta.-secretase is expressed (Smith
et al. (1983) J Virol 46:584; Engelhard E K et al. (1994) Proc Nat
Acad Sci 91:3224-3227).
[0268] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, a .beta.-secretase coding sequence may be
ligated into an adenovirus transcription/translation complex
consisting of the late promoter and tripartite leader sequence.
Insertion in a nonessential E1 or E3 region of the viral genome
will result in a viable virus capable of expressing the enzyme in
infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci
81:3655-3659). In addition, transcription enhancers, such as the
rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells.
[0269] Specific initiation signals may also be required for
efficient translation of a .beta.-secretase coding sequence. These
signals include the ATG initiation codon and adjacent sequences. In
cases where .beta.-secretase coding sequence, its initiation codon
and upstream sequences are inserted into the appropriate expression
vector, no additional translational control signals may be needed.
However, in cases where only coding sequence, or a portion thereof,
is inserted, exogenous transcriptional control signals including
the ATG initiation codon must be provided. Furthermore, the
initiation codon must be in the correct reading frame to ensure
transcription of the entire insert. Exogenous transcriptional
elements and initiation codons can be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers appropriate to the cell system in use
(Scharf D et al. (1994) Results Probl Cell Differ 20:125-62;
Bittner et al. (1987) Methods in Enzymol 153:516-544).
[0270] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M., and Battey, I. (1986) Basic
Methods in Molecular Biology) or newer methods, including lipid
transfection with "FUGENE" (Roche Molecular Biochemicals,
Indianapolis, Ind.) or "EFFECTENE" (Quiagen, Valencia, Calif.), or
other DNA carrier molecules. Cell-free translation systems can also
be employed to produce polypeptides using RNAs derived from the DNA
constructs of the present invention.
[0271] A host cell strain may be chosen for its ability to modulate
the expression of the inserted sequences or to process the
expressed protein in the desired fashion. Such modifications of the
protein include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation and
acylation. Post-translational processing which cleaves a "prepro"
form of the protein may also be important for correct insertion,
folding and/or function. For example, in the case of
.beta.-secretase, it is likely that the N-terminus of SEQ ID NO: 2
is truncated, so that the protein begins at amino acid 22, 46 or
57-58 of SEQ ID NO: 2. Different host cells such as CHO, HeLa, BHK,
MDCK, 293, WI38, etc. have specific cellular machinery and
characteristic mechanisms for such post-translational activities
and may be chosen to ensure the correct modification and processing
of the introduced, foreign protein.
[0272] For long-term, high-yield production of recombinant
proteins, stable expression may be preferred. For example, cell
lines that stably express .beta.-secretase may be transformed using
expression vectors which contain viral origins of replication or
endogenous expression elements and a selectable marker gene.
Following the introduction of the vector, cells may be allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells that successfully express the introduced
sequences. Resistant clumps of stably transformed cells can be
proliferated using tissue culture techniques appropriate to the
cell type. For example, in experiments carried out in support of
the present invention, overexpression of the "452stop" form of the
enzyme has been achieved.
[0273] Host cells transformed with a nucleotide sequence encoding
.beta.-secretase may be cultured under conditions suitable for the
expression and recovery of the encoded protein from cell culture.
The protein or fragment thereof produced by a recombinant cell may
be secreted, membrane-bound, or contained intracellularly,
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides encoding .beta.-secretase can be
designed with signal sequences which direct secretion of
.beta.-secretase polypeptide through a prokaryotic or eukaryotic
cell membrane.
[0274] .beta.-secretase may also be expressed as a recombinant
protein with one or more additional polypeptide domains added to
facilitate protein purification. Such purification facilitating
domains include, but are not limited to, metal chelating peptides
such as histidine-tryptophan modules that allow purification on
immobilized metals, protein A domains that allow purification on
immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle,
Wash.). The inclusion of a protease-cleavable polypeptide linker
sequence between the purification domain and .beta.-secretase is
useful to facilitate purification. One such expression vector
provides for expression of a fusion protein comprising
.beta.-secretase (e.g., a soluble .beta.-secretase fragment) fused
to a polyhistidine region separated by an enterokinase cleavage
site. The histidine residues facilitate purification on IMIAC
(immobilized metal ion affinity chromatography, as described in
Porath et al. (1992) Protein Expression and Purification 3:263-281)
while the enterokinase cleavage site provides a means for isolating
.beta.-secretase from the fusion protein. pGEX vectors (Promega,
Madison, Wis.) may also be used to express foreign polypeptides as
fusion proteins with glutathione S-transferase (GST). In general,
such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption to ligand-agarose beads (e.g.,
glutathione-agarose in the case of GST-fusions) followed by elution
in the presence of free ligand.
[0275] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period. Cells are typically harvested by
centrifugation, disrupted by physical or chemical means, and the
resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted
by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents, or
other methods, which are well know to those skilled in the art.
[0276] .beta.-secretase can be recovered and purified from
recombinant cell cultures by any of a number of methods well known
in the art, or, preferably, by the purification scheme described
herein. Protein refolding steps can be used, as necessary, in
completing configuration of the mature protein. Details of methods
for purifying naturally occurring as well as purified forms of
.beta.-secretase are provided in the Examples.
B. Methods of Selecting .beta.-Secretase Inhibitors
[0277] The present invention also includes methods for identifying
molecules, such as synthetic drugs, antibodies, peptides, or other
molecules, which have an inhibitory effect on the activity of
.beta.-secretase described herein, generally referred to as
inhibitors, antagonists or blockers of the enzyme. Such an assay
includes the steps of providing a human .beta.-secretase, such as
the .beta.-secretase which comprises SEQ ID NO: 2, SEQ ID NO: 43,
or more particularly in reference to the present invention, an
isolated protein, about 450 amino acid residues in length, which
includes an amino acid sequence that is at least 90% identical to
SEQ ID NO: 75 [63-423] including conservative substitutions
thereof, which exhibits .beta.-secretase activity, as described
herein. The .beta.-secretase enzyme is contacted with a test
compound to determine whether it has a modulating effect on the
activity of the enzyme, as discussed below, and selecting from test
compounds capable of modulating .beta.-secretase activity. In
particular, inhibitory compounds (antagonists) are useful in the
treatment of disease conditions associated with amyloid deposition,
particularly Alzheimer's disease. Persons skilled in the art will
understand that such assays may be conveniently transformed into
kits.
[0278] Particularly useful screening assays employ cells which
express both .beta.-secretase and APP. Such cells can be made
recombinantly by co-transfection of the cells with polynucleotides
encoding the proteins, as described in Section III, above, or can
be made by transfecting a cell which naturally contains one of the
proteins with the second protein. In a particular embodiment, such
cells are grown up in multi-well culture dishes and are exposed to
varying concentrations of a test compound or compounds for a
pre-determined period of time, which can be determined empirically.
Whole cell lysates, cultured media or cell membranes are assayed
for .beta.-secretase activity. Test compounds which significantly
inhibit activity compared to control (as discussed below) are
considered therapeutic candidates.
[0279] Isolated .beta.-secretase, its ligand-binding, catalytic, or
immunogenic fragments, or oligopeptides thereof, can be used for
screening therapeutic compounds in any of a variety of drug
screening techniques. The protein employed in such a test may be
membrane-bound, free in solution, affixed to a solid support, borne
on a cell surface, or located intracellularly. The formation of
binding complexes between .beta.-secretase and the agent being
tested can be measured. Compounds that inhibit binding between
.beta.-secretase and its substrates, such as APP or APP fragments,
may be detected in such an assay. Preferably, enzymatic activity
will be monitored, and candidate compounds will be selected on the
basis of ability to inhibit such activity. More specifically, a
test compound will be considered as an inhibitor of
.beta.-secretase if the measured .beta.-secretase activity is
significantly lower than .beta.-secretase activity measured in the
absence of test compound. In this context, the term "significantly
lower" means that in the presence of the test compound the enzyme
displays an enzymatic activity which, when compared to enzymatic
activity measured in the absence of test compound, is measurably
lower, within the confidence limits of the assay method. Such
measurements can be assessed by a change in K.sub.m and/or
V.sub.max, single assay endpoint analysis, or any other method
standard in the art. Exemplary methods for assaying
.beta.-secretase are provided in Example 4 herein.
[0280] For example, in studies carried out in support of the
present invention, compounds were selected based on their ability
to inhibit .beta.-secretase activity in the MBP-C125 assay.
Compounds that inhibited the enzyme activity at a concentration
lower than about 50 .mu.M were selected for further screening.
[0281] The groups of compounds that are most likely candidates for
inhibitor activity comprise a further aspect of the present
invention. Based on studies carried out in support of the
invention, it has been determined that the peptide compound
described herein as P10-P4'staD->V (SEQ ID NO: 72) is a
reasonably potent inhibitor of the enzyme. Further studies based on
this sequence and peptidomimetics of portions of this sequence have
revealed a number of small molecule inhibitors.
[0282] Random libraries of peptides or other compounds can also be
screened for suitability as .beta.-secretase inhibitors.
Combinatorial libraries can be produced for many types of compounds
that can be synthesized in a step-by-step fashion. Such compounds
include polypeptides, beta-turn mimetics, polysaccharides,
phospholipids, hormones, prostaglandins, steroids, aromatic
compounds, heterocyclic compounds, benzodiazepines, oligomeric
N-substituted glycines and oligocarbamates. Large combinatorial
libraries of the compounds can be constructed by the encoded
synthetic libraries (ESL) method described in Affymax, WO 95/12608,
Affymax, WO 93/06121, Columbia University, WO 94/08051,
Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each of which
is incorporated by reference for all purposes).
[0283] A preferred source of test compounds for use in screening
for therapeutics or therapeutic leads is a phage display library.
See, e.g., Devlin, WO 91/18980; Key, B. K., et al., eds., Phage
Display of Peptides and Proteins, A Laboratory Manual, Academic
Press, San Diego, Calif., 1996. Phage display is a powerful
technology that allows one to use phage genetics to select and
amplify peptides or proteins of desired characteristics from
libraries containing 10.sup.8-10.sup.9 different sequences.
Libraries can be designed for selected variegation of an amino acid
sequence at desired positions, allowing bias of the library toward
desired characteristics. Libraries are designed so that peptides
are expressed fused to proteins that are displayed on the surface
of the bacteriophage. The phage displaying peptides of the desired
characteristics are selected and can be regrown for expansion.
Since the peptides are amplified by propagation of the phage, the
DNA from the selected phage can be readily sequenced facilitating
rapid analyses of the selected peptides.
[0284] Phage encoding peptide inhibitors can be selected by
selecting for phage that bind specifically to .beta.-secretase
protein. Libraries are generated fused to proteins such as gene II
that are expressed on the surface of the phage. The libraries can
be composed of peptides of various lengths, linear or constrained
by the inclusion of two Cys amino acids, fused to the phage protein
or may also be fused to additional proteins as a scaffold. One may
start with libraries composed of random amino acids or with
libraries that are biased to sequences in the .beta.APP substrate
surrounding the .beta.-secretase cleavage site or preferably, to
the D.fwdarw.V substituted site exemplified in SEQ ID NO: 72. One
may also design libraries biased toward the peptidic inhibitors and
substrates described herein or biased toward peptide sequences
obtained from the selection of binding phage from the initial
libraries provide additional test inhibitor compound.
[0285] The .beta.-secretase is immobilized and phage specifically
binding to the .beta.-secretase selected for. Limitations, such as
a requirement that the phage not bind in the presence of a known
active site inhibitor of .beta.-secretase (e.g. the inhibitors
described herein), serve to further direct phage selection active
site specific compounds. This can be complicated by a differential
selection format. Highly purified .beta.-secretase, derived from
brain or preferably from recombinant cells can be immobilized to 96
well plastic dishes using standard techniques (reference phage
book). Recombinant .beta.-secretase, designed to be fused to a
peptide that can bind (e.g. strepaviden binding motifs, His, FLAG
or myc tags) to another protein immobilized (such as streptavidin
or appropriate antibodies) on the plastic petri dishes can also be
used. Phage are incubated with the bound .beta.-secretase and
unbound phage removed by washing. The phage are eluted and this
selection is repeated until a population of phage binding to
.beta.-secretase is recovered. Binding and elution are carried out
using standard techniques.
[0286] Alternatively .beta.-secretase can be "bound" by expressing
it in Cos or other mammalian cells growing on a petri dish. In this
case one would select for phage binding to the .beta.-secretase
expressing cells, and select against phage that bind to the control
cells, that are not expressing .beta.-secretase.
[0287] One can also use phage display technology to select for
preferred substrates of .beta.-secretase, and incorporate the
identified features of the preferred substrate peptides obtained by
phage display into inhibitors.
[0288] In the case of .beta.-secretase, knowledge of the amino acid
sequence surrounding the cleavage site of APP and of the cleavage
site of APPsw has provided information for purposes of setting up
the phage display screening library to identify preferred
substrates of .beta.-secretase. As mentioned above, knowledge of
the sequence of a particularly good peptide inhibitor,
P10-P4staD.fwdarw.V (SEQ ID NO:72), as described herein, provides
information for setting up a "biased" library toward this
sequence.
[0289] For example, the peptide substrate library containing
10.sup.8 different sequences is fused to a protein (such as a gene
III protein) expressed on the surface of the phage and a sequence
that can be used for binding to streptavidin, or another protein,
such as His tag and antibody to His. The phage are digested with
protease, and undigested phage are removed by binding to
appropriate immobilized binding protein, such as streptavidin. This
selection is repeated until a population of phage encoding
substrate peptide sequences is recovered. The DNA in the phage is
sequenced to yield the substrate sequences. These substrates are
then used for further development of peptidomimetics, particularly
peptidomimetics having inhibitory properties.
[0290] Combinatorial libraries and other compounds are initially
screened for suitability by determining their capacity to bind to,
or preferably, to inhibit .beta.-secretase activity in any of the
assays described herein or otherwise known in the art. Compounds
identified by such screens are then further analyzed for potency in
such assays. Inhibitor compounds can then be tested for
prophylactic and therapeutic efficacy in transgenic animals
predisposed to an amyloidogenic disease, such as various rodents
bearing a human APP-containing transgene, e.g., mice bearing a 717
mutation of APP described by Games et al., Nature 373: 523-527,
1995 and Wadsworth et al. (U.S. Pat. No. 5,811,633, U.S. Pat. No.
5,604,131, U.S. Pat. No. 5,720,936), and mice bearing a Swedish,
mutation of APP such as described by McConlogue et al. (U.S. Pat.
No. 5,612,486) and Hsiao et al. (U.S. Pat. No. 5,877,399);
Staufenbiel et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292
(1997); Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94,
13287-13292 (1997); Borchelt et al., Neuron 19, 939-945 (1997), all
of which are incorporated herein by reference. Compounds or agents
found to be efficacious and safe in such animal models will be
further tested in standard toxicological assays. Compounds showing
appropriate toxicological and pharmacokinetic profiles will be
moved into human clinical trials for treatment of Alzheimer's
disease and related diseases. The same screening approach can be
used on other potential agents such as peptidomimetics described
above.
[0291] In general, in selecting therapeutic compounds based on the
foregoing assays, it is useful to determine whether the test
compound has an acceptable toxicity profile, e.g., in a variety of
in vitro cells and animal model(s). It may also be useful to search
the tested and identified compound(s) against existing compound
databases to determine whether the compound or analogs thereof have
been previously employed for pharmaceutical purposes, and if so,
optimal routes of administration and dose ranges. Alternatively,
routes of administration and dosage ranges can be determined
empirically, using methods well known in the art (see, e.g. Benet,
L. Z., et al Pharmacokinetics in Goodman & Gilman's The
Pharmacological Basis of Therapeutics, Ninth Edition, Hardman, J.
G., et al., Eds., McGraw-Hill, New York, 1966) applied to standard
animal models, such as a transgenic PDAPP animal model (e.g.,
Games, D., et al. Nature 373: 523-527, 1995; Johnson-Wood, K., et
al., Proc. Natl. Acad. Sci. USA 94:1550-1555, 1997). To optimize
compound activity and/or specificity, it may be desirable to
construct a library of near-neighbor analogs to search for analogs
with greater specificity and/or activity. Methods for synthesizing
near-neighbor and/or targeted compound libraries are well-known in
the combinatorial library field.
C. Inhibitors and Therapeutics
[0292] Part B, above, describes method of screening for compounds
having .beta.-secretase inhibitory activity. To summarize, guidance
is provided for specific methods of screening for potent and
selective inhibitors of .beta.-secretase enzyme. Significantly, the
practitioner is directed to specific peptide substrate/inhibitor
sequences, such as P10-P4'staD.fwdarw.V (SEQ ID NO:72), on which
drug design can be based and additional sources, such as biased
phage display libraries, that should provide additional lead
compounds.
[0293] The practitioner is also provided ample guidance for further
refinement of the binding site of the enzyme, for example, by
crystallizing the purified enzyme in accord with the methods
provide herein. Noting the success in this area that has been
enjoyed in the area of HIV protease inhibitor development, it is
contemplated that such efforts will lead to further optimization of
the test compounds described herein. With optimized compounds in
hand, it is possible to define a compound pharmacophore, and
further search existing pharmacophore databases, e.g., as provided
by Tripos, to identify other compounds that may differ in 2-D
structural formulae with the originally discovered compounds, but
which share a common pharmacophore structure and activity. Test
compounds are assayed in any of the inhibitor assays described
herein, at various stages in development. Therefore, the present
invention includes .beta.-secretase inhibitory agents discovered by
any of the methods described herein, particularly the inhibitor
assays and the crystallization/optimization protocols. Such
inhibitory agents are therapeutic candidates for treatment of
Alzheimer's disease, as well as other amyloidoses characterized by
A.beta. peptide deposition. The considerations concerning
therapeutic index (toxicology), bioavailability and dosage
discussed in Part B above are also important to consider with
respect to these therapeutic candidates.
D. Methods of Diagnosis
[0294] The present invention also provides methods of diagnosing
individuals who carry mutations that provide enhanced
.beta.-secretase activity. For example, there are forms of familial
Alzheimer's disease in which the underlying genetic disorder has
yet to be recognized. Members of families possessing this genetic
predisposition can be monitored for alterations in the nucleotide
sequence that encodes .beta.-secretase and/or promoter regions
thereof, since it is apparent, in view of the teachings herein,
that individuals who overexpress of the enzyme or possess
catalytically more efficient forms of the enzyme would be likely to
produce relatively more A.beta. peptide. Support for this
supposition is provided by the observation, reported herein, that
the amount of .beta.-secretase enzyme is rate limiting for
production of A.beta. in cells.
[0295] More specifically, persons suspected to have a predilection
for developing for developing or who already have the disease, as
well as members of the general population, may be screened by
obtaining a sample of their cells, which may be blood cells or
fibroblasts, for example, and testing the samples for the presence
of genetic mutations in the .beta.-secretase gene, in comparison to
SEQ ID NO: 1 described herein, for example. Alternatively or in
addition, cells from such individuals can be tested for
.beta.-secretase activity. According to this embodiment, a
particular enzyme preparation might be tested for increased
affinity and/or Vmax with respect to a .beta.-secretase substrate
such as MBP-C125, as described herein, with comparisons made to the
normal range of values measured in the general population.
Individuals whose .beta.-secretase activity is increased compared
to normal values are susceptible to developing Alzheimer's disease
or other amyloidogenic diseases involving deposition of A.beta.
peptide.
E. Therapeutic Animal Models
[0296] A further utility of the present invention is in creation of
certain transgenic and/or knockout animals that are also useful in
the screening assays described herein. Of particular use is a
transgenic animal that overexpresses the .beta.-secretase enzyme,
such as by adding an additional copy of the mouse enzyme or by
adding the human enzyme. Such an animal can be made according to
methods well known in the art (e.g., Cordell, U.S. Pat. No.
5,387,742; Wadsworth et al., U.S. Pat. No. 5,811,633, U.S. Pat. No.
5,604,131, U.S. Pat. No. 5,720,936; McConlogue et al., U.S. Pat.
No. 5,612,486; Hsiao et al., U.S. Pat. No. 5,877,399; and
"Manipulating the Mouse Embryo, A Laboratory Manual," B. Hogan, F.
Costantini and E. Lacy, Cold Spring Harbor Press, 1986)),
substituting the one or more of the constructs described with
respect to .beta.-secretase, herein, for the APP constructs
described in the foregoing references, all of which are
incorporated by reference.
[0297] An overexpressing .beta.-secretase transgenic mouse will
make higher levels of A.beta. and s.beta.APP from APP substrates
than a mouse expressing endogenous .beta.-secretase. This would
facilitate analysis of APP processing and inhibition of that
processing by candidate therapeutic agents. The enhanced production
of A.beta. peptide in mice transgenic for .beta.-secretase would
allow acceleration of AD-like pathology seen in APP transgenic
mice. This result can be achieved by either crossing the
.beta.-secretase expressing mouse onto a mouse displaying AD-like
pathology (such as the PDAPP or Hsiao mouse) or by creating a
transgenic mouse expressing both the .beta.-secretase and APP
transgene.
[0298] Such transgenic animals are used to screen for
.beta.-secretase inhibitors, with the advantage that they will test
the ability of such inhibitors to gain entrance to the brain and to
effect inhibition in vivo.
[0299] Another animal model contemplated by the present invention
is a so-called "knock-out mouse" in which the endogenous enzyme is
either permanently (as described in U.S. Pat. Nos. 5,464,764,
5,627,059 and 5,631,153, which are incorporated by reference in
their entity) or inducibly deleted (as described in U.S. Pat. No.
4,959,317, which is incorporated by reference in its entity), or
which is inactivated, as described in further detail below. Such
mice serve as controls for .beta.-secretase activity and/or can be
crossed with APP mutant mice, to provide validation of the
pathological sequelae. Such mice can also provide a screen for
other drug targets, such as drugs specifically directed at A.beta.
deposition events.
[0300] .beta.-secretase knockout mice provide a model of the
potential effects of .beta.-secretase inhibitors in vivo.
Comparison of the effects of .beta.-secretase test inhibitors in
vivo to the phenotype of the .beta.-secretase knockout can help
guide drug development. For example, the phenotype may or may not
include pathologies seen during drug testing of .beta.-secretase
inhibitors. If the knockout does not show pathologies seen in the
drug-treated mice, one could infer that the drug is interacting
non-specifically with another target in addition to the
.beta.-secretase target. Tissues from the knockout can be used to
set up drug binding assays or to carry out expression cloning to
find the targets that are responsible for these toxic effects. Such
information can be used to design further drugs that do not
interact with these undesirable targets. The knockout mice will
facilitate analyses of potential toxicities that are inherent to
.beta.-secretase inhibition. Knowledge of potential toxicities will
help guide the design of design drugs or drug-delivery systems to
reduce such toxicities. Inducible knockout mice are particularly
useful in distinguishing toxicity in an adult animal from embryonic
effects seen in the standard knockout. If the knockout confers
fetal-lethal effects, the inducible knockout will be
advantageous.
[0301] Methods and technology for developing knock-out mice have
matured to the point that a number of commercial enterprises
generate such mice on a contract basis (e.g., Lexicon Genetics,
Woodland Tex.; Cell & Molecular Technologies, Lavallette, N.J.;
Crysalis, DNX Transgenic Sciences, Princeton, N.J.). Methodologies
are also available in the art. (See Galli-Taliadoros, L. A., et
al., J. Immunol. Meth. 181: 1-15, 1995). Briefly, a genomic clone
of the enzyme of interest is required. Where, as in the present
invention, the exons encoding the regions of the protein have been
defined, it is possible to achieve inactivation of the gene without
further knowledge of the regulatory sequences controlling
transcription. Specifically, a mouse strain 129 genomic library can
be screened by hybridization or PCR, using the sequence information
provided herein, according to methods well known in the art.
(Ausubel; Sambrook) The genomic clone so selected is then subjected
to restriction mapping and partial exonic sequencing for
confirmation of mouse homologue and to obtain information for
knock-out vector construction. Appropriate regions are then
sub-cloned into a "knock-out" vector carrying a selectable marker,
such as a vector carrying a neo.sup.r cassette, which renders cells
resistant to aminoglycoside antibiotics such as gentamycin. The
construct is further engineered for disruption of the gene of
interest, such as by insertion of a sequence replacement vector, in
which a selectable marker is inserted into an exon of the gene,
where it serves as a mutagen, disrupting the coordinated
transcription of the gene. Vectors are then engineered for
transfection into embryonic stem (ES) cells, and appropriate
colonies are isolated. Positive ES cell clones are micro-injected
into isolated host blastocysts to generate chimeric animals, which
are then bred and screened for germline transmission of the mutant
allele.
[0302] According to a further preferred embodiment,
.beta.-secretase knock-out mice can be generated such that the
mutation is inducible, such as by inserting in the knock-out mice a
lox region flanking the .beta.-secretase gene region. Such mice are
then crossed with mice bearing a "Cre" gene under an inducible
promoter, resulting in at least some off-spring bearing both the
"Cre" and the lox constructs. When expression of "Cre" is induced,
it serves to disrupt the gene flanked by the lox constructs. Such a
"Cre-lox" mouse is particularly useful, when it is suspected that
the knock-out mutation may be lethal. In addition, it provides the
opportunity for knocking out the gene in selected tissues, such as
the brain. Methods for generating Cre-lox constructs are provided
by U.S. Pat. No. 4,959,317, incorporated herein by reference, and
are made on a contractual basis by Lexicon Genetics, Woodlands,
Tex., among others.
[0303] The following examples illustrate, but in no way are
intended to limit the present invention.
EXAMPLE 1
Isolation of Coding Sequences for Human .beta.-Secretase
A. PCR Cloning
[0304] Poly A+ RNA from IMR human neuroblastoma cells was reverse
transcribed using the Perkin-Elmer kit. Eight degenerate primer
pools, each 8 fold degenerate, encoding the N and C terminal
portions of the amino acid sequence obtained from the purified
protein were designed (shown in Table 4; oligos 3407 through 3422)
(SEQ ID NOS:3-21). PCR reactions were composed of cDNA from 10 ng
of RNA, 1.5 mM MgCl.sub.2, 0.125 .mu.l AmpliTaq.RTM. Gold, 160
.mu.M each dNTP (plus 20 .mu.M additional from the reverse
transcriptase reaction), Perkin-Elmer TAQ buffer (from
AmpliTaq.RTM. Gold kit, Perkin-Elmer, Foster City, Calif.), in a 25
.mu.l reaction volume. Each of oligonucleotide primers 3407 through
3414 was used in combination with each of oligos 3415 through 3422
for a total for 64 reactions. Reactions were run on the
Perkin-Elmer 7700 Sequence Detection machine under the following
conditions: 10 min at 95.degree. C., 4 cycles of, 45.degree. C.
annealing for 15 second, 72.degree. C. extension for 45 second and
95.degree. C. denaturation for 15 seconds followed by 35 cycles
under the same conditions with the exception that the annealing
temperature was raised to 55.degree. C. (The foregoing conditions
are referred to herein as "Reaction 1 conditions.") PCR products
were visualized on 4% agarose gel (Northern blots) and a prominent
band of the expected size (68 bp) was seen in reactions,
particularly with the primers 3515-3518. The 68 kb band was
sequenced and the internal region coded for the expected amino acid
sequence. This gave the exact DNA sequence for 22 bp of the
internal region of this fragment.
[0305] Additional sequence was deduced from the efficiency of
various primer pools of discrete sequence in generating this PCR
product. Primer pools 3419 to 3422 (SEQ ID NOS: 15-18) gave very
poor or no product, whereas pools 3415 to 3418 (SEQ ID NOS:11-14
respectively) gave robust signal. The difference between these
pools is a CTC (3415 to 3418) (SEQ ID NOS:11-14) vs. TTC (3419 to
3422) (SEQ ID NOS:15-18) in the 3' most end of the pools. Since CTC
primed more efficiently we can conclude that the reverse complement
GAG is the correct codon. Since Met coding is unique it was
concluded that the following codon is ATG. Thus the exact DNA
sequence obtained is:
TABLE-US-00004 (SEQ ID NO: 49)
CCC.GGC.CGG.AGG.GGC.AGC.TTT.GTG.GAG.ATG.GT
encoding the amino acid sequence P G R R G S F V E M V (SEQ ID NO:
50). This sequence can be used to design exact oligonucleotides for
3 and 5' RACE PCR on either cDNA or libraries or to design specific
hybridization probes to be used to screen libraries. Since the
degenerate PCR product was found to be so robust, this reaction may
also be used as a diagnostic for the presence of clones containing
this sequence. Pools of libraries can be screened using this PCR
product to indicate the presence of a clone in the pool. The pools
can be broken out to identify individual clones. Screening pools of
known complexity and or size can provide information on the
abundance of this clone in a library or source and can approximate
the size of the full length clone or message.
[0306] For generation of a probe, PCR reactions using
oligonucleotides 3458 (SEQ ID NO:19) and 3469 (SEQ ID NO:21) or
3458 (SEQ ID NO: 19) and 3468 (SEQ ID NO: 20) (Table 4) can be
carried out using the 23 RACE product, clone 9C7E.35 (30 ng, clone
9C7E.35 was isolated from origene library, see Example 2), or cDNA
generated from brain, using the standard PCR conditions
(Perkin-Elmer, rtPCR and AmpliTaq.RTM. Gold kits) with the
following: 25 .mu.l reaction volume 1.5 mM MgCl2, 0.125 .mu.l of
AmpliTaq.RTM. Gold (Perkin-Elmer), initial 95.degree. for 10 min to
activate the AmpliTaq.RTM. Gold, 36 cycles of 65.degree. 15 sec
72.degree. 45 sec 95.degree. for 15 sec, followed by 3 min at
72.degree.. Product was purified on a Quiagen PCR purification kit
and used as a substrate for random priming to generate a
radiolabelled probe (Sambrook, et al., supra; Amersham
RediPrime.RTM. kit). This probe was used to isolate full length
close pCEK clone 27 shown in FIGS. 12 and 13 (A-E) (SEQ ID
NO:48).
Derivation of Full Length Clone pCEK Clone 27
[0307] A human primary neuronal cell library in the mammalian
expression vector pCEK2 vector was generated using size selected
cDNA, and pools of clones generated from different sized inserts.
The cDNA library for .beta.-secretase screening was made with
poly(A).sup.+ RNA isolated from primary human neuronal cells. The
cloning vector was pCEK2 (FIG. 12).
pCEK2
[0308] Double-stranded cDNA inserts were synthesized using the cDNA
Synthesis Kit from Stratagene with some modifications. The inserts
were then fractionated according to their sizes. A total of five
fractions were individually ligated with double-cut (NotI and XhoI)
pCEK2 and subsequently transformed into the E. Coli strain XL-10
Gold which is designed to accept very large plasmids.
[0309] The fractions of transformed E. coli were plated on Terrific
Broth agar plates containing ampicillin and let grown for 18 hours.
Each fraction yielded about 200,000 colonies to give a total of one
million colonies. The colonies were then scraped from the plates
and plasmids isolated from them in pools of approximately 70,000
clones/pool.
[0310] 70,000 clones from each pool of the library was screened for
the presence of the putative .beta.-secretase gene using the
diagnostic PCR reaction (degenerate primers 3411 (SEQ ID NO:7) and
3417 (SEQ ID NO:13) shown above).
[0311] 70,000 clones from each pool of the library was screened for
the presence of the putative .beta.-secretase gene using the
diagnostic PCR reaction (degenerate primers 3411 and 3417 shown
above).
[0312] Clones from the 1.5 kb pool were screened using a
radiolabeled probe generated from a 390 b.p. PCR product generated
from clone 9C7E.35. For generation of a probe, PCR product was
generated using 3458 (SEQ ID NO:19) and 3468 (SEQ ID NO:20) as
primers and clone 9C7E.35 (30 ng) as substrate.
[0313] PCR product was used as a substrate for random priming to
generate a radiolabeled probe. 180,000 clones from the 1.5 kb pool
(70,000 original clones in this pool), were screened by
hybridization with the PCR probe and 9 positive clones identified.
Four of these clones were isolated and by restriction mapping these
appear to encode two independent clones of 4 to 5 kb (clone 27) and
6 to 7 kb (clone 53) length. Sequencing of clone 27 verified that
it contains a coding region of 1.5 kb. FIG. 13 (A-E) shows the
sequence of pCEK clone27 (clone 27) (SEQ ID NO:48).
TABLE-US-00005 TABLE 4 SEQ ID Nucleotide Sequence NO. Pool No.
(Degenerate substitutions are shown in parentheses) 3 3407
G.AGA.GAC.GA(GA).GA(GA).CC(AT).GAG.GAG.CC 4 3408
G.AGA.GAC.GA(GA).GA(GA).CC(AT).GAA.GAG.CC 5 3409
G.AGA.GAC.GA(GA).GA(GA).CC(AT).GAA.GAA.CC 6 3410
G.AGA.GAC.GA(GA).GA(GA).CC(AT).GAG.GAA.CC 7 3411
AGA.GAC.GA(GA).GA(GA).CC(CG).GAG.GAG.CC 8 3412
AGA.GAC.GA(GA).GA(GA).CC(CG).GAA.GAG.CC 9 3413
AGA.GAC.GA(GA).GA(GA).CC(CG).GAA.GAA.CC 10 3414
AGA.GAC.GA(GA).GA(GA).CC(CG).GAG.GAA.CC 11 3415
CG.TCA.CAG.(GA)TT.(GA)TC.AAC.CAT.CTC 12 3416
CG.TCA.CAG.(GA)TT.(GA)TC.TAC.CAT.CTC 13 3417
CG.TCA.CAG.(GA)TT.(GA)TC.CAC.CAT.CTC 14 3418
CG.TCA.CAG.(GA)TT.(GA)TC.GAC.CAT.CTC 15 3419
CG.TCA.CAG.(GA)TT.(GA)TC.AAC.CAT.TTC 16 3420
CG.TCA.CAG.(GA)TT.(GA)TC.TAC.CAT.TTC 17 3421
CG.TCA.CAG.(GA)TT.(GA)TC.CAC.CAT.TTC 18 3422
CG.TCA.CAG.(GA)TT.(GA)TC.GAC.CAT.TTC 19 3458 GAG GGG CAG CTT TGT
GGA GA 20 3468 CAG.CAT.AGG.CCA.GCC.CCA.GGA.TGC.CT 21 3469
GTG.ATG.GCA.GCA.ATG.TTG.GCA.CGC
EXAMPLE 2
Screening of Human Fetal Brain cDNA Library
[0314] The Origene human fetal brain Rapid-Screen.TM. cDNA Library
Panel is provided as a 96-well format array consisting of 5000
clones (plasmid DNA) per well from a human fetal brain library.
Subplates are available for each well consisting of 96 wells of 50
clones each in E. coli. This is an oligo-dT primed library,
size-selected and unidirectionally inserted into the vector
pCMV-XL3.
[0315] 94 wells from the master plate were screened using PCR. The
Reaction 1 Conditions described in Example 1, above, were followed,
using only primers 3407 (SEQ ID NO:3) and 3416 (SEQ ID NO:12) with
30 ng of plasmid DNA from each well. Two pools showed the positive
70 bp band. The same primers and conditions were used to screen 1
.mu.l E. coli from each well of one of the subplates. E. coli from
the single positive well was then plated onto LB/amp plates and
single colonies screened using the same PCR conditions. The
positive clone, about 1 Kb in size, was labeled 9C7E.35. It
contained the original peptide sequence as well as 5' sequence that
included a methionine. The 3' sequence did not contain a stop
codon, suggesting that this was not a full-length clone, consistent
with Northern blot data.
EXAMPLE 3
PCR Cloning Methods
[0316] 3'RACE was used in experiments carried out in support of the
present invention to elucidate the polynucleotide encoding human
.beta.-secretase. Methods and conditions appropriate for
replicating the experiments described herein and/or determining
polynucleotide sequences encoding additional members of the novel
family of aspartyl proteases described herein may be found, for
example, in White, B. A., ed., PCR Cloning Protocols; Humana Press,
Totowa, N.J., 1997, or Ausubel, supra, both of which are
incorporated herein by reference.
RT-PCR
[0317] For reverse transcription polymerase chain reaction
(RT-PCR), two partially degenerate primer sets used for RT-PCR
amplification of a cDNA fragment encoding this peptide. Primer set
1 consisted of DNA's #3427-3434 (SEQ ID NOS:22-29 respectively),
the sequences of which are shown in Table 5, below. Matrix RT-PCR
using combinations of primers from this set with cDNA reverse
transcribed from primary human neuronal cultures as template
yielded the predicted 54 bp cDNA product with primers #3428+3433
(SEQ ID NOS:23-28 respectively). All RT-PCR reactions employed
10-50 ng input poly-A+ RNA equivalents per reaction, and were
carried out for 35 cycles employing step cycle conditions with a
95.degree. C. denaturation for 1 minute, 50.degree. C. annealing
for 30 sec, and a 72.degree. C. extension for 30 sec.
[0318] The degeneracy of primers #3428+3433 (SEQ ID NOS:23-28) was
further broken down, resulting in primer set 2, comprising DNAs
#3448-3455 (SEQ ID NOS:30-37) (Table 5). Matrix RT-PCR was repeated
using primer set 2, and cDNA reverse transcribed from poly-A+ RNA
from IMR-32 human neuroblastoma cells (American Type Culture
Collection, Manassas, Va.), as well as primary human neuronal
cultures, as template for amplification. Primers #3450 (SEQ ID
NO:32) and 3454 (SEQ ID NO:36) from set 2 most efficiently
amplified a cDNA fragment of the predicted size (72 bp), although
primers 3450+3453 (SEQ ID NOS:32 and 35), and 3450+3455 (SEQ ID
NOS:32 and 37) also amplified the same product, albeit at lower
efficiency. A 72 bp PCR product was obtained by amplification of
cDNA from IMR-32 cells and primary human neuronal cultures with
primers 3450 (SEQ ID NO:32) and 3454 (SEQ ID NO:36).
5' and 3' RACE-PCR
[0319] Internal primers matching the upper (coding) strand for 3'
Rapid Amplification of 5' Ends (RACE) PCR, and lower (non-coding)
strand for 5' RACE PCR were designed and made according to methods
known in the art (e.g., Frohman, M. A., M. K. Dush and G. R. Martin
(1988). "Rapid production of full-length cDNAs from rare
transcripts: amplification using a single gene specific
oligo-nucleotide primer." Proc. Natl. Acad. Sci. U.S.A. 85 (23):
8998-9002.) The DNA primers used for this experiment (#3459 &
#3460) (SEQ ID NOS:38 and 39) are illustrated schematically in FIG.
4, and the exact sequence of these primers is presented in Table 3.
These primers can be utilized in standard RACE-PCR methodology
employing commercially available templates (e.g. Marathon Ready
cDNA.RTM., Clontech Labs), or custom tailored cDNA templates
prepared from RNAs of interest as described by Frohman et al.
(ibid.).
[0320] In experiments carried out in support of the present
invention, a variation of RACE was employed to exploit an IMR-32
cDNA library cloned in the retrovirus expression vector pLPCXlox, a
derivative of pLNCX. As the vector junctions provide unique anchor
sequences abutting the cDNA inserts in this library, they serve the
purpose of 5' and 3' anchor primers in RACE methodology. The
sequences of the specific 5' and 3' anchor primers we employed to
amplify .beta.-secretase cDNA clones from the library, primers
#3475 (SEQ ID NO:40) and #3476 (SEQ ID NO:41), are derived from the
DNA sequence of the vector provided by Clontech Labs, Inc., and are
shown in Table 3.
[0321] Primers #3459 (SEQ ID NO:38) and #3476 (SEQ ID NO:41) were
used for 3' RACE amplification of downstream sequences from our
IMR-32 cDNA library in the vector pLPCXlox. The library had
previously been sub-divided into 100 pools of 5,000 clones per
pool, and plasmid DNA was isolated from each pool. A survey of the
100 pools with the primers identified as diagnostic for presence of
the .beta.-secretase clone, according to methods described in
Example 1, above, provided individual pools from the library for
RACE-PCR. 100 ng template plasmid from pool 23 was used for PCR
amplification with primers 3459+3476 (SEQ ID NOS:38 and 41
respectively). Amplification was carried out for 40 cycles using
ampli-Taq Gold.RTM., under the following conditions: denaturation
at 95.degree. C. for 1 min, annealing at 65.degree. C. for 45 sec.,
and extension at 72.degree. C. for 2 min. Reaction products were
fractionated by agarose gel chromatography, according to methods
known in the art (Ausubel; Sambrook).
[0322] An approximately 1.8 Kb PCR fragment was revealed by agarose
gel fractionation of the reaction products. The PCR product was
purified from the gel and subjected to DNA sequence analysis using
primer #3459 (SEQ ID NO:38). The resulting sequence, designated
23A, and the predicted amino acid sequence deduced from the DNA
sequence are shown in FIG. 5. Six of the first seven deduced
amino-acids from one of the reading frames of 23A were an exact
match with the last 7 amino-acids of the N-terminal sequence
determined from the purified protein, purified and sequenced in
further experiments carried out in support of the present
invention, from natural sources.
TABLE-US-00006 TABLE 5 SEQ ID DNA NO. # NUCLEOTIDE SEQUENCE
COMMENTS 22 3427 GAY GAR GAG CCN GAG GA 23 3428 GAY GAR GAG CCN GAa
GA 24 3429 GAY GAR GAa CCN GAg GA 25 3430 GAY GAR GAa CCN GAa GA 26
3431 RTT RTC NAC CAT TTC 27 3432 RTT RTC NAC CAT cTC 28 3433 TCN
ACC ATY TCN ACA AA 29 3434 TCN ACC ATY TCN ACG AA 30 3448 ata ttc
tag a GAY GAR GAg CCa GAa 5' primer, break down of 3428 w/ 5' XbaI
tail, GA 1 of 4 31 3449 ata ttc tag a GAY GAR GAg CCg GAa 5'
primer, break down of 3428 w/ 5' XbaI tail, GA 2 of 4 32 3450 ata
ttc tag a GAY GAR GAg CCc GAa 5' primer, break down of 3428 w/ 5'
XbaI tail, GA 3 of 4 33 4571 ata ttc tag a GAY GAR GAg CCt GAa 5'
primer, break down of 3428 w/ 5' XbaI tail, GA 4 of 4 34 3452 aca
cga att c TT RTC NAC CAT YTC breakdown of 3433, 1 of 4; tm = 50 aAC
AAA 35 3453 aca cga att c TT RTC NAC CAT YTC breakdown of 3433 w/
5' Eco RI tail, 2 of 4; gAC AAA tm = 50 36 3454 aca cga att c TT
RTO NAC CAT YTC breakdown of 3433 w/ 5' Eco RI tail, 3 of 4; cAC
AAA tm = 50 37 3455 aca cga att c TT RTC NAC CAT YTC breakdown of
3433 w/ 5' Eco RI tail, 4 of 4; tAC AAA tm = 50 38 3459 aa gaG CCC
GGC CGG AGG GGC A 5' upper strand primer for 3' race encodes
eEPGRRG 39 3460 aaa GCT GCC CCT CCG GCG GGG 3' lower strand primer
for 5' RACE 40 3475 AGC TCG TTT AGT GAA CCG TCA pLNCX 5' primer GAT
CG 41 3476 ACC TAC AGG TGG GGT CTT TCA pLNCX, 3' primer TTC CC
EXAMPLE 4
.beta.-Secretase Inhibitor Assays
[0323] Assays for measuring .beta.-secretase activity are well
known in the art. Particularly useful assays, summarized below, are
detailed in allowed U.S. Pat. No. 5,744,346, incorporated herein by
reference.
A. Preparation of MBP-C125sw
[0324] 1. Preparation of Cells
[0325] Two 250 ml cell culture flasks containing 50 ml LBamp100 per
flask were seeded with one colony per flask of E. coli pMAL-C125SW
cl. 2 (E. coli expressing MBP-C125sw fusion protein). Cells were
allowed to grow overnight at 37.degree. C. Aliqouts (25 ml) were
seeded in 500 ml per flask of LBamp100 in 2 liter flasks, which
were then allowed to grow at 30.degree.. Optical densities were
measured at 600 nm (OD600) vs. LB broth; 1.5 ml 100 mM IPTG was
added when the OD was .about.0.5. At this point, a pre-incubation
aliquot was removed for SDS-PAGE ("-I"). Of this aliquot, 0.5 ml
was centrifuged for 1 min in a Beckman microfuge, and the resulting
pellet was dissolved in 0.5 ml 1.times.LSB. The cells were
incubated/induced for 5-6 hours at 30 C, after which a
post-incubation aliquot ("+I") was removed. Cells were then
centrifuged at 9,000 rpm in a KA9.1 rotor for 10 min at 4.degree.
C. Pellets were retained and stored at -20 C.
[0326] 2. Extraction of Bacterial Cell Pellets
[0327] Frozen cell pellets were resuspended in 50 ml 0.2 M NaCl, 50
mM Tris, pH 7.5, then sonicated in rosette vessel for 5.times.20
sec bursts, with min rests between bursts. The extract was
centrifuged at 16,500 rpm in a KA18.5 rotor 30 min
(39,000.times.g). Using pipette as a pestle, the sonicated pellet
was suspended in 50 ml urea extraction buffer (7.6 M urea, 50 mM
Tris pH 7.5, 1 mM EDTA, 0.5% TX-100). The total volume was about 25
ml per flask. The suspension was then sonicated 6.times.20 sec,
with 1 min rests between bursts. The suspension was then
centrifuged again at 16,500 rpm 30 min in the KA18.5 rotor. The
resulting supernatant was added to 1.5 L of buffer consisting of
0.2 M NaCl 50 mM Tris buffer, pH 7.5, with 1% Triton X-100 (0.2M
NaCl-Tris-1% Tx), and was stirred gently at 4 degrees C. for 1
hour, followed by centrifugation at 9,000 rpm in KA9.1 for 30 min
at 4.degree. C. The supernatant was loaded onto a column of washed
amylose (100 ml of 50% slurry; New England BioLabs). The column was
washed with 0.2 M NaCl-Tris-1% TX to baseline (+10 column volumes),
then with 2 column volumes 0.2M NaCl-Tris-1% reduced Triton X-100.
The protein was then eluted with 10 mM maltose in the same buffer.
An equal volume of 6 M guanidine HCl/0.5% TX-100 was added to each
fraction. Peak fractions were pooled and diluted to a final
concentration of about 2 mg/ml. The fractions were stored at -40
degrees C., before dilution (20-fold, to 0.1 mg/ml in 0.15% Triton
X-100). Diluted aliquots were also stored at -40 C.
B. Antibody-Based Assays
[0328] The assays described in this section are based on the
ability of certain antibodies, hereinafter "cleavage-site
antibodies," to distinguish cleavage of APP by .beta.-secretase,
based on the unique cleavage site and consequent exposure of a
specific C-terminus formed by the cleavage. The recognized sequence
is a sequence of usually about 3-5 residues is immediately amino
terminal of the .beta. amyloid peptide (.beta.AP) produced by
.beta.-secretase cleavage of .beta.-APP, such as Val-Lys-Met in
wild-type or Val-Asn-Leu- in the Swedish double mutation variant
form of APP. Recombinantly-expressed proteins, described below,
were used as substrates for .beta.-secretase.
MBP-C125 Assay:
[0329] MBP-C125 substrates were expressed in E. Coli as a fusion
protein of the last 125 amino acids of APP fused to the
carboxy-terminal end of maltose-binding protein (MBP), using
commercially available vectors from New England Biolabs. The
.beta.-cleavage site was thus 26 amino acids downstream of the
start of the C-125 region. This latter site is recognized by
monoclonal antibody SW192.
[0330] Recombinant proteins were generated with both the wild-type
APP sequence (MBP-C125 wt) at the cleavage site
(..Val-Lys-Met-Asp-Ala..) (SEQ ID NO:54) or the "Swedish" double
mutation (MBP-C125 sw) (..Val-Asn-Leu-Asp-Ala..) (SEQ ID NO:51). As
shown schematically in FIG. 19A, cleavage of the intact MBP-fusion
protein results in the generation of a truncated amino-terminal
fragment, with the new SW-192 Ab-positive epitope uncovered at the
carboxy terminus. This amino-terminal fragment can be recognized on
Western blots with the same Ab, or, quantitatively, using an
anti-MBP capture-biotinylated SW-192 reporter sandwich format, as
shown in FIG. 19A. Anti-MBP polyclonal antibodies were raised in
rabbits (Josman Labs, Berkeley) by immunization with purified
recombinantly expressed MBP (New England Biolabs). Antisera were
affinity purified on a column of immobilized MBP. MBP-C125 SW and
WT substrates were expressed in E. coli, then purified as described
above.
[0331] Microtiter 96-well plates were coated with purified anti-MBP
antibody (at a concentration of 5-10 .mu.g/ml), followed by
blocking with 2.5 g/liter human serum albumin in 1 g/liter sodium
phosphate monobasic, 10.8 g/liter sodium phosphate dibasic, 25
g/liter sucrose, 0.5 g/liter sodium azide, pH 7.4. Appropriately
diluted .beta.-secretase enzyme (5 .mu.l) was mixed with 2.5 .mu.l
of 2.2 .mu.M MBP-C125sw substrate stock, in a 50 .mu.l reaction
mixture with a final buffer concentration of 20 mM acetate buffer,
pH 4.8, 0.06% Triton X-100, in individual wells of a 96-well
microtiter plate, and incubated for 1 hour at 37 degrees C. Samples
were then diluted 5-fold with Specimen Diluent (0.2 g/l sodium
phosphate monobasic, 2.15 g/l sodium phosphate dibasic, 0.5 g/l
sodium azide, 8.5 g/l sodium chloride, 0.05% Triton X-405, 6 g/l
BSA), further diluted 5-10 fold into Specimen Diluent on anti-MBP
coated plates, and incubated for 2 hours at room temperature.
Following incubations with samples or antibodies, plates were
washed at least four times in TTBS (0.15 M NaCl, 50 mM Tris,
ph&.5, 0.05% Tween-20). Biotinylated SW192 antibodies were used
as the reporter. SW192 polyclonal antibodies were biotinylated
using NHS-biotin (Pierce), following the manufacturer's
instruction. Usually, the biotinylated antibodies were used at
about 240 ng/ml, the exact concentration varying with the lot of
antibodies used. Following incubation of the plates with the
reporter, the ELISA was developed using streptavidin-labeled
alkaline phosphatase (Boeringer-Mannheim) and 4-methyl-umbelliferyl
phosphate as fluorescent substrate. Plates were read in a Cytofluor
2350 Fluorescent Measurement System. Recombinantly generated
MBP-26SW (product analog) was used as a standard to generate a
standard curve, which allowed the conversion of fluorescent units
into amount of product generated.
[0332] This assay protocol was used to screen for inhibitor
structures, using "libraries" of compounds assembled onto 96-well
microtiter plates. Compounds were added, in a final concentration
of 20 .mu.g/ml in 2% DMSO, in the assay format described above, and
the extent of product generated compared with control (2% DMSO
only) .beta.-secretase incubations, to calculate "% inhibition."
"Hits" were defined as compounds which result in >35% inhibition
of enzyme activity at test concentration. This assay can also be
used to provide IC.sub.50 values for inhibitors, by varying the
concentration of test compound over a range to calculate from a
dose-response curve the concentration required to inhibit the
activity of the enzyme by 50%.
[0333] Generally, inhibition is considered significant as compared
to control activity in this assay if it results in activity that is
at least 1 standard deviation, and preferably 2 standard deviations
lower than a mean activity value determined over a range of
samples. In addition, a reduction of activity that is greater than
about 25%, and preferably greater than about 35% of control
activity may also be considered significant.
[0334] Using the foregoing assay system, 24 "hits" were identified
(>30% inhibition at 50 .mu.M concentration) from the first 6336
compounds tested (0.4% hit rate). Of these 12 compounds had
IC.sub.50s less than 50 .mu.M, including re-P26-P4'sw assay,
below.
[0335] P26-P4'sw assay The P26-P4'sw substrate is a biotin-linked
peptide of the sequence (biotin)CGGADRGLTTRPGSGLTNIKTEEISEVNLDAEF
(SEQ ID NO:63). The P26-P1 standard has the sequence
(biotin)CGGADRGLTTRPGSGLTNIKTEEISEVNL (SEQ ID NO:64), where the
N-terminal "CGG" serves as a linker between biotin and the
substrate in both cases. Peptides were prepared by Anaspec, Inc.
(San Jose, Calif.) using solid phase synthesis with boc-amino
acids. Biotin was coupled to the terminal cysteine sulfhydryl by
Anaspec, Inc. after synthesis of the peptide, using EZ-link
Iodoacetyl-LC-Biotin (Pierce). Peptides are stored as 0.8-1.0 mM
stocks in 5 mM Tris, with the pH adjucted to around neutral (pH
6.5-7.5) with sodium hydroxide.
[0336] For the enzyme assay, the substrate concentration can vary
from 0-200 .mu.M. Specifically for testing compounds for inhibitory
activity, substrate concentration is 1.0 .mu.M. Compounds to be
tested were added in DMSO, with a final DMSO concentration of 5%;
in such experiments, the controls also receive 5% DMSO.
Concentration of enzyme was varied, to give product concentrations
within the linear range of the ELISA assay (125-2000 pM, after
dilution). These components were incubated in 20 mM sodium acetate,
pH 4.5, 0.06% Triton X-100, at 37.degree. C. for 1 to 3 hours.
Samples were diluted 5-fold in specimen diluent (145.4 mM sodium
chloride, 9.51 mM sodium phosphate, 7.7 mM sodium azide, 0.05%
Triton X-405, 6 gm/liter bovine serum albumin, pH 7.4) to quench
the reaction, then diluted further for the ELISA as needed. For the
ELISA, Costar High Binding 96-well assay plates (Corning, Inc.,
Corning, N.Y.) were coated with SW 192 monoclonal antibody from
clone 16A7, or a clone of similar affinity. Biotin-P26-P4'
standards were diluted in specimen diluent to a final concentration
of 0 to 2 nM. Diluted samples and standards (100 .mu.l) are
incubated on the SW192 plates at 4.degree. C. for 24 hours. The
plates are washed 4 times in TTBS buffer (150 mM sodium chloride,
25 mM Tris, 0.05% Tween 20, pH 7.5), then incubated with 0.1
ml/well of streptavidin-alkaline phosphatase (Roche Molecular
Biochemicals, Indianapolis, Ind.) diluted 1:3000 in specimen
diluent. After incubating for one hour at room temperature, the
plate was washed 4 times in TTBS, as described in the previous
section, and incubated with fluorescent substrate solution A (31.2
gm/liter 2-amino-2-methyl-1-propanol, 30 mg/liter, adjusted to pH
9.5 with HCl). Fluorescent values were read after 30 minutes.
C. Assays Using Synthetic Oligopeptide Substrates
[0337] This assay format is particularly useful for measuring
activity of partially purified .beta.-secretase preparations.
Synthetic oligopeptides are prepared which incorporate the known
cleavage site of .beta.-secretase, and optional detectable tags,
such as fluorescent or chromogenic moieties. Examples of such
peptides, as well as their production and detection methods are
described in allowed U.S. Pat. No. 5,942,400, herein incorporated
by reference. Cleavage products can be detected using high
performance liquid chromatography, or fluorescent or chromogenic
detection methods appropriate to the peptide to be detected,
according to methods well known in the art. By way of example, one
such peptide has the sequence SEVNL DAEF (SEQ ID NO: 52), and the
cleavage site is between residues 5 and 6. Another preferred
substrate has the sequence ADRGLTTRPGSGLTNIKTEEISEVNLDAE F (SEQ ID
NO: 53), and the cleavage site is between residues 26 and 27.
D. .beta.-Secretase Assays of Crude Cell or Tissue Extracts
[0338] Cells or tissues were extracted in extraction buffer (20 mM
HEPES, pH 7.5, 2 mM EDTA, 0.2% Triton X-100, 1 mM PMSF, 20 .mu.g/ml
pepstatin, 10 .mu.g/ml E-64). The volume of extraction buffer will
vary between samples, but should be at least 200 .mu.l per 10.sup.6
cells. Cells can be suspended by trituration with a micropipette,
while tissue may require homogenization. The suspended samples were
incubated for 30 minutes on ice. If necessary to allow pipetting,
unsolubilized material was removed by centrifugation at 4 degrees
C., 16,000.times.g (14,000 rpm in a Beckman microfuge) for 30
minutes. The supernate was assayed by dilution into the final assay
solution. The dilution of extract will vary, but should be
sufficient so that the protein concentration in the assay is not
greater than 60 .mu.g/ml. The assay reaction also contained 20 mM
sodium acetate, pH 4.8, and 0.06% Triton X-100 (including Triton
contributed by the extract and substrate), and 220-110 nM MBP-C125
(a 1:10 or 1:20 dilution of the 0.1 mg/ml stock described in the
protocol for substrate preparation). Reactions were incubated for
1-3 hours at 37 degrees C. before quenching with at least 5-fold
dilution in specimen diluent and assaying using the standard
protocol.
EXAMPLE 5
Purification of .beta.-Secretase
A. Purification of Naturally Occurring .beta.-Secretase
[0339] Human 293 cells were obtained and processed as described in
U.S. Pat. No. 5,744,346, incorporated herein by reference. (293
cells are available from the American Type Culture Collection,
Manassas, Va.). Frozen tissue (293 cell paste or human brain) was
cut into pieces and combined with five volumes of homogenization
buffer (20 mM Hepes, pH 7.5, 0.25 M sucrose, 2 mM EDTA). The
suspension was homogenized using a blender and centrifuged at
16,000.times.g for 30 min at 4.degree. C. The supernatants were
discarded and the pellets were suspended in extraction buffer (20
mM MES, pH 6.0, 0.5% Triton X-100, 150 mM NaCl, 2 mM EDTA, 5
.mu.g/ml leupeptin, 5 .mu.g/ml E64, 1 .mu.g/ml pepstatin, 0.2 mM
PMSF) at the original volume. After vortex-mixing, the extraction
was completed by agitating the tubes at 4.degree. C. for a period
of one hour. The mixtures were centrifuged as above at
16,000.times.g, and the supernatants were pooled. The pH of the
extract was adjusted to 7.5 by adding .about.1% (v/v) of 1 M Tris
base (not neutralized).
[0340] The neutralized extract was loaded onto a wheat germ
agglutinin-agarose (WGA-agarose) column pre-equilibrated with 10
column volumes of 20 mM Tris, pH 7.5, 0.5% Triton X-100, 150 mM
NaCl, 2 mM EDTA, at 4.degree. C. One milliliter of the agarose
resin was used for every 1 g of original tissue used. The
WGA-column was washed with 1 column volume of the equilibration
buffer, then 10 volumes of 20 mM Tris, pH 7.5, 100 mM NaCl, 2 mM
NaCl, 2 mM EDTA, 0.2% Triton X-100 and then eluted as follows.
Three-quarter column volumes of 10% chitin hydrolysate in 20 mM
Tris, pH 7.5, 0.5%, 150 mM NaCl, 0.5% Triton X-100, 2 mM EDTA were
passed through the column after which the flow was stopped for
fifteen minutes. An additional five column volumes of 10% chitin
hydrolysate solution were then used to elute the column. All of the
above eluates were combined (pooled WGA-eluate).
[0341] The pooled WGA-eluate was diluted 1:4 with 20 mM NaOAc, pH
5.0, 0.5% Triton X-100, 2 mM EDTA. The pH of the diluted solution
was adjusted to 5.0 by adding a few drops of glacial acetic acid
while monitoring the pH. This "SP load" was passed through a 5-ml
Pharmacia HiTrap SP-column equilibrated with 20 mM NaOAc, pH 5.0,
0.5% Triton X-100, 2 mM EDTA, at 4 ml/min at 4.degree. C.
[0342] The foregoing methods provided peak activity having a
specific activity of greater than 253 nM product/ml/h/.mu.g protein
in the MBP-C125-SW assay, where specific activity is determined as
described below, with about 1500-fold purification of the protein.
Specific activity of the purified .beta.-secretase was measured as
follows. MBP C125-SW substrate was combined at approximately 220 nM
in 20 mM sodium acetate, pH 4.8, with 0.06% Triton X-100. The
amount of product generated was measured by the .beta.-secretase
assay, also described below. Specific activity was then calculated
as:
Specific Activity = ( Product conc . nM ) ( Dilution factor ) (
Enzyme sol . vol ) ( Incub . time h ) ( Enzyme conc . mg / vol )
##EQU00002##
[0343] The Specific Activity is thus expressed as pmoles of product
produced per .mu.g of .beta.-secretase per hour. Further
purification of human brain enzyme was achieved by loading the SP
flow through fraction on to the P10-P4'sta D->V affinity column,
according to the general methods described below. Results of this
purification step are summarized in Table 1, above.
B. Purification of .beta.-Secretase from Recombinant Cells
[0344] Recombinant cells produced by the methods described herein
generally were made to over-express the enzyme; that is, they
produced dramatically more enzyme per cell than is found to be
endogenously produced by the cells or by most tissues. It was found
that some of the steps described above could be omitted from the
preparation of purified enzyme under these circumstances, with the
result that even higher levels of purification were achieved.
[0345] CosA2 or 293 T cells transfected with .beta.-secretase gene
construct (see Example 6) were pelleted, frozen and stored at -80
degrees until use. The cell pellet was resuspended by homogenizing
for 30 seconds using a handheld homogenizer (0.5 ml/pellet of
approximately 10.sup.6 cells in extraction buffer consisting of 20
mM TRIS buffer, pH 7.5, 2 mM EDTA, 0.2% Triton X-100, plus protease
inhibitors: 5 .mu.g/ml E-64, 10 .mu.g/ml pepstatin, 1 mM PMSF),
centrifuged as maximum speed in a microfuge (40 minutes at 4
degrees C.). Pellets were suspended in original volume of
extraction buffer, then stirred at 1 hour at 4 degrees C. with
rotation, and centrifuged again in a microfuge at maximum speed for
40 minutes. The resulting supernatant was saved as the "extract."
The extract was then diluted with 20 mM sodium acetate, pH 5.0, 2
mM EDTA and 0.2% Triton X-100 (SP buffer A), and 5M NaCl was added
to a final concentration of 60 mM NaCl. The pH of the solution was
then adjusted to pH 5.0 with glacial acetic acid diluted 1:10 in
water. Aliquots were saved ("SP load"). The SP load was passed
through a 1 ml SP HiTrap column which was pre-washed with 5 ml SP
buffer A, 5 ml SP buffer B (SP buffer A with 1 M NaCl) and 10 ml SP
buffer A. An additional 2 ml of 5% SP buffer B was passed through
the column to dissplace any remaining sample from the column. The
pH of the SP flow-through was adjusted to pH 4.5 with 10.times.
diluted acetic acid. This flow-through was then applied to a
P10-P4'staD->V-Sepharose Affinity column, as described below.
The column (250 .mu.l bed size) was pre-equilibrated with at least
20 column volumes of equilibration buffer (25 mM NaCl, 0.2% Triton
X-100, 0.1 mM EDTA, 25 mM sodium acetate, pH 4.5), then loaded with
the diluted supernatant. After loading, subsequent steps were
carried out at room temperature. The column was washed with washing
buffer (125 mM NaCl, 0.2% Triton X-100, 25 mM sodium acetate, pH
4.5) before addition of 0.6 column bed volumes of borate elution
buffer (200 mM NaCl, 0.2% reduced Triton X-100, 40 mM sodium
borate, pH 9.5). The column was then capped, and an additional 0.2
ml elution buffer was added. The column was allowed to stand for 30
minutes. Two bed volumes elution buffer were added, and column
fractions (250 .mu.l) were collected. The protein peak eluted in
two fractions. 0.5 ml of 10 mg/ml peptstatin was added per
milliliter of collected fractions.
[0346] Cell extracts made from cells transfected with full length
clone 27 (encoding SEQ ID NO: 2; 1-501), 419stop (SEQ ID NO:57) and
452stop (SEQ ID NO: 59) were detected by Western blot analysis
using antibody 264A (polyclonal antibody directed to amino acids
46-67 of .beta.-secretase with reference to SEQ ID NO: 2).
EXAMPLE 6
Preparation of Heterologous Cells Expressing Recombinant
.beta.-Secretase
[0347] Two separate clones (pCEKclone27 and pCEKclone53) were
transfected into 293T or COS(A2) cells using Fugene and Effectene
methods known in the art. 293T cells were obtained from Edge
Biosystems (Gaithersburg, Md.). They are KEK293 cells transfected
with SV40 large antigen. COSA2 are a subclone of COS1 cells;
subcloned in soft agar.
[0348] FuGENE Method: 293T cells were seeded at 2.times.10.sup.5
cells per well of a 6 well culture plate. Following overnight
growth, cells were at approximately 40-50% confluency. Media was
changed a few hours before transfection (2 ml/well). For each
sample, 3 .mu.l of FuGENE 6 Transfection Reagent (Roche Molecular
Biochemicals, Indianapolis, Ind.) was diluted into 0.1 ml of
serum-free culture medium (DME with 10 mM Hepes) and incubated at
room temperature for 5 min. One microgram of DNA for each sample
(0.5-2 mg/ml) was added to a separate tube. The diluted FuGENE
reagent was added drop-wise to the concentrated DNA. After gentle
tapping to mix, this mixture was incubated at room temperature for
15 minutes. The mixture was added dropwise onto the cells and
swirled gently to mix. The cells were then incubated at 37 degrees
C., in an atmosphere of 7.5% CO.sub.2. The conditioned media and
cells were harvested after 48 hours. Conditioned media was
collected, centrifuged and isolated from the pellet. Protease
inhibitors (5 .mu.g/ml E64, 2 .mu.g/ml peptstatin, 0.2 mM PMSF)
were added prior to freezing. The cell monolayer was rinsed once
with PBS, then 0.5 ml of lysis buffer (1 mM HIPIS, pH 7.5, 1 mM
EDTA, 0.5% Triton X-100, 1 mM PMSF, 10 .mu.g/ml E64) was added. The
lysate was frozen and thawed, vortex mixed, then centrifuged, and
the supernatant was frozen until assayed.
[0349] Effective Method: DNA (0.6 .mu.g) was added with "EFFECTENE"
reagent (Qiagen, Valencia, Calif.) into a 6-well culture plate
using a standard transfection protocol according to manufacturer's
instructions. Cells were harvested 3 days after transfection and
the cell pellets were snap frozen. Whole cell lysates were prepared
and various amounts of lysate were tested for .beta.-secretase
activity using the MBP-C125sw substrate. FIG. 14B shows the results
of these experiments, in which picomoles of product formed is
plotted against micrograms of COS cell lysate added to the
reaction. The legend to the figure describes the enzyme source,
where activity from cells transfected with DNA from pCEKclone27 and
PCEKclone53 (clones 27 and 53) using Effective are shown as closed
diamonds and solid squares, respectively, activity from cells
transfected with DNA from clone 27 prepared with FuGENE are shown
as open triangles, and mock transfected and control plots show no
activity (closed triangles and "X" markers). Values greater than
700 pM product are out of the linear range of the assay.
EXAMPLE 7
Preparation of P10-P4'sta(D->V) Sepharose Affinity Matrix
[0350] A. Preparation of P10-P4'sta(D->V) Inhibitor Peptide
[0351] P10-P4'sta(D->V) has the sequence
NH.sub.2-KTEEISEVN[sta]VAEF-COOH (SEQ ID NO: 72), where "sta"
represents a statine moiety. The synthetic peptide was synthesized
in a peptide synthesizer using boc-protected amino acids for chain
assembly. All chemicals, reagents, and boc amino acids were
purchased from Applied Biosystems (ABI; Foster City, Calif.) with
the exception of dichloromethane and N,N-dimethylformamide which
were from Burdick and Jackson. The starting resin, boc-Phe-OCH2-Pam
resin was also purchased from ABI. All amino acids were coupled
following preactivation to the corresponding HOBT ester using 1.0
equivalent of 1-hydroxybenzotriazole (HOBT), and 1.0 equivalent of
N,N-dicyclohexylcarbodiimide (DCC) in dimethylformamide. The boc
protecting group on the amino acid .alpha.-amine was removed with
50% trifluoroacetic acid in dichloromethane after each coupling
step and prior to Hydrogen Fluoride cleavage.
[0352] Amino acid side chain protection was as follows: Glu(Bzl),
Lys(Cl-CBZ), Ser(OBzl), Thr(OBzl). All other amino acids were used
with no further side chain protection including boc-Statine.
[(Bzl) benzyl, (CBZ) carbobenzoxy, (Cl-CBZ) chlorocarbobenzoxy,
(OBzl) O-benzyl]
[0353] The side chain protected peptide resin was deprotected and
cleaved from the resin by reacting with anhydrous hydrogen fluoride
(HF) at 0.degree. C. for one hour. This generates the fully
deprotected crude peptide as a C-terminal carboxylic acid.
[0354] Following HF treatment, the peptide was extracted from the
resin in acetic acid and lyophilized. The crude peptide was then
purified using preparative reverse phase HPLC on a Vydac C4, 330
.ANG., 10 .mu.m column 2.2 cm I.D..times.25 cm in length. The
solvent system used with this column was 0.1% TFA/H2O ([A] buffer)
and 0.1% TFA/CH3CN ([B] buffer) as the mobile phase. Typically the
peptide was loaded onto the column in 2% [B] at 8-10 mL/min. and
eluted using a linear gradient of 2% [B] to 60% [B] in 174
minutes.
[0355] The purified peptide was subjected to mass spectrometry, and
analytical reverse phase HPLC to confirm its composition and
purity.
B. Incorporation into Affinity Matrix
[0356] All manipulations were carried out at room temperature. 12.5
ml of 80% slurry of NHS-Sepharose (i.e. 10 ml packed volume;
Pharmacia, Piscataway, N.J.) was poured into a Bio-Rad EconoColumn
(BioRad, Richmond, Calif.) and washed with 165 ml of ice-cold 1.0
mM HCl. When the bed was fully drained, the bottom of the column
was closed off, and 5.0 ml of 7.0 mg/ml P10-P4'sta(D->V) peptide
(SEQ ID NO:72) (dissolved in 0.1 M HEPES, pH 8.0) was added. The
column was capped and incubated with rotation for 24 hours. After
incubation, the column was allowed to drain, then washed with 8 ml
of 1.0 M ethanolamine, pH 8.2. An additional 10 ml of the
ethanolamine solution was added, and the column was again capped
and incubated overnight with rotation. The column bed was washed
with 20 ml of 1.5 M sodium chloride, 0.5 M Tris, pH 7.5, followed
by a series of buffers containing 0.1 mM EDTA, 0.2% Triton X-100,
and the following components; 20 mM sodium acetate, pH 4.5 (100
ml); 20 mM sodium acetate, pH 4.5, 1.0 M sodium chloride (100 ml);
20 mM sodium borate, pH 9.5, 1.0 M sodium chloride (200 ml); 20 mM
sodium borate, pH 9.5 (100 ml). Finally, the column bed was washed
with 15 ml of 2 mM Tris, 0.01% sodium azide (no Triton or EDTA),
and stored in that buffer, at 4.degree. C.
EXAMPLE 8
Co-Transfection of Cells with .beta.-Secretase and APP
[0357] 293T cells were co-transfected with equivalent amounts
plasmids encoding APPsw or wt and .beta.-secretase or control
.beta.-galactosidase (.beta.-gal) cDNA using FuGene 6 Reagent, as
described in Example 4, above. Either pCEKclone27 or pohCJ
containing full length .beta.-secretase were used for expression of
.beta.-secretase. The plasmid construct pohCK751 used for the
expression of APP in these transfections was derived as described
in Dugan et al., JBC, 270 (18) 10982-10989 (1995) and shown
schematically in FIG. 21. A .beta.-gal control plasmid was added so
that the total amount of plasmid transfected was the same for each
condition. .beta.-gal expressing pCEK and pohCK vectors do not
replicate in 293T or COS cells. Triplicate wells of cells were
transfected with the plasmid, according to standard methods
described above, then incubated for 48 hours, before collection of
conditioned media and cells. Whole cell lysates were prepared and
tested for the .beta.-secretase enzymatic activity. The amount of
.beta.-secretase activity expressed by transfected 293T cells was
comparable to or higher than that expressed by CosA2 cells used in
the single transfection studies. Western blot assays were carried
out on conditioned media and cell lysates, using the antibody 13G8,
and A.beta. ELISAs carried out on the conditioned media to analyze
the various APP cleavage products.
[0358] While the invention has been described with reference to
specific methods and embodiments, it will be appreciated that
various modifications and changes may be made without departing
from the invention. All patent and literature references referred
to herein are herein incorporated by reference.
Sequence CWU 1
1
10411503DNAHomo sapiens 1atggcccaag ccctgccctg gctcctgctg
tggatgggcg cgggagtgct gcctgcccac 60ggcacccagc acggcatccg gctgcccctg
cgcagcggcc tggggggcgc ccccctgggg 120ctgcggctgc cccgggagac
cgacgaagag cccgaggagc ccggccggag gggcagcttt 180gtggagatgg
tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc
240gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag
taactttgca 300gtgggtgctg ccccccaccc cttcctgcat cgctactacc
agaggcagct gtccagcaca 360taccgggacc tccggaaggg tgtgtatgtg
ccctacaccc agggcaagtg ggaaggggag 420ctgggcaccg acctggtaag
catcccccat ggccccaacg tcactgtgcg tgccaacatt 480gctgccatca
ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg
540gggctggcct atgctgagat tgccaggcct gacgactccc tggagccttt
ctttgactct 600ctggtaaagc agacccacgt tcccaacctc ttctccctgc
agctttgtgg tgctggcttc 660cccctcaacc agtctgaagt gctggcctct
gtcggaggga gcatgatcat tggaggtatc 720gaccactcgc tgtacacagg
cagtctctgg tatacaccca tccggcggga gtggtattat 780gaggtgatca
ttgtgcgggt ggagatcaat ggacaggatc tgaaaatgga ctgcaaggag
840tacaactatg acaagagcat tgtggacagt ggcaccacca accttcgttt
gcccaagaaa 900gtgtttgaag ctgcagtcaa atccatcaag gcagcctcct
ccacggagaa gttccctgat 960ggtttctggc taggagagca gctggtgtgc
tggcaagcag gcaccacccc ttggaacatt 1020ttcccagtca tctcactcta
cctaatgggt gaggttacca accagtcctt ccgcatcacc 1080atccttccgc
agcaatacct gcggccagtg gaagatgtgg ccacgtccca agacgactgt
1140tacaagtttg ccatctcaca gtcatccacg ggcactgtta tgggagctgt
tatcatggag 1200ggcttctacg ttgtctttga tcgggcccga aaacgaattg
gctttgctgt cagcgcttgc 1260catgtgcacg atgagttcag gacggcagcg
gtggaaggcc cttttgtcac cttggacatg 1320gaagactgtg gctacaacat
tccacagaca gatgagtcaa ccctcatgac catagcctat 1380gtcatggctg
ccatctgcgc cctcttcatg ctgccactct gcctcatggt gtgtcagtgg
1440cgctgcctcc gctgcctgcg ccagcagcat gatgactttg ctgatgacat
ctccctgctg 1500aag 15032501PRTHomo sapiens 2Met Ala Gln Ala Leu Pro
Trp Leu Leu Leu Trp Met Gly Ala Gly Val1 5 10 15Leu Pro Ala His Gly
Thr Gln His Gly Ile Arg Leu Pro Leu Arg Ser20 25 30Gly Leu Gly Gly
Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp35 40 45Glu Glu Pro
Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val50 55 60Asp Asn
Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr65 70 75
80Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser85
90 95Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg
Tyr100 105 110Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg
Lys Gly Val115 120 125Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly
Glu Leu Gly Thr Asp130 135 140Leu Val Ser Ile Pro His Gly Pro Asn
Val Thr Val Arg Ala Asn Ile145 150 155 160Ala Ala Ile Thr Glu Ser
Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp165 170 175Glu Gly Ile Leu
Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp180 185 190Ser Leu
Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr His Val Pro195 200
205Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro Leu Asn
Gln210 215 220Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile
Gly Gly Ile225 230 235 240Asp His Ser Leu Tyr Thr Gly Ser Leu Trp
Tyr Thr Pro Ile Arg Arg245 250 255Glu Trp Tyr Tyr Glu Val Ile Ile
Val Arg Val Glu Ile Asn Gly Gln260 265 270Asp Leu Lys Met Asp Cys
Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val275 280 285Asp Ser Gly Thr
Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu Ala290 295 300Ala Val
Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp305 310 315
320Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Gln Ala Gly Thr
Thr325 330 335Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu Met
Gly Glu Val340 345 350Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu Pro
Gln Gln Tyr Leu Arg355 360 365Pro Val Glu Asp Val Ala Thr Ser Gln
Asp Asp Cys Tyr Lys Phe Ala370 375 380Ile Ser Gln Ser Ser Thr Gly
Thr Val Met Gly Ala Val Ile Met Glu385 390 395 400Gly Phe Tyr Val
Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala405 410 415Val Ser
Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu420 425
430Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn Ile
Pro435 440 445Gln Thr Asp Glu Ser Thr Leu Met Thr Ile Ala Tyr Val
Met Ala Ala450 455 460Ile Cys Ala Leu Phe Met Leu Pro Leu Cys Leu
Met Val Cys Gln Trp465 470 475 480Arg Cys Leu Arg Cys Leu Arg Gln
Gln His Asp Asp Phe Ala Asp Asp485 490 495Ile Ser Leu Leu
Lys500324DNAHomo sapiens 3gagagacgar garccwgagg agcc
24424DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 4gagagacgar garccwgaag agcc
24524DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 5gagagacgar garccwgaag aacc
24624DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 6gagagacgar garccwgagg aacc
24723DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 7agagacgarg arccsgagga gcc
23823DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 8agagacgarg arccsgaaga gcc
23923DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 9agagacgarg arccsgaaga acc
231023DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 10agagacgarg arccsgagga acc
231123DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 11cgtcacagrt trtcaaccat ctc
231223DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 12cgtcacagrt trtctaccat ctc
231323DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 13cgtcacagrt trtccaccat ctc
231423DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 14cgtcacagrt trtcgaccat ctc
231523DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 15cgtcacagrt trtcaaccat ttc
231623DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 16cgtcacagrt trtctaccat ttc
231723DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 17cgtcacagrt trtccaccat ttc
231823DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 18cgtcacagrt trtcgaccat ttc
231920DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 19gaggggcagc tttgtggaga
202026DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 20cagcataggc cagccccagg atgcct
262124DNAArtificial SequenceDegenerate oligonucleotide primer
derived from SEQ ID NO 2 21gtgatggcag caatgttggc acgc
242217DNAArtificial SequenceDegenerate oligonucleotide primer
22gaygargagc cngagga 172317DNAArtificial SequenceDegenerate
oligonucleotide primer 23gaygargagc cngaaga 172417DNAArtificial
SequenceDegenerate oligonucleotide primer 24gaygargaac cngagga
172517DNAArtificial SequenceDegenerate oligonucleotide primer
25gaygargaac cngaaga 172615DNAArtificial SequenceDegenerate
oligonucleotide primer 26rttrtcnacc atttc 152715DNAArtificial
SequenceDegenerate oligonucleotide primer 27rttrtcnacc atctc
152817DNAArtificial SequenceDegenerate oligonucleotide primer
28tcnaccatyt cnacaaa 172917DNAArtificial SequenceDegenerate
oligonucleotide primer 29tcnaccatyt cnacgaa 173027DNAArtificial
SequenceDegenerate oligonucleotide primer 30atattctaga gaygargagc
cagaaga 273127DNAArtificial SequenceDegenerate oligonucleotide
primer 31atattctaga gaygargagc cggaaga 273227DNAArtificial
SequenceDegenerate oligonucleotide primer 32atattctaga gaygargagc
ccgaaga 273327DNAArtificial SequenceDegenerate oligonucleotide
primer 33atattctaga gaygargagc ctgaaga 273430DNAArtificial
SequenceDegenerate oligonucleotide primer 34acacgaattc ttrtcnacca
tytcaacaaa 303530DNAArtificial SequenceDegenerate oligonucleotide
primer 35acacgaattc ttrtcnacca tytcgacaaa 303630DNAArtificial
SequenceDegenerate oligonucleotide primer 36acacgaattc ttrtcnacca
tytccacaaa 303730DNAArtificial SequenceDegenerate oligonucleotide
primer 37acacgaattc ttrtcnacca tytctacaaa 303821DNAArtificial
SequenceDegenerate oligonucleotide primer 38aagagcccgg ccggaggggc a
213921DNAArtificial SequenceDegenerate oligonucleotide primer
39aaagctgccc ctccggccgg g 214026DNAArtificial SequenceDegenerate
oligonucleotide primer 40agctcgttta gtgaaccgtc agatcg
264126DNAArtificial SequenceDegenerate oligonucleotide primer
41acctacaggt ggggtctttc attccc 26421368DNAHomo sapiens 42gagaccgacg
aagagcccga ggagcccggc cggaggggca gctttgtgga gatggtggac 60aacctgaggg
gcaagtcggg gcagggctac tacgtggaga tgaccgtggg cagccccccg
120cagacgctca acatcctggt ggatacaggc agcagtaact ttgcagtggg
tgctgccccc 180caccccttcc tgcatcgcta ctaccagagg cagctgtcca
gcacataccg ggacctccgg 240aagggtgtgt atgtgcccta cacccagggc
aagtgggaag gggagctggg caccgacctg 300gtaagcatcc cccatggccc
caacgtcact gtgcgtgcca acattgctgc catcactgaa 360tcagacaagt
tcttcatcaa cggctccaac tgggaaggca tcctggggct ggcctatgct
420gagattgcca ggcctgacga ctccctggag cctttctttg actctctggt
aaagcagacc 480cacgttccca acctcttctc cctgcagctt tgtggtgctg
gcttccccct caaccagtct 540gaagtgctgg cctctgtcgg agggagcatg
atcattggag gtatcgacca ctcgctgtac 600acaggcagtc tctggtatac
acccatccgg cgggagtggt attatgaggt gatcattgtg 660cgggtggaga
tcaatggaca ggatctgaaa atggactgca aggagtacaa ctatgacaag
720agcattgtgg acagtggcac caccaacctt cgtttgccca agaaagtgtt
tgaagctgca 780gtcaaatcca tcaaggcagc ctcctccacg gagaagttcc
ctgatggttt ctggctagga 840gagcagctgg tgtgctggca agcaggcacc
accccttgga acattttccc agtcatctca 900ctctacctaa tgggtgaggt
taccaaccag tccttccgca tcaccatcct tccgcagcaa 960tacctgcggc
cagtggaaga tgtggccacg tcccaagacg actgttacaa gtttgccatc
1020tcacagtcat ccacgggcac tgttatggga gctgttatca tggagggctt
ctacgttgtc 1080tttgatcggg cccgaaaacg aattggcttt gctgtcagcg
cttgccatgt gcacgatgag 1140ttcaggacgg cagcggtgga aggccctttt
gtcaccttgg acatggaaga ctgtggctac 1200aacattccac agacagatga
gtcaaccctc atgaccatag cctatgtcat ggctgccatc 1260tgcgccctct
tcatgctgcc actctgcctc atggtgtgtc agtggcgctg cctccgctgc
1320ctgcgccagc agcatgatga ctttgctgat gacatctccc tgctgaag
136843456PRTHomo sapiens 43Glu Thr Asp Glu Glu Pro Glu Glu Pro Gly
Arg Arg Gly Ser Phe Val1 5 10 15Glu Met Val Asp Asn Leu Arg Gly Lys
Ser Gly Gln Gly Tyr Tyr Val20 25 30Glu Met Thr Val Gly Ser Pro Pro
Gln Thr Leu Asn Ile Leu Val Asp35 40 45Thr Gly Ser Ser Asn Phe Ala
Val Gly Ala Ala Pro His Pro Phe Leu50 55 60His Arg Tyr Tyr Gln Arg
Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg65 70 75 80Lys Gly Val Tyr
Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu85 90 95Gly Thr Asp
Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg100 105 110Ala
Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly115 120
125Ser Asn Trp Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala
Arg130 135 140Pro Asp Asp Ser Leu Glu Pro Phe Phe Asp Ser Leu Val
Lys Gln Thr145 150 155 160His Val Pro Asn Leu Phe Ser Leu Gln Leu
Cys Gly Ala Gly Phe Pro165 170 175Leu Asn Gln Ser Glu Val Leu Ala
Ser Val Gly Gly Ser Met Ile Ile180 185 190Gly Gly Ile Asp His Ser
Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro195 200 205Ile Arg Arg Glu
Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile210 215 220Asn Gly
Gln Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys225 230 235
240Ser Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys
Val245 250 255Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser Ser
Thr Glu Lys260 265 270Phe Pro Asp Gly Phe Trp Leu Gly Glu Gln Leu
Val Cys Trp Gln Ala275 280 285Gly Thr Thr Pro Trp Asn Ile Phe Pro
Val Ile Ser Leu Tyr Leu Met290 295 300Gly Glu Val Thr Asn Gln Ser
Phe Arg Ile Thr Ile Leu Pro Gln Gln305 310 315 320Tyr Leu Arg Pro
Val Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr325 330 335Lys Phe
Ala Ile Ser Gln Ser Ser Thr Gly Thr Val Met Gly Ala Val340 345
350Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg
Ile355 360 365Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu Phe
Arg Thr Ala370 375 380Ala Val Glu Gly Pro Phe Val Thr Leu Asp Met
Glu Asp Cys Gly Tyr385 390 395 400Asn Ile Pro Gln Thr Asp Glu Ser
Thr Leu Met Thr Ile Ala Tyr Val405 410 415Met Ala Ala Ile Cys Ala
Leu Phe Met Leu Pro Leu Cys Leu Met Val420 425 430Cys Gln Trp Arg
Cys Leu Arg Cys Leu Arg Gln Gln His Asp Asp Phe435 440 445Ala Asp
Asp Ile Ser Leu Leu Lys450 455442348DNAHomo sapiens 44ccatgccggc
ccctcacagc cccgccggga gcccgagccc gctgcccagg ctggccgccg 60ccgtgccgat
gtagcgggct ccggatccca gcctctcccc tgctcccgtg ctctgcggat
120ctcccctgac cgctctccac agcccggacc cgggggctgg cccagggccc
tgcaggccct 180ggcgtcctga tgcccccaag ctccctctcc tgagaagcca
ccagcaccac ccagacttgg 240gggcaggcgc cagggacgga cgtgggccag
tgcgagccca gagggcccga aggccggggc 300ccaccatggc ccaagccctg
ccctggctcc tgctgtggat gggcgcggga gtgctgcctg 360cccacggcac
ccagcacggc atccggctgc ccctgcgcag cggcctgggg ggcgcccccc
420tggggctgcg gctgccccgg gagaccgacg aagagcccga ggagcccggc
cggaggggca 480gctttgtgga gatggtggac aacctgaggg gcaagtcggg
gcagggctac tacgtggaga 540tgaccgtggg cagccccccg cagacgctca
acatcctggt ggatacaggc agcagtaact 600ttgcagtggg tgctgccccc
caccccttcc tgcatcgcta ctaccagagg cagctgtcca 660gcacataccg
ggacctccgg aagggtgtgt atgtgcccta cacccagggc aagtgggaag
720gggagctggg caccgacctg gtaagcatcc cccatggccc caacgtcact
gtgcgtgcca 780acattgctgc catcactgaa tcagacaagt tcttcatcaa
cggctccaac tgggaaggca 840tcctggggct ggcctatgct gagattgcca
ggcctgacga ctccctggag cctttctttg 900actctctggt aaagcagacc
cacgttccca acctcttctc cctgcagctt tgtggtgctg 960gcttccccct
caaccagtct gaagtgctgg cctctgtcgg agggagcatg atcattggag
1020gtatcgacca ctcgctgtac acaggcagtc tctggtatac acccatccgg
cgggagtggt 1080attatgaggt gatcattgtg cgggtggaga tcaatggaca
ggatctgaaa atggactgca 1140aggagtacaa ctatgacaag agcattgtgg
acagtggcac caccaacctt cgtttgccca 1200agaaagtgtt tgaagctgca
gtcaaatcca tcaaggcagc ctcctccacg gagaagttcc 1260ctgatggttt
ctggctagga gagcagctgg tgtgctggca agcaggcacc accccttgga
1320acattttccc agtcatctca ctctacctaa tgggtgaggt taccaaccag
tccttccgca 1380tcaccatcct tccgcagcaa tacctgcggc cagtggaaga
tgtggccacg tcccaagacg 1440actgttacaa gtttgccatc tcacagtcat
ccacgggcac
tgttatggga gctgttatca 1500tggagggctt ctacgttgtc tttgatcggg
cccgaaaacg aattggcttt gctgtcagcg 1560cttgccatgt gcacgatgag
ttcaggacgg cagcggtgga aggccctttt gtcaccttgg 1620acatggaaga
ctgtggctac aacattccac agacagatga gtcaaccctc atgaccatag
1680cctatgtcat ggctgccatc tgcgccctct tcatgctgcc actctgcctc
atggtgtgtc 1740agtggcgctg cctccgctgc ctgcgccagc agcatgatga
ctttgctgat gacatctccc 1800tgctgaagtg aggaggccca tgggcagaag
atagagattc ccctggacca cacctccgtg 1860gttcactttg gtcacaagta
ggagacacag atggcacctg tggccagagc acctcaggac 1920cctccccacc
caccaaatgc ctctgccttg atggagaagg aaaaggctgg caaggtgggt
1980tccagggact gtacctgtag gaaacagaaa agagaagaaa gaagcactct
gctggcggga 2040atactcttgg tcacctcaaa tttaagtcgg gaaattctgc
tgcttgaaac ttcagccctg 2100aacctttgtc caccattcct ttaaattctc
caacccaaag tattcttctt ttcttagttt 2160cagaagtact ggcatcacac
gcaggttacc ttggcgtgtg tccctgtggt accctggcag 2220agaagagacc
aagcttgttt ccctgctggc caaagtcagt aggagaggat gcacagtttg
2280ctatttgctt tagagacagg gactgtataa acaagcctaa cattggtgca
aagattgcct 2340cttgaatt 2348458PRTArtificial SequenceFlag sequence
45Asp Tyr Lys Asp Asp Asp Asp Lys1 54621PRTHomo sapiens 46Met Ala
Gln Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val1 5 10 15Leu
Pro Ala His Gly204724PRTHomo sapiens 47Thr Gln His Gly Ile Arg Leu
Pro Leu Arg Ser Gly Leu Gly Gly Ala1 5 10 15Pro Leu Gly Leu Arg Leu
Pro Arg204816080DNAArtificial SequenceExpression Vector pCEK
48ttctcatgtt tgacagctta tcatcgcaga tccgggcaac gttgttgcat tgctgcaggc
60gcagaactgg taggtatgga agatccgatg tacgggccag atatacgcgt tgacattgat
120tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
ccatatatgg 180agttccgcgt tacataactt acggtaaatg gcccgcctgg
ctgaccgccc aacgaccccc 240gcccattgac gtcaataatg acgtatgttc
ccatagtaac gccaataggg actttccatt 300gacgtcaatg ggtggactat
ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 360atatgccaag
tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
420cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
ttagtcatcg 480ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
gcgtggatag cggtttgact 540cacggggatt tccaagtctc caccccattg
acgtcaatgg gagtttgttt tggcaccaaa 600atcaacggga ctttccaaaa
tgtcgtaaca actccgcccc attgacgcaa atgggcggta 660ggcgtgtacg
gtgggaggtc tatataagca gagctctctg gctaactaga gaacccactg
720cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
ctgttgggct 780cgcggttgag gacaaactct tcgcggtctt tccagtactc
ttggatcgga aacccgtcgg 840cctccgaacg gtactccgcc accgagggac
ctgagcgagt ccgcatcgac cggatcggaa 900aacctctcga ctgttggggt
gagtactccc tctcaaaagc gggcatgact tctgcgctaa 960gattgtcagt
ttccaaaaac gaggaggatt tgatattcac ctggcccgcg gtgatgcctt
1020tgagggtggc cgcgtccatc tggtcagaaa agacaatctt tttgttgtca
agcttgaggt 1080gtggcaggct tgagatctgg ccatacactt gagtgacaat
gacatccact ttgcctttct 1140ctccacaggt gtccactccc aggtccaact
gcaggtcgac tctagacccg gggaattctg 1200cagatatcca tcacactggc
cgcactcgtc cccagcccgc ccgggagctg cgagccgcga 1260gctggattat
ggtggcctga gcagccaacg cagccgcagg agcccggagc ccttgcccct
1320gcccgcgccg ccgcccgccg gggggaccag ggaagccgcc accggcccgc
catgcccgcc 1380cctcccagcc ccgccgggag cccgcgcccg ctgcccaggc
tggccgccgc cgtgccgatg 1440tagcgggctc cggatcccag cctctcccct
gctcccgtgc tctgcggatc tcccctgacc 1500gctctccaca gcccggaccc
gggggctggc ccagggccct gcaggccctg gcgtcctgat 1560gcccccaagc
tccctctcct gagaagccac cagcaccacc cagacttggg ggcaggcgcc
1620agggacggac gtgggccagt gcgagcccag agggcccgaa ggccggggcc
caccatggcc 1680caagccctgc cctggctcct gctgtggatg ggcgcgggag
tgctgcctgc ccacggcacc 1740cagcacggca tccggctgcc cctgcgcagc
ggcctggggg gcgcccccct ggggctgcgg 1800ctgccccggg agaccgacga
agagcccgag gagcccggcc ggaggggcag ctttgtggag 1860atggtggaca
acctgagggg caagtcgggg cagggctact acgtggagat gaccgtgggc
1920agccccccgc agacgctcaa catcctggtg gatacaggca gcagtaactt
tgcagtgggt 1980gctgcccccc accccttcct gcatcgctac taccagaggc
agctgtccag cacataccgg 2040gacctccgga agggtgtgta tgtgccctac
acccagggca agtgggaagg ggagctgggc 2100accgacctgg taagcatccc
ccatggcccc aacgtcactg tgcgtgccaa cattgctgcc 2160atcactgaat
cagacaagtt cttcatcaac ggctccaact gggaaggcat cctggggctg
2220gcctatgctg agattgccag gcctgacgac tccctggagc ctttctttga
ctctctggta 2280aagcagaccc acgttcccaa cctcttctcc ctgcagcttt
gtggtgctgg cttccccctc 2340aaccagtctg aagtgctggc ctctgtcgga
gggagcatga tcattggagg tatcgaccac 2400tcgctgtaca caggcagtct
ctggtataca cccatccggc gggagtggta ttatgaggtc 2460atcattgtgc
gggtggagat caatggacag gatctgaaaa tggactgcaa ggagtacaac
2520tatgacaaga gcattgtgga cagtggcacc accaaccttc gtttgcccaa
gaaagtgttt 2580gaagctgcag tcaaatccat caaggcagcc tcctccacgg
agaagttccc tgatggtttc 2640tggctaggag agcagctggt gtgctggcaa
gcaggcacca ccccttggaa cattttccca 2700gtcatctcac tctacctaat
gggtgaggtt accaaccagt ccttccgcat caccatcctt 2760ccgcagcaat
acctgcggcc agtggaagat gtggccacgt cccaagacga ctgttacaag
2820tttgccatct cacagtcatc cacgggcact gttatgggag ctgttatcat
ggagggcttc 2880tacgttgtct ttgatcgggc ccgaaaacga attggctttg
ctgtcagcgc ttgccatgtg 2940cacgatgagt tcaggacggc agcggtggaa
ggcccttttg tcaccttgga catggaagac 3000tgtggctaca acattccaca
gacagatgag tcaaccctca tgaccatagc ctatgtcatg 3060gctgccatct
gcgccctctt catgctgcca ctctgcctca tggtgtgtca gtggcgctgc
3120ctccgctgcc tgcgccagca gcatgatgac tttgctgatg acatctccct
gctgaagtga 3180ggaggcccat gggcagaaga tagagattcc cctggaccac
acctccgtgg ttcactttgg 3240tcacaagtag gagacacaga tggcacctgt
ggccagagca cctcaggacc ctccccaccc 3300accaaatgcc tctgccttga
tggagaagga aaaggctggc aaggtgggtt ccagggactg 3360tacctgtagg
aaacagaaaa gagaagaaag aagcactctg ctggcgggaa tactcttggt
3420cacctcaaat ttaagtcggg aaattctgct gcttgaaact tcagccctga
acctttgtcc 3480accattcctt taaattctcc aacccaaagt attcttcttt
tcttagtttc agaagtactg 3540gcatcacacg caggttacct tggcgtgtgt
ccctgtggta ccctggcaga gaagagacca 3600agcttgtttc cctgctggcc
aaagtcagta ggagaggatg cacagtttgc tatttgcttt 3660agagacaggg
actgtataaa caagcctaac attggtgcaa agattgcctc ttgaattaaa
3720aaaaaaaact agattgacta tttatacaaa tgggggcggc tggaaagagg
agaaggagag 3780ggagtacaaa gacagggaat agtgggatca aagctaggaa
aggcagaaac acaaccactc 3840accagtccta gttttagacc tcatctccaa
gatagcatcc catctcagaa gatgggtgtt 3900gttttcaatg ttttcttttc
tgtggttgca gcctgaccaa aagtgagatg ggaagggctt 3960atctagccaa
agagctcttt tttagctctc ttaaatgaag tgcccactaa gaagttccac
4020ttaacacatg aatttctgcc atattaattt cattgtctct atctgaacca
ccctttattc 4080tacatatgat aggcagcact gaaatatcct aaccccctaa
gctccaggtg ccctgtggga 4140gagcaactgg actatagcag ggctgggctc
tgtcttcctg gtcataggct cactctttcc 4200cccaaatctt cctctggagc
tttgcagcca aggtgctaaa aggaataggt aggagacctc 4260ttctatctaa
tccttaaaag cataatgttg aacattcatt caacagctga tgccctataa
4320cccctgcctg gatttcttcc tattaggcta taagaagtag caagatcttt
acataattca 4380gagtggtttc attgccttcc taccctctct aatggcccct
ccatttattt gactaaagca 4440tcacacagtg gcactagcat tataccaaga
gtatgagaaa tacagtgctt tatggctcta 4500acattactgc cttcagtatc
aaggctgcct ggagaaagga tggcagcctc agggcttcct 4560tatgtcctcc
accacaagag ctccttgatg aaggtcatct ttttccccta tcctgttctt
4620cccctccccg ctcctaatgg tacgtgggta cccaggctgg ttcttgggct
aggtagtggg 4680gaccaagttc attacctccc tatcagttct agcatagtaa
actacggtac cagtgttagt 4740gggaagagct gggttttcct agtataccca
ctgcatccta ctcctacctg gtcaacccgc 4800tgcttccagg tatgggacct
gctaagtgtg gaattacctg ataagggaga gggaaataca 4860aggagggcct
ctggtgttcc tggcctcagc cagctgccca caagccataa accaataaaa
4920caagaatact gagtcagttt tttatctggg ttctcttcat tcccactgca
cttggtgctg 4980ctttggctga ctgggaacac cccataacta cagagtctga
caggaagact ggagactgtc 5040cacttctagc tcggaactta ctgtgtaaat
aaactttcag aactgctacc atgaagtgaa 5100aatgccacat tttgctttat
aatttctacc catgttggga aaaactggct ttttcccagc 5160cctttccagg
gcataaaact caaccccttc gatagcaagt cccatcagcc tattattttt
5220ttaaagaaaa cttgcacttg tttttctttt tacagttact tccttcctgc
cccaaaatta 5280taaactctaa gtgtaaaaaa aagtcttaac aacagcttct
tgcttgtaaa aatatgtatt 5340atacatctgt atttttaaat tctgctcctg
aaaaatgact gtcccattct ccactcactg 5400catttggggc ctttcccatt
ggtctgcatg tcttttatca ttgcaggcca gtggacagag 5460ggagaaggga
gaacaggggt cgccaacact tgtgttgctt tctgactgat cctgaacaag
5520aaagagtaac actgaggcgc tcgctcccat gcacaactct ccaaaacact
tatcctcctg 5580caagagtggg ctttccgggt ctttactggg aagcagttaa
gccccctcct caccccttcc 5640ttttttcttt ctttactcct ttggcttcaa
aggattttgg aaaagaaaca atatgcttta 5700cactcatttt caatttctaa
atttgcaggg gatactgaaa aatacggcag gtggcctaag 5760gctgctgtaa
agttgagggg agaggaaatc ttaagattac aagataaaaa acgaatcccc
5820taaacaaaaa gaacaataga actggtcttc cattttgcca cctttcctgt
tcatgacagc 5880tactaacctg gagacagtaa catttcatta accaaagaaa
gtgggtcacc tgacctctga 5940agagctgagt actcaggcca ctccaatcac
cctacaagat gccaaggagg tcccaggaag 6000tccagctcct taaactgacg
ctagtcaata aacctgggca agtgaggcaa gagaaatgag 6060gaagaatcca
tctgtgaggt gacaggcacg gatgaaagac aaagacggaa aagagtatca
6120aaggcagaaa ggagatcatt tagttgggtc tgaaaggaaa agtntttgct
atccgacatg 6180tactgctagt wcctgtaagc attttaggtc ccagaatgga
aaaaaaaatc aagctatngg 6240ttatataata atgnnnnnnn nnnnnnnnnn
nntcgagcat gcatctagag ggccctattc 6300tatagtgtca cctaaatgct
agagctcgct gatcagcctc gactgtgcct tctagttgcc 6360agccatctgt
tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
6420ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
tgtcattcta 6480ttctgggggg tggggtgggg caggacagca agggggagga
ttgggaagac aatagcaggc 6540atgctgggga tgcggtgggc tctatggctt
ctgaggcgga aagaaccagc tggggctcta 6600gggggtatcc ccacgcgccc
tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 6660gcagcgtgac
cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
6720cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggc
atccctttag 6780ggttccgatt tagtgcttta cggcacctcg accccaaaaa
acttgattag ggtgatggtt 6840cacgtagtgg gccatcgccc tgatagacgg
tttttcgccc tttgacgttg gagtccacgt 6900tctttaatag tggactcttg
ttccaaactg gaacaacact caaccctatc tcggtctatt 6960cttttgattt
ataagggatt ttggggattt cggcctattg gttaaaaaat gagctgattt
7020aacaaaaatt taacgcgaat tctagagccc cgccgccgga cgaactaaac
ctgactacgg 7080catctctgcc ccttcttcgc ggggcagtgc atgtaatccc
ttcagttggt tggtacaact 7140tgccaactgg gccctgttcc acatgtgaca
cgggggggga ccaaacacaa aggggttctc 7200tgactgtagt tgacatcctt
ataaatggat gtgcacattt gccaacactg agtggctttc 7260atcctggagc
agactttgca gtctgtggac tgcaacacaa cattgccttt atgtgtaact
7320cttggctgaa gctcttacac caatgctggg ggacatgtac ctcccagggg
cccaggaaga 7380ctacgggagg ctacaccaac gtcaatcaga ggggcctgtg
tagctaccga taagcggacc 7440ctcaagaggg cattagcaat agtgtttata
aggccccctt gttaacccta aacgggtagc 7500atatgcttcc cgggtagtag
tatatactat ccagactaac cctaattcaa tagcatatgt 7560tacccaacgg
gaagcatatg ctatcgaatt agggttagta aaagggtcct aaggaacagc
7620gatatctccc accccatgag ctgtcacggt tttatttaca tggggtcagg
attccacgag 7680ggtagtgaac cattttagtc acaagggcag tggctgaaga
tcaaggagcg ggcagtgaac 7740tctcctgaat cttcgcctgc ttcttcattc
tccttcgttt agctaataga ataactgctg 7800agttgtgaac agtaaggtgt
atgtgaggtg ctcgaaaaca aggtttcagg tgacgccccc 7860agaataaaat
ttggacgggg ggttcagtgg tggcattgtg ctatgacacc aatataaccc
7920tcacaaaccc cttgggcaat aaatactagt gtaggaatga aacattctga
atatctttaa 7980caatagaaat ccatggggtg gggacaagcc gtaaagactg
gatgtccatc tcacacgaat 8040ttatggctat gggcaacaca taatcctagt
gcaatatgat actggggtta ttaagatgtg 8100tcccaggcag ggaccaagac
aggtgaacca tgttgttaca ctctatttgt aacaagggga 8160aagagagtgg
acgccgacag cagcggactc cactggttgt ctctaacacc cccgaaaatt
8220aaacggggct ccacgccaat ggggcccata aacaaagaca agtggccact
cttttttttg 8280aaattgtgga gtgggggcac gcgtcagccc ccacacgccg
ccctgcggtt ttggactgta 8340aaataagggt gtaataactt ggctgattgt
aaccccgcta accactgcgg tcaaaccact 8400tgcccacaaa accactaatg
gcaccccggg gaatacctgc ataagtaggt gggcgggcca 8460agataggggc
gcgattgctg cgatctggag gacaaattac acacacttgc gcctgagcgc
8520caagcacagg gttgttggtc ctcatattca cgaggtcgct gagagcacgg
tgggctaatg 8580ttgccatggg tagcatatac tacccaaata tctggatagc
atatgctatc ctaatctata 8640tctgggtagc ataggctatc ctaatctata
tctgggtagc atatgctatc ctaatctata 8700tctgggtagt atatgctatc
ctaatttata tctgggtagc ataggctatc ctaatctata 8760tctgggtagc
atatgctatc ctaatctata tctgggtagt atatgctatc ctaatctgta
8820tccgggtagc atatgctatc ctaatagaga ttagggtagt atatgctatc
ctaatttata 8880tctgggtagc atatactacc caaatatctg gatagcatat
gctatcctaa tctatatctg 8940ggtagcatat gctatcctaa tctatatctg
ggtagcatag gctatcctaa tctatatctg 9000ggtagcatat gctatcctaa
tctatatctg ggtagtatat gctatcctaa tttatatctg 9060ggtagcatag
gctatcctaa tctatatctg ggtagcatat gctatcctaa tctatatctg
9120ggtagtatat gctatcctaa tctgtatccg ggtagcatat gctatcctca
tgcatataca 9180gtcagcatat gatacccagt agtagagtgg gagtgctatc
ctttgcatat gccgccacct 9240cccaaggggg cgtgaatttt cgctgcttgt
ccttttcctg catgctggtt gctcccattc 9300ttaggtgaat ttaaggaggc
caggctaaag ccgtcgcatg tctgattgct caccaggtaa 9360atgtcgctaa
tgttttccaa cgcgagaagg tgttgagcgc ggagctgagt gacgtgacaa
9420catgggtatg cccaattgcc ccatgttggg aggacgaaaa tggtgacaag
acagatggcc 9480agaaatacac caacagcacg catgatgtct actggggatt
tattctttag tgcgggggaa 9540tacacggctt ttaatacgat tgagggcgtc
tcctaacaag ttacatcact cctgcccttc 9600ctcaccctca tctccatcac
ctccttcatc tccgtcatct ccgtcatcac cctccgcggc 9660agccccttcc
accataggtg gaaaccaggg aggcaaatct actccatcgt caaagctgca
9720cacagtcacc ctgatattgc aggtaggagc gggctttgtc ataacaaggt
ccttaatcgc 9780atccttcaaa acctcagcaa atatatgagt ttgtaaaaag
accatgaaat aacagacaat 9840ggactccctt agcgggccag gttgtgggcc
gggtccaggg gccattccaa aggggagacg 9900actcaatggt gtaagacgac
attgtggaat agcaagggca gttcctcgcc ttaggttgta 9960aagggaggtc
ttactacctc catatacgaa cacaccggcg acccaagttc cttcgtcggt
10020agtcctttct acgtgactcc tagccaggag agctcttaaa ccttctgcaa
tgttctcaaa 10080tttcgggttg gaacctcctt gaccacgatg ctttccaaac
caccctcctt ttttgcgcct 10140gcctccatca ccctgacccc ggggtccagt
gcttgggcct tctcctgggt catctgcggg 10200gccctgctct atcgctcccg
ggggcacgtc aggctcacca tctgggccac cttcttggtg 10260gtattcaaaa
taatcggctt cccctacagg gtggaaaaat ggccttctac ctggaggggg
10320cctgcgcggt ggagacccgg atgatgatga ctgactactg ggactcctgg
gcctcttttc 10380tccacgtcca cgacctctcc ccctggctct ttcacgactt
ccccccctgg ctctttcacg 10440tcctctaccc cggcggcctc cactacctcc
tcgaccccgg cctccactac ctcctcgacc 10500ccggcctcca ctgcctcctc
gaccccggcc tccacctcct gctcctgccc ctcctgctcc 10560tgcccctcct
cctgctcctg cccctcctgc ccctcctgct cctgcccctc ctgcccctcc
10620tgctcctgcc cctcctgccc ctcctgctcc tgcccctcct gcccctcctc
ctgctcctgc 10680ccctcctgcc cctcctcctg ctcctgcccc tcctgcccct
cctgctcctg cccctcctgc 10740ccctcctgct cctgcccctc ctgcccctcc
tgctcctgcc cctcctgctc ctgcccctcc 10800tgctcctgcc cctcctgctc
ctgcccctcc tgcccctcct gcccctcctc ctgctcctgc 10860ccctcctgct
cctgcccctc ctgcccctcc tgcccctcct gctcctgccc ctcctcctgc
10920tcctgcccct cctgcccctc ctgcccctcc tcctgctcct gcccctcctg
cccctcctcc 10980tgctcctgcc cctcctcctg ctcctgcccc tcctgcccct
cctgcccctc ctcctgctcc 11040tgcccctcct gcccctcctc ctgctcctgc
ccctcctcct gctcctgccc ctcctgcccc 11100tcctgcccct cctcctgctc
ctgcccctcc tcctgctcct gcccctcctg cccctcctgc 11160ccctcctgcc
cctcctcctg ctcctgcccc tcctcctgct cctgcccctc ctgctcctgc
11220ccctcccgct cctgctcctg ctcctgttcc accgtgggtc cctttgcagc
caatgcaact 11280tggacgtttt tggggtctcc ggacaccatc tctatgtctt
ggccctgatc ctgagccgcc 11340cggggctcct ggtcttccgc ctcctcgtcc
tcgtcctctt ccccgtcctc gtccatggtt 11400atcaccccct cttctttgag
gtccactgcc gccggagcct tctggtccag atgtgtctcc 11460cttctctcct
aggccatttc caggtcctgt acctggcccc tcgtcagaca tgattcacac
11520taaaagagat caatagacat ctttattaga cgacgctcag tgaatacagg
gagtgcagac 11580tcctgccccc tccaacagcc cccccaccct catccccttc
atggtcgctg tcagacagat 11640ccaggtctga aaattcccca tcctccgaac
catcctcgtc ctcatcacca attactcgca 11700gcccggaaaa ctcccgctga
acatcctcaa gatttgcgtc ctgagcctca agccaggcct 11760caaattcctc
gtcccccttt ttgctggacg gtagggatgg ggattctcgg gacccctcct
11820cttcctcttc aaggtcacca gacagagatg ctactggggc aacggaagaa
aagctgggtg 11880cggcctgtga ggatcagctt atcgatgata agctgtcaaa
catgagaatt cttgaagacg 11940aaagggcctc gtgatacgcc tatttttata
ggttaatgtc atgataataa tggtttctta 12000gacgtcaggt ggcacttttc
ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 12060aatacattca
aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
12120ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
ccttttttgc 12180ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
gtgaaagtaa aagatgctga 12240agatcagttg ggtgcacgag tgggttacat
cgaactggat ctcaacagcg gtaagatcct 12300tgagagtttt cgccccgaag
aacgttttcc aatgatgagc acttttaaag ttctgctatg 12360tggcgcggta
ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
12420ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
cggatggcat 12480gacagtaaga gaattatgca gtgctgccat aaccatgagt
gataacactg cggccaactt 12540acttctgaca acgatcggag gaccgaagga
gctaaccgct tttttgcaca acatggggga 12600tcatgtaact cgccttgatc
gttgggaacc ggagctgaat gaagccatac caaacgacga 12660gcgtgacacc
acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
12720actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
ataaagttgc 12780aggaccactt ctgcgctcgg cccttccggc tggctggttt
attgctgata aatctggagc 12840cggtgagcgt gggtctcgcg gtatcattgc
agcactgggg ccagatggta agccctcccg 12900tatcgtagtt atctacacga
cggggagtca ggcaactatg gatgaacgaa atagacagat 12960cgctgagata
ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
13020tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
tgaagatcct 13080ttttgataat ctcatgacca aaatccctta acgtgagttt
tcgttccact gagcgtcaga 13140ccccgtagaa aagatcaaag gatcttcttg
agatcctttt tttctgcgcg taatctgctg 13200cttgcaaaca aaaaaaccac
cgctaccagc ggtggtttgt ttgccggatc aagagctacc 13260aactcttttt
ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
13320agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
catacctcgc 13380tctgctaatc ctgttaccag tggctgctgc cagtggcgat
aagtcgtgtc ttaccgggtt 13440ggactcaaga cgatagttac cggataaggc
gcagcggtcg ggctgaacgg ggggttcgtg 13500cacacagccc agcttggagc
gaacgaccta caccgaactg agatacctac agcgtgagct 13560atgagaaagc
gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
13620ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
atctttatag 13680tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
ttgtgatgct cgtcaggggg
13740gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
ccttttgctg 13800cgccgcgtgc ggctgctgga gatggcggac gcgatggata
tgttctgcca agggttggtt 13860tgcgcattca cagttctccg caagaattga
ttggctccaa ttcttggagt ggtgaatccg 13920ttagcgaggt gccgccggct
tccattcagg tcgaggtggc ccggctccat gcaccgcgac 13980gcaacgcggg
gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc
14040atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccagtg
atcgaagtta 14100ggctggtaag agccgcgagc gatccttgaa gctgtccctg
atggtcgtca tctacctgcc 14160tggacagcat ggcctgcaac gcgggcatcc
cgatgccgcc ggaagcgaga agaatcataa 14220tggggaaggc catccagcct
cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg 14280ccgccatgcc
ctgcttcatc cccgtggccc gttgctcgcg tttgctggcg gtgtccccgg
14340aagaaatata tttgcatgtc tttagttcta tgatgacaca aaccccgccc
agcgtcttgt 14400cattggcgaa ttcgaacacg cagatgcagt cggggcggcg
cggtcccagg tccacttcgc 14460atattaaggt gacgcgtgtg gcctcgaaca
ccgagcgacc ctgcagcgac ccgcttaaca 14520gcgtcaacag cgtgccgcag
atcccgggca atgagatatg aaaaagcctg aactcaccgc 14580gacgtctgtc
gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct
14640ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
gatatgtcct 14700gcgggtaaat agctgcgccg atggtttcta caaagatcgt
tagtgggatc ggcactttgc 14760atcggccgcg ctccccgatt ccggaagtgc
ttgacattgg ggaattcagc gagagcctga 14820cctattgcat ctcccgccgt
gcacagggtg tcacgttgca agacctgcct gaaaccgaac 14880tgcccgctgt
tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta
14940gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
actacatggc 15000gtgatttcat atgcgcgatt gctgatcccc atgtgtatca
ctggcaaact gtgatggacg 15060acaccgtcag tgcgtccgtc gcgcaggctc
tcgatgagct gatgctttgg gccgaggact 15120gccccgaagt ccggcacctc
gtgcacgcgg atttcggctc caacaatgtc ctgacggaca 15180atggccgcat
aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg
15240aggtcgccaa catcttcttc tggaggccgt ggttggcggg tatggagcag
cagacgcgct 15300acttcgagcg gaggcatccg gagcttgcag gatcgccgcg
gctccgggcg tatatgctcc 15360gcattggtct tgaccaactc tatcagagct
tggttgacgg caatttcgat gatgcagctt 15420gggcgcaggg tcgatgcgac
gcaatcgtcc gatccggagc cgggactgtc gggcgtacac 15480aaatcgcccg
cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata
15540gtggaaacgg gagatggggg aggctaactg aaacacggaa ggagacaata
ccggaaggaa 15600cccgcgctat gacggcaata aaaagacaga ataaaacgca
cgggtgttgg gtcgtttgtt 15660cataaacgcg gggttcggtc ccagggctgg
cactctgtcg ataccccacc gagaccccat 15720tggggccaat acgcccgcgt
ttcttccttt tccccacccc accccccaag ttcgggtgaa 15780ggcccagggc
tcgcagccaa cgtcggggcg gcaggccctg ccatagccac tggccccgtg
15840ggttagggac ggggtccccc atggggaatg gtttatggtt cgtgggggtt
attattttgg 15900gcgttgcgtg gggtctggtc cacgactgga ctgagcagac
agacccatgg tttttggatg 15960gcctgggcat ggaccgcatg tactggcgcg
acacgaacac cgggcgtctg tggctgccaa 16020acacccccga cccccaaaaa
ccaccgcgcg gatttctggc gtgccaagct agtcgaccaa 160804932DNAHomo
sapiens 49cccggccgga ggggcagctt tgtggagatg gt 325011PRTHomo sapiens
50Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val1 5 10515PRTHomo
sapiens 51Val Asn Leu Asp Ala1 5529PRTArtificial SequenceSynthetic
oligopeptide substrate 52Ser Glu Val Asn Leu Asp Ala Glu Phe1
55330PRTArtificial SequenceSynthetic oligopeptide substrate 53Ala
Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile1 5 10
15Lys Thr Glu Glu Ile Ser Glu Val Asn Leu Asp Ala Glu Phe20 25
30545PRTHomo sapiensWild type Amyloid Precursor Protein cleavage
site (fragment) 54Val Lys Met Asp Ala1 55524PRTHomo sapiens 55Glu
Thr Asp Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val1 5 10
15Glu Met Val Asp Asn Leu Arg Gly205615PRTHomo sapiens 56Ile Gly
Phe Ala Val Ser Ala Cys His Val His Asp Glu Phe Arg1 5 10
1557419PRTHomo sapiens 57Met Ala Gln Ala Leu Pro Trp Leu Leu Leu
Trp Met Gly Ala Gly Val1 5 10 15Leu Pro Ala His Gly Thr Gln His Gly
Ile Arg Leu Pro Leu Arg Ser20 25 30Gly Leu Gly Gly Ala Pro Leu Gly
Leu Arg Leu Pro Arg Glu Thr Asp35 40 45Glu Glu Pro Glu Glu Pro Gly
Arg Arg Gly Ser Phe Val Glu Met Val50 55 60Asp Asn Leu Arg Gly Lys
Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr65 70 75 80Val Gly Ser Pro
Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser85 90 95Ser Asn Phe
Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr100 105 110Tyr
Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val115 120
125Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly Thr
Asp130 135 140Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg
Ala Asn Ile145 150 155 160Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe
Ile Asn Gly Ser Asn Trp165 170 175Glu Gly Ile Leu Gly Leu Ala Tyr
Ala Glu Ile Ala Arg Pro Asp Asp180 185 190Ser Leu Glu Pro Phe Phe
Asp Ser Leu Val Lys Gln Thr His Val Pro195 200 205Asn Leu Phe Ser
Leu Gln Leu Cys Gly Ala Gly Phe Pro Leu Asn Gln210 215 220Ser Glu
Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile225 230 235
240Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg
Arg245 250 255Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile
Asn Gly Gln260 265 270Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr
Asp Lys Ser Ile Val275 280 285Asp Ser Gly Thr Thr Asn Leu Arg Leu
Pro Lys Lys Val Phe Glu Ala290 295 300Ala Val Lys Ser Ile Lys Ala
Ala Ser Ser Thr Glu Lys Phe Pro Asp305 310 315 320Gly Phe Trp Leu
Gly Glu Gln Leu Val Cys Trp Gln Ala Gly Thr Thr325 330 335Pro Trp
Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val340 345
350Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr Leu
Arg355 360 365Pro Val Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr
Lys Phe Ala370 375 380Ile Ser Gln Ser Ser Thr Gly Thr Val Met Gly
Ala Val Ile Met Glu385 390 395 400Gly Phe Tyr Val Val Phe Asp Arg
Ala Arg Lys Arg Ile Gly Phe Ala405 410 415Val Ser Ala58407PRTHomo
sapiens 58Glu Thr Asp Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser
Phe Val1 5 10 15Glu Met Val Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly
Tyr Tyr Val20 25 30Glu Met Thr Val Gly Ser Pro Pro Gln Thr Leu Asn
Ile Leu Val Asp35 40 45Thr Gly Ser Ser Asn Phe Ala Val Gly Ala Ala
Pro His Pro Phe Leu50 55 60His Arg Tyr Tyr Gln Arg Gln Leu Ser Ser
Thr Tyr Arg Asp Leu Arg65 70 75 80Lys Gly Val Tyr Val Pro Tyr Thr
Gln Gly Lys Trp Glu Gly Glu Leu85 90 95Gly Thr Asp Leu Val Ser Ile
Pro His Gly Pro Asn Val Thr Val Arg100 105 110Ala Asn Ile Ala Ala
Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly115 120 125Ser Asn Trp
Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg130 135 140Pro
Asp Asp Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr145 150
155 160His Val Pro Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe
Pro165 170 175Leu Asn Gln Ser Glu Val Leu Ala Ser Val Gly Gly Ser
Met Ile Ile180 185 190Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser
Leu Trp Tyr Thr Pro195 200 205Ile Arg Arg Glu Trp Tyr Tyr Glu Val
Ile Ile Val Arg Val Glu Ile210 215 220Asn Gly Gln Asp Leu Lys Met
Asp Cys Lys Glu Tyr Asn Tyr Asp Lys225 230 235 240Ser Ile Val Asp
Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val245 250 255Phe Glu
Ala Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys260 265
270Phe Pro Asp Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Gln
Ala275 280 285Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser Leu
Tyr Leu Met290 295 300Gly Glu Val Thr Asn Gln Ser Phe Arg Ile Thr
Ile Leu Pro Gln Gln305 310 315 320Tyr Leu Arg Pro Val Glu Asp Val
Ala Thr Ser Gln Asp Asp Cys Tyr325 330 335Lys Phe Ala Ile Ser Gln
Ser Ser Thr Gly Thr Val Met Gly Ala Val340 345 350Ile Met Glu Gly
Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile355 360 365Gly Phe
Ala Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr Ala370 375
380Ala Val Glu Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly
Tyr385 390 395 400Asn Ile Pro Gln Thr Asp Glu40559452PRTHomo
sapiens 59Met Ala Gln Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala
Gly Val1 5 10 15Leu Pro Ala His Gly Thr Gln His Gly Ile Arg Leu Pro
Leu Arg Ser20 25 30Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro
Arg Glu Thr Asp35 40 45Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser
Phe Val Glu Met Val50 55 60Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly
Tyr Tyr Val Glu Met Thr65 70 75 80Val Gly Ser Pro Pro Gln Thr Leu
Asn Ile Leu Val Asp Thr Gly Ser85 90 95Ser Asn Phe Ala Val Gly Ala
Ala Pro His Pro Phe Leu His Arg Tyr100 105 110Tyr Gln Arg Gln Leu
Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val115 120 125Tyr Val Pro
Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp130 135 140Leu
Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile145 150
155 160Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn
Trp165 170 175Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg
Pro Asp Asp180 185 190Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys
Gln Thr His Val Pro195 200 205Asn Leu Phe Ser Leu Gln Leu Cys Gly
Ala Gly Phe Pro Leu Asn Gln210 215 220Ser Glu Val Leu Ala Ser Val
Gly Gly Ser Met Ile Ile Gly Gly Ile225 230 235 240Asp His Ser Leu
Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg245 250 255Glu Trp
Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly Gln260 265
270Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile
Val275 280 285Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val
Phe Glu Ala290 295 300Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr
Glu Lys Phe Pro Asp305 310 315 320Gly Phe Trp Leu Gly Glu Gln Leu
Val Cys Trp Gln Ala Gly Thr Thr325 330 335Pro Trp Asn Ile Phe Pro
Val Ile Ser Leu Tyr Leu Met Gly Glu Val340 345 350Thr Asn Gln Ser
Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr Leu Arg355 360 365Pro Val
Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr Lys Phe Ala370 375
380Ile Ser Gln Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile Met
Glu385 390 395 400Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg
Ile Gly Phe Ala405 410 415Val Ser Ala Cys His Val His Asp Glu Phe
Arg Thr Ala Ala Val Glu420 425 430Gly Pro Phe Val Thr Leu Asp Met
Glu Asp Cys Gly Tyr Asn Ile Pro435 440 445Gln Thr Asp
Glu45060420PRTHomo sapiens 60Met Ala Gln Ala Leu Pro Trp Leu Leu
Leu Trp Met Gly Ala Gly Val1 5 10 15Leu Pro Ala His Gly Thr Gln His
Gly Ile Arg Leu Pro Leu Arg Ser20 25 30Gly Leu Gly Gly Ala Pro Leu
Gly Leu Arg Leu Pro Arg Glu Thr Asp35 40 45Glu Glu Pro Glu Glu Pro
Gly Arg Arg Gly Ser Phe Val Glu Met Val50 55 60Asp Asn Leu Arg Gly
Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr65 70 75 80Val Gly Ser
Pro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser85 90 95Ser Asn
Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr100 105
110Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly
Val115 120 125Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu
Gly Thr Asp130 135 140Leu Val Ser Ile Pro His Gly Pro Asn Val Thr
Val Arg Ala Asn Ile145 150 155 160Ala Ala Ile Thr Glu Ser Asp Lys
Phe Phe Ile Asn Gly Ser Asn Trp165 170 175Glu Gly Ile Leu Gly Leu
Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp180 185 190Ser Leu Glu Pro
Phe Phe Asp Ser Leu Val Lys Gln Thr His Val Pro195 200 205Asn Leu
Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro Leu Asn Gln210 215
220Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly
Ile225 230 235 240Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr
Pro Ile Arg Arg245 250 255Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg
Val Glu Ile Asn Gly Gln260 265 270Asp Leu Lys Met Asp Cys Lys Glu
Tyr Asn Tyr Asp Lys Ser Ile Val275 280 285Asp Ser Gly Thr Thr Asn
Leu Arg Leu Pro Lys Lys Val Phe Glu Ala290 295 300Ala Val Lys Ser
Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp305 310 315 320Gly
Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Gln Ala Gly Thr Thr325 330
335Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu
Val340 345 350Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu Pro Gln Gln
Tyr Leu Arg355 360 365Pro Val Glu Asp Val Ala Thr Ser Gln Asp Asp
Cys Tyr Lys Phe Ala370 375 380Ile Ser Gln Ser Ser Thr Gly Thr Val
Met Gly Ala Val Ile Met Glu385 390 395 400Gly Phe Tyr Val Val Phe
Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala405 410 415Val Ser Ala
Cys420617PRTArtificial SequenceSynthetic peptide inhibitor 61Glu
Val Met Xaa Ala Glu Phe1 56226PRTHomo sapiens 62Leu Met Thr Ile Ala
Tyr Val Met Ala Ala Ile Cys Ala Leu Phe Met1 5 10 15Leu Pro Leu Cys
Leu Met Val Cys Gln Trp20 256333PRTHomo sapiensP26-P4'sw peptide
substrate 63Cys Gly Gly Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser
Gly Leu1 5 10 15Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Asn Leu
Asp Ala Glu20 25 30Phe6429PRTHomo sapiensP26-P1' peptide substrate
with CGG linker 64Cys Gly Gly Ala Asp Arg Gly Leu Thr Thr Arg Pro
Gly Ser Gly Leu1 5 10 15Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val
Asn Leu20 2565501PRTMus musculus 65Met Ala Pro Ala Leu His Trp Leu
Leu Leu Trp Val Gly Ser Gly Met1 5 10 15Leu Pro Ala Gln Gly Thr His
Leu Gly Ile Arg Leu Pro Leu Arg Ser20 25 30Gly Leu Ala Gly Pro Pro
Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp35 40 45Glu Glu Ser Glu Glu
Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val50 55 60Asp Asn Leu Arg
Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr65 70 75 80Val Gly
Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser85 90 95Ser
Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr100 105
110Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly
Val115 120 125Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu
Gly Thr Asp130 135 140Leu Val Ser Ile Pro His Gly Pro Asn Val Thr
Val Arg Ala Asn Ile145 150 155 160Ala Ala Ile Thr Glu Ser Asp Lys
Phe Phe Ile Asn Gly Ser Asn Trp165 170 175Glu Gly Ile Leu Gly Leu
Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp180 185 190Ser Leu Glu Pro
Phe Phe Asp Ser Leu Val Lys Gln Thr His Ile Pro195 200 205Asn Ile
Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro Leu Asn Gln210 215
220Thr Glu Ala Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly
Ile225 230 235 240Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr
Pro Ile Arg Arg245 250
255Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly
Gln260 265 270Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys
Ser Ile Val275 280 285Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys
Lys Val Phe Glu Ala290 295 300Ala Val Lys Ser Ile Lys Ala Ala Ser
Ser Thr Glu Lys Phe Pro Asp305 310 315 320Gly Phe Trp Leu Gly Glu
Gln Leu Val Cys Trp Gln Ala Gly Thr Thr325 330 335Pro Trp Asn Ile
Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val340 345 350Thr Asn
Gln Ser Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr Leu Arg355 360
365Pro Val Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr Lys Phe
Ala370 375 380Val Ser Gln Ser Ser Thr Gly Thr Val Met Gly Ala Val
Ile Met Glu385 390 395 400Gly Phe Tyr Val Val Phe Asp Arg Ala Arg
Lys Arg Ile Gly Phe Ala405 410 415Val Ser Ala Cys His Val His Asp
Glu Phe Arg Thr Ala Ala Val Glu420 425 430Gly Pro Phe Val Thr Ala
Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro435 440 445Gln Thr Asp Glu
Ser Thr Leu Met Thr Ile Ala Tyr Val Met Ala Ala450 455 460Ile Cys
Ala Leu Phe Met Leu Pro Leu Cys Leu Met Val Cys Gln Trp465 470 475
480Arg Cys Leu Arg Cys Leu Arg His Gln His Asp Asp Phe Gly Asp
Asp485 490 495Ile Ser Leu Leu Lys50066480PRTHomo sapiens 66Thr Gln
His Gly Ile Arg Leu Pro Leu Arg Ser Gly Leu Gly Gly Ala1 5 10 15Pro
Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp Glu Glu Pro Glu Glu20 25
30Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val Asp Asn Leu Arg Gly35
40 45Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr Val Gly Ser Pro
Pro50 55 60Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser Ser Asn Phe
Ala Val65 70 75 80Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr Tyr
Gln Arg Gln Leu85 90 95Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val
Tyr Val Pro Tyr Thr100 105 110Gln Gly Lys Trp Glu Gly Glu Leu Gly
Thr Asp Leu Val Ser Ile Pro115 120 125His Gly Pro Asn Val Thr Val
Arg Ala Asn Ile Ala Ala Ile Thr Glu130 135 140Ser Asp Lys Phe Phe
Ile Asn Gly Ser Asn Trp Glu Gly Ile Leu Gly145 150 155 160Leu Ala
Tyr Ala Glu Ile Ala Arg Pro Asp Asp Ser Leu Glu Pro Phe165 170
175Phe Asp Ser Leu Val Lys Gln Thr His Val Pro Asn Leu Phe Ser
Leu180 185 190Gln Leu Cys Gly Ala Gly Phe Pro Leu Asn Gln Ser Glu
Val Leu Ala195 200 205Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile
Asp His Ser Leu Tyr210 215 220Thr Gly Ser Leu Trp Tyr Thr Pro Ile
Arg Arg Glu Trp Tyr Tyr Glu225 230 235 240Val Ile Ile Val Arg Val
Glu Ile Asn Gly Gln Asp Leu Lys Met Asp245 250 255Cys Lys Glu Tyr
Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr260 265 270Asn Leu
Arg Leu Pro Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile275 280
285Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu
Gly290 295 300Glu Gln Leu Val Cys Trp Gln Ala Gly Thr Thr Pro Trp
Asn Ile Phe305 310 315 320Pro Val Ile Ser Leu Tyr Leu Met Gly Glu
Val Thr Asn Gln Ser Phe325 330 335Arg Ile Thr Ile Leu Pro Gln Gln
Tyr Leu Arg Pro Val Glu Asp Val340 345 350Ala Thr Ser Gln Asp Asp
Cys Tyr Lys Phe Ala Ile Ser Gln Ser Ser355 360 365Thr Gly Thr Val
Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val370 375 380Phe Asp
Arg Ala Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His385 390 395
400Val His Asp Glu Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val
Thr405 410 415Leu Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro Gln Thr
Asp Glu Ser420 425 430Thr Leu Met Thr Ile Ala Tyr Val Met Ala Ala
Ile Cys Ala Leu Phe435 440 445Met Leu Pro Leu Cys Leu Met Val Cys
Gln Trp Arg Cys Leu Arg Cys450 455 460Leu Arg Gln Gln His Asp Asp
Phe Ala Asp Asp Ile Ser Leu Leu Lys465 470 475 48067444PRTHomo
sapiens 67Gly Ser Phe Val Glu Met Val Asp Asn Leu Arg Gly Lys Ser
Gly Gln1 5 10 15Gly Tyr Tyr Val Glu Met Thr Val Gly Ser Pro Pro Gln
Thr Leu Asn20 25 30Ile Leu Val Asp Thr Gly Ser Ser Asn Phe Ala Val
Gly Ala Ala Pro35 40 45His Pro Phe Leu His Arg Tyr Tyr Gln Arg Gln
Leu Ser Ser Thr Tyr50 55 60Arg Asp Leu Arg Lys Gly Val Tyr Val Pro
Tyr Thr Gln Gly Lys Trp65 70 75 80Glu Gly Glu Leu Gly Thr Asp Leu
Val Ser Ile Pro His Gly Pro Asn85 90 95Val Thr Val Arg Ala Asn Ile
Ala Ala Ile Thr Glu Ser Asp Lys Phe100 105 110Phe Ile Asn Gly Ser
Asn Trp Glu Gly Ile Leu Gly Leu Ala Tyr Ala115 120 125Glu Ile Ala
Arg Pro Asp Asp Ser Leu Glu Pro Phe Phe Asp Ser Leu130 135 140Val
Lys Gln Thr His Val Pro Asn Leu Phe Ser Leu Gln Leu Cys Gly145 150
155 160Ala Gly Phe Pro Leu Asn Gln Ser Glu Val Leu Ala Ser Val Gly
Gly165 170 175Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu Tyr Thr
Gly Ser Leu180 185 190Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr
Glu Val Ile Ile Val195 200 205Arg Val Glu Ile Asn Gly Gln Asp Leu
Lys Met Asp Cys Lys Glu Tyr210 215 220Asn Tyr Asp Lys Ser Ile Val
Asp Ser Gly Thr Thr Asn Leu Arg Leu225 230 235 240Pro Lys Lys Val
Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser245 250 255Ser Thr
Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu Gln Leu Val260 265
270Cys Trp Gln Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile
Ser275 280 285Leu Tyr Leu Met Gly Glu Val Thr Asn Gln Ser Phe Arg
Ile Thr Ile290 295 300Leu Pro Gln Gln Tyr Leu Arg Pro Val Glu Asp
Val Ala Thr Ser Gln305 310 315 320Asp Asp Cys Tyr Lys Phe Ala Ile
Ser Gln Ser Ser Thr Gly Thr Val325 330 335Met Gly Ala Val Ile Met
Glu Gly Phe Tyr Val Val Phe Asp Arg Ala340 345 350Arg Lys Arg Ile
Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu355 360 365Phe Arg
Thr Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp Met Glu370 375
380Asp Cys Gly Tyr Asn Ile Pro Gln Thr Asp Glu Ser Thr Leu Met
Thr385 390 395 400Ile Ala Tyr Val Met Ala Ala Ile Cys Ala Leu Phe
Met Leu Pro Leu405 410 415Cys Leu Met Val Cys Gln Trp Arg Cys Leu
Arg Cys Leu Arg Gln Gln420 425 430His Asp Asp Phe Ala Asp Asp Ile
Ser Leu Leu Lys435 44068395PRTHomo sapiens 68Gly Ser Phe Val Glu
Met Val Asp Asn Leu Arg Gly Lys Ser Gly Gln1 5 10 15Gly Tyr Tyr Val
Glu Met Thr Val Gly Ser Pro Pro Gln Thr Leu Asn20 25 30Ile Leu Val
Asp Thr Gly Ser Ser Asn Phe Ala Val Gly Ala Ala Pro35 40 45His Pro
Phe Leu His Arg Tyr Tyr Gln Arg Gln Leu Ser Ser Thr Tyr50 55 60Arg
Asp Leu Arg Lys Gly Val Tyr Val Pro Tyr Thr Gln Gly Lys Trp65 70 75
80Glu Gly Glu Leu Gly Thr Asp Leu Val Ser Ile Pro His Gly Pro Asn85
90 95Val Thr Val Arg Ala Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys
Phe100 105 110Phe Ile Asn Gly Ser Asn Trp Glu Gly Ile Leu Gly Leu
Ala Tyr Ala115 120 125Glu Ile Ala Arg Pro Asp Asp Ser Leu Glu Pro
Phe Phe Asp Ser Leu130 135 140Val Lys Gln Thr His Val Pro Asn Leu
Phe Ser Leu Gln Leu Cys Gly145 150 155 160Ala Gly Phe Pro Leu Asn
Gln Ser Glu Val Leu Ala Ser Val Gly Gly165 170 175Ser Met Ile Ile
Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu180 185 190Trp Tyr
Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val195 200
205Arg Val Glu Ile Asn Gly Gln Asp Leu Lys Met Asp Cys Lys Glu
Tyr210 215 220Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn
Leu Arg Leu225 230 235 240Pro Lys Lys Val Phe Glu Ala Ala Val Lys
Ser Ile Lys Ala Ala Ser245 250 255Ser Thr Glu Lys Phe Pro Asp Gly
Phe Trp Leu Gly Glu Gln Leu Val260 265 270Cys Trp Gln Ala Gly Thr
Thr Pro Trp Asn Ile Phe Pro Val Ile Ser275 280 285Leu Tyr Leu Met
Gly Glu Val Thr Asn Gln Ser Phe Arg Ile Thr Ile290 295 300Leu Pro
Gln Gln Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser Gln305 310 315
320Asp Asp Cys Tyr Lys Phe Ala Ile Ser Gln Ser Ser Thr Gly Thr
Val325 330 335Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe
Asp Arg Ala340 345 350Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys
His Val His Asp Glu355 360 365Phe Arg Thr Ala Ala Val Glu Gly Pro
Phe Val Thr Leu Asp Met Glu370 375 380Asp Cys Gly Tyr Asn Ile Pro
Gln Thr Asp Glu385 390 39569439PRTHomo sapiens 69Met Val Asp Asn
Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu1 5 10 15Met Thr Val
Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr20 25 30Gly Ser
Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His35 40 45Arg
Tyr Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys50 55
60Gly Val Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly65
70 75 80Thr Asp Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg
Ala85 90 95Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn
Gly Ser100 105 110Asn Trp Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu
Ile Ala Arg Pro115 120 125Asp Asp Ser Leu Glu Pro Phe Phe Asp Ser
Leu Val Lys Gln Thr His130 135 140Val Pro Asn Leu Phe Ser Leu Gln
Leu Cys Gly Ala Gly Phe Pro Leu145 150 155 160Asn Gln Ser Glu Val
Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly165 170 175Gly Ile Asp
His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile180 185 190Arg
Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn195 200
205Gly Gln Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys
Ser210 215 220Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys
Lys Val Phe225 230 235 240Glu Ala Ala Val Lys Ser Ile Lys Ala Ala
Ser Ser Thr Glu Lys Phe245 250 255Pro Asp Gly Phe Trp Leu Gly Glu
Gln Leu Val Cys Trp Gln Ala Gly260 265 270Thr Thr Pro Trp Asn Ile
Phe Pro Val Ile Ser Leu Tyr Leu Met Gly275 280 285Glu Val Thr Asn
Gln Ser Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr290 295 300Leu Arg
Pro Val Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr Lys305 310 315
320Phe Ala Ile Ser Gln Ser Ser Thr Gly Thr Val Met Gly Ala Val
Ile325 330 335Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys
Arg Ile Gly340 345 350Phe Ala Val Ser Ala Cys His Val His Asp Glu
Phe Arg Thr Ala Ala355 360 365Val Glu Gly Pro Phe Val Thr Leu Asp
Met Glu Asp Cys Gly Tyr Asn370 375 380Ile Pro Gln Thr Asp Glu Ser
Thr Leu Met Thr Ile Ala Tyr Val Met385 390 395 400Ala Ala Ile Cys
Ala Leu Phe Met Leu Pro Leu Cys Leu Met Val Cys405 410 415Gln Trp
Arg Cys Leu Arg Cys Leu Arg Gln Gln His Asp Asp Phe Ala420 425
430Asp Asp Ile Ser Leu Leu Lys43570390PRTHomo sapiens 70Met Val Asp
Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu1 5 10 15Met Thr
Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr20 25 30Gly
Ser Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His35 40
45Arg Tyr Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys50
55 60Gly Val Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu
Gly65 70 75 80Thr Asp Leu Val Ser Ile Pro His Gly Pro Asn Val Thr
Val Arg Ala85 90 95Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe
Ile Asn Gly Ser100 105 110Asn Trp Glu Gly Ile Leu Gly Leu Ala Tyr
Ala Glu Ile Ala Arg Pro115 120 125Asp Asp Ser Leu Glu Pro Phe Phe
Asp Ser Leu Val Lys Gln Thr His130 135 140Val Pro Asn Leu Phe Ser
Leu Gln Leu Cys Gly Ala Gly Phe Pro Leu145 150 155 160Asn Gln Ser
Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly165 170 175Gly
Ile Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile180 185
190Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile
Asn195 200 205Gly Gln Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr
Asp Lys Ser210 215 220Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu
Pro Lys Lys Val Phe225 230 235 240Glu Ala Ala Val Lys Ser Ile Lys
Ala Ala Ser Ser Thr Glu Lys Phe245 250 255Pro Asp Gly Phe Trp Leu
Gly Glu Gln Leu Val Cys Trp Gln Ala Gly260 265 270Thr Thr Pro Trp
Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu Met Gly275 280 285Glu Val
Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr290 295
300Leu Arg Pro Val Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr
Lys305 310 315 320Phe Ala Ile Ser Gln Ser Ser Thr Gly Thr Val Met
Gly Ala Val Ile325 330 335Met Glu Gly Phe Tyr Val Val Phe Asp Arg
Ala Arg Lys Arg Ile Gly340 345 350Phe Ala Val Ser Ala Cys His Val
His Asp Glu Phe Arg Thr Ala Ala355 360 365Val Glu Gly Pro Phe Val
Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn370 375 380Ile Pro Gln Thr
Asp Glu385 39071374PRTHomo sapiens 71Glu Thr Asp Glu Glu Pro Glu
Glu Pro Gly Arg Arg Gly Ser Phe Val1 5 10 15Glu Met Val Asp Asn Leu
Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val20 25 30Glu Met Thr Val Gly
Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp35 40 45Thr Gly Ser Ser
Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu50 55 60His Arg Tyr
Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg65 70 75 80Lys
Gly Val Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu85 90
95Gly Thr Asp Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val
Arg100 105 110Ala Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe
Ile Asn Gly115 120 125Ser Asn Trp Glu Gly Ile Leu Gly Leu Ala Tyr
Ala Glu Ile Ala Arg130 135 140Pro Asp Asp Ser Leu Glu Pro Phe Phe
Asp Ser Leu Val Lys Gln Thr145 150 155 160His Val Pro Asn Leu Phe
Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro165 170 175Leu Asn Gln Ser
Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile180 185 190Gly Gly
Ile Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro195 200
205Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu
Ile210 215 220Asn Gly Gln Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn
Tyr Asp Lys225 230 235 240Ser Ile Val Asp Ser Gly Thr Thr Asn Leu
Arg Leu Pro Lys Lys Val245
250 255Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu
Lys260 265 270Phe Pro Asp Gly Phe Trp Leu Gly Glu Gln Leu Val Cys
Trp Gln Ala275 280 285Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile
Ser Leu Tyr Leu Met290 295 300Gly Glu Val Thr Asn Gln Ser Phe Arg
Ile Thr Ile Leu Pro Gln Gln305 310 315 320Tyr Leu Arg Pro Val Glu
Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr325 330 335Lys Phe Ala Ile
Ser Gln Ser Ser Thr Gly Thr Val Met Gly Ala Val340 345 350Ile Met
Glu Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile355 360
365Gly Phe Ala Val Ser Ala3707214PRTArtificial
SequenceP10-P4'staD-V peptide inhibitor 72Lys Thr Glu Glu Ile Ser
Glu Val Asn Xaa Val Ala Glu Phe1 5 10739PRTArtificial
SequenceP4-P4'staD-V peptide inhibitor 73Ser Glu Val Asn Xaa Val
Ala Glu Phe1 574431PRTHomo sapiens 74Thr Gln His Gly Ile Arg Leu
Pro Leu Arg Ser Gly Leu Gly Gly Ala1 5 10 15Pro Leu Gly Leu Arg Leu
Pro Arg Glu Thr Asp Glu Glu Pro Glu Glu20 25 30Pro Gly Arg Arg Gly
Ser Phe Val Glu Met Val Asp Asn Leu Arg Gly35 40 45Lys Ser Gly Gln
Gly Tyr Tyr Val Glu Met Thr Val Gly Ser Pro Pro50 55 60Gln Thr Leu
Asn Ile Leu Val Asp Thr Gly Ser Ser Asn Phe Ala Val65 70 75 80Gly
Ala Ala Pro His Pro Phe Leu His Arg Tyr Tyr Gln Arg Gln Leu85 90
95Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val Tyr Val Pro Tyr
Thr100 105 110Gln Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp Leu Val
Ser Ile Pro115 120 125His Gly Pro Asn Val Thr Val Arg Ala Asn Ile
Ala Ala Ile Thr Glu130 135 140Ser Asp Lys Phe Phe Ile Asn Gly Ser
Asn Trp Glu Gly Ile Leu Gly145 150 155 160Leu Ala Tyr Ala Glu Ile
Ala Arg Pro Asp Asp Ser Leu Glu Pro Phe165 170 175Phe Asp Ser Leu
Val Lys Gln Thr His Val Pro Asn Leu Phe Ser Leu180 185 190Gln Leu
Cys Gly Ala Gly Phe Pro Leu Asn Gln Ser Glu Val Leu Ala195 200
205Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu
Tyr210 215 220Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg Glu Trp
Tyr Tyr Glu225 230 235 240Val Ile Ile Val Arg Val Glu Ile Asn Gly
Gln Asp Leu Lys Met Asp245 250 255Cys Lys Glu Tyr Asn Tyr Asp Lys
Ser Ile Val Asp Ser Gly Thr Thr260 265 270Asn Leu Arg Leu Pro Lys
Lys Val Phe Glu Ala Ala Val Lys Ser Ile275 280 285Lys Ala Ala Ser
Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly290 295 300Glu Gln
Leu Val Cys Trp Gln Ala Gly Thr Thr Pro Trp Asn Ile Phe305 310 315
320Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val Thr Asn Gln Ser
Phe325 330 335Arg Ile Thr Ile Leu Pro Gln Gln Tyr Leu Arg Pro Val
Glu Asp Val340 345 350Ala Thr Ser Gln Asp Asp Cys Tyr Lys Phe Ala
Ile Ser Gln Ser Ser355 360 365Thr Gly Thr Val Met Gly Ala Val Ile
Met Glu Gly Phe Tyr Val Val370 375 380Phe Asp Arg Ala Arg Lys Arg
Ile Gly Phe Ala Val Ser Ala Cys His385 390 395 400Val His Asp Glu
Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr405 410 415Leu Asp
Met Glu Asp Cys Gly Tyr Asn Ile Pro Gln Thr Asp Glu420 425
43075361PRTHomo sapiens 75Met Val Asp Asn Leu Arg Gly Lys Ser Gly
Gln Gly Tyr Tyr Val Glu1 5 10 15Met Thr Val Gly Ser Pro Pro Gln Thr
Leu Asn Ile Leu Val Asp Thr20 25 30Gly Ser Ser Asn Phe Ala Val Gly
Ala Ala Pro His Pro Phe Leu His35 40 45Arg Tyr Tyr Gln Arg Gln Leu
Ser Ser Thr Tyr Arg Asp Leu Arg Lys50 55 60Gly Val Tyr Val Pro Tyr
Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly65 70 75 80Thr Asp Leu Val
Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala85 90 95Asn Ile Ala
Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser100 105 110Asn
Trp Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro115 120
125Asp Asp Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr
His130 135 140Val Pro Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly
Phe Pro Leu145 150 155 160Asn Gln Ser Glu Val Leu Ala Ser Val Gly
Gly Ser Met Ile Ile Gly165 170 175Gly Ile Asp His Ser Leu Tyr Thr
Gly Ser Leu Trp Tyr Thr Pro Ile180 185 190Arg Arg Glu Trp Tyr Tyr
Glu Val Ile Ile Val Arg Val Glu Ile Asn195 200 205Gly Gln Asp Leu
Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser210 215 220Ile Val
Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val Phe225 230 235
240Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys
Phe245 250 255Pro Asp Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp
Gln Ala Gly260 265 270Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser
Leu Tyr Leu Met Gly275 280 285Glu Val Thr Asn Gln Ser Phe Arg Ile
Thr Ile Leu Pro Gln Gln Tyr290 295 300Leu Arg Pro Val Glu Asp Val
Ala Thr Ser Gln Asp Asp Cys Tyr Lys305 310 315 320Phe Ala Ile Ser
Gln Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile325 330 335Met Glu
Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly340 345
350Phe Ala Val Ser Ala Cys His Val His355 3607663DNAHomo
sapiensmisc_feature(1)...(63)n = A,T,C or G 76garacngayg argarccnga
rgarccnggn mgnmgnggnw snttygtnga ratggtngay 60aay 637721PRTHomo
sapiens 77Glu Thr Asp Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser
Phe Val1 5 10 15Glu Met Val Asp Asn20787PRTArtificial
SequencePeptide inhibitor P3-P4' XD-V 78Val Met Xaa Val Ala Glu
Phe1 57911PRTHomo sapiens 79Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe
Val1 5 1080419DNAArtificial Sequencenucleotide insert in vector pCF
80ctgttgggct cgcggttgag gacaaactct tcgcggtctt tccagtactc ttggatcgga
60aacccgtcgg cctccgaacg gtactccgcc accgagggac ctgagcgagt ccgcatcgac
120cggatcggaa aacctctcga ctgttggggt gagtactccc tctcaaaagc
gggcatgact 180tctgcgctaa gattgtcagt ttccaaaaac gaggaggatt
tgatattcac ctggcccgcg 240gtgatgcctt tgagggtggc cgcgtccatc
tggtcagaaa agacaatctt tttgttgtca 300agcttgaggt gtggcaggct
tgagatctgg ccatacactt gagtgacaat gacatccact 360ttgcctttct
ctccacaggt gtccactccc aggtccaact gcaggtcgac tctagaccc
419818PRTArtificial SequencePeptide inhibitor P4-P4' XD-V 81Glu Val
Met Xaa Val Ala Glu Phe1 5829PRTHomo sapiensAPP fragment P5-P4' wt
82Ser Glu Val Lys Met Asp Ala Glu Phe1 5839PRTHomo sapiensAPP
fragment P5-P4'wt 83Ser Glu Val Asn Leu Asp Ala Glu Phe1
5849PRTArtificial SequenceAPP fragment 84Ser Glu Val Lys Leu Asp
Ala Glu Phe1 5859PRTArtificial SequenceAPP fragment 85Ser Glu Val
Lys Phe Asp Ala Glu Phe1 5869PRTArtificial SequenceAPP fragment
86Ser Glu Val Asn Phe Asp Ala Glu Phe1 5879PRTArtificial
SequenceAPP fragment 87Ser Glu Val Lys Met Ala Ala Glu Phe1
5889PRTArtificial SequenceAPP fragment 88Ser Glu Val Asn Leu Ala
Ala Glu Phe1 5899PRTArtificial SequenceAPP fragment 89Ser Glu Val
Lys Leu Ala Ala Glu Phe1 5909PRTArtificial SequenceAPP fragment
90Ser Glu Val Lys Met Leu Ala Glu Phe1 5919PRTArtificial
SequenceAPP fragment 91Ser Glu Val Asn Leu Leu Ala Glu Phe1
5929PRTArtificial SequenceAPP fragment 92Ser Glu Val Lys Leu Leu
Ala Glu Phe1 5939PRTArtificial SequenceAPP fragment 93Ser Glu Val
Lys Phe Ala Ala Glu Phe1 5949PRTArtificial SequenceAPP fragment
94Ser Glu Val Asn Phe Ala Ala Glu Phe1 5959PRTArtificial
SequenceAPP fragment 95Ser Glu Val Lys Phe Leu Ala Glu Phe1
5969PRTArtificial SequenceAPP fragment 96Ser Glu Val Asn Phe Leu
Ala Glu Phe1 59714PRTArtificial SequenceAPP-derived fragment
P10-P4'(D-V) 97Lys Thr Glu Glu Ile Ser Glu Val Asn Leu Val Ala Glu
Phe1 5 109835DNAHomo sapiens 98cccgaagagc ccggccggag gggcagcttt
gtcga 359911PRTHomo sapiens 99Glu Thr Asp Glu Glu Pro Glu Glu Pro
Gly Arg1 5 1010010PRTArtificial SequenceRecombinant 293T cells
100Thr Gln His Gly Ile Arg Leu Pro Leu Arg1 5 101019PRTArtificial
SequenceRecombinant 293T cells 101Met Val Asp Asn Leu Arg Gly Lys
Ser1 510210PRTArtificial SequenceRecombinant CosA2 cells 102Gly Ser
Phe Val Glu Met Val Asp Asn Leu1 5 101034PRTArtificial SequenceAPP
substrate fragmentWT Sequence 103Val Lys Met Asp11044PRTArtificial
SequenceAPP substrate fragmentSwedish Sequence 104Val Asn Leu
Asp1
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