U.S. patent application number 17/406051 was filed with the patent office on 2022-03-10 for recombinant clusterin and use thereof in the treatment and prevention of disease.
This patent application is currently assigned to BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM. The applicant listed for this patent is BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM. Invention is credited to Yong-Jian GENG.
Application Number | 20220073591 17/406051 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-10 |
United States Patent
Application |
20220073591 |
Kind Code |
A1 |
GENG; Yong-Jian |
March 10, 2022 |
RECOMBINANT CLUSTERIN AND USE THEREOF IN THE TREATMENT AND
PREVENTION OF DISEASE
Abstract
Recombinant clusterin polypeptides and compositions comprising
the same are provided. In some aspects, recombinant clusterin or
nucleic acids encoding the same may be used for treating and
preventing an abnormality of morphology and function in a mammal
with disease (e.g., cardiovascular diseases or alcoholism).
Inventors: |
GENG; Yong-Jian; (Houston,
TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM |
Austin |
TX |
US |
|
|
Assignee: |
BOARD OF REGENTS OF THE UNIVERSITY
OF TEXAS SYSTEM
Austin
TX
|
Appl. No.: |
17/406051 |
Filed: |
August 18, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16597400 |
Oct 9, 2019 |
11104719 |
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17406051 |
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14957030 |
Dec 2, 2015 |
10464994 |
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16597400 |
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62087364 |
Dec 4, 2014 |
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International
Class: |
C07K 14/775 20060101
C07K014/775; A61P 9/10 20060101 A61P009/10 |
Claims
1. (canceled)
2. The method of claim 47, comprising a Clusterin coding sequence
having a deletion of the nuclear localization signal and/or
transmembrane domain.
3. The method of claim 47, comprising a Clusterin coding sequence
and a heterologous polypeptide sequence fused to said Clusterin
coding sequence.
4. The method of claim 3, wherein the heterologous polypeptide
sequence comprises a protease cleavage site.
5. The method of claim 3, wherein the protease cleavage site is a
thrombin cleavage site.
6. The method of claim 47, wherein the polypeptide is a fusion
protein comprising the Clusterin coding sequence and a heterologous
polypeptide sequence.
7. The method of claim 47, wherein the polypeptide is
aglycosylated.
8. The method of claim 47, wherein the Clusterin coding sequence
has a deletion of the nuclear localization signal.
9. The method of claim 47, wherein the Clusterin coding sequence
has a deletion of the transmembrane domain.
10. The method of claim 9, wherein the deletion of the
transmembrane domain disrupts endoplasmic reticulum (ER)-targeting
of the polypeptide.
11. The method of claim 47, further comprising a tag sequence.
12. The method of claim 11, further comprising a protease cleavage
site positioned between the tag sequence and the clusterin coding
sequence.
13. The method of claim 11, wherein the protease cleavage site is a
thrombin or enteropeptidase cleavage site.
14. The method of claim 11, wherein the tag sequence is a
polyhistidine tag.
15. The method of claim 11, wherein the tag sequence is positioned
N-terminally relative to the Clusterin coding sequence.
16. The method of claim 11, wherein the tag sequence is positioned
C-terminally relative to the Clusterin coding sequence.
17-46. (canceled)
47. A method of treating or preventing alcoholism in a subject
comprising administering an effective amount of a composition
comprising a polypeptide comprising: (i) a Clusterin coding
sequence, said coding sequence having a deletion of the nuclear
localization signal and/or transmembrane domain; or (ii) a
Clusterin coding sequence and a heterologous polypeptide sequence
fused to said Clusterin coding sequence.
48. The method of claim 47, wherein the composition is administered
by intravenous injection, intratissue injection and/or catheter
delivery.
49. The method of claim 47, wherein the nucleic acid sequence
encoding the Clusterin polypeptide is operably linked to a
promoter.
50. The method of claim 49, wherein the promoter is a gene promoter
functional in mammalian cells.
51. The method of claim 50, wherein the polynucleotide molecule is
part of a gene expression vector, such as a viral expression
vector.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/597,400, now U.S. Pat. No. 11,104,719,
filed Oct. 9, 2019, which is a continuation of U.S. patent
application Ser. No. 14/957,030, now U.S. Pat. No. 10,464,994,
filed Dec. 2, 2015, which claims the benefit of U.S. Provisional
Patent Application No. 62/087,364, filed Dec. 4, 2014, the entirety
of each of which is incorporated herein by reference.
INCORPORATION OF SEQUENCE LISTING
[0002] The sequence listing that is contained in the file named
"UTSHP0306USC2_ST25.txt", which is 61 KB (as measured in Microsoft
Windows.RTM.) and was created on Aug. 17, 2021, is filed herewith
by electronic submission and is incorporated by reference
herein.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0003] The present invention generally relates production of
recombinant clusterin and the use thereof for prevention and
treatment of pathological conditions.
2. Description of Related Art
[0004] Hypercholesterolemia represents the most defined risk factor
for atherosclerosis, an arterial disease that causes myocardial and
cerebral infarctions. Low density lipoprotein (LDL) carries
cholesterol from the liver to peripheral tissues, and when elevated
in blood, LDL deposits the lipid in the arterial wall, which in
turn develops atherosclerotic plaques and increases the risk for
thrombogenic events in the arteries. In contrast, high density
lipoprotein (HDL) functions as a reverse cholesterol-transporter
that removes the lipid from the arterial wall to the liver where
cholesterol is metabolized. In essence, LDL is pro-atherogenic
while HDL anti-atherogenic (Nicholls et al., 2005; Ansell et al.,
2004; von Eckardstein et al., 2005) LDL mainly contains ApoB 100
and HDL apoE and apoA-I. In spite of the success of lowering
LDL-cholesterol therapy with statins, raising HDL levels with
torcetrapib (an inhibitor of cholesterol ester transfer protein
(CETP) has shown little benefit to patients with atherosclerosis
(Kastelein et al., 2007; Nissen et al., 2007). The failure of
torcetrapib therapy underscores the incompleteness of our
fundamental understanding of HDL function. HDL particles are
heterogeneous in shape, density, size, composition and have
multiple functional properties such as reverse cholesterol
transport, as well as anti-oxidant, anti-inflammatory, and
anti-thrombotic activities. Indeed, dysfunctional, proinflammatory
HDL has been found in several pathological conditions, including
atherosclerosis (Smith et al., 2010), diabetes (Hoofnagle et al.
2010) and autoimmune disorders (McMahon et al., 2009; Volkmann et
al., 2010). Thus, the development of an anti-atherogenic,
anti-apoptotic, and anti-inflammatory agent that enables HDL
beneficial action is of clinical significance.
[0005] Clusterin is a sulfated, heterodimeric glycoprotein
containing two 40 kDa chains joined by a unique five disulfide bond
motif 1). It contains several domains, such as amphipathic helix,
heparin-binding domain, and lipid-binding domain. This protein was
initially identified from ram rete testes fluid and named for its
ability to elicit clustering of Sertoli cells supporting sperm
maturation and development (NCBI/GenBank Accession No. NM_203339,
NM_001831) Thereafter, species homologues have been isolated and
cloned by a number of groups working in widely divergent areas,
generating various synonyms of clusterin, including testosterone
repressed prostate message-2 (TRPM-2), sulfated glycoprotein-2
(SGP-2), apolipoprotein-J (Apo-J), SP-40, 40, complement cytolysis
inhibitor (CLI), and dimeric acidic glycoprotein (DAG), gp 80,
NA1/NA2, glycoprotein III, etc.
[0006] Encoded on a 2-kb mRNA, clusterin is transcribed from a
single copy gene located on mouse chromosome 14 and human 8p21
(Fink et al., 1993), and translated as a 51 kD or so protein
comprising 427 amino acid sequence (Jordan-Starck et al., 1994). In
the blood stream, clusterin circulates mainly with HDL as one of
apolipoproteins (Choi-Miura et al., 1992; Stuart et al., 1992) but
a small portion of clusterin may exist in LDL (Karlsson et al.,
2005). Clusterin expression is induced by stress responses (Wilson
et al., 2000). Clusterin binds megalin/LRP-2 receptor, members of
LDL receptor family. Increased expression of clusterin occurs in
both human (Mackness et al., 1997; Ishikawa et al., 2001; Ishikawa
et al., 1998) and experimental animal (Jordan-Starck et al., 1994;
Navab et al., 1997) atherosclerotic lesions. Reported functions of
clusterin include apoptosis inhibition (Kowolik et al. 2006),
complement factor inactivation (Correa-Rotter et al., 1992), lipid
recycling and transport (Gelissen et al., 1998), membrane
protection, and maintenance of cell-cell or cell-substratum
contacts. It can effectively bind to lipids and promote efflux of
cholesterol and oxysterols from lipid-laden foam cells, a
hall-marker of atherosclerosis (Gelissen et al., 1998). Clusterin
has a high-affinity to a wide array of biological ligands. The
presence of both hydrophilic and hydrophobic domains enables
clusterin to act as a chaperone or a "biological detergent".
[0007] Clusterin plays a role in regulation of metabolism and
function of various tissues and organs, particularly in the
cardiovascular system. HDL with decreased levels of clusterin has
been found in association with a high incidence of myocardiac
infarction in patients with insulin-resistant metabolic syndromes
(Hoofnagle et al., 2010). Administration of an oral clusterin
peptide was reported to reduce atherosclerosis in ApoE-null mice
(Navab et al., 2005), and intravenous injection of clusterin
diminishes rat myocardiac infarction (Van Dijk et al., 2010).
Transduction of clusterin can restore the mitochondrial membrane
potential and prevent the release of cytochrome-c from mitochondria
into cytoplasma in cardiac myoblasts damaged by ethanol (Li et al.,
2007). Furthermore, increased clusterin expression in myoblasts
enhances the cell capacity of migration and homing through
induction of CXCR4, a chemokin-receptor for stromal cell-derived
factor (SDF) (Li et al., 2010).
[0008] Human clusterin gene located in chromosome 8 (location
8p21-p18) with 17876 bp long contains 10 exons in total. Exon one
and exon two are alternative yielding two different transcript
isoforms. Other exons (Ansell et al., 2004; von Eckardstein et al.,
2005; Kastelein et al., 2007; Nissen et al., 2007; Smith et al.,
2010; Hoofnagle et al. 2010; McMahon et al., 2009; Volkmann et al.,
2010) are shared with both isoforms. clusterin transcripts contain
3 different translation start sites (ATG), all in-frame. The best
characterized protein isoform is produced from transcript isoform
2, where translation starts at the second ATG present in exon 2,
right before ER-targeting signal. clusterin protein precursor
(NP-976084) consists of 449 amino acids. There is evidence
suggesting that two nuclear protein isoforms can be produced from
this transcript isoform, one in which translation starts at ATG in
exon 3 (417 aa), and another with translation starting from ATG in
exon 1 (459 aa). Secreted clusterin is produced from the transcript
isoform 2. The initial protein precursor, presecretory psCLU (-60
kDa), becomes heavily glycosylated and cleaved in the ER, and the
resulting alpha and beta peptide chains are held together by 5
disulfide bonds in the mature secreted heterodimer protein form,
sCLU (-75-80 kDa).
[0009] Under stimulation by ionic radiation and oxidative stress,
the nuclear clusterin is first translated as a non-glycosylated
protein precursor, pnCLU (.about.49 kDa), that is then translocated
into nucleus. There is evidence of two distinct sized nuclear
clusterin proteins (.about.50 kDa and .about.60 kDa) (Pajak et al.,
2007), that could result from translation started either at ATG
present in exon 3 or in exon 1, respectively. Secreted clusterin is
cytoprotective but nuclear clusterin cytotoxic. The controversy of
clusterin functions mainly results from the not well-established
role of the two different protein isoforms with distinct
subcellular localization and somewhat opposing functionalities.
Some known functions include involvement in apoptosis through
complexing with Ku70 autoantigen (nCLU, proapoptotic) or
interfering with Bax-activation (sCLU, antiapoptotic) (Araki et
al., 2005; Klokov et al., 2004; Leskov et al., 2003; Yang et al.,
2000). Clusterin has also been linked to spermatogenesis,
epithelial cell differentiation, TGF-beta signaling through
Smad2/Smad3 (Shin et al., 2008; Ahn et al., 2008; Lee et al.,
1992), complement activation (Dietzsch et al., 1992; O'Bryan et
al., 1990). Secreted native clusterin contains the sequence domains
of nuclear clusterin critical for nuclear translocation and binding
to nuclear death signaling proteins such as Ku70.
[0010] Despite the various roles in cellular regulation ascribed to
clusterin, there remains a need for the development of recombinant
clusterin and clusterin analogs as potential therapeutics.
Embodiments of this invention disclose technology of producing
recombinant clusterin with a high homology to the secreted form of
native clusterin with a protective function, and compositions of
recombinant clusterin that can be used for prevention and treatment
of diseases in a mammal.
SUMMARY OF THE INVENTION
[0011] In a first embodiment, a recombinant polypeptide is provided
that comprises a mammalian clusterin coding sequence. In various
aspects, the clusterin coding sequence may have a deletion of a
nuclear localization signal and/or transmembrane domain (TMD). In
some aspects, the polypeptide may be a fusion protein comprising
the clusterin coding sequence and a heterologous polypeptide
sequence. For example, the polypeptide may further comprise a tag
sequence. In further aspects, the polypeptide may comprise a
protease cleavage site (e.g., a thrombin cleavage site
(Leu-Val-Pro-Arg-Gly-Ser; SEQ ID NO: 16) or enteropeptidase
cleavage site (Asp-Asp-Asp-Asp-Lys; SEQ ID NO: 17)). For example,
protease cleavage site can be positioned between the tag sequence
and the clusterin coding sequence. In various aspects, the tag
sequence may be a polyhistidine tag. In some aspects, the tag
sequence may be positioned N-terminally relative to the Clusterin
coding sequence, while in other aspects the tag sequence may be
positioned C-terminally relative to the Clusterin coding
sequence.
[0012] In a further embodiment, a composition is provided that
comprises a clusterin polypeptide of the present embodiments in a
pharmaceutically acceptable carrier. In various aspects, the
composition may be frozen or lyophilized.
[0013] In yet a further embodiment, an isolated polynucleotide
molecule is provided that comprises a nucleic acid sequence
encoding a clusterin polypeptide of the present embodiments. In
some aspects, the nucleic acid sequence encoding the polypeptide
may be operably linked to a promoter. In certain aspects, the
promoter may be a promoter functional in mammalian, bacterial or
insect cells. In some aspects, the polynucleotide molecule may be
part of an expression vector, such as, a plasmid, an episomal
expression vector or a viral expression vector.
[0014] In a further embodiment, a host cell is provided that
comprises a polynucleotide molecule encoding a clusterin
polypeptide of the present embodiments. In some aspect, the host
cell may be a bacterial cell, an insect cell, or a mammalian cell.
In some specific aspects, the host cell is a human cell, such as a
pluripotent cell, a cardiac cell, and endothelial cell, or a
cardiac or endothelial precursor cell.
[0015] In yet a further embodiment, a method of manufacture a
recombinant clusterin polypeptide is provided that comprises (a)
expressing a polynucleotide molecule encoding a clusterin
polypeptide of the present embodiments in a cell; and (b) purifying
the polypeptide from the cell. In various aspects, the polypeptide
may comprise a purification tag, and purifying the polypeptide may
comprise use of a matrix having an affinity for the purification
tag. In some aspects, the purification tag may be a polyhistidine
tag, and purifying the polypeptide may comprise purifying the
polypeptide using a metal affinity column. In certain aspects, the
purification tag may further comprise a protease cleavage site
positioned between the tag sequence and the clusterin coding
sequence, and purifying the polypeptide may comprise contacting the
polypeptide with a protease that cleaves at the cleavage site.
[0016] In certain aspects, methods of the embodiments concern
construction and/or transfection of a nucleotide encoding clusterin
into cells of a mammalian cell or a non-mammalian cell causes
sufficient expression of clusterin polypeptides. In certain
embodiments, the step of causing the expression of an amount of a
nucleotide encoding clusterin includes transfecting cells of the
tissue with a DNA sequence encoding the entire clusterin
polypeptide sequence, or a biologically active portion of the
clusterin sequence, operably linked to a promoter and capable of
being expressed in the cells to provide an amount of clusterin
sufficient to be identified, concentrated, extracted, and
purified.
[0017] In still a further embodiment, a method of treating or
preventing a cardiovascular disease in a subject is provided that
comprises administering an effective amount of a clusterin
composition comprising (a) a recombinant clusterin polypeptide, (b)
a polynucleotide (e.g., an expression vector) encoding a clusterin
polypeptide, and/or (c) cells expressing exogenous clusterin
polypeptide of the present embodiments. In some aspects, the
cardiovascular disease may be hypertension, hyperlipidemia,
hypercholesterolemia, hyperglycemia, hypertension, atherosclerosis
and atherosclerosis-associated ischemic heart failure, stenosis,
calcification of cardiovascular tissues, stroke, myocardial
infarction or cerebral infarction. In some aspects, the
cardiovascular disease is hyperlipidemia, hypercholesterolemia or
atherosclerosis. In still further aspects, the cardiovascular
disease may be diabetes. In various aspects, an effective amount of
a clusterin composition may be an amount effective to reduce blood
cholesterol, reduce blood glucose, reduce blood triglyceride,
increase efflux of intracellular cholesterol, and/or increase
vascular or cardiac cell survival. In some aspects an effective
amount of a clusterin composition provides enhancement or promotion
of cell survival and growth against cytotoxic or cytostatic
factors, including but not limited to, oxysterols, oxidized
lipoproteins, and proinflammatory cytokines.
[0018] In accordance with certain embodiments, a method of treating
or preventing atherosclerosis, or a complication thereof in a
mammal, is provided. For the purposes of this disclosure, the term
"preventing" atherosclerosis has its usual meaning in the art and
includes "deterring" and "reducing the risk of" atherosclerosis.
This method comprises carrying out an above-described method
wherein the tissue is a cardiac or vascular or brain region
comprising an atherosclerotic lesion, or an area that is at risk of
forming an atherosclerotic lesion, and wherein the contacting of
cells in the tissue with clusterin polypeptides deters or prevents
apoptotic cell death sufficiently to prevent, or reduce the risk
of, formation of an atherosclerotic lesion. In some embodiments,
the contacting of cells in the tissue with clusterin polypeptides
deters or prevents tissue injury or degeneration sufficiently to
prevent, or reduce the risk of, rupture of an atherosclerotic
lesion.
[0019] In certain aspects, the atherosclerotic lesion comprises
calcification in a vessel or a valve with inflammation, which
causes vascular tissue stiffness and cardiac or aortic valve
malfunction, comprising stenosis or insufficient closure. In
certain of the above-described methods, the amounts of clusterin
compositions are effective to deter or prevent calcification and/or
protect against inflammatory injury, induced by at least one
condition chosen from the group consisting of:
hypercholesterolemia, hyperglycemia, hyperphosphatemia, and/or
hypertension.
[0020] In some aspects, the atherosclerotic lesion or plaque
comprises an unstable plaque caused by hyperlipidemia and
inflammation, and the amount of clusterin contacting a treatment
site are effective to stabilize the plaque (e.g., reduce the risk
of rupture of the plaque, thrombus formation, or other
complications).
[0021] Another embodiment of the present invention provides a
method of treating acute vascular syndromes and heart failure in a
mammal, which comprises delivery of an amount of clusterin
composition into the heart of the mammal to protect and improve
heart function.
[0022] In yet a further embodiment, a method of treating or
preventing alcoholism in a subject is provided that comprises
administering an effective amount of a clusterin composition
comprising (a) a recombinant clusterin polypeptide, (b) a
polynucleotide (e.g., an expression vector) encoding a clusterin
polypeptide, and/or (c) cells expressing exogenous clusterin
polypeptide of the present embodiments. In some aspects, the
effective amount of the clusterin composition is an amount
effective to reduce withdrawal symptoms, alcohol intact or markers
of liver damage in a subject.
[0023] In some aspects, a clusterin composition of the embodiments
(e.g., recombinant clusterin polypeptide, a polynucleotide encoding
a clusterin polypeptide, and/or cells expressing exogenous
clusterin polypeptide) may be administered by intravenous injection
or catheter delivery. In some aspects, clusterin compositions are
delivered into one or more tissue or organs of a mammal suffering
from, or at risk of being subjected to, physical and/or chemical
injury. In some embodiments the tissue is heart or vascular or
brain tissue. In certain embodiments, the vascular or cardiac
tissue is affected with atherosclerosis or heart failure. In other
embodiments, the vascular or cardiac tissue is not affected with
atherosclerosis, acute vascular syndromes, or heart failure. In
some embodiments, the cells are one or more of the cell types:
vascular endothelial cells, smooth muscle cells, cardiac myocytes
and brain cells.
[0024] Thus, in certain aspects, there is provided a method of
administering a mammalian cell, such as a stem cell, with enhanced
expression of recombinant clusterin, or that have been treated with
recombinant clusterin, into a tissue or organ. In some embodiments,
the tissue or organ comprises a failing heart or an atherosclerotic
blood vessel. In some embodiments, the stem cells are administered
by intravenous injection, intra-arterial catheter, or by
intramuscular or intratissue injection. In certain embodiments, the
stem cells are delivered or injected together with an agent that
causes vascular dilation and/or are co-administered with an
anti-thrombotic agent. These and other embodiments, features and
advantages embodiments of the present invention will be recognized
by those of skill in the art from the following detailed
description and drawings.
[0025] In further aspects, delivering an amount of recombinant
clusterin comprises dissolving the polypeptide in a solution or
buffer and injecting the clusterin containing solution into blood
stream or tissues of a mammal or apply said clusterin solution on
the surface of tissues or organs with injury.
[0026] As used herein, the terms "clusterin", "Apolipoprotein J",
and "Apo J" are used interchangeably.
[0027] As used herein, "essentially free," in terms of a specified
component, is used herein to mean that none of the specified
component has been purposefully formulated into a composition
and/or is present only as a contaminant or in trace amounts. The
total amount of the specified component resulting from any
unintended contamination of a composition is therefore well below
0.05%. In some aspects, most preferred is a composition in which no
amount of the specified component can be detected with standard
analytical methods.
[0028] As used herein in the specification and claims, "a" or "an"
may mean one or more. As used herein in the specification and
claims, when used in conjunction with the word "comprising", the
words "a" or "an" may mean one or more than one. As used herein, in
the specification and claim, "another" or "a further" may mean at
least a second or more.
[0029] As used herein in the specification and claims, the term
"about" is used to indicate that a value includes the inherent
variation of error for the device, the method being employed to
determine the value, or the variation that exists among the study
subjects.
[0030] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating certain
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0032] FIG. 1--A schematic representation of the clusterin analog
plasmid pCluAg. Representation of clusterin cDNA subcloning and
construction of an expression vector with an insert of clusterin
analog-encoding cDNA is shown (SEQ ID NO: 26).
[0033] FIGS. 2A-2E--Polypeptide sequences and structures of various
clusterin polypeptides of the embodiments.
[0034] FIG. 3--Coomassie blue (G250) dye-stained polyacrylamide gel
(SDS-PAGE) of recombinant human clusterin protein stored in
solution and in dry powder for 3 months. The results show long term
stability of the polypeptides.
[0035] FIG. 4.--Immunoblotting analysis of the recombinant human
clusterin polypeptide. Clusterin polypeptides (1 .mu.g/lane) were
loaded into SDS-PAGE (7%). After electropherosis, protein bands
were transferred onto PVDF membrane, probed with rabbit anti-human
clusterin antibodies (1:200), and immunostained bands were
developed by chemiluminescence. The results again show long term
stability of the polypeptides.
[0036] FIG. 5. --Ion exchange fast protein liquid chromatography
(FPLC) of recombinant human clusterin proteins. Clusterin
polypeptides were separated from other components in an aqueous
solution, or buffer. The buffer flow rate was controlled by a
positive-displacement pump and is normally kept constant, while the
composition of the buffer can be varied by drawing fluids in
different proportions from two or more external reservoirs. Ion
Exchange FPLC (BioRad UnoQ12 column); Buffer A: 20 mM Tris (pH8.0)
0.5 mM EDTA; Buffer B: 20 mM Tris (pH8.0) 0.5 mM EDTA+1N NaCl; Flow
rate: 2 ml/min; Injected amount: 250 ug; Result: peak eluted after
80 minutes.
[0037] FIGS. 6A-6C--Immunoblotting analysis of levels of
recombinant clusterin injected intraveneously into the blood stream
of wild type and ApoE-null mice. Immunoblotting for clusterin and
ApoAI proteins in the plasma of WT and ApoE-null mice with
different recombinant clusterin analog (CluAg) or saline control
injection (7 days) (FIG. 6A). Densitometry showed increased
clusterin levels in Clu injected mice as compared to saline
injected one (FIG. 6B). Co-precipitation of clusterin (Clu) in the
plasma of C57BL/6 mice injected with recombinant human clusterin
analogs (FIG. 6C). Apolipoprotein-A1, a known HDL component, was
pulled-down with anti-ApoA1 antibody, and Western blot analysis of
the ApoA1 pull-down was conducted with anti-clusterin
antibodies.
[0038] FIG. 7--Immunoblotting analysis of clusterin and HisTR tag
in proteins extracted from the supernatants of human smooth muscle
cell (SMC) cultures treated with oxLDL and recombinant clusterin
analog (CluAg) with HisTR tag.
[0039] FIG. 8--Growth curves evidencing that incubation with
recombinant clusterin analog stimulates proliferation of human
vascular smooth muscle cells. Human smooth muscle cells (SMC) were
incubated in DMEM media containing CluAg-I (1-6 .mu.g/ml).
[0040] FIG. 9--Growth curves evidencing that incubation with
recombinant clusterin analog blocks inhibitory effect of oxidized
lipoprotein and stimulates proliferation of human vascular smooth
muscle cells (SMCs). Human SMCs were incubated in DMEM media
containing CluAg-I (1-6 .mu.g/ml) in the presence of 50 .mu.g/ml
oxidized low density lipoprotein (oxLDL).
[0041] FIG. 10--Blood plasma comparison of WT mice (upper panel)
and ApoE.sup.-/- mice (lower panel). Graphs show injection of human
recombinant clusterin, CluAg-I, reducing blood levels of glucose,
cholesterol, triglyceride, and LDL in ApoE.sup.-/-
atherosclerosis-prone mice but not in age and sex-matched normal
wild type (WT) control mice.
[0042] FIG. 11--Blood pressure comparison of WT mice and
ApoE.sup.-/- mice. Graphs show intravenous injection of human
recombinant clusterin analog, CluAg-I, for 3 months reducing both
systolic and blood pressure in ApoE.sup.-/- atherosclerosis-prone
mice but has no effect on that in age (6-8 months old) and sex
(male)-matched normal wild type (WT) control mice.
[0043] FIG. 12--Echocardiography of normal wild type (WT) and
ApoE-/- mice with CluAg-I injection. WT and ApoE.sup.-/- mice were
injected intravenously with CluAg for 3 months and then subjected
to ultrasound examination using the Visualsonics.TM. echo
device.
[0044] FIG. 13--Left ventricle ejection fraction comparison of WT
mice and ApoE.sup.-/- mice. Intravenous injection of human
recombinant clusterin analog, CluAg-I, for 3 months reduces blood
pressure in ApoE.sup.-/- atherosclerosis-prone mice but not in age
(6-8 months old) and sex (male)-matched normal wild type (WT)
control mice.
[0045] FIGS. 14A-14D--Oil Red 0 staining of WT and ApoE.sup.-/-
aortas, evidencing that weekly intravenous injection of human
recombinant clusterin analog, CluAg-I, for 3 months reduces plaque
sizes in ApoE.sup.-/- atherosclerosis-prone mice, but not in age
(6-8 months old) and sex (male)-matched normal wild type (WT)
control mice. Reduced Oil red 0 staining in ApoE-/- mice treated
with CluAg is observed (FIG. 14D).
[0046] FIG. 15--Alizarin Red staining of murine vascular smooth
muscle cells evidence that CluAg reduces phosphate-induced
calcification of ApoE.sup.-/- SMCs in a matter dependent upon
expression of ApoER2 and VLDLR. SMCs treated with ApoER2 or VLDLR
shRNA were incubated with Pi (3.6 mmol/L)+/-CluAg in DMEM
containing 5% FBS for 6 days. At the end of culture cells were
washed in PBS, fixed in 10% formalin, and stained in 2% Alizarin
Red at 37.degree. C. for 10 min. Reduced Alizarin Red staining
could be partly blocked by ApoER2 and VLDLR shRNA.
[0047] FIGS. 16A-16C--Graphs show that CluAg treatment reduces
phosphate-induced (Pi) calcification in ApoE.sup.-/- vascular
smooth muscle cells (SMCs) in a manner dependent upon expression of
ApoER2 and VLDLR. SMCs treated with ApoER2 or VLDLR shRNA were
incubated with Pi (3.6 mmol/L) in the presence or absence of CluAg
(6 .mu.g/ml) in DMEM containing 5% FBS for 6 days. In the end of
culture cells were washed in PBS, incubated in 0.6M HCl overnight.
Cell lysates were mixed with 0.1N NaOH was used to lysis cells and
concentrations of proteins were measured. Supernatants were
collected, and calcium contents measured using a calcium assay;
were mixed with 90 .mu.l of the Chromogenic Reagent, and 60 .mu.l
of the Calcium Assay Buffer, mix gently; Incubated the reaction for
5-10 mins at room temperature, protected from light; Measured OD at
575 nm. 16A, SMCs treated with negative control ShRNA; 16B, with
ApoER2 ShRNA; and 16C, with VLDLR ShRNA. Data represent
means+/-S.D., **, p<0.01 and #p<0.05.
[0048] FIGS. 17A-17D--Alizarin Red staining showing that weekly
intravenous injection of human recombinant clusterin analog, CluAg
for 3 months inhibits atherosclerosis as well as calcification in
ApoE.sup.-/- atherosclerosis-prone mice. 17A shows wild type (WT)
control mice. Aortas were stained in 2% Alizarin Red at 37.degree.
C. for 10 min. Images were taken using .times.4 objective for 17A
and 17B, and .times.10 objective for 17C and 17D. Reduced plaque
size and Alizarin staining in ApoE-/- mice treated with CluAg was
observed.
[0049] FIGS. 18A-18H--Immunofluorescence of bone morphogenic
protein-2 (BMP2) in aortas of wild type (WT) mice and ApoE-/- mice
treated with or without CluAg. Again, results show that CluAg
treatment is able to reduce BMP2 expression in ApoE-/- mice.
[0050] FIGS. 19A-19B--Calcium deposit in smooth muscle cells (SMC)
isolated from aorta of WT and ApoE-null mice. ApoE-/- VSMCs are
more prone to Pi-induced calcification. Alizarin red staining (19A)
and calcium assay (19B) of VSMCs isolated from aorta of WT and
ApoE-/- mice. Scale bar=200 .mu.m. VSMCs from WT and ApoE-/- mice
were treated with inorganic phosphate (Pi) with or without Apo J (6
.mu.g/mL). Bars represent means.+-.SD, n=5, *P<0.05 vs. control;
**P<0.01 vs. control; #P<0.05 vs. Pi group; ##P<0.01 vs.
Pi group.
[0051] FIGS. 20A-20B--Calcium deposit in SMC treating with or
without Apo J. Inhibitory effect of Apo J on calcification in
ApoE-/- VSMCs is correlated to concentration of Apo J. a: Alizarin
S staining of VSMCs from ApoE-/- mice. VSMCs were treated with
different concentrations of Apo J (0-12 .mu.g/mL) during
calcification. Scale bar=200 .mu.m. b: Calcium assay of ApoE-/-
VSMCs treated with various concentrations of Apo J (0-12 .mu.g/mL)
and Pi. Bars represent means.+-.SD; n=5 per group. **P<0.01 vs.
control; ##P<0.01 vs. Pi group.
[0052] FIGS. 21A-21D--Apo J modulates the protein expression of
smooth muscle lineage-specific markers and calcification-related
genes. VSMCs were treated with Pi and Apo J (0-12 .mu.g/mL). 21A:
40 .mu.L cell culture medium was used to detect the level of Apo J
in culture medium. Levels of SM22.alpha., .alpha.SMA and Runx2
protein expressions were assessed by western blot. GAPDH was used
as the loading control. 21B-21D: Densitometry analysis shows the
quantification of SM22.alpha., .alpha.SMA and Runx2. Bars represent
means.+-.SD; n=5 per group. *P<0.05 vs. control; **P<0.01 vs.
control; #P<0.05 vs. Pi group; ##P<0.01 vs. Pi group.
[0053] FIGS. 22A-22B--Expression of Apo J during calcification.
22A: 40 .mu.l cell culture medium was used to detect the expression
of Apo J by Western blot. 22B: Apo J mRNA expression was detected
by qRT-PCR. GAPDH was used as an internal control. Bars represent
means.+-.SD; n=3 per group. *P<0.05 vs. control; **P<0.01 vs.
control.
[0054] FIGS. 23A-23D--Apo J affects osteogenetic markers on mRNA
levels during calcification. VSMCs were treated with Pi in
combination with Apo J (0-12 .mu.g/mL). Runx2 (23A), BMP-2 (23B),
ALP (23C), and OPN (23D) mRNA expressions were quantified by
qRT-PCR and normalized to GAPDH as an internal control. Bars
represent means.+-.SD; n=5 per group. **P<0.01 vs. control;
#P<0.05 vs. Pi group; ##P<0.01 vs. Pi group.
[0055] FIGS. 24A-24F--Lentiviral shRNA knockdown of ApoER2 and
VLDLR receptors. 24A-24B: ApoE-/- cells were transfected with
ApoER2 shRNA, VLDLR shRNA or negative control shRNA to collect
whole-cell lysates for western blot analysis of ApoER2 or VLDLR.
24C-24D: Densitometry analysis shows the quantification of ApoER2
or VLDLR expression. 24E-24F: Bar graph illustrating real-time PCR
data demonstrating that ApoER2 or VLDLR was successfully knocked
down on mRNA level. Relative mRNA expressions were normalized to
GAPDH as an internal control. Bars represent means.+-.SD; n=3 per
group. *P<0.05 vs. control, **P<0.01 vs. control; #P<0.05
vs. Pi group, ##P<0.01 vs. Pi group.
[0056] FIGS. 25A-25B--Knockdown of ApoER2 or VLDLR gene abolishes
the inhibitory effect of Apo J on calcification. Alizarin red
staining (25A) and calcium assay (25B) of receptor knockdown VSMCs
under the treatment of Pi with or without Apo J (6 .mu.g/mL). Scale
bar=200 .mu.m. Bars represent means.+-.SD; n=3 per group.
**P<0.01 vs. control; ##P<0.01 vs. Pi group.
[0057] FIGS. 26A-26G--Knockdown of ApoER2 gene abolishes the effect
of Apo J on calcification markers and smooth muscle
lineage-specific markers. ApoE-/- VSMCs were treated with Pi and
Apo J (6 .mu.g/mL). 26A: SM22.alpha. and Runx2 protein expressions
in ApoER2 knockdown cells were detected by western blot. GAPDH was
used as the loading control. 26A-C: Densitometry analysis shows the
quantification of SM22.alpha. and Runx2 in ApoER2 knockdown cells.
26D-G: Bar graph illustrating real-time PCR data showing the mRNA
expression of Runx2, BMP-2, ALP and OPN. Relative mRNA expressions
were normalized to GAPDH as an internal control. Bars represent
means.+-.SD; n=3 per group. +P<0.05 means negative control shRNA
infected cells vs. ApoER2 shRNA infected cells, ++P<0.01;
*P<0.05 vs. control, **P<0.01 vs. control; #P<0.05 vs. Pi
group, ##P<0.01 vs. Pi group.
[0058] FIGS. 27A-27G--Knockdown of VLDLR gene abolishes the effect
of Apo J on calcification markers and smooth muscle
lineage-specific markers. ApoE-/- VSMCs were treated with Pi and
Apo J (6 .mu.g/mL). 27A: SM22.alpha. and Runx2 protein expressions
in VLDLR knockdown cells were detected by western blot. GAPDH was
used as the loading control. 27B-C: Densitometry analysis shows the
quantification of SM22.alpha. and Runx2 in VLDLR knockdown cells.
27D-G: Bar graph illustrating real-time PCR data showing the mRNA
expression of Runx2, BMP-2, ALP and OPN. Bars represent
means.+-.SD; n=3 per group. +P<0.05 means negative control shRNA
infected cells vs. VLDLR shRNA infected cells, ++P<0.01;
*P<0.05 vs. control, **P<0.01 vs. control; #P<0.05 vs. Pi
group, ##P<0.01 vs. Pi group.
[0059] FIGS. 28a-28d--ApoJ mutants with a peptide comprising a
thrombin-specific cleavage site exert an anti-coagulational effect
by competitively inhibiting the conversion of fibrinogen into time
of pig blood with (A) or without (B) ApoJ mutant. Fresh blood (0.3
ml) from adult pig at different dilutions (0, 1/2, and 1/4 in PBS)
was mixed with recombinant ApoJ mutant at 10 or 10 mg/ml),
incubated at 37.degree. C., and then subjected to rotational
thromboelastometry (ROTEM.TM.). a, clotting time; b, Alpha Angle
clotting rapidity; c, maximum clot firmness; and d, maximum clot
firmness at 10 minutes. Data represented from average of two
separate experiments with the ApoJ mutant, Clusterin-ATMD-TRHis
(SEQ ID NO: 8).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0060] Clusterin is a multifunctional protein that may play an
important role in regulation of survival, proliferation and
differentiation of a diversity of cell types in a mammal. The
present disclosure provides recombinant clusterin polypeptides and
demonstrates the therapeutic efficacy of such recombinant
polypeptides. In particular, data presented herein demonstrates
that recombinant clusterin can be effectively synthesized and
purified and that the clusterin polypeptide preparations are highly
stable. The recombinant polypeptides appear to be essentially
non-toxic, when administered to animals. However, in murine models
for cardiovascular disease the clusterin polypeptides are able to
reduce markers of cardiovascular disease such as hyperglycemia,
hypertension and calcification as well as to normalize serum lipid
levels. These data indicate that the recombinant clusterin
polypeptides of the embodiments (and cells and nucleic acids
encoding the same) could be used as safe and effective therapeutics
for treatment and prevention of cardiovascular diseases and
alcoholism.
I. RECOMBINANT CLUSTERIN POLYPEPTIDES
[0061] For the purposes of this disclosure, the terms "clusterin"
or "recombinant clusterin" refers to proteins, whose sequence is
based on a mammalian clusterin sequence. In preferred aspects a
recombinant clusterin polypeptide comprises a deletion of a nuclear
localization signal, a transmembrane domain and/or is fused with a
heterologous polypeptide sequence (e.g., a purification tag). A
skilled artisan will recognize that deletions of the clusterin TMD
likewise can disrupt the endoplasmic reticulum (ER)-targeting of
recombinant clusterin. The terms "ApoJ" and "Clusterin" as used
interchangeably herein. Examples of specific clusterin polypeptides
include, without limitation, polypeptides provided as NCBI Acc. No.
NM_203339, NM_001831, NM_013492, NM_053021, NM_012679, each of
which is incorporated herein by reference. In certain aspects, the
recombinant clusterin is about or at least about 90%, 91% 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to the clusterin
polypeptide sequence of SEQ ID NOs: 1-3. For examples, in some
aspects, the recombinant clusterin polypeptide about or at least
about 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to SEQ ID NO:1, but comprises a deletion of a nuclear localization
signal, ER-targeting sequence and/or a transmembrane domain. In yet
further aspects, the recombinant clusterin is about or at least
about 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical
to the clusterin polypeptide sequence of SEQ ID NOs: 4-15.
[0062] Clusterin polypeptides and fragments, mutated, truncated or
deleted forms of the clusterin and/or clusterin fusion proteins can
be prepared for a variety of uses, including but not limited to the
generation of antibodies, as reagents in diagnostic assays, as
reagents in assays for the identification of other cellular gene
products involved in the regulation of clusterin mediated
disorders, as reagents in assays for screening for compounds that
can be used in the treatment of clusterin mediated disorders, and
as pharmaceutical reagents, useful in the treatment of disorders
related to clusterin.
[0063] Embodiments of the present invention also encompasses
proteins that are functionally equivalent to the clusterin encoded
by the nucleotide sequences described, as judged by any of a number
of criteria, including but not limited to resulting in the
biological effect of clusterin, a change in phenotype when the
clusterin equivalent is present in an appropriate cell type. Such
functionally equivalent clusterin proteins include but are not
limited to additions or substitutions of amino acid residues within
the amino acid sequence encoded by the clusterin nucleotide
sequences described, but which result in a silent change, thus
producing a functionally equivalent gene product.
[0064] In additional aspects, clusterin polypeptides may be further
modified by one or more other amino substitutions while maintaining
their biological activity. For example, amino acid substitutions
can be made at one or more positions wherein the substitution is
for an amino acid having a similar hydrophilicity. The importance
of the hydropathic amino acid index in conferring interactive
biologic function on a protein is generally understood in the art
(Kyte and Doolittle, 1982). It is accepted that the relative
hydropathic character of the amino acid contributes to the
secondary structure of the resultant protein, which in turn defines
the interaction of the protein with other molecules, for example,
enzymes, substrates, receptors, DNA, antibodies, antigens, and the
like. Thus such conservative substitution can be made in GrB and
will likely only have minor effects on their activity. As detailed
in U.S. Pat. No. 4,554,101, the following hydrophilicity values
have been assigned to amino acid residues: arginine (+3.0); lysine
(+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine
(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine
(-0.4); proline (-0.5.+-.1); alanine (0.5); histidine -0.5);
cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5);
tryptophan (-3.4). These values can be used as a guide and thus
substitution of amino acids whose hydrophilicity values are within
.+-.2 are preferred, those that are within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred. Thus, any of the GrB polypeptides described herein may
be modified by the substitution of an amino acid, for different,
but homologous amino acid with a similar hydrophilicity value.
Amino acids with hydrophilicities within +/-1.0, or +/-0.5 points
are considered homologous. Furthermore, it is envisioned that
clusterin sequences may be modified by amino acid deletions,
substitutions, additions or insertions while retaining its
biological activity.
[0065] In some aspects, a clusterin polypeptide is fused with a
heterologous polypeptide sequence. For example, heterologous
polypeptide sequences may be included to aid production or
purification of a cell targeting construct. Some specific examples
of amino acid sequences that may be attached to clusterin include,
but are not limited to, purification tags (e.g., a T7, MBP. GST,
HA, or polyHis tag), proteolytic cleavage sites, such as a thrombin
or furin cleavage site, intracellular localization signals or
secretion signals. In some aspects, a clusterin further comprises a
cell-penetrating peptide (CPP). As used herein the terms CPP and
membrane translocation peptide (MTP) as used interchangeably to
refer to peptide sequences that enhance the ability of a protein to
be internalized by a cell. Examples for CPPs for use according to
the embodiments include, without limitation, peptide segments
derived from HIV Tat, herpes virus VP22, the Drosophila
Antennapedia homeobox gene product, protegrin I, as well as the T1,
T2, and INF7 peptides.
[0066] Other mutations to the coding sequences described above can
be made to generate polypeptides that are better suited for
expression, scale up, etc. in the host cells chosen. For example,
the triplet code for each amino acid can be modified to conform
more closely to the preferential codon usage of the host cell's
translational machinery, or, for example, to yield a messenger RNA
molecule with a longer half-life. Those skilled in the art would
readily know what modifications of the nucleotide sequence would be
desirable to conform the nucleotide sequence to preferential codon
usage or to make the messenger RNA more stable. Such information
would be obtainable, for example, through use of computer programs,
through review of available research data on codon usage and
messenger RNA stability, and through other means known to those of
skill in the art.
[0067] Polypeptides corresponding to one or more portions of
clusterin, truncated or deleted clusterin as well as fusion
proteins in which the full length clusterin or truncated clusterin
is fused to an unrelated protein are also within the scope of the
invention and can be designed on the basis of the clusterin
nucleotide and clusterin amino acid sequences disclosed above. Such
fusion proteins include but are not limited to IgFc fusions which
stabilize the clusterin polypeptide and prolong half-life in vivo
or in in vitro assays; fusions to any amino acid sequence that
allows the fusion protein to be anchored to the cell membrane; or
fusions to an enzyme, fluorescent protein, or luminescent protein
which provide a marker function.
[0068] Additionally, the clusterin gene can be subcloned into a
recombinant plasmid such that the gene's open reading frame is
translationally fused to an amino-terminal tag consisting of
multiple (generally about six) histidine residues. Extracts from
cells infected or transfected with such constructs are loaded onto
Ni.sup.2+ nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
[0069] While clusterin polypeptides can be chemically synthesized
(e.g., see Creighton, 1983), large polypeptides derived from the
clusterin and the full length clusterin itself may advantageously
be produced by recombinant DNA technology using techniques well
known in the art for expressing nucleic acids. Such methods can be
used to construct expression vectors containing the clusterin
nucleotide sequences and appropriate transcriptional and
translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination. See, for example, the techniques
described in Sambrook et al., 1989, supra, and Ausubel et al.,
1989, supra. Alternatively, RNA capable of encoding Clusterin
nucleotide sequences may be chemically synthesized using, for
example, synthesizers. See, for example, the techniques described
in "Oligonucleotide Synthesis", 1984, Gait, M. J., ed., IRL Press,
Oxford, which is incorporated by reference herein in its
entirety.
[0070] A variety of host-expression vector systems can be utilized
to express the clusterin nucleotide sequences of embodiments of the
invention. Where clusterin polypeptide is a soluble derivative
(e.g., with a deleted TMD), the polypeptide can be recovered from
the culture, i.e., from the host cell in cases where clusterin
polypeptide is not secreted, and from the culture media in cases
where clusterin polypeptide is secreted by the cells. However, the
expression systems also encompass engineered host cells that
express clusterin or functional equivalents in situ, i.e., anchored
in the cell membrane. Purification or enrichment of clusterin from
such expression systems can be accomplished using appropriate
detergents and lipid micelles and methods well known to those
skilled in the art. However, such engineered host cells themselves
can be used in situations where it is important not only to retain
the structural and functional characteristics of clusterin, but to
assess biological activity, e.g., in drug screening assays.
[0071] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express clusterin sequences can be engineered, for
example, as described in SEQ ID NOs: 4-15 and in the examples
below. Rather than using expression vectors which contain viral
origins of replication, host cells can be transformed with DNA
controlled by appropriate expression control elements (e.g.,
promoter, enhancer sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the Clusterin gene product. Such engineered
cell lines may be particularly useful in screening and evaluation
of compounds that affect the endogenous activity of the Clusterin
gene product. A number of selection systems can be used, including
but not limited to the herpes simplex virus thymidine kinase
(Wigler, et al., 1977), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962), and
adenine phosphoribosyltransferase (Lowy, et al., 1980) genes can be
employed in tk-, hgprt- or aprt-cells, respectively. Also,
anti-metabolite resistance can be used as the basis of selection
for the following genes: dhfr, which confers resistance to
methotrexate (Wigler, et al., 1980; O'Hare, et al., 1981); gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg,
1981); neo, which confers resistance to the aminoglycoside G-418
(Colberre-Garapin, et al., 1981); and hygro, which confers
resistance to hygromycin (Santerre, et al., 1984).
[0072] The expression systems that can be used for purposes of the
embodiments include but are not limited to microorganisms such as
bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing clusterin nucleotide sequences; yeast (e.g.,
Saccharomyces, Pichia) transformed with recombinant yeast
expression vectors containing the clusterin nucleotide sequences;
insect cell systems infected with recombinant virus expression
vectors (e.g., baculovirus) containing the Clusterin sequences;
plant cell systems infected with recombinant virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or transformed with recombinant plasmid expression
vectors (e.g., Ti plasmid) containing Clusterin nucleotide
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293,
3T3) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
[0073] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
clusterin gene product being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of clusterin protein or for raising
antibodies to the clusterin protein, for example, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., 1983), in which the clusterin coding sequence may
be ligated individually into the vector in frame with the lacZ
coding region so that a fusion protein is produced; pIN vectors
(Inouye & Inouye, 1985; Van Heeke & Schuster, 1989); and
the like. pGEX vectors 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 glutathione-agarose
beads followed by elution in the presence of free glutathione. The
pGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0074] In an insect system, Autographa californica nuclear
polyhidrosis virus (AcNPV) is used as a vector to express foreign
sequences. The virus grows in Spodoptera frugiperda cells. The
clusterin gene coding sequence may be cloned individually into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter). Successful insertion of clusterin coding
sequence will result in inactivation of the polyhedrin gene and
production of non-occluded recombinant virus, (i.e., virus lacking
the proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted polynucleotide is expressed (e.g., see
Smith et al., 1983 and U.S. Pat. No. 4,215,051).
[0075] In mammalian host cells, a number of viral-based expression
systems can be utilized. In cases where an adenovirus is used as an
expression vector, the clusterin nucleotide sequence of interest
may be ligated to an adenovirus transcription/translation control
complex, e.g., the late promoter and tripartite leader sequence.
This chimeric gene may then be inserted in the adenovirus genome by
in vitro or in vivo recombination. Insertion in a non-essential
region of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
clusterin gene product in infected hosts (e.g., See Logan &
Shenk, 1984). Specific initiation signals may also be important for
efficient translation of inserted clusterin nucleotide sequences.
These signals include the ATG initiation codon and adjacent
sequences. In cases where an entire clusterin gene or cDNA,
including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where only a portion of the clusterin coding sequence is inserted,
exogenous translational control signals, including, perhaps, the
ATG initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (See Bitter, et al., 1987).
[0076] In yeast, a number of vectors containing constitutive or
inducible promoters may be used. For a review, see Current
Protocols in Molecular Biology, 1988; Grant, et al., 1987; Wu &
Grossman, 1987; Bitter, 1987; and "The Molecular Biology of the
Yeast Saccharomyces", 1982.
[0077] In cases where plant expression vectors are used, the
expression of the coding sequence may be driven by any of a number
of promoters. For example, viral promoters such as the 35S RNA and
19S RNA promoters of CaMV (Brisson et al., 1984), or the coat
protein promoter of TMV (Takamatsu et al., 1987) may be used;
alternatively, plant promoters such as the small subunit of RUBISCO
(Coruzzi et al., 1984; Broglie et al., 1984); or heat shock
promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al.,
1986) may be used. These constructs can be introduced into plant
cells using Ti plasmids, Ri plasmids, plant virus vectors, direct
DNA transformation, microinjection, electroporation, etc. For
reviews of such techniques see, for example, Methods for Plant
Molecular Biology 1988; and Grierson & Corey, 1988.
[0078] In cases where an adenovirus is used as an expression
vector, the nucleotide sequence of interest can be ligated to an
adenovirus transcription/translation control complex, e.g., the
late promoter and tripartite leader sequence. This chimeric gene
cam then be inserted in the adenovirus genome by in vitro or in
vivo recombination. Insertion in a non-essential region of the
viral genome (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing the gene product of
interest in infected hosts (e.g., See Logan & Shenk, 1984).
Specific initiation signals such as those described above can also
be important for efficient translation of inserted nucleotide
sequences of interest.
[0079] In addition, a host cell strain can be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products can be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product can be used. Such mammalian
host cells include but are not limited to CHO, VERO, BHK, HeLa,
COS, MDCK, 293, 3T3, WI38 and U937 cells.
II. THERAPEUTIC FORMULATION AND ADMINISTRATION
[0080] Therapeutics comprising clusterin polypeptides and/or
nucleic acids encoding clusterin polypeptides, such as those
described in SEQ ID NOs: 4-15 can be administered to a patient at
therapeutically effective doses of pharmaceutical preparations used
to treat or ameliorate conditions such as, but not limited to,
cardiovascular disease may be hypertension, hyperlipidemia,
hypercholesterolemia, hyperglycemia, hypertension, atherosclerosis
and atherosclerosis-associated ischemic heart failure, stenosis,
calcification of cardiovascular tissues, stroke, myocardial
infarction or cerebral infarction. In some aspects, the
cardiovascular disease is hyperlipidemia, hypercholesterolemia or
atherosclerosis. In still further aspects, the cardiovascular
disease may be diabetes. In various aspects, an effective amount of
a clusterin composition may be an amount effective to reduce blood
cholesterol, reduce blood glucose, reduce blood triglyceride,
increase efflux of intracellular cholesterol, and/or increase
vascular or cardiac cell survival. In some aspects an effective
amount of a clusterin composition provides enhancement or promotion
of cell survival and growth against cytotoxic or cytostatic
factors, including but not limited to, oxysterols, oxidized
lipoproteins, and proinflammatory cytokines, for examples those
induced by alcoholism. In further aspects a therapeutically
effective dose refers to that amount of the compound sufficient to
result in any amelioration or retardation of disease symptoms or
progression in a mammal with acute vascular syndromes, to prevent
and treat degeneration, stenosis and calcification of
cardiovasulcar tissues, comprising valve tissues. In further
aspects, clusterin derived polypeptides can be used to prevent or
treat male infertility by, for example, supporting sperm maturation
and development, as has been described for native clusterin.
[0081] Toxicity and therapeutic efficacy of such clusterin derived
compositions can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Compositions which
exhibit large therapeutic indices are preferred. While compositions
that exhibit toxic side effects may be used, care should be taken
to design a delivery system that targets such compounds to the site
of affected tissue in order to minimize potential damage to
uninfected cells and, thereby, reduce side effects.
[0082] Data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compositions are preferably within a
range of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compositions used in the methods
of the embodiments, the therapeutically effective dose can be
estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range that includes the IC.sub.50 (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0083] When the therapeutic treatment of disease is contemplated,
the appropriate dosage can also be determined using animal studies
to determine the maximal tolerable dose, or MTD, of a bioactive
agent per kilogram weight of the test subject. In general, at least
one animal species tested is mammalian. Those skilled in the art
regularly extrapolate doses for efficacy and avoiding toxicity to
other species, including human. Before human studies of efficacy
are undertaken, Phase I clinical studies in normal subjects help
establish safe doses.
[0084] Additionally, the bioactive agent (e.g., a clusterin
polypeptide) may be complexed with a variety of well-established
compounds or structures that, for instance, enhance the stability
of the bioactive agent, or otherwise enhance its pharmacological
properties (e.g., increase in vivo half-life, reduce toxicity,
etc.).
[0085] Pharmaceutical compositions for use in accordance with the
present embodiments can be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
[0086] The above therapeutic agents will be administered by any
number of methods known to those of ordinary skill in the art
including, but not limited to, administration by inhalation; by
subcutaneous (sub-q), intravenous (I.V.), intraperitoneal (I.P.),
intramuscular (I.M.), or intrathecal injection; or as a topically
applied agent (transderm, ointments, creams, salves, eye drops, and
the like). Thus, the compositions can be formulated for
administration by inhalation or insufflation (either through the
mouth or the nose) or oral, buccal, parenteral or rectal
administration.
[0087] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets can be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate. Preparations for oral administration can be
suitably formulated to give controlled release of the active
composition.
[0088] For buccal administration the compositions can take the form
of tablets or lozenges formulated in conventional manner.
[0089] For administration by inhalation, the compositions for use
according to the embodiments are conveniently delivered in the form
of an aerosol spray presentation from pressurized packs or a
nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0090] The compositions can be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0091] The compositions can also be formulated for rectal
administration such as suppositories or retention enemas, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0092] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt. The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
III. EXAMPLES
[0093] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1--General Methods and Results
[0094] Preparation of native and recombinant clusterin analogs. To
treat stem cells with clusterin, two forms of clusterin were
prepared. Native clusterin (NCBI Accession No. NM_0134921,
incorporated herein by reference). Clusterin was prepared from
blood plasma using affinity chromatography with anti-clusterin
antibody. In addition, a plasmid was constructed in which mouse or
human clusterin cDNA is fused with a His-tag and inserted under a
promoter that will be activated in a mammalian (e.g., human cells)
or non-mammalian (e.g., bacteria) cell. Schematic presentation of
the plasmid pCluAg with a clusterin cDNA-His insert is shown in
FIG. 1. pCluAg was constructed by inserting clusterin-TR/EK-His
cDNA using a TOPO plasmid (Invitrogen). Recombinant clusterin
produced by transfected mammalian cells (e.g., human 293 cells) or
bacterial cells (E. coli) was purified. Different clusterin analog
cDNAs were generated by RT-PCR and sequences predicting the
encoding of different clusterin analog polypeptide sequences (FIGS.
2A-D and Table 1, below).
TABLE-US-00001 TABLE 1 Sequence Descriptions SEQ ID Description:
NO: CluAg-a (recombinant Clusterin) 1 CluAg-b (recombinant
Clusterin-.DELTA.TMD) 2 CluAg-c (recombinant
Clusterin-.DELTA.TMD-.DELTA.NLS) 3 CluAg-Ia (HisTR-Clusterin) 4
CluAg-Ib (HisTR-Clusterin-.DELTA.TMD) 5 CluAg-Ic
(HisTR-Clusterin-.DELTA.TMD-.DELTA.NLS) 6 CluAg-IIa
(Clusterin-TR-His) 7 CluAg-IIb (Clusterin-TR-His-.DELTA.TMD) 8
CluAg-IIc (Clusterin-TR-His-.DELTA.TMD-.DELTA.NLS) 9 CluAg-IIIa
(HisEK-Clusterin) 10 CluAg-IIIb (HisEK-Clusterin-.DELTA.TMD) 11
CluAg-IIIc (HisEK-Clusterin-.DELTA.TMD-.DELTA.NLS) 12 CluAg-IVa
(HisEK-Clusterin) 13 CluAg-IVb (HisEK-Clusterin-.DELTA.TMD) 14
CluAg-IVc (HisEK-Clusterin-.DELTA.TMD-.DELTA.NLS) 15
[0095] Analysis of recombinant clusterin analog protein properties.
The purity of recombinant clusterin analogs was examined by
polyacrylamide gel electrophoresis. Clusterin analog proteins are
isolated from pCluAg transfected cells, stored in solution or in
dry powder, and loaded into SDS-PAGE. After electrophoresis,
fractions of proteins will be electrotransferred onto a PVDF
membrane, and probed with anti-clusterin antibodies. The membrane
will be developed by using a chemiluminescence kit. Single bands of
clusterin analog CluAg-I are visualized in SDS-PAGE stained with
Commassia blue dye G250 (FIG. 3) and confirmed by immunoblotting
with anti-Clu antibody (FIG. 4). Furthermore, the analogs were
examined by ion exchange chromatography, showing a clear eluent of
clusterin analogs (FIG. 5).
[0096] Clusterin analogs in the HDL fraction in mice.
Apolipoprotein E deficient (ApoE.sup.-/-) mice are widely used
murine mode for atherosclerosis. To evaluate the levels of
clusterin association with ApoAI in the blood, an
immunoprecipitation method was developed with monoclonal antibody
against apoAI and clusterin. In the plasma of blood from CluAg
injected mice, ApoAI antibody co-precipitates ApoAI and CluAg,
indicating that ApoAI is binding to CluAg (FIG. 6). To further
confirm the presence of CluAg and compare them to native clusterin,
CluAg was added to SMC culture and incubated for 2 days with 5%
serum containing native {00939480}-29-clusterin in the presence of
oxLDL. Supernatants of the cultures were subjected to analysis of
poly-His tag in CluAg. In the culture with or without oxLDL, CluAg
apparently are free of the poly-His tag while CluAg (FIG. 7) in
cell free cultures contains the poly-His tag (FIG. 7).
[0097] Clusterin analog regulation of vascular cell proliferation
in culture. An in vitro system was first employed to examine the
protective effects of clusterin analogs on survival and growth of
vascular cells. Cells were treated with clusterin analogs at
different concentrations in the cultures with or without oxidized
LDL. After 2-4 days of stimulation, cell survival and proliferation
were examined using a combination of techniques including flow
cytometry, fluorescent microscopy, and radioactive isotope
labeling, as described below. Control experiments were set up using
other types of proteins, such as bovine albumin. FIGS. 8 and 9 show
growth curves of SMCs treated with or without CluAg (1-6 .mu.g/nl)
in the presence or absence of oxLDL (50 .mu.g/ml). Treatment with
the analog increased SMC proliferation even under the condition of
oxLDL exposure. CluAg dose-dependently increases SMC proliferation
even in the presence of oxLDL (FIGS. 8 and 9).
[0098] Analysis of lipid profiles, cholesterol, glucose, and
triglyceride in wild type and atherosclerosis prone mice injected
with clusterin. Weekly injection of CluAg (30-40 .mu.g) for 3
months was conducted in wild type (WT) and apoE-null mice. The
blood samples were collected, during tail DNA sampling, from the
mice injected with CluAg. Serum was prepared from the blood
samples. Cholesterol levels, lipoprotein profiles and clusterin
concentrations were determined respectively. In brief, serum
diluted in PBS was incubated in a 96 well plate coated with a
rabbit polyclonal antibody to clusterin. After incubation and
washing in PBS, bound clusterin was detected by incubating with
mouse monoclonal antibody to clusterin. Goat anti-mouse IgG
conjugated with peroxidase was used as the second antibody.
Cholesterol and HDL was determined in the laboratory of Department
of Laboratory Medicine. The ratio of clusterin vs. HDL was
calculated after normalization with the lipid content. In addition
to ELISA, immunoblotting assays were performed to verify the
results. CluAg injection significantly reduced blood levels of
total cholesterol, glucose, triglyceride, and LDL in apoE-null mice
but no changes were found in WT mice (FIG. 10).
[0099] Analysis of blood pressure. Blood pressure measurement was
performed in the tail artery using a tail-cuff method. Each
measurement was repeated 3 times to ensure reproducibility. Weekly
injection of CluAg (30-40 .mu.g) for 3 months led to reduction in
the tail arterial blood pressure of both systolic and diastolic
phases was conducted in apoE-null mice (FIG. 11, left panel) but no
changes in blood pressure were found in WT mice (FIG. 11, right
panel).
[0100] Echocardiography of mice injected with CluAg. After weekly
injection of CluAg, morphological and functional changes were
monitored using echocardiography. B- and M-Mode echocardiography
was performed one, 6, 10, and 18 weeks after injection. The
echocardiography studies were conducted actually using
ultrasonography as the murine heart is small. Mice were
anesthetized with ketamine and xylazine, chests shaved and a layer
of acoustic coupling gel will be applied to the thorax. A
dynamically focused 9-MHz annual array transducer was applied using
a warmed saline bag as a standoff. All echo studies were performed
using a state of the art echo machine (HP Model Sonos 5500 HP).
Area fractions were determined by planimetry of diastolic and
systolic volumes in parasternal short axis. The LV end-diastolic
and end-systolic dimensions were measured using the M-Mode from
>3 beats by two independent investigators blinded to the
research animals. LVEF (left ventricular ejection fraction) was
calculated as follows: LVEF=[(LVIDd)-(LVIDs)]/(LVIDd), LVIDd:
end-diastolic left ventricular internal diameter; LVIDs:
end-systolic left ventricular diameter. FIG. 12 shows ultrasound
images of murine hearts with or without CluAg injection in both 2D
and M-mode. FIG. 13 demonstrates that weekly administration of
CluAg (30-40 .mu.g/mouse) increases ejection fraction in the hearts
of ApoE-/- atherosclerosis-prone mice but no changes in wild type
mice with the same dose CluAg.
[0101] Oil red O staining of aortic wall. Mice were sacrificed 3
months after CluAg treatment. Aortas were opened across the long
axis and fixed in 10% buffered formalin for histological
evaluation. Aortas were stained with Oil Red 0 solution for
assessing neutral lipid contents. Little staining was detected in
WT aortas. However, compared to WT aortas, the ApoE-null aortas
were stained very intensively with Oil Red 0, which visualizes
atherosclerotic plaques. Decreased Oil Red 0 stains were found in
ApoE-null mice injected with CluAg protein (FIG. 14).
[0102] Analysis of aortic tissue and cell calcification. Aortic
smooth muscle cells (SMCs) were incubated with CluAg in the
presence of sodium phosphate (3.6 mmol/L) for 6 days. Alizarin red
S (Sigma, St. Louis, Mo., USA) staining assesses calcium deposition
in a reaction in which Alizarin red S dye binds with Ca ions in
cell layer matrix. Cells were fixed with 2% paraformaldehyde and
stained with 1% Alizarin red S (pH 4.2). The culture plates were
photographed under a light microscope and assessed for the
mineralized nodules which shown as red (FIG. 15). CluAg treatment
markedly reduces calcification in SMCs. To determine if clusterin
receptors mediate the inhibitory effect of CluAg on phosphate
induced calcification in SMCs, snRNA for ApoER2 and VLDLR, two
known receptors for clusterin, was constructed and used to knock
down ApoER2 and VLDLR. CluAg treatment had no inhibitory effect on
calcification in SMCs with the receptor knockdown with the snRNA
(FIG. 15). This result was confirmed by direct calcium assays
(FIGS. 16A-C). For assessing in vivo calcification of aortic
tissue, Alizarin red S staining was performed in the aortas of WT
and apoE-null mice with CluAg injection. Sections of aortas from
apoE-null mice but not wild type control mice show the development
of atherosclerotic plaques. Calcium deposits were highly abundant
in ApoE-null mice. Treatment with CluAg reduces calcification in
ApoE-null mice (FIGS. 18A-H).
[0103] Immunohistochemistry of aortic tissue. In order to assess
whether CluAg injection alters expression of
calcification-regulatory proteins, such as bone morphogenic protein
(BMP)-2, in the aortic tissue, aortic sections were stained with
anti-BMP-2 antibodies. Immunofluorescence for BMP-2 was developed
with rhodamine-conjugated second antibodies (Sigma, St. Louis,
Mo.). The slides were mounted in the Vectashield mounting medium
with 4',6 diamidino-2-phenylindole (DAPI) (Vector, Burlingame,
Calif.), and examined under an Olympus fluorescence microscope.
Intensive BMP-2 immunofluorescence was found in ApoE-null aorta but
CluAg injection reduced the intensity of the BMP-2
fluorescence.
Example 2--Further Analysis of Clusterin In Vivo Methods and
Materials
[0104] Cell culture. Aortic VSMCs were isolated from 5- to
6-week-old male ApoE-/- or WT mice with C57BL/6 background. The
cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM;
Invitrogen, Carlsbad, Calif., USA) supplemented with 10% fetal
bovine serum (FBS) with 100 ng/mL penicillin and streptomycin
(Invitrogen) at 37.degree. C. in a humidified atmosphere with 5%
CO2. Calcification medium was made by adding NaH.sub.2PO.sup.4 (pH
adjusted to 7.4) into 5% FBS medium to obtain a final concentration
of 3.6 mM inorganic phosphate. VSMCs from passage 5-10 were used.
Apo J Medium was replaced every 2 or 3 days for up to 9 days. Cells
maintained in regular culture medium with 0.9 mM phosphate were
used as controls.
[0105] Lentivirus infection and selection. To generate a stable
(long-term) knockdown of ApoER2 or VLDLR gene expression in VSMCs,
the VSMCs were infected with lentivirus particles containing a
pLKO.1 vector with the information to express a shRNA against mouse
ApoER2 or VLDLR. This plasmid also has a puromycin resistance gene,
thereby allowing for the selection of cells stably expressing
desired shRNA by addition of puromycin into culture medium. The
optimal puromycin concentration for VSMCs before initiating the
experiments (titration assay) was determined to be 1 .mu.g/ml. To
perform lentivirus infection, cells approximately 80% confluent
were used. 1.5 ml of fresh culture media containing virus was added
onto cells. Fresh media was changed every 3 days 48 h after
infection. To select the infected cells, selection media containing
puromycin was used for culture 48 h after infection. ApoER2 and
VLDLR knockdown were monitored by western blot analysis and was
achieved after four passages. One plate of cells infected with
pLKO.1 vector with shRNA verified to contain no homology to known
mammalian genes was maintained in parallel. This plate served as a
negative control for following experiments.
[0106] Calcium determination. Calcium content in VSMCs was
determined by colorimetric calcium detection kit (Abcam). Cells
were washed with PBS and then incubated with 0.6M HCl under
37.degree. C. overnight to be decalcified. The calcium content in
the supernatants was measured using spectrometer. Then cells were
lysed with 0.1 mol/LNaOH/0.1% SDS. Protein content was determined
with BCA protein assay kit (Thermo Scientific) and calcium content
was normalized to total protein content.
[0107] Alizarin Red S staining. Cells were fixed in 2.5%
glutaraldehyde and incubated with 2% Alizarin Red S under
37.degree. C. for 15 minutes. Then VSMCs were rinsed with PBS three
times. Calcium mineralization visualized by red staining was
observed under microscope.
[0108] Real-time RT-PCR. Total RNA was extracted from VSMCs using
Trizol (Invitrogen). 4 .mu.g total RNA was used for cDNA synthesis
in a reaction mixture of 20 .mu.L with SuperScript III First-Strand
Synthesis SuperMix (Invitrogen). Real-time PCR amplication was
performed with IQ.TM. SYBR Green Supermix (Bio-Rad) in a
ICYCLERIQ.TM. thermocycler (Bio-Rad). The following primers sets
were used: ALP, 5'-CACAATATCAAGGATATCGACGTGA-3'(sense; SEQ ID NO:
18) and 5'-ACATCAGTTCTGTTCTTCGGGTACA-3'(antisense; SEQ ID NO: 19);
BMP-2, 5'-TTGTATGTGGACTTCAGTGATGTG-3'(sense; SEQ ID NO: 20) and
5'-AGTTCAGGTGGTCAGCAAGG-3' (antisense; SEQ ID NO: 21); Osteopontin,
5'-TGGCTATAGGATCTGGGTGC-3' (sense; SEQ ID NO: 22) and
5'-ATTTGCTTTTGCCTGTTTGG-3' (antisense; SEQ ID NO: 23); and Runx2,
5'-TTACCTACACCCCGCCAGTC-3' (sense; SEQ ID NO: 24) and
5'-TGCTGGTCTGGAAGGGTCC-3' (antisense; SEQ ID NO: 25).
[0109] Western blot. Proteins were isolated from VSMCs using RIPA
buffer (Pierce) containing protease and phosphatase inhibitors
(Sigma-Aldrich). Proteins separated on 10% SDS-Polyacrylamide gel
were transferred to polyvinylidenedifluoride (PVDF) membranes
(Millipore) and Western blot was performed using the standard
protocol. Membranes were blocked with 5% bovine serum albumin (BSA)
in TBS containing 0.1% Tween-20 (TBST). Primary antibodies were
diluted in 3% BSA [goat anti-ApoJ 1:1000 (Santa Cruz
Biotechnology), rabbit anti-Runx2 1:1000 (Cell Signaling), goat
anti-SM22.alpha. 1:5000 (Abcam), rabbit anti-.alpha.SMA 1:1600
(Abcam), rabbit anti-GAPDH 1:30000 (Abcam)] and were detected using
HRP-conjugated secondary antibodies [donkey anti-goat 1:5000 (Santa
Cruz Biotechnology), goat anti-rabbit 1:20000 (Santa Cruz
Biotechnology)] diluted in 3% BSA in TB ST.
[0110] Data analysis and statistics. Western blot results were
analyzed by densitometry using Scion Image (Scion Corp). Real-time
polymerase chain reaction data was quantified using EXCEL.RTM.
software (Microsoft Corp). Values were graphed as mean.+-.SD of at
least triplicates determinations. Statistics (t test and ANOVA)
were performed using Graphpad software (Graphpad Software Inc). A
value of P<0.05 was considered statistically significant.
[0111] Results
[0112] Apo J attenuates calcification in both ApoE-/- and WT smooth
muscle cells. Because ApoE-/- mice are more prone to vascular
calcification than WT ones, the responses of ApoE-/- and WT VSMCs
to inorganic phosphate (Pi) were examined as well as the effect of
Apo J on calcification of these two groups of cells. The study
shows that when induced with Pi, ApoE-/- VSMCs exhibited much
higher levels of calcification on day 6 of treatment than WT cells.
The addition of Apo J (6 m/mL) into culture medium reduced calcium
level in both groups, with a more dramatic inhibition on
calcification in ApoE-/- VSMCs, shown by both Alizarin S staining
(FIGS. 19A and 20A) and calcium assay (FIGS. 19B and 20B).
Therefore, additional experiments were conducted using ApoE-/-
VSMCs. Various concentrations (3 m/mL, 6 m/mL, 9 m/mL, and 12 m/mL)
of Apo J were then applied to test if the suppressive effect of the
Apo J on vascular cell calcification is dose dependent. The data
demonstrated that 6 m/mL Apo J in culture is sufficient to
significantly decrease calcium deposition level in ApoE-/- VSMCs,
while 12 .mu.g/mL is incapable to further attenuate calcification
compared to 9 m/mL group (FIG. 20B), possibly due to saturation of
Apo J receptors.
[0113] Apo J modulates osteogenesis-related genes during
calcification in ApoE-/- VSMCs. To confirm the effect of Apo J on
calcification, the change of osteogenesis regulator Runx2 was
assessed, as well as the smooth muscle lineage markers SM22.alpha.
and .alpha.SMA, with or without added Apo J during calcification of
ApoE-/- VSMCs, which were treated with Pi for 6 days.
Concentrations from 3 m/mL to 12 .mu.g/mL of Apo J were used. Equal
volumes (40 .mu.L) of culture medium were immunoblotted with Apo J
antibody at the endpoint of the experiment to verify the existence
of Apo J protein in medium. The data suggested that both the mRNA
level and the native secreted form of Apo J increased in calcifying
cells (FIGS. 21A and 22A-B); addition of Apo J resulted in a much
stronger signal detected by Apo J antibody, indicating Apo J in
medium was sustained on a potent level compared to control group
and not subject to bulk degradation through the period of
experiment. It was found Runx2 was increased in response to Pi and
this increase was attenuated in all Apo J treated groups, with 6
m/mL Apo J sufficient to keep Runx2 protein expression down to
approximately the same level in uncalcified cells. No significant
differences of Runx2 levels were observed between 6 .mu.g/mL, 9
.mu.g/mL, and 12 .mu.g/mL groups (FIGS. 21A-21B). SM22.alpha. and
.alpha.SMA protein expressions were downregulated by Pi compared to
control group; Apo J rescued the decreased level of SM22.alpha. and
.alpha.SMA at a concentration as small as 3 .mu.g/mL (FIGS. 21A,
21C and 21D). mRNA expressions of osteogenic genes including Runx2,
BMP-2, OPN and ALP were increased during calcification while Apo J
weakened this effect (FIGS. 23A-23D).
[0114] Knockdown of apolipoprotein-E receptor 2 (ApoER2 or very low
density lipoprotein receptor (VLDLR) abolishes the inhibitory
effect of Apo J on calcification. Because 6 .mu.g/mL of Apo J has
been shown to inhibit calcium mineralization as well as activation
of calcification-associated genes, this same concentration of Apo J
was used to treat VSMCs in remaining experiments. Apo J receptors
ApoER2 and VLDLR were knocked down in ApoE-/- VSMCs by lentiviral
shRNA. Knockdown effect was confirmed by western blots and qRT-PCR.
The data demonstrated that the knockdown of VLDLR or ApoER2 itself
didn't change calcification level under the influence of Pi. The
negative control shRNA didn't alter the sensitivity of VSMCs to Apo
J in calcification and calcium amount still decreased in Apo J
treated negative control group. In contrast, VLDLR or ApoER2 shRNA
mediated knockdown of these receptors eliminated the remissive
impact of Apo J on calcification, shown by Alizarin S and
quantitative calcium assay (FIGS. 24A-24F), indicating Apo J
functions through ApoER2 and VLDLR to regulate calcification
process.
[0115] Knockdown of ApoER2 or VLDLR partially abolishes the effect
of Apo J on osteogenesis related genes in calcification. Knockdown
of ApoER2 or VLDLR weakened the negative regulation of Apo J on the
enhanced expression of Runx2 in calcifying ApoE-/- VSMCs on protein
level (FIGS. 25A and 26C and mRNA level (FIG. 27C). SM22.alpha.
protein expression decreased in Pi treated cells with ApoER2 or
VLDLR knockdown despite the presence of Apo J in culture medium
(FIGS. 25B and 26D). Osteogenic markers alkaline phosphatase (ALP),
bone morphogenic protein (BMP)-2, and Osteopontin (OPN) remained
upregulated compared to uncalcified control group in ApoER2 or
VLDLR knockdown cells; no significant differences were found in
mRNA expressions of these markers between Apo J treated group and
group without Apo J (FIGS. 27D-27F). This implies that knockdown of
ApoER2 or VLDLR partially abolished the effect of Apo J on
osteogenesis related genes in calcification in ApoE-/- VSMCs.
[0116] Studies were undertaken to assess the effect recombinant
Clusterin on blood coagulation. ApoJ mutants with a peptide
comprising a thrombin-specific cleavage site (e.g.,
Clusterin-TRHis, Clusterin-.DELTA.TMD-TRHis,
Clusterin-.DELTA.TMD-.DELTA.NLS-TRHis,
HisTR-Clusterin-.DELTA.TMD-.DELTA.NLS, or
HisTR-Clusterin-.DELTA.TMD) exert an anti-coagulational effect by
competitively inhibiting the conversion of fibrinogen into time of
pig blood with (A) or without (B) ApoJ mutant. Fresh blood (0.3 ml)
from adult pig at different dilutions (0, 1/2, and 1/4 in PBS) was
mixed with recombinant ApoJ mutant at 10 or 10 mg/ml), incubated at
37.degree. C., and then subjected to rotational thromboelastometry
(ROTEM.TM.). a, clotting time; b, Alpha Angle clotting rapidity; c,
maximum clot firmness; and d, maximum clot firmness at 10 minutes.
Data represented from average of two separate experiments with the
ApoJ mutant, Clusterin-.DELTA.TMD-TRHis (SEQ ID NO: 8). Thus, these
data indicate that recombinant Clusterin with thrombin-cleavable
peptide target, such as Clusterin-TRHis,
Clusterin-.DELTA.TMD-TRHis, Clusterin-.DELTA.TMD-.DELTA.NLS-TRHis,
HisTR-Clusterin-.DELTA.TMD-.DELTA.NLS, or HisTR-Clusterin-ATMD can
exert an anti-coagulation effect by competitively inhibiting the
conversion of fibrinogen into fibrin and thus decreasing clot
firmness and increasing the clotting time of blood. Thus,
recombinant Clusterin may serve as a multi-functional agent for the
treatment of patients who suffer from a heart disease, such as
heart disease complicated by hypercholesterolemia, hypertension,
hyperglycemia and thrombogenesis.
* * *
[0117] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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Sequence CWU 1
1
281449PRTArtificial SequenceSynthetic peptide 1Met Met Lys Thr Leu
Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu1 5 10 15Ser Gly Gln Val
Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln 20 25 30Glu Met Ser
Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn 35 40 45Ala Val
Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn 50 55 60Glu
Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys65 70 75
80Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys
85 90 95Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu
Glu 100 105 110Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr
Ala Arg Val 115 120 125Cys Arg Ser Gly Ser Gly Leu Val Gly Arg Gln
Leu Glu Glu Phe Leu 130 135 140Asn Gln Ser Ser Pro Phe Tyr Phe Trp
Met Asn Gly Asp Arg Ile Asp145 150 155 160Ser Leu Leu Glu Asn Asp
Arg Gln Gln Thr His Met Leu Asp Val Met 165 170 175Gln Asp His Phe
Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln 180 185 190Asp Arg
Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro 195 200
205Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg
210 215 220Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu
Asn Phe225 230 235 240His Ala Met Phe Gln Pro Phe Leu Glu Met Ile
His Glu Ala Gln Gln 245 250 255Ala Met Asp Ile His Phe His Ser Pro
Ala Phe Gln His Pro Pro Thr 260 265 270Glu Phe Ile Arg Glu Gly Asp
Asp Asp Arg Thr Val Cys Arg Glu Ile 275 280 285Arg His Asn Ser Thr
Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys 290 295 300Cys Arg Glu
Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln305 310 315
320Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg
325 330 335Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp
Lys Met 340 345 350Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu
Gln Phe Asn Trp 355 360 365Val Ser Arg Leu Ala Asn Leu Thr Gln Gly
Glu Asp Gln Tyr Tyr Leu 370 375 380Arg Val Thr Thr Val Ala Ser His
Thr Ser Asp Ser Asp Val Pro Ser385 390 395 400Gly Val Thr Glu Val
Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr 405 410 415Val Thr Val
Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu 420 425 430Thr
Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu 435 440
445Glu2436PRTArtificial SequenceSynthetic peptide 2Thr Trp Glu Ser
Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn1 5 10 15Glu Leu Gln
Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu 20 25 30Ile Gln
Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu 35 40 45Lys
Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala 50 55
60Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr65
70 75 80Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala
Leu 85 90 95Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys
Phe Tyr 100 105 110Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly
Arg Gln Leu Glu 115 120 125Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr
Phe Trp Met Asn Gly Asp 130 135 140Arg Ile Asp Ser Leu Leu Glu Asn
Asp Arg Gln Gln Thr His Met Leu145 150 155 160Asp Val Met Gln Asp
His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu 165 170 175Leu Phe Gln
Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His 180 185 190Tyr
Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro 195 200
205Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro
210 215 220Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu Met Ile
His Glu225 230 235 240Ala Gln Gln Ala Met Asp Ile His Phe His Ser
Pro Ala Phe Gln His 245 250 255Pro Pro Thr Glu Phe Ile Arg Glu Gly
Asp Asp Asp Arg Thr Val Cys 260 265 270Arg Glu Ile Arg His Asn Ser
Thr Gly Cys Leu Arg Met Lys Asp Gln 275 280 285Cys Asp Lys Cys Arg
Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn 290 295 300Pro Ser Gln
Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val305 310 315
320Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln
325 330 335Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn
Glu Gln 340 345 350Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln
Gly Glu Asp Gln 355 360 365Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser
His Thr Ser Asp Ser Asp 370 375 380Val Pro Ser Gly Val Thr Glu Val
Val Val Lys Leu Phe Asp Ser Asp385 390 395 400Pro Ile Thr Val Thr
Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys 405 410 415Phe Met Glu
Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys 420 425 430His
Arg Glu Glu 4353428PRTArtificial SequenceSynthetic peptide 3Thr Trp
Glu Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn1 5 10 15Glu
Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu 20 25
30Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu
35 40 45Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala
Leu 50 55 60Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro
Gly Val65 70 75 80Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys
Lys Pro Cys Leu 85 90 95Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val
Cys Arg Ser Gly Ser 100 105 110Gly Leu Val Gly Arg Gln Leu Glu Glu
Phe Leu Asn Gln Ser Ser Pro 115 120 125Phe Tyr Phe Trp Met Asn Gly
Asp Arg Ile Asp Ser Leu Leu Glu Asn 130 135 140Asp Arg Gln Gln Thr
His Met Leu Asp Val Met Gln Asp His Phe Ser145 150 155 160Arg Ala
Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr 165 170
175Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His
180 185 190Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg
Ser Leu 195 200 205Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His
Ala Met Phe Gln 210 215 220Pro Phe Leu Glu Met Ile His Glu Ala Gln
Gln Ala Met Asp Ile His225 230 235 240Phe His Ser Pro Ala Phe Gln
His Pro Pro Thr Glu Phe Ile Arg Glu 245 250 255Gly Asp Asp Asp Arg
Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr 260 265 270Gly Cys Leu
Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu 275 280 285Ser
Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg 290 295
300Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys
Tyr305 310 315 320Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu
Asn Thr Ser Ser 325 330 335Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn
Trp Val Ser Arg Leu Ala 340 345 350Asn Leu Thr Gln Gly Glu Asp Gln
Tyr Tyr Leu Arg Val Thr Thr Val 355 360 365Ala Ser His Thr Ser Asp
Ser Asp Val Pro Ser Gly Val Thr Glu Val 370 375 380Val Val Lys Leu
Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val385 390 395 400Glu
Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys 405 410
415Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu 420
4254461PRTArtificial SequenceSynthetic peptide 4His His His His His
His Leu Val Pro Arg Gly Ser Met Met Lys Thr1 5 10 15Leu Leu Leu Phe
Val Gly Leu Leu Leu Thr Trp Glu Ser Gly Gln Val 20 25 30Leu Gly Asp
Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn 35 40 45Gln Gly
Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly 50 55 60Val
Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys65 70 75
80Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys Glu Asp Ala
85 90 95Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro
Gly 100 105 110Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys
Lys Pro Cys 115 120 125Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg
Val Cys Arg Ser Gly 130 135 140Ser Gly Leu Val Gly Arg Gln Leu Glu
Glu Phe Leu Asn Gln Ser Ser145 150 155 160Pro Phe Tyr Phe Trp Met
Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu 165 170 175Asn Asp Arg Gln
Gln Thr His Met Leu Asp Val Met Gln Asp His Phe 180 185 190Ser Arg
Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe 195 200
205Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro
210 215 220His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val
Arg Ser225 230 235 240Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn
Phe His Ala Met Phe 245 250 255Gln Pro Phe Leu Glu Met Ile His Glu
Ala Gln Gln Ala Met Asp Ile 260 265 270His Phe His Ser Pro Ala Phe
Gln His Pro Pro Thr Glu Phe Ile Arg 275 280 285Glu Gly Asp Asp Asp
Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser 290 295 300Thr Gly Cys
Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile305 310 315
320Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg
325 330 335Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu Thr
Arg Lys 340 345 350Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met
Leu Asn Thr Ser 355 360 365Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe
Asn Trp Val Ser Arg Leu 370 375 380Ala Asn Leu Thr Gln Gly Glu Asp
Gln Tyr Tyr Leu Arg Val Thr Thr385 390 395 400Val Ala Ser His Thr
Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu 405 410 415Val Val Val
Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro 420 425 430Val
Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala Glu 435 440
445Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu 450 455
4605448PRTArtificial SequenceSynthetic peptide 5His His His His His
His Leu Val Pro Arg Gly Ser Thr Trp Glu Ser1 5 10 15Gly Gln Val Leu
Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu 20 25 30Met Ser Asn
Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala 35 40 45Val Asn
Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu 50 55 60Glu
Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys65 70 75
80Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu
85 90 95Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu
Cys 100 105 110Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala
Arg Val Cys 115 120 125Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu
Glu Glu Phe Leu Asn 130 135 140Gln Ser Ser Pro Phe Tyr Phe Trp Met
Asn Gly Asp Arg Ile Asp Ser145 150 155 160Leu Leu Glu Asn Asp Arg
Gln Gln Thr His Met Leu Asp Val Met Gln 165 170 175Asp His Phe Ser
Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp 180 185 190Arg Phe
Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe 195 200
205Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile
210 215 220Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn
Phe His225 230 235 240Ala Met Phe Gln Pro Phe Leu Glu Met Ile His
Glu Ala Gln Gln Ala 245 250 255Met Asp Ile His Phe His Ser Pro Ala
Phe Gln His Pro Pro Thr Glu 260 265 270Phe Ile Arg Glu Gly Asp Asp
Asp Arg Thr Val Cys Arg Glu Ile Arg 275 280 285His Asn Ser Thr Gly
Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys 290 295 300Arg Glu Ile
Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala305 310 315
320Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg Leu
325 330 335Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys
Met Leu 340 345 350Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln
Phe Asn Trp Val 355 360 365Ser Arg Leu Ala Asn Leu Thr Gln Gly Glu
Asp Gln Tyr Tyr Leu Arg 370 375 380Val Thr Thr Val Ala Ser His Thr
Ser Asp Ser Asp Val Pro Ser Gly385 390 395 400Val Thr Glu Val Val
Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val 405 410 415Thr Val Pro
Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr 420 425 430Val
Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu 435 440
4456440PRTArtificial SequenceSynthetic peptide 6His His His His His
His Leu Val Pro Arg Gly Ser Thr Trp Glu Ser1 5 10 15Gly Gln Val Leu
Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu 20 25 30Met Ser Asn
Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala 35 40 45Val Asn
Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu 50 55 60Glu
Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala Leu Asn Glu Thr Arg65 70 75
80Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr
85 90 95Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr
Cys 100 105 110Met Lys Phe Tyr Ala Arg Val Cys Arg Ser Gly Ser Gly
Leu Val Gly 115 120 125Arg Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser
Pro Phe Tyr Phe Trp 130 135 140Met Asn Gly Asp Arg Ile Asp Ser Leu
Leu Glu Asn Asp Arg Gln Gln145 150 155 160Thr His Met Leu Asp Val
Met Gln Asp His Phe Ser Arg Ala Ser Ser 165 170 175Ile Ile Asp Glu
Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln 180 185 190Asp Thr
Tyr His Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro His 195 200
205Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser
210
215 220Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu
Glu225 230 235 240Met Ile His Glu Ala Gln Gln Ala Met Asp Ile His
Phe His Ser Pro 245 250 255Ala Phe Gln His Pro Pro Thr Glu Phe Ile
Arg Glu Gly Asp Asp Asp 260 265 270Arg Thr Val Cys Arg Glu Ile Arg
His Asn Ser Thr Gly Cys Leu Arg 275 280 285Met Lys Asp Gln Cys Asp
Lys Cys Arg Glu Ile Leu Ser Val Asp Cys 290 295 300Ser Thr Asn Asn
Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu305 310 315 320Ser
Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu 325 330
335Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln
340 345 350Leu Asn Glu Gln Phe Asn Trp Val Ser Arg Leu Ala Asn Leu
Thr Gln 355 360 365Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val
Ala Ser His Thr 370 375 380Ser Asp Ser Asp Val Pro Ser Gly Val Thr
Glu Val Val Val Lys Leu385 390 395 400Phe Asp Ser Asp Pro Ile Thr
Val Thr Val Pro Val Glu Val Ser Arg 405 410 415Lys Asn Pro Lys Phe
Met Glu Thr Val Ala Glu Lys Ala Leu Gln Glu 420 425 430Tyr Arg Lys
Lys His Arg Glu Glu 435 4407461PRTArtificial SequenceSynthetic
peptide 7Met Met Lys Thr Leu Leu Leu Phe Val Gly Leu Leu Leu Thr
Trp Glu1 5 10 15Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn
Glu Leu Gln 20 25 30Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys
Glu Ile Gln Asn 35 40 45Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu
Ile Glu Lys Thr Asn 50 55 60Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu
Glu Glu Ala Lys Lys Lys65 70 75 80Lys Glu Asp Ala Leu Asn Glu Thr
Arg Glu Ser Glu Thr Lys Leu Lys 85 90 95Glu Leu Pro Gly Val Cys Asn
Glu Thr Met Met Ala Leu Trp Glu Glu 100 105 110Cys Lys Pro Cys Leu
Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val 115 120 125Cys Arg Ser
Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu 130 135 140Asn
Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp145 150
155 160Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val
Met 165 170 175Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu
Leu Phe Gln 180 185 190Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr
Tyr His Tyr Leu Pro 195 200 205Phe Ser Leu Pro His Arg Arg Pro His
Phe Phe Phe Pro Lys Ser Arg 210 215 220Ile Val Arg Ser Leu Met Pro
Phe Ser Pro Tyr Glu Pro Leu Asn Phe225 230 235 240His Ala Met Phe
Gln Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln 245 250 255Ala Met
Asp Ile His Phe His Ser Pro Ala Phe Gln His Pro Pro Thr 260 265
270Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile
275 280 285Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys
Asp Lys 290 295 300Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn
Asn Pro Ser Gln305 310 315 320Ala Lys Leu Arg Arg Glu Leu Asp Glu
Ser Leu Gln Val Ala Glu Arg 325 330 335Leu Thr Arg Lys Tyr Asn Glu
Leu Leu Lys Ser Tyr Gln Trp Lys Met 340 345 350Leu Asn Thr Ser Ser
Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp 355 360 365Val Ser Arg
Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu 370 375 380Arg
Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser385 390
395 400Gly Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile
Thr 405 410 415Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys
Phe Met Glu 420 425 430Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg
Lys Lys His Arg Glu 435 440 445Glu Leu Val Pro Arg Gly Ser His His
His His His His 450 455 4608448PRTArtificial SequenceSynthetic
peptide 8Thr Trp Glu Ser Gly Gln Val Leu Gly Asp Gln Thr Val Ser
Asp Asn1 5 10 15Glu Leu Gln Glu Met Ser Asn Gln Gly Ser Lys Tyr Val
Asn Lys Glu 20 25 30Ile Gln Asn Ala Val Asn Gly Val Lys Gln Ile Lys
Thr Leu Ile Glu 35 40 45Lys Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser
Asn Leu Glu Glu Ala 50 55 60Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu
Thr Arg Glu Ser Glu Thr65 70 75 80Lys Leu Lys Glu Leu Pro Gly Val
Cys Asn Glu Thr Met Met Ala Leu 85 90 95Trp Glu Glu Cys Lys Pro Cys
Leu Lys Gln Thr Cys Met Lys Phe Tyr 100 105 110Ala Arg Val Cys Arg
Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu 115 120 125Glu Phe Leu
Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp 130 135 140Arg
Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu145 150
155 160Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp
Glu 165 170 175Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp
Thr Tyr His 180 185 190Tyr Leu Pro Phe Ser Leu Pro His Arg Arg Pro
His Phe Phe Phe Pro 195 200 205Lys Ser Arg Ile Val Arg Ser Leu Met
Pro Phe Ser Pro Tyr Glu Pro 210 215 220Leu Asn Phe His Ala Met Phe
Gln Pro Phe Leu Glu Met Ile His Glu225 230 235 240Ala Gln Gln Ala
Met Asp Ile His Phe His Ser Pro Ala Phe Gln His 245 250 255Pro Pro
Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg Thr Val Cys 260 265
270Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln
275 280 285Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp Cys Ser Thr
Asn Asn 290 295 300Pro Ser Gln Ala Lys Leu Arg Arg Glu Leu Asp Glu
Ser Leu Gln Val305 310 315 320Ala Glu Arg Leu Thr Arg Lys Tyr Asn
Glu Leu Leu Lys Ser Tyr Gln 325 330 335Trp Lys Met Leu Asn Thr Ser
Ser Leu Leu Glu Gln Leu Asn Glu Gln 340 345 350Phe Asn Trp Val Ser
Arg Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln 355 360 365Tyr Tyr Leu
Arg Val Thr Thr Val Ala Ser His Thr Ser Asp Ser Asp 370 375 380Val
Pro Ser Gly Val Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp385 390
395 400Pro Ile Thr Val Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro
Lys 405 410 415Phe Met Glu Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr
Arg Lys Lys 420 425 430His Arg Glu Glu Leu Val Pro Arg Gly Ser His
His His His His His 435 440 4459444PRTArtificial SequenceSynthetic
peptide 9Met Met Lys Thr Thr Trp Glu Ser Gly Gln Val Leu Gly Asp
Gln Thr1 5 10 15Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln Gly
Ser Lys Tyr 20 25 30Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly Val
Lys Gln Ile Lys 35 40 45Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg Lys
Thr Leu Leu Ser Asn 50 55 60Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser
Glu Thr Lys Leu Lys Glu65 70 75 80Leu Pro Gly Val Cys Asn Glu Thr
Met Met Ala Leu Trp Glu Glu Cys 85 90 95Lys Pro Cys Leu Lys Gln Thr
Cys Met Lys Phe Tyr Ala Arg Val Cys 100 105 110Arg Ser Gly Ser Gly
Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn 115 120 125Gln Ser Ser
Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser 130 135 140Leu
Leu Glu Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln145 150
155 160Asp His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln
Asp 165 170 175Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr
Leu Pro Phe 180 185 190Ser Leu Pro His Arg Arg Pro His Phe Phe Phe
Pro Lys Ser Arg Ile 195 200 205Val Arg Ser Leu Met Pro Phe Ser Pro
Tyr Glu Pro Leu Asn Phe His 210 215 220Ala Met Phe Gln Pro Phe Leu
Glu Met Ile His Glu Ala Gln Gln Ala225 230 235 240Met Asp Ile His
Phe His Ser Pro Ala Phe Gln His Pro Pro Thr Glu 245 250 255Phe Ile
Arg Glu Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg 260 265
270His Asn Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys
275 280 285Arg Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser
Gln Ala 290 295 300Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val
Ala Glu Arg Leu305 310 315 320Thr Arg Lys Tyr Asn Glu Leu Leu Lys
Ser Tyr Gln Trp Lys Met Leu 325 330 335Asn Thr Ser Ser Leu Leu Glu
Gln Leu Asn Glu Gln Phe Asn Trp Val 340 345 350Ser Arg Leu Ala Asn
Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg 355 360 365Val Thr Thr
Val Ala Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly 370 375 380Val
Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val385 390
395 400Thr Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu
Thr 405 410 415Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His
Arg Glu Glu 420 425 430Leu Val Pro Arg Gly Ser His His His His His
His 435 44010460PRTArtificial SequenceSynthetic peptide 10His His
His His His His Asp Asp Asp Asp Lys Met Met Lys Thr Leu1 5 10 15Leu
Leu Phe Val Gly Leu Leu Leu Thr Trp Glu Ser Gly Gln Val Leu 20 25
30Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met Ser Asn Gln
35 40 45Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val Asn Gly
Val 50 55 60Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu Glu Arg
Lys Thr65 70 75 80Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys Lys
Glu Asp Ala Leu 85 90 95Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys
Glu Leu Pro Gly Val 100 105 110Cys Asn Glu Thr Met Met Ala Leu Trp
Glu Glu Cys Lys Pro Cys Leu 115 120 125Lys Gln Thr Cys Met Lys Phe
Tyr Ala Arg Val Cys Arg Ser Gly Ser 130 135 140Gly Leu Val Gly Arg
Gln Leu Glu Glu Phe Leu Asn Gln Ser Ser Pro145 150 155 160Phe Tyr
Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn 165 170
175Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp His Phe Ser
180 185 190Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg Phe
Phe Thr 195 200 205Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro Phe
Ser Leu Pro His 210 215 220Arg Arg Pro His Phe Phe Phe Pro Lys Ser
Arg Ile Val Arg Ser Leu225 230 235 240Met Pro Phe Ser Pro Tyr Glu
Pro Leu Asn Phe His Ala Met Phe Gln 245 250 255Pro Phe Leu Glu Met
Ile His Glu Ala Gln Gln Ala Met Asp Ile His 260 265 270Phe His Ser
Pro Ala Phe Gln His Pro Pro Thr Glu Phe Ile Arg Glu 275 280 285Gly
Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr 290 295
300Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile
Leu305 310 315 320Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala
Lys Leu Arg Arg 325 330 335Glu Leu Asp Glu Ser Leu Gln Val Ala Glu
Arg Leu Thr Arg Lys Tyr 340 345 350Asn Glu Leu Leu Lys Ser Tyr Gln
Trp Lys Met Leu Asn Thr Ser Ser 355 360 365Leu Leu Glu Gln Leu Asn
Glu Gln Phe Asn Trp Val Ser Arg Leu Ala 370 375 380Asn Leu Thr Gln
Gly Glu Asp Gln Tyr Tyr Leu Arg Val Thr Thr Val385 390 395 400Ala
Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val Thr Glu Val 405 410
415Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val
420 425 430Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu Thr Val Ala
Glu Lys 435 440 445Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu Glu
450 455 46011447PRTArtificial SequenceSynthetic peptide 11His His
His His His His Asp Asp Asp Asp Lys Thr Trp Glu Ser Gly1 5 10 15Gln
Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln Glu Met 20 25
30Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn Ala Val
35 40 45Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu
Glu 50 55 60Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys
Lys Glu65 70 75 80Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys
Leu Lys Glu Leu 85 90 95Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu
Trp Glu Glu Cys Lys 100 105 110Pro Cys Leu Lys Gln Thr Cys Met Lys
Phe Tyr Ala Arg Val Cys Arg 115 120 125Ser Gly Ser Gly Leu Val Gly
Arg Gln Leu Glu Glu Phe Leu Asn Gln 130 135 140Ser Ser Pro Phe Tyr
Phe Trp Met Asn Gly Asp Arg Ile Asp Ser Leu145 150 155 160Leu Glu
Asn Asp Arg Gln Gln Thr His Met Leu Asp Val Met Gln Asp 165 170
175His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln Asp Arg
180 185 190Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro
Phe Ser 195 200 205Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys
Ser Arg Ile Val 210 215 220Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu
Pro Leu Asn Phe His Ala225 230 235 240Met Phe Gln Pro Phe Leu Glu
Met Ile His Glu Ala Gln Gln Ala Met 245 250 255Asp Ile His Phe His
Ser Pro Ala Phe Gln His Pro Pro Thr Glu Phe 260 265 270Ile Arg Glu
Gly Asp Asp Asp Arg Thr Val Cys Arg Glu Ile Arg His 275 280 285Asn
Ser Thr Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys Cys Arg 290 295
300Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln Ala
Lys305 310 315 320Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala
Glu Arg Leu Thr 325 330 335Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr
Gln Trp Lys Met Leu Asn 340 345 350Thr Ser Ser Leu Leu Glu Gln Leu
Asn Glu Gln Phe Asn Trp Val Ser 355 360 365Arg Leu Ala Asn Leu Thr
Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val 370 375 380Thr Thr Val Ala
Ser His Thr Ser Asp Ser Asp Val Pro Ser Gly Val385 390 395 400Thr
Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val
Thr 405 410 415Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met
Glu Thr Val 420 425 430Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys
His Arg Glu Glu 435 440 44512439PRTArtificial SequenceSynthetic
peptide 12His His His His His His Asp Asp Asp Asp Lys Thr Trp Glu
Ser Gly1 5 10 15Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu
Gln Glu Met 20 25 30Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile
Gln Asn Ala Val 35 40 45Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu
Lys Thr Asn Glu Glu 50 55 60Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala
Leu Asn Glu Thr Arg Glu65 70 75 80Ser Glu Thr Lys Leu Lys Glu Leu
Pro Gly Val Cys Asn Glu Thr Met 85 90 95Met Ala Leu Trp Glu Glu Cys
Lys Pro Cys Leu Lys Gln Thr Cys Met 100 105 110Lys Phe Tyr Ala Arg
Val Cys Arg Ser Gly Ser Gly Leu Val Gly Arg 115 120 125Gln Leu Glu
Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr Phe Trp Met 130 135 140Asn
Gly Asp Arg Ile Asp Ser Leu Leu Glu Asn Asp Arg Gln Gln Thr145 150
155 160His Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser
Ile 165 170 175Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr Arg Glu
Pro Gln Asp 180 185 190Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His
Arg Arg Pro His Phe 195 200 205Phe Phe Pro Lys Ser Arg Ile Val Arg
Ser Leu Met Pro Phe Ser Pro 210 215 220Tyr Glu Pro Leu Asn Phe His
Ala Met Phe Gln Pro Phe Leu Glu Met225 230 235 240Ile His Glu Ala
Gln Gln Ala Met Asp Ile His Phe His Ser Pro Ala 245 250 255Phe Gln
His Pro Pro Thr Glu Phe Ile Arg Glu Gly Asp Asp Asp Arg 260 265
270Thr Val Cys Arg Glu Ile Arg His Asn Ser Thr Gly Cys Leu Arg Met
275 280 285Lys Asp Gln Cys Asp Lys Cys Arg Glu Ile Leu Ser Val Asp
Cys Ser 290 295 300Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg Glu
Leu Asp Glu Ser305 310 315 320Leu Gln Val Ala Glu Arg Leu Thr Arg
Lys Tyr Asn Glu Leu Leu Lys 325 330 335Ser Tyr Gln Trp Lys Met Leu
Asn Thr Ser Ser Leu Leu Glu Gln Leu 340 345 350Asn Glu Gln Phe Asn
Trp Val Ser Arg Leu Ala Asn Leu Thr Gln Gly 355 360 365Glu Asp Gln
Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser His Thr Ser 370 375 380Asp
Ser Asp Val Pro Ser Gly Val Thr Glu Val Val Val Lys Leu Phe385 390
395 400Asp Ser Asp Pro Ile Thr Val Thr Val Pro Val Glu Val Ser Arg
Lys 405 410 415Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys Ala Leu
Gln Glu Tyr 420 425 430Arg Lys Lys His Arg Glu Glu
43513461PRTArtificial SequenceSynthetic peptide 13Met Met Lys Thr
Leu Leu Leu Phe Val Gly Leu Leu Leu Thr Trp Glu1 5 10 15Ser Gly Gln
Val Leu Gly Asp Gln Thr Val Ser Asp Asn Glu Leu Gln 20 25 30Glu Met
Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu Ile Gln Asn 35 40 45Ala
Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn 50 55
60Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala Lys Lys Lys65
70 75 80Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu
Lys 85 90 95Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala Leu Trp
Glu Glu 100 105 110Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe
Tyr Ala Arg Val 115 120 125Cys Arg Ser Gly Ser Gly Leu Val Gly Arg
Gln Leu Glu Glu Phe Leu 130 135 140Asn Gln Ser Ser Pro Phe Tyr Phe
Trp Met Asn Gly Asp Arg Ile Asp145 150 155 160Ser Leu Leu Glu Asn
Asp Arg Gln Gln Thr His Met Leu Asp Val Met 165 170 175Gln Asp His
Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu Leu Phe Gln 180 185 190Asp
Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His Tyr Leu Pro 195 200
205Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro Lys Ser Arg
210 215 220Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro Leu
Asn Phe225 230 235 240His Ala Met Phe Gln Pro Phe Leu Glu Met Ile
His Glu Ala Gln Gln 245 250 255Ala Met Asp Ile His Phe His Ser Pro
Ala Phe Gln His Pro Pro Thr 260 265 270Glu Phe Ile Arg Glu Gly Asp
Asp Asp Arg Thr Val Cys Arg Glu Ile 275 280 285Arg His Asn Ser Thr
Gly Cys Leu Arg Met Lys Asp Gln Cys Asp Lys 290 295 300Cys Arg Glu
Ile Leu Ser Val Asp Cys Ser Thr Asn Asn Pro Ser Gln305 310 315
320Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val Ala Glu Arg
325 330 335Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln Trp
Lys Met 340 345 350Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu
Gln Phe Asn Trp 355 360 365Val Ser Arg Leu Ala Asn Leu Thr Gln Gly
Glu Asp Gln Tyr Tyr Leu 370 375 380Arg Val Thr Thr Val Ala Ser His
Thr Ser Asp Ser Asp Val Pro Ser385 390 395 400Gly Val Thr Glu Val
Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr 405 410 415Val Thr Val
Pro Val Glu Val Ser Arg Lys Asn Pro Lys Phe Met Glu 420 425 430Thr
Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys His Arg Glu 435 440
445Glu Leu Asp Asp Asp Asp Lys His His His His His His 450 455
46014448PRTArtificial SequenceSynthetic peptide 14Thr Trp Glu Ser
Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn1 5 10 15Glu Leu Gln
Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu 20 25 30Ile Gln
Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu 35 40 45Lys
Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Leu Glu Glu Ala 50 55
60Lys Lys Lys Lys Glu Asp Ala Leu Asn Glu Thr Arg Glu Ser Glu Thr65
70 75 80Lys Leu Lys Glu Leu Pro Gly Val Cys Asn Glu Thr Met Met Ala
Leu 85 90 95Trp Glu Glu Cys Lys Pro Cys Leu Lys Gln Thr Cys Met Lys
Phe Tyr 100 105 110Ala Arg Val Cys Arg Ser Gly Ser Gly Leu Val Gly
Arg Gln Leu Glu 115 120 125Glu Phe Leu Asn Gln Ser Ser Pro Phe Tyr
Phe Trp Met Asn Gly Asp 130 135 140Arg Ile Asp Ser Leu Leu Glu Asn
Asp Arg Gln Gln Thr His Met Leu145 150 155 160Asp Val Met Gln Asp
His Phe Ser Arg Ala Ser Ser Ile Ile Asp Glu 165 170 175Leu Phe Gln
Asp Arg Phe Phe Thr Arg Glu Pro Gln Asp Thr Tyr His 180 185 190Tyr
Leu Pro Phe Ser Leu Pro His Arg Arg Pro His Phe Phe Phe Pro 195 200
205Lys Ser Arg Ile Val Arg Ser Leu Met Pro Phe Ser Pro Tyr Glu Pro
210 215 220Leu Asn Phe His Ala Met Phe Gln Pro Phe Leu Glu Met Ile
His Glu225 230 235 240Ala Gln Gln Ala Met Asp Ile His Phe His Ser
Pro Ala Phe Gln His 245 250 255Pro Pro Thr Glu Phe Ile Arg Glu Gly
Asp Asp Asp Arg Thr Val Cys 260 265 270Arg Glu Ile Arg His Asn Ser
Thr Gly Cys Leu Arg Met Lys Asp Gln 275 280 285Cys Asp Lys Cys Arg
Glu Ile Leu Ser Val Asp Cys Ser Thr Asn Asn 290 295 300Pro Ser Gln
Ala Lys Leu Arg Arg Glu Leu Asp Glu Ser Leu Gln Val305 310 315
320Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys Ser Tyr Gln
325 330 335Trp Lys Met Leu Asn Thr Ser Ser Leu Leu Glu Gln Leu Asn
Glu Gln 340 345 350Phe Asn Trp Val Ser Arg Leu Ala Asn Leu Thr Gln
Gly Glu Asp Gln 355 360 365Tyr Tyr Leu Arg Val Thr Thr Val Ala Ser
His Thr Ser Asp Ser Asp 370 375 380Val Pro Ser Gly Val Thr Glu Val
Val Val Lys Leu Phe Asp Ser Asp385 390 395 400Pro Ile Thr Val Thr
Val Pro Val Glu Val Ser Arg Lys Asn Pro Lys 405 410 415Phe Met Glu
Thr Val Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys 420 425 430His
Arg Glu Glu Leu Asp Asp Asp Asp Lys His His His His His His 435 440
44515440PRTArtificial SequenceSynthetic peptide 15Thr Trp Glu Ser
Gly Gln Val Leu Gly Asp Gln Thr Val Ser Asp Asn1 5 10 15Glu Leu Gln
Glu Met Ser Asn Gln Gly Ser Lys Tyr Val Asn Lys Glu 20 25 30Ile Gln
Asn Ala Val Asn Gly Val Lys Gln Ile Lys Thr Leu Ile Glu 35 40 45Lys
Thr Asn Glu Glu Arg Lys Thr Leu Leu Ser Asn Glu Asp Ala Leu 50 55
60Asn Glu Thr Arg Glu Ser Glu Thr Lys Leu Lys Glu Leu Pro Gly Val65
70 75 80Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys Lys Pro Cys
Leu 85 90 95Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys Arg Ser
Gly Ser 100 105 110Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn
Gln Ser Ser Pro 115 120 125Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile
Asp Ser Leu Leu Glu Asn 130 135 140Asp Arg Gln Gln Thr His Met Leu
Asp Val Met Gln Asp His Phe Ser145 150 155 160Arg Ala Ser Ser Ile
Ile Asp Glu Leu Phe Gln Asp Arg Phe Phe Thr 165 170 175Arg Glu Pro
Gln Asp Thr Tyr His Tyr Leu Pro Phe Ser Leu Pro His 180 185 190Arg
Arg Pro His Phe Phe Phe Pro Lys Ser Arg Ile Val Arg Ser Leu 195 200
205Met Pro Phe Ser Pro Tyr Glu Pro Leu Asn Phe His Ala Met Phe Gln
210 215 220Pro Phe Leu Glu Met Ile His Glu Ala Gln Gln Ala Met Asp
Ile His225 230 235 240Phe His Ser Pro Ala Phe Gln His Pro Pro Thr
Glu Phe Ile Arg Glu 245 250 255Gly Asp Asp Asp Arg Thr Val Cys Arg
Glu Ile Arg His Asn Ser Thr 260 265 270Gly Cys Leu Arg Met Lys Asp
Gln Cys Asp Lys Cys Arg Glu Ile Leu 275 280 285Ser Val Asp Cys Ser
Thr Asn Asn Pro Ser Gln Ala Lys Leu Arg Arg 290 295 300Glu Leu Asp
Glu Ser Leu Gln Val Ala Glu Arg Leu Thr Arg Lys Tyr305 310 315
320Asn Glu Leu Leu Lys Ser Tyr Gln Trp Lys Met Leu Asn Thr Ser Ser
325 330 335Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser Arg
Leu Ala 340 345 350Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg
Val Thr Thr Val 355 360 365Ala Ser His Thr Ser Asp Ser Asp Val Pro
Ser Gly Val Thr Glu Val 370 375 380Val Val Lys Leu Phe Asp Ser Asp
Pro Ile Thr Val Thr Val Pro Val385 390 395 400Glu Val Ser Arg Lys
Asn Pro Lys Phe Met Glu Thr Val Ala Glu Lys 405 410 415Ala Leu Gln
Glu Tyr Arg Lys Lys His Arg Glu Glu Leu Asp Asp Asp 420 425 430Asp
Lys His His His His His His 435 440166PRTArtificial
SequenceProtease cleavage site 16Leu Val Pro Arg Gly Ser1
5175PRTArtificial SequenceProtease cleavage site 17Asp Asp Asp Asp
Lys1 51825DNAArtificial SequenceSynthetic primer 18cacaatatca
aggatatcga cgtga 251925DNAArtificial SequenceSynthetic primer
19acatcagttc tgttcttcgg gtaca 252024DNAArtificial SequenceSynthetic
primer 20ttgtatgtgg acttcagtga tgtg 242120DNAArtificial
SequenceSynthetic primer 21agttcaggtg gtcagcaagg
202220DNAArtificial SequenceSynthetic primer 22tggctatagg
atctgggtgc 202320DNAArtificial SequenceSynthetic primer
23atttgctttt gcctgtttgg 202420DNAArtificial SequenceSynthetic
primer 24ttacctacac cccgccagtc 202519DNAArtificial
SequenceSynthetic primer 25tgctggtctg gaagggtcc 192615DNAArtificial
sequenceSynthetic oligonucleotide 26gcccttggtg aaggg 15278PRTHomo
sapiens 27Leu Glu Glu Ala Lys Lys Lys Lys1 5289PRTHomo sapiens
28Leu Leu Leu Phe Val Gly Leu Leu Leu1 5
* * * * *