U.S. patent application number 12/245944 was filed with the patent office on 2010-02-25 for treatment for dark adaptation.
Invention is credited to Kathy R. Bailey, Keith G. Duncan, Brian Y. Ishida, John P. Kane, Daniel M. Schwartz.
Application Number | 20100047330 12/245944 |
Document ID | / |
Family ID | 35056677 |
Filed Date | 2010-02-25 |
United States Patent
Application |
20100047330 |
Kind Code |
A1 |
Schwartz; Daniel M. ; et
al. |
February 25, 2010 |
TREATMENT FOR DARK ADAPTATION
Abstract
The present invention addresses the treatment of age-related
macular degeneration using regulation of pathogenic mechanisms
similar to atherosclerosis. In further specific embodiments,
compositions that increase reverse cholesterol transport are
utilized as therapeutic targets for age-related macular
degeneration. In a specific embodiment, the lipid content of the
retinal pigment epithelium, and/or Bruch's membrane is reduced by
delivering Apolipoprotein A1, particularly a mimetic peptide.
Inventors: |
Schwartz; Daniel M.; (San
Francisco, CA) ; Duncan; Keith G.; (San Francisco,
CA) ; Bailey; Kathy R.; (San Francisco, CA) ;
Kane; John P.; (Hillsborough, CA) ; Ishida; Brian
Y.; (Walnut Creek, CA) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY, SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
35056677 |
Appl. No.: |
12/245944 |
Filed: |
October 6, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11055309 |
Feb 10, 2005 |
7470660 |
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12245944 |
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10794198 |
Mar 5, 2004 |
7470659 |
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11055309 |
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10428551 |
May 2, 2003 |
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10794198 |
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10313641 |
Dec 6, 2002 |
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10428551 |
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60340498 |
Dec 7, 2001 |
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60415864 |
Oct 3, 2002 |
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Current U.S.
Class: |
424/450 ;
514/1.1 |
Current CPC
Class: |
A61K 31/685 20130101;
A61K 38/1709 20130101; A61P 27/02 20180101; A61K 38/17 20130101;
A61K 31/66 20130101; A61K 38/17 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/450 ;
514/12 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 38/17 20060101 A61K038/17; A61P 27/02 20060101
A61P027/02 |
Claims
1. A method of treating macular degeneration in an individual,
comprising the step of increasing reverse cholesterol transport in
an ocular tissue of the individual, wherein said increasing reverse
cholesterol transport comprises increasing Apolipoprotein A-I
(ApoA-I) level in the ocular tissue.
2. The method of claim 1, wherein said increasing ApoA-I level
comprises administering to the individual a therapeutically
effective amount of an ApoA-I composition.
3. The method of claim 2, wherein the ApoA-I composition comprises
an ApoA-I polypeptide or peptide.
4. The method of claim 3, wherein the ApoA-I peptide comprises SEQ
ID NO:15.
5. The method of claim 3, wherein the ApoA-I peptide comprises SEQ
ID NO:16.
6. The method of claim 3, wherein the ApoA-I polypeptide or peptide
comprises D-amino acids.
7. The method of claim 1, wherein increasing the ApoA-I level
comprises upregulating expression of ApoA-I.
8. The method of claim 1, wherein increasing the ApoA-I level is
further defined as administering an agent to the individual, such
that said agent increases the level of circulating ApoA-I in the
individual.
9. The method of claim 8, wherein the agent comprises 1,2
dimyristoyl-.alpha.-glycero-3-phosphocholine (DMPC).
10. The method of claim 1, wherein the composition is administered
to the individual locally or systemically.
11. The method of claim 1, wherein the composition is administered
orally, parenterally, topically, intradermally, subcutaneously,
intramuscularly, intraperitoneally, or intravenously.
12. The method of claim 6, wherein said composition is administered
to an individual orally, wherein said composition is further
defined as comprising a liposome.
13. A method of increasing lipid efflux from an ocular tissue
comprising the step of increasing the level of ApoA-I in said
tissue.
14. A method of treating macular degeneration (AMD) in an
individual, comprising the step of delivering a therapeutically
effective amount of a liposome comprising ApoA-I to at least one
tissue of the individual.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present invention is a continuation application of U.S.
Nonprovisional patent application Ser. No. 11/055,309, filed Feb.
10, 2005, which is a divisional application of U.S. Nonprovisional
patent application Ser. No. 10/794,198, filed Mar. 5, 2004, which
is a continuation-in-part of U.S. Nonprovisional patent application
Ser. No. 10/428,551, filed May 2, 2003, which is a
continuation-in-part of U.S. Nonprovisional patent application Ser.
No. 10/313,641, filed Dec. 6, 2002, which claims the benefit of
U.S. Provisional Patent Application 60/340,498, filed Dec. 7, 2001;
and which also claims the benefit of U.S. Provisional Patent
Application 60/415,864, filed Oct. 3, 2002; all priority
applications are incorporated by reference herein in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed to the fields of
opthalmology and cell biology. Specifically, the invention regards
increasing reverse cholesterol transport in the retinal pigment
epithelium and Bruch's membrane. More specifically, the invention
relates to treatment of age-related macular degeneration (AMD)
utilizing regulation of reverse cholesterol transport.
BACKGROUND OF THE INVENTION
[0003] Age-related macular degeneration (AMD) is the leading cause
of severe visual loss in the developed world (Taylor et al., 2001;
VanNewkirk et al., 2000). In the early stages of the disease,
before visual loss occurs from choroidal neovascularization, there
is progressive accumulation of lipids in Bruch's membrane
(Pauleikhoff et al., 1990; Holz et al., 1994; Sheraidah et al.,
1993; Spaide et al., 1999). Bruch's membrane lies at the critical
juncture between the outer retina and its blood supply, the
choriocapillaris. Lipid deposition causes reduced hydraulic
conductivity and macromolecular permeability in Bruch's membrane
and is thought to impair retinal metabolism (Moore et al., 1995;
Pauleikhoff et al., 1990; Starita et al., 1996). Retina and/or RPE
may respond by elaboration of angiogenic factors (e.g. VEGF, vFGF)
that promote growth of choroidal neovascularization.
[0004] Interestingly, lipid accumulation in Bruch's membrane
similar to that in AMD has been observed in apolipoprotein E (apo
E) null mice (Dithmar et al., 2000; Kliffen et al., 2000). Because
of the additional association between apo E alleles and other
age-related degenerations, Alzheimer's disease and atherosclerosis,
there has been recent investigation into a potential role for apo E
in AMD.
[0005] Several studies on apo E polymorphism in AMD have been
conducted (Simonelli et al., 2001; Klayer et al., 1998; Souied et
al., 1998). In contrast to Alzheimer's disease, the apo E-4 allele
has been associated with reduced prevalence of AMD. Apo E-2 allele
is slightly increased in patients with AMD. Further supporting a
role in AMD pathogenesis, apo E has been detected in drusen, the
Bruch's membrane deposits that are the hallmark of AMD (Klayer et
al., 1998; Anderson et al., 2001). Immunohistochemistry on
post-mortem eyes has demonstrated apo E in the basal aspect of the
retinal pigment epithelium (RPE) (Anderson et al., 2001). Cultured
RPE cells synthesize high levels of apo E mRNA, comparable to
levels found in brain (Anderson et al., 2001).
[0006] While the role of apo E in AMD is not established, this
apolipoprotein has several functions that may affect the course of
this disease. Apo E has anti-angiogenic (Browning et al., 1994),
anti-inflammatory (Michael et al., 1994), and anti-oxidative
effects (Tangirala et al., 2001). These are all considered
atheroprotective attributes of Apo E, but may also be important in
protecting against progression of AMD. While atheroprotective
effects of apo E were initially thought to stem from effects on
plasma lipid levels, local effects on vascular macrophages are
probably equally important. Thus, selective enhanced expression of
macrophage apo E in the arterial wall reduces atherosclerosis in
spite of hyperlipidemia (Shimano et al., 1995; Bellosta et al.,
1995; Hasty et al., 1999). Conversely, reconstitution of apo E null
macrophages in C57BL/6 wild type mice induces atherosclerosis
(Fazio et al., 1994). Atheroprotective effects of arterial apo E
expression are thought to derive in part from facilitation of
reverse cholesterol transport (Mazzone et al., 1992; Lin et al.,
1999). The mechanisms by which apo E facilitates reverse
cholesterol transport are incompletely understood. Apo E expression
increases cholesterol efflux to HDL3 in J774 macrophages (Mazzone
and Reardon, 1994) and lipid free apolipoprotein A1 (Langer et al.,
2000). Cell surface apo E is also hypothesized to induce efflux
from the plasma membrane (Lin et al., 1999).
[0007] Reverse cholesterol transport may be important in the
pathogenesis of AMD because of lipid efflux from RPE into Bruch's
membrane. Very much like intimal macrophages, RPE cells
progressively accumulate lipid deposits throughout life; however,
unlike vessel wall macrophages, the source of RPE lipid is thought
to be retinal photoreceptor outer segments (POS) (Kennedy et al.,
1995). Every day, each RPE cell phagocytoses and degrades more than
one thousand POS via lyzosmal enzymes. These POS are enriched in
phospholipid and contain the photoreactive pigment, rhodopsin.
Incompletely digested POS accumulate as lipofuscin in RPE. By age
80, approximately 20% of RPE cell volume is occupied by lipofuscin
(Feeney-Burns et al., 1984).
[0008] Analysis of Bruch's membrane lipid reveals an age-related
accumulation of phospholipid, triglyceride, cholesterol, and
cholesterol ester (Holz et al., 1994; Curcio et al., 2001). The
origin of these lipids also is thought to derive principally from
POS rather than from the circulation (Holz et al., 1994; Spaide et
al., 1999). POS lipids are hypothesized to efflux from the RPE into
Bruch's membrane. Although cholesterol ester deposition in Bruch's
suggests contribution from plasma lipids, biochemical analysis of
these esters suggests esterification of intracellular cholesterol
by RPE cell derived ACAT (Curcio et al., 2002). While trafficking
of lipids from the retina to RPE cells has been studied
extensively, mechanisms of lipid efflux from RPE to Bruch's
membrane are not well understood. Furthermore, from a pathogenic
standpoint, regulation of lipid efflux into Bruch's membrane may be
important in determining the rate of lipid-induced thickening that
occurs in aging.
[0009] In AS, similar to AMD, lipids accumulate in the
extracellular matrix and within phagocytic cells, primarily
macrophages. Mechanisms of lipid metabolism in AS have been
investigated in detail. Similar investigations into lipid
processing by RPE and subsequent lipid efflux into BM and the
circulation have not been conducted with the same depth as those
for AS. As a consequence, potential therapeutic approaches to dry
AMD are wonting.
[0010] Navab et al. (2003) describe ApoA-I mimetic peptides
comprising D-amino acids for oral delivery for the treatment of
atherosclerosis.
[0011] U.S. Patent Application Publication US 2002/0142953 relates
to human compositions encoding apolipoproteins that are related to
lipid metabolism and cardiovascular disease.
[0012] Thus, the present invention provides a novel approach to
reduce lipid content of ocular tissue, such as Bruch's membrane and
further provides methods and compositions for the treatment of
macular degeneration, such as AMD.
SUMMARY OF THE INVENTION
[0013] In the present invention, there are methods and compositions
that relate to increasing reverse cholesterol transport in the
retinal pigment epithelium (RPE). Particularly, the increase in
reverse cholesterol transport is mediated, enhanced, facilitated,
and/or triggered by administration of a composition. More
particularly, one or more compositions promote efflux of lipids
from Bruch's membrane and/or enhances binding of effluxed lipids
from Bruch's membrane, thereby reducing accumulation of lipids in
both retinal pigment epithelium and Bruch's membrane. This is
beneficial in these regions, given that in aging Bruch's membrane,
there is progressive accumulation of lipid and cross-linked protein
that impedes hydraulic conductivity and macromolecular
permeability. This abnormal deposition, in specific embodiments,
also impairs the ability of some larger molecular weight species of
HDL, a preferred cholesterol and phospholipids acceptor for lipids
effluxed by cultured human RPE, to act as a lipoprotein acceptor.
As HDL is unable to pass through BM and promote efflux and/or bind
effluxed lipids, more lipids accumulate in both RPE and BM. A
skilled artisan recognizes that such accumulations are a major
finding in age-related macular degeneration (AMD), and, therefore,
recognizes the need for novel compositions for the treatment of
this debilitating disease.
[0014] Apolipoprotein A1 (ApoA-I) is the major lipoprotein
component of HDL, and it has a mass of approximately 28 kDaltons.
ApoA-I bound to phospholipids comprises nascent HDL particles that
bind to ABCA1 on the RPE basal membrane and promote lipid efflux.
Because of the low molecular weight of ApoA-I, it can penetrate an
aged BM more easily than larger molecular weight species of HDL to
bind to the RPE. In addition to its role in promoting reverse
cholesterol transport from RPE, ApoA-I also is a potent
anti-oxidant, which is known to reduce visual loss in patients with
AMD.
[0015] In some embodiments, the present invention is directed to a
system, method, and/or composition(s) related to treating AMD.
Treatments for dry AMD have been lacking, because the pathogenesis
of this common condition is poorly understood, and the inventors
have demonstrated analogous biological behavior between human
retinal pigment epithelial (RPE) cells and macrophages that point
toward similar pathogenic mechanisms of AMD and atherosclerosis.
Specifically, reverse cholesterol transport (RCT) is exploited in
the present invention for the treatment of AMD. The present
inventors provide the novel demonstration of RCT in RPE cells in
the eye. More specifically, RCT is regulated through manipulation
of levels of cholesterol and/or phospholipid transporters (ABCA-1,
Apo E, SRB-1, SRB-2) by nuclear hormone receptor ligands such as
agonists of thyroid hormone (TR), liver X receptor (LXR), and/or
retinoid X receptor (RXR). A goal for the present invention is the
reduction of lipid content of RPE Bruch's membrane to facilitate an
improvement in visual function and/or, in some embodiments, prevent
ocular disease, such as AMD. Reduction of the lipid content of
Bruch's membrane preferably results in at least one or more of the
following: reduction in development of CNV; improvement in dark
adaptation; improvement in night vision; improved visual acuity;
and/or improved recovery to bright flash stimulus.
[0016] In an additional embodiment of the present invention, there
is a method of treating macular degeneration (AMD) in an
individual, comprising the step of delivering to the individual a
therapeutically effective amount of an ApoA-I composition. In a
specific embodiment, the delivering occurs under conditions wherein
reverse cholesterol transport is upregulated, wherein lipid
accumulations in BM or RPE are reduced, wherein efflux of lipids
from BM is increased, and/or wherein therapeutic anti-oxidant
applications are achieved. In further specific embodiments, the
administration of the ApoA-I composition results in effective
treatment for AMD or any ocular disease, such as be ameliorating at
least one symptom of the disease. Delivery of the ApoA-I
composition may occur by any method in the art so long as it
provides a therapeutically effective amount to the tissue or
tissues in need thereof. The delivery may be local or systemic. In
preferred embodiments, the delivery is intravenously, as has been
done for ApoA-I in mouse models of atherosclerosis and in patients
with coronary artery disease. In other embodiments, the delivery is
oral.
[0017] In particular aspects of the invention, the ApoA-I
composition may be any ApoA-I composition that upon delivery to an
individual suffering from an ocular disease such as AMD, said
disease has amelioration of at least one symptom. For example, the
ApoA-I composition may be comprised of one or more L-amino acids or
one or more D-amino acids, or mixtures thereof. In particular
embodiments, there is an ApoA-I mimetic peptide comprised of
D-amino acids, which is not recognized as readily by human
proteases, and thus can be administered orally. In specific
embodiments, this is more convenient than parenteral administration
with an intravenous formulation comprising the L-amino acid ApoA-I
or its mimetic peptide. In specific embodiments, exemplary mimetic
ApoA-I peptides are used, such as are described by Navab et al.
(2003), incorporated by reference herein in its entirety.
[0018] By way of example, patients with AMD (atrophic or exudative)
are administered an ApoA-I composition, such as intravenous ApoA-I,
ApoA-I mimetic peptide, or compositions that increase circulating
ApoA-I, such as the exemplary oral synthetic phospholipid (1,2
Dimyristoyl-sn-glycero-3-phosphocholine) (DMPC). Administration
could occur in any frequency so long as there is at least one
therapeutic effect, and in preferred embodiments the effect is
detectable. The administration, in specific but exemplary
embodiments, is as frequent as daily or less frequently as in every
other month depending on the method of administration and the
clinical response.
[0019] In another embodiment of the present invention, there is a
kit for the treatment of macular degeneration, housed in a suitable
container, comprising a ApoA-I composition. In particular
embodiments, the ApoA-I composition may be ApoA-I from any
organism, but particularly human ApoA-I, a mimetic ApoA-I peptide,
or an agent that increases ApoA-I circulating levels, such as DMPC.
In a specific embodiment, the kit comprises a pharmaceutically
acceptable excipient. In another specific embodiment, the ApoA-I
composition is comprised in the pharmaceutically acceptable
excipient. In other specific embodiments, the ApoA-I composition is
comprised in a liposome and delivered orally to an individual.
[0020] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] For a more complete understanding of the present invention,
reference is now made to the following descriptions taken in
conjunction with the accompanying drawing, in which:
[0022] FIG. 1 shows that RPE cells express Apo E, ABCA1, and LXR
.alpha..
[0023] FIG. 2 shows RPE cell expression of SR-BI and SR-BII.
[0024] FIG. 3 illustrates SR-BI and SR-BII immunofluorescence in
RPE cells.
[0025] FIG. 4 demonstrates ABCA1 immunofluorescence in RPE
cells.
[0026] FIG. 5 demonstrates that basal Apo E expression is greater
than apical Apo E expression in cultured human RPE cells.
[0027] FIG. 6 shows regulation of Apo E expression by nuclear
hormone receptor ligands.
[0028] FIG. 7 provides a non-denatured polyacrylamide gel of
lipoprotein fractions.
[0029] FIG. 8 shows .sup.14C distribution of the fractions from
FIG. 7.
[0030] FIG. 9 demonstrates thin layer chromatography illustrating
the identification of six out of seventeen spots of an HDL
fraction. Note: HDL is the high density lipoprotein fraction; POS
is labeled POS starting material; PC is phophatidylcholine; PI is
phosphatidylinisotol; PE is phosphatidylethanolamine; C is
cholesterol; TRL is TG rich lipid, including triglycerides and
cholesterol ester.
[0031] FIG. 10 demonstrates that .sup.14C counts increase following
drug treatments that increase RCT.
[0032] FIG. 11 illustrates ABCA1 regulation by RXR and LXR
ligands.
[0033] FIG. 12 shows HDL, LDL and plasma stimulation of
.sup.14C-labeled lipid transport the identification of HDL from RPE
cells.
[0034] FIG. 13 shows stimulation of CD36 expression by oxidized
lipid.
[0035] FIG. 14 illustrates apical and basal secretion from RPE
cells of apoe in the presence of T.sub.3 (T), 22(R)
hydroxycholesterol, or cis retinoic acid (RA).
[0036] FIG. 15 shows that apoe secreted from RPE cells binds to
HDL.
[0037] FIG. 16 demonstrates that HDL stimulates lipid efflux from
RPE cells in culture.
[0038] FIG. 17 shows characterization of HDL and plasma bound POS
lipids by thin layer chromatography.
[0039] FIG. 18 shows plasma and HDL levels of species identified in
FIG. 17.
[0040] FIG. 19 shows measurement of .sup.14C-labeled lipid efflux
for no human high density lipoprotein (HDL) (Control); 100 .mu.g/ml
of human HDL; pure human apoA-I; or human apoA-I vesicles.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0041] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one. As used herein "another" may mean at least a second
or more.
[0042] The term "age-related macular degeneration" as used herein
refers to macular degeneration in an individual over the age of
about 50. In one specific embodiment, it is associated with
destruction and loss of the photoreceptors in the macula region of
the retina resulting in decreased central vision and, in advanced
cases, legal blindness.
[0043] The term "Bruch's membrane" as used herein refers to a
five-layered structure separating the choriocapillaris from the
RPE.
[0044] The term "HDL or subspecies thereof" refers to the fact that
high density lipoproteins (HDL) can be fractionated into
particulate species defined in molecular size and composition. HDL
as prepared by density ultracentrifugation and by native
nondenaturing purification processes including anti-apolipoprotein
A-I immunoaffinity chromatography have been characterized for its
constituent species by two-dimensional nondenaturing polyacrylamide
electrophoresis, immunoblotting, and mass spectroscopy. HDL has
been resolved into more than twenty-five particle species that
differ in charge and molecular size. Each particle is defined by a
unique combination of protein (including apolipoproteins A-I, A-II,
A-IV, A-V, C-III, D, E, J, L, lecithin:cholesterol acyltransferase,
cholesterol ester transferase, phospholipid transfer protein,
alpha-2 macroglobulin) and lipid (including phospholipid,
triglyceride, cholesterol, cholesterol ester, fatty acids). A
partial list of HDL species include HDL alpha-1, HDL alpha-2, HDL
alpha-3, HDL prebeta-1, HDL prebeta-2 (and variants "a", "b", "c",
"d"), HDL prebeta-3, HDL prebeta-4, and HDL prealpha-1.
[0045] The term "increase lipid efflux" or "increasing lipid
efflux" as used herein refers to an increased level and/or rate of
lipid efflux, promoting lipid efflux, enhancing lipid efflux,
facilitating lipid efflux, upregulating lipid efflux, improving
lipid efflux, and/or augmenting lipid efflux. In a specific
embodiment, the efflux comprises efflux of phospholipid,
triglyceride, cholesterol, and/or cholesterol ester.
[0046] A skilled artisan recognizes that the term "lipid
transporter" as used herein refers to a lipoprotein that carries
lipids away from peripheral cells into the circulation, and
examples include HDL and subspecies thereof, or a mixture thereof.
The term "lipid transporter" is also used in the art to refer to,
for example, transmembrane proteins that transport cholesterol and
phospholipids, for example, from inside a cell to outside the cell.
Examples include ABCA1, SR-BI, SR-BII, ABCA4, ABCG5, ABCG8, or a
mixture thereof.
[0047] The term "macula" as used herein refers to the light-sensing
cells of the central region of the retina.
[0048] The term "macular degeneration" as used herein refers to
deterioration of the central portion of the retina, the macula.
[0049] The term "reverse cholesterol transport" as used herein
refers to transport of cholesterol from peripheral tissues to the
liver. In a specific embodiment, it refers to efflux of lipid from
RPE cells. In specific embodiments, it comprises efflux of cellular
cholesterol and/or phospholipid to HDL, and, in further specific
embodiments, it comprises HDL delivery of cholesterol ester to the
liver, such as for biliary secretion.
[0050] The term "therapeutically effective" as used herein refers
to the amount of a compound required to improve some symptom
associated with a disease. For example, in the treatment of macular
degeneration, a compound which improves sight to any degree or
arrests any symptom of impaired sight would be therapeutically
effective. A therapeutically effective amount of a compound is not
required to cure a disease but will provide a treatment for a
disease.
[0051] The term "upregulate" as used herein is defined as
increasing the level and/or rate of an event, process, or
mechanism, such as reverse cholesterol transport and/or the
transcription and/or translation processes of a nucleic acid, such
as a gene.
II. The Present Invention
[0052] The present inventors have shown that HDL is a preferred
cholesterol and phospholipids acceptor for lipids effluxed by
cultured human RPE. In aging BM, there is progressive accumulation
of lipid and cross-linked protein that impedes hydraulic
conductivity and macromolecular permeability. This abnormal
deposition may also impair the ability of some larger molecular
weight species of HDL to act as a lipoprotein acceptor. As HDL is
unable to pass through BM and promote efflux and bind effluxed
lipids, more lipids accumulate in both RPE and BM. Indeed, such
accumulations are a major finding in age-related macular
degeneration.
[0053] Apolipoprotein A1 (ApoA-I), having a mass of approximately
28 kDaltons, is the major lipoprotein component of HDL. When bound
to phospholipids, it comprises nascent HDL particles that bind to
ABCA1 on the RPE basal membrane and promote lipid efflux. Because
of its low molecular weight, it can penetrate an aged BM more
easily than larger molecular weight species of HDL to bind to the
RPE. In addition to it role in promoting reverse cholesterol
transport from RPE, ApoA-I also is a potent anti-oxidant.
Anti-oxidants have been established to reduce visual loss in
patients with AMD.
[0054] Several methods are used to increase ApoA-I delivery to RPE
as a treatment for AMD, including administration intravenously as
has been done in mouse models of atherosclerosis and in patients
with coronary artery disease. ApoA-I, which is normally comprised
of L-amino acids, can be administered as an ApoA-I mimetic peptide
(e.g. amino acid sequence SEQ ID NO:15 and/or SEQ ID NO:16)
comprising D-amino acids. The D-amino acid based ApoA-I mimentic
peptide is not recognized as readily by human proteases, and thus
can be administered orally. In some embodiments, this would be more
convenient than parenteral administration with an intravenous
formulation containing the L-amino acid ApoA-I or its mimetic
peptide. Furthermore, oral synthetic phospholipid (1,2
Dimyristoyl-.alpha.-glycero-3-phosphocholine, DMPC) increases
levels of circulating ApoA-I.
[0055] By way of example, patients with AMD (atrophic or exudative)
are administered intravenously either ApoA-I, ApoA-I mimetic
peptide, an agent to increase levels of circulating ApoA-I, such as
DMPC, or a mixture thereof.
[0056] The histopathology of macula in patients with AMD shows
diffuse thickening of Bruch's membrane, and the overlying RPE is
attenuated and full of lipofuscin granules. Photoreceptors are
shortened and atrophic, and much of the thickened Bruch's membrane
consists of lipid deposition. It is known that following about 50
years of age, the rate of lipid accumulation accelerates (Holz et
al, 1994).
[0057] Using cell culture methods to study lipid metabolism, the
inventors have shown a number of analogous mechanisms for lipid
metabolism that are shared by macrophages and human RPE cells. The
shared biology of these two cell types indicates useful therapeutic
approaches for treatment of AMD. Specifically, the present
inventors are the first to show that RCT occurs in RPE cells, and
enhancement of RCT is beneficial for removing undesired lipid from
the RPE cells and/or Bruch's membrane to facilitate retinal
metabolism. In a specific embodiment, the transporters in the RCT
system are regulated to improve RCT. In a further specific
embodiment, this regulatory aspect of the present invention
provides a novel treatment for AMD.
[0058] Although there has been discussion in the field regarding
mechanisms of lipid accumulation in macula of AMD individuals, the
present invention regards efflux of lipid into the circulation,
which reduces the amount of lipid in RPE and/or Bruch's membrane.
Promotion of this efflux comprises one aspect of the invention and
is an effective therapy for both early and late AMD. A skilled
artisan recgonizes that early AMD comprises the presence of drusen
and late stage AMD comprises visual loss from choroidal
neovascularization or geographic atrophy.
[0059] Thus, the present invention provides the novel idea in the
field in which reverse cholesterol transport occurs in RPE cells.
In specific embodiments, the invention provides methods and
compositions related to facilitating efflux of cholesterol and/or
phospholipids from inside an RPE cell to the outside of the RPE
cell, and further through Bruch's membrane. In another specific
embodiment, following efflux from Bruch's membrane the cholesterol
and/or phospholipids are transported by apolipoprotein E,
apolipoprotein A1, and other transporters, or a combination
thereof, to HDL for removal to the liver.
[0060] A skilled artisan recognizes the important role reverse
cholesterol transport (RCT) plays in lipid homeostasis. HDL levels
are inversely correlated with incidence of coronary artery disease
(CAD). Tangier's disease, which comprises a mutation of ABCA1,
leads to deposition of cholesterol in reticuloendothelial tissues
and premature atherosclerosis. Furthermore, the Apo E null mouse is
an excellent model of atherosclerosis and hyperlipidemia.
Interestingly, supporting an important role of Apo E in RCT,
reconstitution of Apo E positive macrophages via bone marrow
transplant into an Apo E null mouse prevents atherosclerosis. This
occurs in spite of persistent hyperlipidemia.
[0061] In one embodiment of the present invention a transporter of
lipid from RPE cells is enhanced for the transport activity, such
as by an increase in the level of the transporter. Examples of
transporters include apo E, ABCA1, SR-BI, SR-BII, ABCA4, ABCG5,
ABCG8; other proteins that might be involved are LCAT, CETP, PLTP,
LRP receptor, LDL receptor, Lox-1, and lipases. In a specific
embodiment, lox-1 and PLTP are expressed in RPE, as demonstrated by
RT_PCR. In a specific embodiment of the present invention, ApoA-I
is utilized to facilitate RCT from RPE cells. In an additional
specific embodiment, ApoA-I is made by RPE cells.
[0062] In a specific embodiment of the present invention,
strategies for intervention for treatment of AMD are provided in
which reverse cholesterol transport is enhanced at the level of the
RPE by upregulating ApoA-I, ABCA1, Apo E, SR-BI and/or SR-BII
expression. SR-B has been reported to be upregulated by
17beta-Estradiol and testosterone. Additionally, or alone, HDL
binding to effluxed lipids is enhanced, thereby increasing efflux
of lipids from Bruch's membrane into the circulation and providing
therapy for AMD. Although the present invention generally regards
an increase in ApoA-I, a major lipoprotein component of HDL, in one
embodiment, an increase in HDL levels overall is utilized to
facilitate lipid efflux from RPE cells and/or Bruch's membrane, and
in a specific embodiment, levels of specific subspecies of HDL are
utilized to facilitate lipid efflux. For example, effluxed lipids
could bind to pre.beta.-HDL, HDL1, HDL2 or HDL3. Effluxed lipids
could also bind prebeta-1, prebeta-2, prebeta-3, and/or prebeta-4
HDL. In a specific embodiment, the effluxed lipids bind
preferentially to HDL2 that comprises apo E.
[0063] One skilled in the art recognizes particular RCT components
are present in RPE cells (Mullins et al., 2000; Anderson et al.,
2001). Nuclear hormone receptors known to regulate expression of
reverse cholesterol transport proteins are also expressed in
cultured human RPE. Thus, in a preferred embodiment of the present
invention, ligands to at least one of the nuclear hormone receptors
upregulates RCT. In further embodiments, following efflux from RPE
cells, the lipids bind HDL, so in an embodiment of the present
invention there is upregulation of HDL for AMD treatment, such as
by statins and/or niacin.
[0064] In an alternative embodiment, treatment for AMD comprises
reduction of RCT. For example, in individuals past a certain age,
such as about 50, 55, 60, 65, 70, 75, 80, and so on, the
transporters are preferentially inhibited. In one aspect of this
embodiment, HDL is unable to enter Bruch's membrane to remove the
lipids and the RPE continues to efflux lipids. In such cases where
effluxed lipids from RPE cannot be removed by a lipoprotein
acceptor, lipid efflux by RPE is inhibited to maintain
macromolecular transport across Bruch's membrane. Inhibition of RCT
by reducing levels of ABCA-1, apo E, and/or SRB-1, or SRB-2 would
reduce accumulation of lipid in Bruch's membrane.
[0065] In embodiments of the present invention, ligands for nuclear
hormone receptors are utilized as compounds for enhancing RCT for
the reduction of lipid content of RPE and Bruch's membrane. In a
specific embodiment, the nuclear hormone receptor ligands are
utilized for treatment of AMD. In a further specific embodiment,
the nuclear hormone receptors comprise TR, RXR, and/or LXR. In
other specific embodiments, ligands of the nuclear hormone
receptors are delivered to at least one RPE cell to facilitate
efflux of lipids from the RPE cell and/or are delivered to Bruch's
membrane for efflux from Bruch's membrane. Examples of ligands for
TR include T3 (3,5,3'-L-triiodothyronine). Other examples of TR
ligands include but are not limited to TRIAC (3-triiodothyroacetic
acid); KB141 (Karo Bio); GC-1; and 3,5
dimethyl-3-isopropylthyronine. Examples of ligands for RXR include
9 cis-retinoic acid, and other RXR ligands also include but are not
limited to: AGN 191659
[(E)-5-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)propen-1-y-
l]-2-thiophenecarboxylic acid]; AGN 191701 [(E)
2-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)propen-1-yl]-4--
thiophene-carboxylic acid]; AGN 192849
[(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl) (5
carboxypyrid-2-yl)sulfide]; LGD346; LG100268; LG100754; BMS649; and
bexaroteneR (Ligand Pharmaceuticals)
(4-[1-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)ethenyl]be-
nzoic acid). Examples of ligands for LXR include 22 (R)
hydroxycholesterol, acetyl-podocarpic dimer, T0901317, and
GW3965.
[0066] In an embodiment of the present invention, expression of a
sequence is monitored following administration of an upregulator of
its expression or a compound suspected to be an upregulator. A
skilled artisan recognizes how to obtain these sequences, such as
commercially from Celera Genomics, Inc. (Rockville, Md.) or from
the National Center for Biotechnology Information's GenBank
database. Exemplary apo E polynucleotide sequences include the
following, cited with their GenBank Accession number: SEQ ID NO:1
(K00396); SEQ ID NO:2 (M10065); and SEQ ID NO:3 (M12529). Some
exemplary apo E polypeptide sequences include the following, cited
with their GenBank Accession number: SEQ ID NO:4 (AAB59546); SEQ ID
NO:5 (AAB59397); and SEQ ID NO:6 (AAB59518).
[0067] In other embodiments, sequences of ABCA-1 are utilized, such
as to monitor ABCA-1 expression related to methods of the present
invention. Some examples of ABCA1 polynucleotides include SEQ ID
NO:7 (NM.sub.--005502); and SEQ ID NO:8 (AB055982). Some examples
of ABCA1 polypeptides include SEQ ID NO:9 (NP.sub.--005493); and
SEQ ID NO:10 (BAB63210).
[0068] In some methods of the present invention, expression levels
of sequences of SR-BI and SR-B2 polynucleotides are monitored
following administration of a nuclear hormone receptor ligand. An
example of SR-BI polynucleotide is SEQ ID NO:11 (NM.sub.--005505)
and an example of a SR-BI polypeptide is SEQ ID NO:12
(NP.sub.--005496).
III. Pharmaceutical Compositions and Routes of Administration
[0069] Compositions of the present invention may have an effective
amount of a compound for therapeutic administration and, in some
embodiments, in combination with an effective amount of a second
compound that is also an anti-AMD agent. In a specific embodiment,
the compound is a ligand/agonist of a nuclear hormone receptor. In
other embodiments, compounds that upregulate expression of HDL are
the compounds for therapeutic administration. Such compositions
will generally be dissolved or dispersed in a pharmaceutically
acceptable carrier or aqueous medium.
[0070] The phrases "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce an adverse, allergic or other untoward reaction when
administered to an animal, or human, as appropriate. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredients, its use in
the therapeutic compositions is contemplated. Supplementary active
ingredients, such as other anti-AMD agents, can also be
incorporated into the compositions.
[0071] In addition to the compounds formulated for parenteral
administration, such as intravenous or intramuscular injection,
other pharmaceutically acceptable forms include, e.g., tablets or
other solids for oral administration; time release capsules; and
any other form currently used, including cremes, lotions,
mouthwashes, inhalants and the like.
[0072] The delivery vehicles of the present invention may include
classic pharmaceutical preparations. Administration of these
compositions according to the present invention will be via any
common route so long as the target ocular tissue is available via
that route. This includes oral, nasal, buccal, rectal, vaginal or
topical. Alternatively, administration may be by orthotopic,
intradermal, subcutaneous, intramuscular, intraperitoneal or
intravenous injection. Such compositions would normally be
administered as pharmaceutically acceptable compositions. In some
embodiments, the compositions are administered by sustained release
intra- or extra-ocular devices.
[0073] The vehicles and therapeutic compounds therein of the
present invention are advantageously administered in the form of
injectable compositions either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection also may be prepared. These preparations also
may be emulsified. A typical composition for such purposes
comprises a 50 mg or up to about 100 mg of human serum albumin per
milliliter of phosphate buffered saline. Other pharmaceutically
acceptable carriers include aqueous solutions, non-toxic
excipients, including salts, preservatives, buffers and the like.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oil and injectable organic esters, such as
theyloleate. Aqueous carriers include water, alcoholic/aqueous
solutions, saline solutions, parenteral vehicles such as sodium
chloride, Ringer's dextrose, etc. Intravenous vehicles include
fluid and nutrient replenishers. Preservatives include
antimicrobial agents, anti-oxidants, chelating agents and inert
gases. The pH and exact concentration of the various components in
the pharmaceutical are adjusted according to well-known
parameters.
[0074] Additional formulations are suitable for oral
administration. Oral formulations include such typical excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. The compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders. When the route is topical, the form may be
a cream, ointment, salve or spray.
[0075] An effective amount of the therapeutic agent is determined
based on the intended goal. The term "unit dose" refers to a
physically discrete unit suitable for use in a subject, each unit
containing a predetermined quantity of the therapeutic composition
calculated to produce the desired response in association with its
administration, i.e., the appropriate route and treatment regimen.
The quantity to be administered, both according to number of
treatments and unit dose, depends on the subject to be treated, the
state of the subject and the protection desired. Precise amounts of
the therapeutic composition also depend on the judgment of the
practitioner and are peculiar to each individual.
[0076] All of the essential materials and reagents required for AMD
treatment, diagnosis and/or prevention may be assembled together in
a kit. When the components of the kit are provided in one or more
liquid solutions, the liquid solution preferably is an aqueous
solution, with a sterile aqueous solution being particularly
preferred.
[0077] For in vivo use, an anti-AMD agent may be formulated into a
single or separate pharmaceutically acceptable syringeable
composition. In this case, the container means may itself be an
inhalant, syringe, pipette, eye dropper, or other such like
apparatus, from which the formulation may be applied to an infected
area of the body, such as the lungs, injected into an animal, or
even applied to and mixed with the other components of the kit.
[0078] The components of the kit may also be provided in dried or
lyophilized forms. When reagents or components are provided as a
dried form, reconstitution generally is by the addition of a
suitable solvent. It is envisioned that the solvent also may be
provided in another container means. The kits of the invention may
also include an instruction sheet defining administration of the
anti-AMD composition.
[0079] The kits of the present invention also will typically
include a means for containing the vials in close confinement for
commercial sale such as, e.g., injection or blow-molded plastic
containers into which the desired vials are retained. Irrespective
of the number or type of containers, the kits of the invention also
may comprise, or be packaged with, an instrument for assisting with
the injection/administration or placement of the ultimate complex
composition within the body of an animal. Such an instrument may be
an inhalant, syringe, pipette, forceps, measured spoon, eye dropper
or any such medically approved delivery vehicle.
[0080] The active compounds of the present invention will often be
formulated for parenteral administration, e.g. formulated for
injection via the intravenous, intramuscular, subcutaneous, or even
intraperitoneal routes. The preparation of an aqueous composition
that contains a second agent(s) as active ingredients will be known
to those of skill in the art in light of the present disclosure.
Typically, such compositions can be prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for using
to prepare solutions or suspensions upon the addition of a liquid
prior to injection can also be prepared; and the preparations can
also be emulsified.
[0081] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0082] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases the form must be sterile and
must be fluid to the extent that easy syringability exists. It must
be stable under the conditions of manufacture and storage and must
be preserved against the contaminating action of microorganisms,
such as bacteria and fungi.
[0083] The active compounds may be formulated into a composition in
a neutral or salt form. Pharmaceutically acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like.
[0084] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and/or antifungal agents, for example,
parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the
like. In many cases, it will be preferable to include isotonic
agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0085] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0086] In certain cases, the therapeutic formulations of the
invention could also be prepared in forms suitable for topical
administration, such as in eye drops, cremes and lotions.
[0087] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, with even drug release capsules and the
like being employable.
[0088] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intraocular, intravenous, intramuscular, and
subcutaneous administration. In this connection, sterile aqueous
media that can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 mL of isotonic NaCl solution and either
added to 1000 mL of hypodermoclysis fluid or injected at the
proposed site of infusion, (see for example, "Remington's
Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and
1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject.
[0089] Targeting of ocular tissues may be accomplished in any one
of a variety of ways. In one embodiment, there is the use of
liposomes to target a compound of the present invention to the eye,
and preferably to RPE cells and/or Bruch's membrane. For example,
the compound may be complexed with liposomes in the manner
described above, and this compound/liposome complex injected into
patients with AMD, using intravenous injection to direct the
compound to the desired ocular tissue or cell. Directly injecting
the liposome complex into the proximity of the RPE or Bruch's
membrane can also provide for targeting of the complex with some
forms of AMD. In a specific embodiment, the compound is
administered via intra-ocular sustained delivery (such as
Vitrasert.RTM. or Envision.RTM. by Bauch and). In a specific
embodiment, the compound is delivered by posterior subtenons
injection. In another specific embodiment, microemulsion particles
with apo E (such as, recombinant) are delivered to ocular tissue to
take up lipid from Bruch's membrane, RPE cells, or both.
[0090] Those of skill in the art will recognize that the best
treatment regimens for using compounds of the present invention to
treat AMD can be straightforwardly determined. This is not a
question of experimentation, but rather one of optimization, which
is routinely conducted in the medical arts. In vivo studies in nude
mice often provide a starting point from which to begin to optimize
the dosage and delivery regimes. The frequency of injection will
initially be once a week, as has been done in some mice studies.
However, this frequency might be optimally adjusted from one day to
every two weeks to monthly, depending upon the results obtained
from the initial clinical trials and the needs of a particular
patient. Human dosage amounts can initially be determined by
extrapolating from the amount of compound used in mice, as a
skilled artisan recognizes it is routine in the art to modify the
dosage for humans compared to animal models. In certain embodiments
it is envisioned that the dosage may vary from between about 1 mg
compound/Kg body weight to about 5000 mg compound/Kg body weight;
or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight
or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight;
or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight;
or from about 100 mg/Kg body weight to about 1000 mg/Kg body
weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body
weight. In other embodiments this dose may be about 1, 5, 10, 25,
50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,
1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500,
3000, 3500, 4000, 4500, 5000 mg/Kg body weight. In other
embodiments, it is envisaged that higher does may be used, such
doses may be in the range of about 5 mg compound/Kg body to about
20 mg compound/Kg body. In other embodiments the doses may be about
8, 10, 12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage
amount may be adjusted upward or downward, as is routinely done in
such treatment protocols, depending on the results of the initial
clinical trials and the needs of a particular patient.
[0091] In some embodiments of the present invention, the ApoA-I
composition is comprised as a polynucleotide, utilizing delivery
vehicles well known in the art. In other embodiments of the present
invention, ApoA-I composition comprises a polypeptide or peptide.
Any form may be distributed in a delivery composition, such as a
liposome, examples of which are known in the art.
IV. Kits
[0092] Any of the compositions described herein may be comprised in
a kit. In a non-limiting example, an ApoA-I composition, and in
some embodiments, at least one additional agent, may be comprised
in a kit. In other embodiments, a lipid transporter such as HDL or
a subspecies thereof.
[0093] The kits may comprise a suitably aliquoted ApoA-I
composition, and/or additional agent compositions of the present
invention, whether labeled or unlabeled, as may be used to prepare
a standard curve for treatment of macular degeneration, such as
AMD. The components of the kits may be packaged in aqueous media or
in lyophilized form. When reagents and/or components are provided
as a dry powder, the powder can be reconstituted by the addition of
a suitable solvent. It is envisioned that the solvent may also be
provided in another container means.
[0094] Suitable ApoA-I compositions comprise those that are
sufficient to upregulate reverse cholesterol transport, those that
reduce lipid accumulations in BM or RPE, those that increase efflux
of lipids from BM, and/or are sufficient to provide an anti-oxidant
therapeutic effect. In preferred embodiments, the ApoA-I
compositions are suitable to provide therapy for macular
degeneration, such as by ameliorating and/or preventing at least
one symptom.
[0095] The container means of the kits will generally include at
least one vial, test tube, flask, bottle, syringe or other
container means, into which a component may be placed, and
preferably, suitably aliquoted. Where there are more than one
component in the kit, the kit also will generally contain a second,
third or other additional container into which the additional
components may be separately placed. However, various combinations
of components may be comprised in a vial. The kits of the present
invention also will typically include a means for containing the
nuclear hormone receptor ligand, additional agent, and any other
reagent containers in close confinement for commercial sale. Such
containers may include injection or blow-molded plastic containers
into which the desired vials are retained.
V. Biological Functional Equivalents
[0096] As modifications and/or changes may be made in the structure
of ApoA-I polypeptides or peptides according to the present
invention, while obtaining molecules having similar or improved
characteristics, such biologically functional equivalents are also
encompassed within the present invention.
[0097] A. Modified Polynucleotides and Polypeptides
[0098] Although administration of ApoA-I peptides or polypeptides
is preferable, in some embodiments the ApoA-I composition is a
polynucleotide encoding the desired polypeptide or peptide. The
biological functional equivalent may comprise a polynucleotide that
has been engineered to contain distinct sequences while at the same
time retaining the capacity to encode the "wild-type" or standard
protein or other polypeptide or peptide of interest. This can be
accomplished to the degeneracy of the genetic code, i.e., the
presence of multiple codons, which encode for the same amino acids.
In one example, one of skill in the art may wish to introduce a
restriction enzyme recognition sequence into a polynucleotide while
not disturbing the ability of that polynucleotide to encode a
protein.
[0099] In another example, a polynucleotide may encode a biological
functional equivalent with more significant changes. Certain amino
acids may be substituted for other amino acids in a protein
structure without appreciable loss of interactive binding capacity
with structures such as, for example, antigen-binding regions of
antibodies, binding sites on substrate molecules, receptors, and
such like. So-called "conservative" changes do not disrupt the
biological activity of the protein, as the structural change is not
one that impinges of the protein's ability to carry out its
designed function. It is thus contemplated by the inventors that
various changes may be made in the sequence of genes and proteins
disclosed herein, while still fulfilling the goals of the present
invention.
[0100] In terms of functional equivalents, it is well understood by
the skilled artisan that, inherent in the definition of a
"biologically functional equivalent" protein and/or polynucleotide,
is the concept that there is a limit to the number of changes that
may be made within a defined portion of the molecule while
retaining a molecule with an acceptable level of equivalent
biological activity. Biologically functional equivalents are thus
defined herein as those proteins (and polynucleotides) in selected
amino acids (or codons) may be substituted. Functional activity,
such as the ability to bind lipids, is preferably retained in any
natural or synthetic ApoA-I polypeptide or peptide.
[0101] In general, the shorter the length of the molecule, the
fewer changes that can be made within the molecule while retaining
function. Longer domains may have an intermediate number of
changes. The full-length protein will have the most tolerance for a
larger number of changes. However, it must be appreciated that
certain molecules or domains that are highly dependent upon their
structure may tolerate little or no modification.
[0102] Amino acid substitutions are generally based on the relative
similarity of the amino acid side-chain substituents, for example,
their hydrophobicity, hydrophilicity, charge, size, and/or the
like. An analysis of the size, shape and/or type of the amino acid
side-chain substituents reveals that arginine, lysine and/or
histidine are all positively charged residues; that alanine,
glycine and/or serine are all a similar size; and/or that
phenylalanine, tryptophan and/or tyrosine all have a generally
similar shape. Therefore, based upon these considerations,
arginine, lysine and/or histidine; alanine, glycine and/or serine;
and/or phenylalanine, tryptophan and/or tyrosine; are defined
herein as biologically functional equivalents.
[0103] To effect more quantitative changes, the hydropathic index
of amino acids may be considered. Each amino acid has been assigned
a hydropathic index on the basis of their hydrophobicity and/or
charge characteristics, these are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and/or arginine (-4.5).
[0104] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte and Doolittle, 1982,
incorporated herein by reference). It is known that certain amino
acids may be substituted for other amino acids having a similar
hydropathic index and/or score and/or still retain a similar
biological activity. In making changes based upon the hydropathic
index, the substitution of amino acids whose hydropathic indices
are within .+-.2 is preferred, those which are within .+-.1 are
particularly preferred, and/or those within .+-.0.5 are even more
particularly preferred.
[0105] It also is understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biological functional
equivalent protein and/or peptide thereby created is intended for
use in immunological embodiments, as in certain embodiments of the
present invention. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and/or antigenicity,
i.e., with a biological property of the protein.
[0106] 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). In making changes based
upon similar hydrophilicity values, the substitution of amino acids
whose hydrophilicity values are within .+-.2 is preferred, those
which are within .+-.1 are particularly preferred, and/or those
within .+-.0.5 are even more particularly preferred.
[0107] B. Altered Amino Acids
[0108] The present invention, in some aspects, may rely on the
synthesis of peptides and polypeptides in cyto, via transcription
and translation of appropriate polynucleotides. In alternative
embodiments, the polypeptide or peptide is synthesized outside a
cell, such as chemically. These peptides and polypeptides may
include the twenty "natural" amino acids, and, in some embodiments,
post-translational modifications thereof. However, in vitro peptide
synthesis permits the use of modified and/or unusual amino acids. A
table of exemplary, but not limiting, modified and/or unusual amino
acids is provided herein below.
TABLE-US-00001 TABLE 1 Modified and/or Unusual Amino Acids Abbr.
Amino Acid Abbr. Amino Acid Aad 2-Aminoadipic acid EtAsn
N-Ethylasparagine BAad 3-Aminoadipic acid Hyl Hydroxylysine BAla
beta-alanine, beta-Amino- AHyl allo-Hydroxylysine propionic acid
Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline 4Abu 4-Aminobutyric
acid, 4Hyp 4-Hydroxyproline piperidinic acid Acp 6-Aminocaproic
acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid Aile
allo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,
sarcosine BAib 3-Aminoisobutyric acid MeIle N-Methylisoleucine Apm
2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric
acid MeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm
2,2'-Diaminopimelic acid Nle Norleucine Dpr 2,3-Diaminopropionic
acid Orn Ornithine EtGly N-Ethylglycine
[0109] C. Mimetics
[0110] In addition to the biological functional equivalents
discussed above, the present inventors also contemplate that
structurally similar compounds may be formulated to mimic the key
portions of peptide or polypeptides of the present invention. Such
compounds, which may be termed peptidomimetics, may be used in the
same manner as the peptides of the invention and, hence, also are
functional equivalents. In a specific embodiment, the key portion
comprises lipid binding activity.
[0111] Certain mimetics that mimic elements of protein secondary
and tertiary structure are described in Johnson et al. (1993). The
underlying rationale behind the use of peptide mimetics is that the
peptide backbone of proteins exists chiefly to orient amino acid
side chains in such a way as to facilitate molecular interactions,
such as those of antibody and/or antigen. A peptide mimetic is thus
designed to permit molecular interactions similar to the natural
molecule.
[0112] Some successful applications of the peptide mimetic concept
have focused on mimetics of .beta.-turns within proteins, which are
known to be highly antigenic. Likely .beta.-turn structure within a
polypeptide can be predicted by computer-based algorithms, as
discussed herein. Once the component amino acids of the turn are
determined, mimetics can be constructed to achieve a similar
spatial orientation of the essential elements of the amino acid
side chains.
[0113] Other approaches have focused on the use of small,
multidisulfide-containing proteins as attractive structural
templates for producing biologically active conformations that
mimic the binding sites of large proteins. Vita et al. (1998). A
structural motif that appears to be evolutionarily conserved in
certain toxins is small (30-40 amino acids), stable, and high
permissive for mutation. This motif is composed of a beta sheet and
an alpha helix bridged in the interior core by three
disulfides.
[0114] Beta II turns have been mimicked successfully using cyclic
L-pentapeptides and those with D-amino acids. Weisshoff et al.
(1999). Also, Johannesson et al. (1999) report on bicyclic
tripeptides with reverse turn inducing properties.
[0115] Methods for generating specific structures have been
disclosed in the art. For example, alpha-helix mimetics are
disclosed in U.S. Pat. Nos. 5,446,128; 5,710,245; 5,840,833; and
5,859,184. Theses structures render the peptide or protein more
thermally stable, also increase resistance to proteolytic
degradation. Six, seven, eleven, twelve, thirteen and fourteen
membered ring structures are disclosed.
[0116] Methods for generating conformationally restricted beta
turns and beta bulges are described, for example, in U.S. Pat. Nos.
5,440,013; 5,618,914; and 5,670,155. Beta-turns permit changed side
substituents without having changes in corresponding backbone
conformation, and have appropriate termini for incorporation into
peptides by standard synthesis procedures. Other types of mimetic
turns include reverse and gamma turns. Reverse turn mimetics are
disclosed in U.S. Pat. Nos. 5,475,085 and 5,929,237, and gamma turn
mimetics are described in U.S. Pat. Nos. 5,672,681 and
5,674,976.
EXAMPLES
[0117] The following is an illustration of preferred embodiments
for practicing the present invention. However, they are not
limiting examples. Other examples and methods are possible in
practicing the present invention.
Example 1
Materials and Methods
Cell Culture and Drug Treatments
[0118] Primary cultures of normal human RPE cells from passages 5
to 10 were used for the experiments described. RPE cells were grown
to confluence on laminin-coated 6 well Transwell tissue culture
plates (Costar) with DMEM-H21 containing 10% fetal bovine serum, 2
mM glutamine, 50 .mu.g/ml gentamicin and 2.5 mg/ml fungizone in the
top and bottom chambers. For immunofluorescent staining cells were
grown on laminin coated slides in the same medium. Cells were grown
for at least 1 week at confluence prior to drug treatment. Cells to
be treated with drugs were incubated in serum free DMEM-H21 prior
to drug addition. Drug treatments were in serum free DMEM-H21 with
or without 10.sup.-7 M thyroid hormone (T.sub.3),
2.5.times.10.sup.-6 M 22 (R) hydroxycholesterol, or 10.sup.-7 M cis
retinoic acid in both chambers for 36 hours.
RT-PCR
[0119] Confluent cell cultures were harvested and total RNA was
purified using RNAzol (Teltest, Inc., Friendswood, Tex.) according
to the manufacturer's instructions. Equal amounts of purified RNA
were used in each reaction as templates for cDNA synthesis using
the 1st Strand Synthesis Kit for RT-PCR (AMV) (Boehringer,
Indianapolis, Ind.). RT-PCR was carried out on 1 .mu.g of cDNA with
Amplitaq Taq polymerase (Perkin-Elmer, Branchburg, N.J.). In some
experiments apo E RT-PCR products were quantified using the
QuantumRNA assay kit according to the manufacturer's instructions
(Ambion, Austin, Tex.). Briefly, 18S rRNA and apo E cDNAs are
simultaneously amplified in each reaction. The RT-PCR products are
resolved by electrophoresis on 1.4% agarose gels. The apo E mRNA
expression is assessed relative to the internal 18S rRNA expression
by densitometric analysis of photographed agarose gels.
[0120] RT-PCR primers specific to human apo E, ABCA1, SR-BI,
SR-BII, and lxr .alpha. were used. The RT-PCR product of the
predicted sizes for the apo E, ABCA1, SR-BI, and SR-BII RT-PCR
products were excised form the gel and their identities were
confirmed by DNA sequencing (not shown).
Immunofluoresence Microscopy
[0121] RPE cells, grown on slides, were
.sigma..tau..alpha..nu..epsilon..delta. with either antisera to
ABCA1, or with purified antibodies to SR-BI or SR-BII. Cells were
fixed in ice cold 100% MeOH for 20 min. All subsequent steps were
performed at room temperature. Cells were washed in phosphate
buffered saline (PBS) and incubated for in 5% goat serum in PBS for
30 min. Cells were then washed in buffer A (150 mM NaCl, 10 mM
phosphate, pH 7.8) and incubated with the primary antibody in
buffer A for 45 min. After washing with buffer A the cells were
incubated in Avidin Blocking Reagent (Vector Laboratories,
Burlingame, Calif.) for 15 min, washed in buffer A again and
incubated in Biotin Blocking Reagent (Vector Laboratories,
Burlingame, Calif.) for 15 min. After washing in buffer A, cells
were incubated in 10 .mu.g/ml biotinylated goat anti-rabbit IgG
(Vector Laboratories, Burlingame, Calif.) in buffer A for 30 min,
washed in buffer A and incubated in 20 .mu.g/ml fluorescein
conjugated avidin D (Vector Laboratories, Burlingame, Calif.) in
buffer B (150 mM NaCl, 100 mM sodium bicarbonate, pH 8.5) for 30
min. The cells were washed in buffer B and a cover slip was added
to each slide, over a few drops of Vectashield (Vector
Laboratories, Burlingame, Calif.). The slides were stored in the
dark until ready for microscopic examination.
Apo E Western Blotting
[0122] Cells were treated with Media was concentrated 20-fold by
centrifugal ultrafiltration (VIVA SPIN 20, MCO 5,000, Viva
Sciences, Hannover, Germany), dialyzed against 0.15M NaCl, 1 mM
sodium EDTA, 0.025% sodium azide (SalEN). Total protein content was
determined by a modified Lowry assay (Bio-Rad DC kit, Richmond,
Calif.). Concentrated media (50 .mu.g protein) was made to Start
Buffer (0.025 M NaCl, 0.010 M tris (pH 8.5), 5 mM MnCl.sub.2) and
adsorbed onto a 0.1 ml column containing Heparin-Sepharose CL-4B
(Pharmacia, Uppsala, Sweden). Following a 2 ml wash in Start
Buffer, the apo E containing bound fraction was eluted with 0.5M
NaCl in Start buffer. The eluate was concentrated to 20 .mu.l and
buffer-exchanged to SalEN by centrifugal ultrafiltration (Biomax,
5k MCO, Millipore, Bedford, Mass.). Apo E was resolved by
tris-tricine buffered SDS-PAGE (5-25% linear acrylamide gradient)
and proteins electrophoetically transferred (55V, 18 h) to
nitrocelluose membrane filters (Schleicher and Shuell, Keen, N.H.).
Membranes were blocked with 10% bovine serum albumin at room
temperature and probed with 1% goat anti-human apo E antiserum (18
h, 3.degree. C.) prepared in 0.15% NaCl, 1 mM EDTA (pH 7.4), 0.1%
Triton X-100 (SalET). The primary-bound anti-apo E antibodies were
detected calorimetrically with horseradish peroxidase conjugated
rabbit anti-goat Ig (H+L) and NiCl.sub.2-enhanced diaminobenzine
staining. Stained bands were compared densitometrically from the
digitized scanned image (NIH Image, v. 1.62). Anti apo E antibodies
were obtained by hyper-immunization of goats with purified apo E or
obtained from Assay Designs (A299, Ann Arbor, Mich.).
[0123] Lipoprotein fractions were prepared from conditioned media
that was adjusted with solid KBr to a density of 1.21 g/ml. Samples
were ultracentrifuged in a Beckman 42.2 Ti rotor at 40,000 rpm for
18 h at 1.degree. C. The lipoprotein and lipoprotein-free
fractions, the top and bottom 50 .mu.l, respectively, were dialysed
against SalEN prior to analysis.
[.sup.14C] Docosohexanoic Acid (DHA) Labeled POS Uptake and
Transport
[0124] Bovine outer photoreceptor outer segments (POS) were labeled
by incubating for with Coenzyme A, ATP, Mg.sup.2+, and
[.sup.14C]-DHA. Cells grown on laminin coated Transwell plates were
incubated with 12 .mu.g/ml labeled POS in the apical chamber for 36
hours in medium containing 10% lipoprotein deficient fetal bovine
serum. The basal medium was subjected to scintillation counting to
determine the amount of [.sup.14C] labeled lipids transported
through the RPE cells.
Identification of Acceptors for Exported .sup.14C Lipids
[0125] Bovine outer photoreceptor outer segments (POS) were labeled
by incubating for with Coenzyme A, ATP, Mg.sup.2+, and
[.sup.14C]-DHA. Cells grown on laminin coated Transwell plates were
incubated with 12 .mu.g/ml labeled POS in the apical chamber for 36
hours in medium containing 10% lipoprotein deficient fetal bovine
serum. The basal chambers contained either 1 mg/ml human HDL, 1
mg/ml human LDL or 100% human plasma. The basal medium was
collected and lipoproteins were repurified from by potassium
bromide density gradient centrifugation at d=1.21 g/ml (Beckman
42.2 Ti rotor, 40,000 rpm, 18 h, 10.degree. C.), dialyzed, and
resolved by size in nondenaturing 0-35% PAGE. Gels were stained
with coomassie blue R-250. Gel lanes were sectioned into thirty 2
mm slices that were digested (TS-1, Research Products
International) and radioactivity quantified by liquid scintillation
spectrometry.
Example 2
Expression of Transporters in RPE Cells
[0126] One skilled in the art recognizes that certain RCT
components in cultured human RPE cells have been demonstrated
(Mullins et al., 2000; Anderson et al., 2001). Nuclear hormone
receptors known to regulate expression of reverse cholesterol
transport proteins are also expressed in cultured human RPE.
[0127] A skilled artisan recognizes that there is expression of TRs
and RXRs in RPE cells in culture (Duncan et al., 1999). RT-PCR of
human RPE cell cDNA revealed that these cells also express mRNAs
for apo E, ABCA1, SR-BI, SR-BII and lxr .alpha.. As shown in FIG. 1
lane 1, FIG. 1 lane 2 and FIG. 1 lane 3, RPE cells express mRNAs
for apo E, ABCA1 and lxr .alpha., respectively.
[0128] As shown in FIG. 2, lane 1, and FIG. 2, lane 2, RPE cells
express mRNA for SR-BI and SR-BII respectively.
[0129] Furthermore, in immunofluoresence microscopy experiments,
RPE cells stain strongly for SR-BI (FIG. 3A) and SR-BII (FIG. 3B).
Control non-specific IgG or antibody vehicle did not stain RPE
cells (FIGS. 3C and 3D, respectively). Expression of SR-BI and
SR-BII in these cells was confirmed by PCR.
[0130] Expression of ABCA1 protein was demonstrated by
immunofluorescent staining of RPE cells with an antibody to ABCA1
(FIG. 4). Cell nuclei were stained with DAPI.
Example 3
Regulation of Apo E Secretion in RPE Cells
[0131] In order to distinguish apical (A) from basally (B) secreted
apo E, RPE cells were cultured on laminin-coated Transwell plates.
Specifically, human cultured RPE (passage 2-10, 35 y.o. donor) were
placed on laminin-coated Transwell plates, wherein the upper and
lower wells both had serum-free media. Total protein and apo
E-specific protein concentrations were measured from media pooled
and concentrated from 3-6 replicate wells. To assess apo E-specific
secretion, apo E was purified from conditioned media by
heparin-sepharose affinity chromatography and visualized by western
blotting. Apo E concentrations were consistently greater in the
basolateral media (FIG. 5, lane 1 vs. lane 2). These data
demonstrate that RPE cells display polarized secretion of cellular
proteins, including apo E. Thus, this indicated that Apo E is
preferentially secreted basally, supporting its role in RCT.
[0132] Since RPE cells express lxr .alpha. as well as thyroid
hormone receptors (TRs) and retinoid-X-receptors (RXRs), the effect
of 10.sup.-7 M T3, 2.5.times.10.sup.-6 M 22 (R) hydroxycholesterol
(HC) (an lxr .alpha..alpha.agonist), or 10.sup.-7 M cis retinoic
acid (cRA) (an RXR agonist) on apo E secretion from RPE cells was
tested. FIG. 6 illustrates the same experimental procedure as
described above, but with basal and apical media both containing
the following compounds for a 36 hour incubation: T3 (10.sup.-7) M
(T); 9 cis-RA (10.sup.-6) M (RA); and 22 (R) hydroxycholesterol 2.5
(10.sup.-6) M (HC). The basal media was analyzed for Apo E
expression with Western blot, and the results showed increased
basal expression of Apo E with the compound treatments. Thus, as
before, polarized apo E secretion was observed and, in this case,
occurred in the presence of T3, HC or cRA, indicating that an
increase in levels of basally secreted apo E is the result of
administration of these compounds to RPE cells.
Example 4
Assay of Efflux from RPE Cells
[0133] This example characterizes efflux of POS residues from RPE
cells, particularly regarding binding to HDL. Giusto et al. (1986)
describes a method of .sup.14C decoshexanoic acid (DHA) labeling of
bovine photoreceptor outer segment (POS) lipids. Generally, an
approximately 36 hour incubation over human RPE cells wherein the
basal medium contains plasma, HDL, or LDL is followed by
centrifugation of the basal media to collect lipoprotein fraction,
which is then analyzed to determine distribution of
radioactivity.
[0134] Specifically, bovine photoreceptor outer segment (POS) are
labeled with .sup.14C decoshexanoic acid (DHA) and placed in
lipoprotein deficient media. Following this, they are placed over
cultured human RPE on Transwell plates for 36 hours, and the basal
medium contained either 100% plasma, HDL (1 mg/cc) or LDL (1
mg/cc). After 36 hours, basal media was centrifuged to collect
lipoprotein fraction (density 1.2). This fraction was then run on a
non-denaturing gel and stained with Coomassie blue. FIG. 7 shows
LDL and HDL fractions, both separately and together in plasma (PL).
The PL fraction contains the same amount of HDL and LDL as each of
the separated fractions (HDL, LDL).
[0135] The PA gel was cut into about 1 mm pieces, and the
radioactivity distribution was determined (FIG. 8). With either LDL
or HDL alone, counts were observed over respective lipoprotein
fractions. When both LDL and HDL in plasma are present, counts
localize preferentially over HDL fraction. This indicates that
following phagocytosis of POS by RPE, POS residues are effluxed and
preferentially bound by HDL. This is a novel demonstration
illustrating that RCT to an HDL acceptor occurs in RPE cells.
[0136] To characterize the lipids in the lipoprotein fraction, thin
layer chromatography was performed. Acid charring was used to
identify lipid containing spots. The spots were scraped off of the
plate and .sup.14C was quantified by liquid scintillation counting.
Six of 17 .sup.14C-containing spots were identified with standards
shown (FIG. 9). Eleven .sup.14C-containing spots bound to HDL
remain unidentified and could be unique serum marker(s) for
patients with early AMD.
[0137] Thus, in an embodiment of the present invention, a patient
sample is obtained, such as by drawing blood, and the HDL is
examined for bound POS residues. From this, a determination of
their risk of visual loss from AMD is made. In a specific
embodiment, the profile of bound POS residues is indicative of
identifying an individual afflicted with ocular disease and/or of
identifying an individual at risk for developing an ocular
disease.
Example 5
Modulation of RCT by Compound Administration
[0138] This experiment determines whether compound administration
can upregulate efflux of labeled POS residues to HDL, particularly
by showing regulation of .sup.14C-DHA labeled POS efflux into basal
media. An assay similar to that described in Example 4 is utilized;
however, in this Example the cells were treated with T3, 9
cis-retinoic acid, and 22 (R) hydroxycholesterol in the
concentrations described above for 36 hours. Total radioactivity
(cpm) in the absence of HDL purification was determined by liquid
scintillation counting of the basal media. FIG. 10 indicates that
compound treatments increase RCT by cultured human RPE cells.
[0139] Specifically, cells were grown for 1 to 2 weeks at
confluence on Transwell plates. .sup.14C-labeled POS (30 mg/ml)
were added to the apical medium. The apical and basal medium
comprised either 10.sup.-7 M T3, 2.5.times.10.sup.-5 M 22 (R)
hydroxycholesterol, or 10.sup.-7 M cis retinoic acid. The basal
medium contained 1 mg/ml HDL. After 36 hours the basal medium was
collected and .sup.14C counts were determined by scintillation
counting. As stated, all of the compound treatments increased
transport of .sup.14C-labeled POS to the basal medium.
[0140] The effect of T3 on Apo E mRNA levels was also assessed by
RT-PCR. Treatment with 10.sup.-7 M T3 resulted in a 1.5 to 2-fold
increase in apo E mRNA levels, suggesting that T3 is acting, at
least in part, to increase apo E levels at the mRNA level. In
specific embodiments, administration of 9 cis-retinoic acid and 22
(R) hydroxycholesterol similarly upregulates expression of apo
E.
[0141] Thus, in a specific embodiment, RCT is regulated via nuclear
hormone receptor ligands. For example, ABCA1 expression is
upregulated by binding of LXR and RXR agonists to their respective
nuclear hormone receptors (FIG. 11). Since these receptors form
heterodimers bound to the ABCA1 promoter, ligand binding increases
expression of ABCA1 and, hence, RCT.
Example 6
Identification of HDL as Lipid Acceptor from RPE Cells
[0142] In the presence of added purified human LDL and HDL,
radiolabeled lipid efflux is enhanced (FIG. 12). As shown
graphically, efflux (bottoms in graph) was greatly enhanced by the
presence of plasma (PL in graph), HDL or LDL, as compared to no
addition to the bottom medium (left side of graph).
[0143] As shown in FIG. 8, when whole human EDTA-plasma is employed
and lipoproteins are isolated, [.sup.14C]-labeled lipids are
incorporated into LDL and HDL. However, radiolabel preferentially
associated with HDL. Furthermore, the radiolabel in HDL was
localized to the larger HDL 2 subspecies, which include the HDL
particles enriched in apo E. This result suggests that lipid efflux
from RPE is enhanced by the apo E--containing HDL.
Example 7
Reduction of BM Lipids Via Scavenger Receptors (SRS)
[0144] Scavenger receptors in macrophages function to phagocytose
oxLDL molecules. There are types of SRs previously described in
macrophages including SR-A1, SR-A2, SR-B1, SR-B2, CD36, and LOX.
SRs are distinct from LDL receptors in that levels of expression
for SRs are upregulated by oxLDL. This upregulation by
intracellular oxLDL levels is modulated by nuclear hormone
receptors, peroxisome proliferator activated receptor (PPAR) and
retinoic acid X receptor (RXR), that exert transcriptional control
of CD36 expression. Because the earliest lesion of AS, the fatty
streak, consists of macrophages engulfed with excessive oxLDL, and
because RPE cells similarly become filled with lipid inclusions in
AMD, SR expression was studied in RPE cells. Expression of the
following SRs in RPE cells was identified: CD36 (confirmation of
previous investigators), SR-A1, SR-A2 (both first time demonstrated
in RPE), SR-B1, SR-B2 (both first time demonstrated in RPE).
[0145] The inventors have also shown that, like macrophages, oxLDL
upregulates expression of CD36 in RPE cells (FIG. 13).
Additionally, RPE cells express the nuclear hormone receptors, PPAR
and RXR, indicating control mechanisms for SR expression are
analogous between the cell types. Thus, in specific embodiments the
expression of RPE SRs in patients is controlled with PPAR and RXR
ligands (e.g. PG-J2, thiazolidinediones, cis-retinoic acid). This
controls the rate at which RPE cells phagocytose oxidized
photoreceptor outer segments, and hence slows the rate at which
abnormal lipid inclusions accumulate in RPE and BM. In other
specific embodiments, expression of CD36 is downregulated with a
composition such as tamoxifen, TGF-beta or INF-gamma. Similarly,
regulating expression of other RPE SRs would control levels of
lipids in both RPE and BM. For example, for SR-A regulation IGF-1,
TGF-beta, EGF, and/or PDGF is used, and for SR-B regulation cAMP
and/or estradiol (for upregulation) or TNF-alpha, LPS, and/or
INF-gamma (for downregulation) is used.
Example 8
HDL Increases .sup.14C Lipid Efflux from RPE Cells Preferentially
to Other Lipoproteins
[0146] Transcription of the apo E gene is regulated by
liver-X-receptor alpha (LXR .alpha.) that acts as heterodimers with
retinoid-X-receptor alpha (RXR .alpha.) (Mak et al., 2002). The
inventors have previously shown that RPE cells express T.sub.3
receptors (TRs) that also act as heterodimers with RXR .alpha.
(Duncan et al., 1999). The inventors and others, have demonstrated
that primary cultures of RPE cells express mRNA for lxr .alpha.,
RXR .alpha., apo E, and other proteins involved in regulation of
lipid and cholesterol uptake, metabolism and efflux (summarized
herein). In this Example, the inventors show that apo E secreted by
primary cultures of RPE cells can be up-regulated by thyroid
hormone (T.sub.3), 22(R) hydroxycholesterol (HC), and cis retinoic
acid (RA). The inventors also demonstrated that a high density
lipoprotein (HDL) fraction rich in apo E is a preferential acceptor
for labeled POS lipids.
[0147] As shown in Table II, the present inventors and other
investigators have identified mRNAs for the proteins involved in
regulating lipid and cholesterol uptake, metabolism and efflux. The
cells used in the experiments described below express only the apo
E3 allele (E3/E3).
TABLE-US-00002 TABLE II Agents involved in regulating lipid and
cholesterol uptake, metabolism, and efflux TRANSCRIPTION FACTORS
LIGANDS TR .alpha.1 Thyroid hormone Receptor alpha1 T.sub.3 TR
.alpha.2 Thyroid hormone Receptor alpha 2 T.sub.3 TR .beta. 1
Thyroid hormone Receptor beta1 T.sub.3 RXR .alpha. Retinoid-X
Receptor alpha Retinoic Acid RXR .beta. Retinoid-X Receptor beta
Retinoic Acid PPAR .gamma. Peroxisome Proliferator Oxidized lipids
Activator Receptor gamma Lxr .alpha. Liver-X Receptor alpha
Oxysterols CELL SURFACE RECEPTORS LIGANDS SR-BI Scavenger Receptor
BI Oxidized Lipids SR-BII Scavenger Receptor BII Oxidized Lipids
SR-AI Scavenger Receptor AI Oxidized Lipoproteins SR-AII Scavenger
Receptor AII Oxidized Lipoproteins Lox-1 Lectin-like Oxidized LDL
receptor 1 Oxidized Lipoproteins CHOLESTEROL/LIPID TRANSPORT AND
METABOLISM FUNCTIONS SR-BI Scavenger Receptor BI Reverse
Cholesterol Transport SR-BII Scavenger Receptor BII Reverse
Cholesterol Transport ABCA1 ATP Binding Cassette Protein A1 Reverse
Cholesterol Transport ACAT1 Acyl-CoA Cholesterol Cholesterol
Acylation Acyltransferase 1 Apo E Apolipoprotein E
Cholesterol/Lipid Trafficking
[0148] As shown qualitatively in FIG. 6, T.sub.3 (TR agonist), RA
(RXR agonist), HC (LXR agonist) stimulate basal apo E secretion. As
previously indicated, RPE cells were treated for 36 hours on
Transwell.RTM. plates with serum free DMEM in upper and lower
chambers+/-the drugs indicated. Control (C) refers to no drug
addition; T refers to 10.sup.-7 M T.sub.3; RA refers to 10.sup.-7 M
cis retinoic acid; and HC refers to 2.5.times.10.sup.-6 M 22(R)
hydroxycholesterol. Basal media from 3 wells were combined,
concentrated, and apo E was detected by western blotting.
[0149] As shown quantitatively in FIG. 14, TR, LXR and RXR agonists
upregulate apo E secretion alone and in combination, as assessed by
ELISA assays. RPE cells were treated for 36 hours on Transwell.RTM.
plates with serum free DMEM+/-the drugs indicated. Control refers
to no drug addition, T refers to 10.sup.-7 M T.sub.3; HC refers to
2.5.times.10.sup.-6 M 22(R) hydroxycholesterol; RA refers to
10.sup.-7 M cis retinoic acid. N=6, * indicates p.ltoreq.0.05
(two-tailed t-test) compared to Control.
[0150] As shown in FIG. 15, apo E secreted from RPE cells binds to
HDL. RPE cells on Transwell.RTM. plates were grown in DMEM with 5%
FBS for 36 hours (apical chamber). Basal chambers had serum free
DMEM with either 200, 50, or 0 .mu.g/ml mouse HDL (lanes 2, 3, and
4 respectively. Lane 1 illustrates molecular weight markers. HDL
was purified by ultracentrifugation, resolved by polyacrylamide gel
electrophoresis, and human apo E was identified by western
blotting.
[0151] As shown in FIG. 16, HDL stimulates POS lipid efflux from
RPE cells in culture. RPE cells on Transwell.RTM. plates were fed
.sup.14C labeled POS in DMEM with 5% FBS for 36 hours (apical
chamber). Basal chambers had serum free DMEM. Both upper and lower
media contained either no addition (Control), 10% human plasma, 100
.mu.g/ml HDL, 1000 .mu.g/ml LDL or 50 .mu.g/ml HDL+500 .mu.g/ml LDL
as indicated. FIG. 16 left: Basal .sup.14C cpm/130 .mu.l. N=3, *
indicates p.ltoreq.0.05 (two-tailed t-test) compared to Control.
FIG. 16 right: Lipoproteins were purified by ultracentrifugation,
dialyzed to remove soluble .sup.14C, and counted.
[0152] As shown in FIG. 8, .sup.14C labeled POS lipids
preferentially bind to apo E containing high molecular weight HDL
(HDL3). .sup.14C labeled lipoproteins from the lower chamber were
purified by ultracentrifugation and resolved on native
polyacrylamide gels.
[0153] Characterization of HDL and plasma bound POS lipids was made
by thin layer chromatography, as shown in FIG. 17. .sup.14C labeled
lipoproteins from the lower chamber were purified by
ultracentrifugation, and lipids were resolved by thin layer
chromatography followed by acid charring.
[0154] As shown in FIG. 18, six spots in HDL and plasma were
tentatively identified; at least 11 other spots are not yet
identified. Spots identified by charring were cut out and .sup.14C
cpm determined by liquid scintillation counting.
Example 9
Exemplary Methods and Materials for Example 8
Cell Culture
[0155] Primary cultures of normal human RPE cells were prepared
from a 35 year old donor eye as described (Song and Lui, 1990).
Cells from passages 4 to 10 were used. RPE cells were grown on
laminin-coated tissue culture plates, or on laminin coated 0.4
.mu.M cellulose acetate Transwell.RTM. dishes (Costar) in DMEM H21
containing 5-10% fetal bovine serum (FBS), 2 mM glutamine, 5
.mu.g/ml gentamycin, 100 IU/ml penicillin, 100 mg/ml streptomycin,
2.5 mg/ml fungizone, 1 ng/ml bFGF, and 1 ng/ml EGF. Where
indicated, FBS was substituted with: 6 g/L NEAA, 0.39 g/L
methylcellulose (serum free medium). No differences in cell
morphology or protein expression were observed in cultures from
different passages. RPE cells were grown at confluence for at least
14 days prior to undergoing the experimental treatments described
below.
RT-PCR
[0156] RT-PCR was carried out on 1 .mu.g of cDNA. The RT-PCR
products are resolved by electrophoresis on 1.4% agarose gels. The
RT-PCR primer sequences used are given followed by the predicted
apo E RT-PCR product size. apo E forward: 5'-TAA GCT TGG CAC GGC
TGT CCA AGG A (SEQ ID NO:13); apo E reverse: 5'-ACA GAA TTC GCC CCG
GCC TGG TAC AC (SEQ ID NO:14); 241 bp product (detects both apo E3
and apo E4). PCR was conducted for 20-30 cycles at 55.degree. C. in
buffer containing 2.0-5.0 mM MgCl.sub.2. The RT-PCR product of the
predicted size for apo E had its identity confirmed by DNA
sequencing. Only the apo E3 mRNA sequence was detected. Messenger
RNAs for the other proteins listed in the Table II were identified
using similar strategies with primers specific to each cDNA.
Western Blotting
[0157] Briefly, media was concentrated 20-fold by centrifugal
ultrafiltration (VIVA SPIN 20, MCO 5,000; Viva Sciences).
Concentrated media (20 .mu.g protein) was purified over
Heparin-Sepharose CL-4B. The apo E containing (bound) fraction was
eluted and re-concentrated to 20 .mu.l. Apo E was resolved by 5-25%
linear gradient SDS polyacrylamide gel electrophoresis, and
proteins were electrophoretically transferred to nitrocelluose.
Membranes were blocked with 10% BSA and probed with 1% goat
anti-human apo E antiserum. Apo E antibodies were detected
colorimetrically with horseradish peroxidase conjugated rabbit
anti-goat IgG and NiCl.sub.2-enhanced diaminobenzine staining.
ELISA Assay
[0158] Media samples treated with 0.1% Tween-20 containing 1%
bovine serum albumin were incubated (37.degree. C., 4 h) in 96-well
plates previously coated with apo E-affinity purified goat anti-apo
E antibody. Apo E was detected using a secondary
antibody-peroxidase conjugate and
3.3.5.5'-tetramethylethylenediamine (TMB) substrate. Optical
density was measured at 450 nm. The assay was calibrated with
purified plasma apo E. The dynamic range of the assay was 1-40
ng/ml apo E with a CV<5%.
POS Lipid Transport and Lipoprotein Gel Analysis
[0159] Briefly, purified POS lipids were labeled with .sup.14C
docosohexanoic acid as described (Guisto et al., 1986). Twenty
.mu.g/ml (protein) of POS were added to the top chambers of 6 well
Transwell.RTM. plates. The bottom chambers contained serum free
medium with or without human high density lipoprotein (HDL), human
low density lipoprotein (LDL), or human plasma in the amounts
indicated. After 36 hours, cell culture medium was harvested from
the bottom chambers, adjusted to a density of 1.25 g/ml with solid
potassium bromide and underlayed over a KBr solution of d=1.21.
Samples were ultracentrifuged (Beckman 50.2Ti, 45,000 rpm,
10.degree. C.) for 20 hours. The top (lipoprotein) layer was
removed, dialyzed, and subjected to non-denaturing gel
electrophoresis. The gels were stained with Coomassie Blue and
photographed, after which 2 mM slices were subjected to
scintillation counting.
Thin Layer Chromatography
[0160] Lipoprotein samples were extracted for lipid by the method
of Bligh-Dyer, which is well known in the art. Lipids were resolved
by silica gel K6 thin layer chromtography using sequential
developments in Solvent 1: chloroform/methanol/acetic acid/water
(25:15:4:2) and Solvent 2: n-hexane/diethylether/acetic acid
(65:35:2). Lipid species were detected by acid charring, plates
were immersed in 7.5% copper acetate, 2.5% copper sulfate, 8%
phosphoric acid and heated on a hot plate for 1 hour. .sup.14C
radioactivity was measured by liquid scintillation counting in
standard methods known in the art.
Example 10
APOA-I Delivery to Increase Reverse Cholesterol Transport
[0161] The present inventors have shown that HDL is a preferred
cholesterol and phospholipids acceptor for lipids effluxed by
cultured human RPE. In aging BM, there is progressive accumulation
of lipid and cross-linked protein that impedes hydraulic
conductivity and macromolecular permeability. This abnormal
deposition may also impair the ability of some larger molecular
weight species of HDL to act as a lipoprotein acceptor. As HDL is
unable to pass through BM and promote efflux and bind effluxed
lipids, more lipids would accumulate in both RPE and BM. Indeed
such accumulations are a major finding in age-related macular
degeneration (AMD).
[0162] Apolipoprotein A1 (ApoA-I) is the major lipoprotein
component of HDL. It has a mass of approximately 28 kDaltons.
ApoA-I bound to phospholipids comprises nascent HDL particles that
bind to ABCA1 on the RPE basal membrane and promote lipid efflux.
Because of ApoA-I's low molecular weight, it can penetrate an aged
BM more easily than larger molecular weight species of HDL to bind
to the RPE. In addition to it role in promoting reverse cholesterol
transport from RPE, ApoA-I also in a potent anti-oxidant.
Anti-oxidants have been established to reduce visual loss in
patients with AMD.
[0163] Several methods are used to increase ApoA-I delivery to RPE
as a treatment for AMD:
[0164] 1. ApoA-I is administered intravenously as has been done in
mouse models of atherosclerosis and in patients with coronary
artery disease.
[0165] 2. ApoA-I, which is normally comprised on L-amino acids, can
be administered as an ApoA-I mimetic peptide consisting of D-amino
acids. The D-amino acid based ApoA-I mimetic peptide is not
recognized as readily by human proteases, and thus can be
administered orally. This would be more convenient than parenteral
administration with an intravenous formulation containing the
L-amino acid ApoA-I or its mimetic peptide.
[0166] 3. Oral synthetic phospholipid (1,2
Dimyristoyl-.alpha.-glycero-3-phosphocholine, DMPC) increases
levels of circulating ApoA-I.
[0167] By way of example, patients with AMD (atrophic or exudative)
are administered either intravenous ApoA-I, ApoA-I mimetic peptide,
or DMPC to increase levels of circulating ApoA-I. Administration
could occur as frequent as daily or less frequently as in every
other month depending on the method of administration and the
clinical response.
[0168] Apo A-I mimetic peptides may be synthesized according to
standard methods in the art, and in some embodiments one or more
amino acids in the peptide are the D-stereoisomer. Methods to
synthesize mimetic peptides are known in the art, including those
described in de Beer, M. C., et al. (2001) and Matz and Jonas,
1982.
[0169] The peptides are based on the sequence
Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH.sub.2 (Ac-18A-NH2 or 2F)
(SEQ ID NO:15) (Navab et al., 2003), where Ac symbolizes
acetylated. Thus, in specific embodiments, the C-terminus is
carboxylated. The 2F peptide or an analog of 2F with the primary
amino acid sequence Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (4F)
(SEQ ID NO:16) is used (Navab et al., 2003). An example of a human
apolipoprotein A1 includes: LSPLGEEMRD RARAHVDALR THLAPYSDEL
RQPLAARLEA LKENGGARLA EYHAKATEHL STLSEKA (SEQ ID NO:17). Other
apolipoprotein A1 sequences, including those from organisms other
than human, are available to the skilled artisan at the National
Center for Biotechnology Information's GenBank database on the
World Wide Web.
[0170] In some embodiments of the present invention, a mouse model
is utilized to characterize administration of ApoA-I compositions.
For example, the model generated by Dithmar et al. (2000) or an
analogous model generated by similar methods in the art may be used
in optimizing the present invention. In this model, ApoE.sup.- mice
demonstrate ultrastructural changes in Bruch's membrane, such as
accumulation of material similar to basal linear deposit and an
increase in membrane-bound material.
Example 11
APOA-I and RPE Cells
[0171] The present inventors have created liposomes comprising
apoA-I (artificial pre.beta..sub.1 HDL). Artificial discoidal
apoA-I liposomes comprising purified human plasma apoAI, the
saturated phospholipid dimyristoyl-L-.alpha.-phoshatidylcholine
(DMPC) and cholesterol were constructed by the sodium cholate
dialysis method. Liposomes were prepared using a molar ratio of
approximately Jan. 5, 1995, apoA-I/free cholesterol/DMPC. Human
retinal pigment epithelial cells grown on 6 well laminin coated
Transwell.RTM. plates were fed .sup.14C-docosahexaenoic acid
labeled photoreceptor outer segments in medium containing 5%
lipoprotein free fetal bovine serum for 36 hours (apical chamber).
Basal chambers contained serum free medium and either no human high
density lipoprotein (HDL) (Control), 100 .mu.g/ml of human HDL,
pure human apoA-I, or human apoA-I vesicles. An aliquot of the
basal medium was subjected to liquid scintillation counting. The
results are shown in FIG. 19. Wells were treated in triplicate. HDL
stimulated .sup.14C-labeled lipid efflux by about 60%. ApoA-1
appeared to stimulate 14C-labeled lipid efflux. The apoA-I vesicles
(apoA-I V) did not stimulate .sup.14C-labeled lipid efflux.
REFERENCES
[0172] All patents and publications mentioned in the specification
are indicative of the level of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference. The references, to the extent that they
provide exemplary procedural or other details supplementary to
those set forth herein, are specifically incorporated herein by
reference.
PATENTS
[0173] U.S. Pat. No. 6,071,924 [0174] U.S. Pat. No. 5,846,711
[0175] U.S. Pat. No. 5,440,013 [0176] U.S. Pat. No. 5,618,914
[0177] U.S. Pat. No. 5,670,155 [0178] U.S. Pat. No. 5,446,128
[0179] U.S. Pat. No. 5,710,245 [0180] U.S. Pat. No. 5,840,833
[0181] U.S. Pat. No. 5,859,184 [0182] U.S. Pat. No. 5,929,237
[0183] U.S. Pat. No. 5,475,085 [0184] U.S. Pat. No. 5,672,681
[0185] U.S. Pat. No. 5,674,976 [0186] U.S. Pat. No. 4,554,101
[0187] U.S. Patent Application Publication US 2002/0142953 [0188]
WO 00/52479 [0189] WO 01/58494 [0190] WO 02/13812 [0191] WO
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[0239] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined by the
appended claims. Moreover, the scope of the present application is
not intended to be limited to the particular embodiments of the
process, machine, manufacture, composition of matter, means,
methods and steps described in the specification. As one of
ordinary skill in the art will readily appreciate from the
disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the present invention. Accordingly, the
appended claims are intended to include within their scope such
processes, machines, manufacture, compositions of matter, means,
methods, or steps.
Sequence CWU 1
1
1711156DNAHUMAN 1cgcagcggag gtgaaggacg tccttcccca ggagccgact
ggccaatcac aggcaggaag 60atgaaggttc tgtgggctgc gttgctggtc acattcctgg
caggatgcca ggccaaggtg 120gagcaagcgg tggagacaga gccggagccc
gagctgcgcc agcagaccga gtggcagagc 180ggccagcgct gggaactggc
actgggtcgc ttttgggatt acctgcgctg ggtgcagaca 240ctgtctgagc
aggtgcagga ggagctgctc agctcccagg tcacccagga actgagggcg
300ctgatggacg agaccatgaa ggagttgaag gcctacaaat cggaactgga
ggaacaactg 360accccggtgg cggaggagac gcgggcacgg ctgtccaagg
agctgcaggc ggcgcaggcc 420cggctgggcg cggacatgga ggacgtgtgc
ggccgcctgg tgcagtaccg cggcgaggtg 480caggccatgc tcggccagag
caccgaggag ctgcgggtgc gcctcgcctc ccacctgcgc 540aagctgcgta
agcggctcct ccgcgatgcc gatgacctgc agaagcgcct ggcagtgtac
600caggccgggg cccgcgaggg cgccgagcgc ggcctcagcg ccatccgcga
gcgcctgggg 660cccctggtgg aacagggccg cgtgcgggcc gccactgtgg
gctccctggc cggccagccg 720ctacaggagc gggcccaggc ctggggcgag
cggctgcgcg cgcggatgga ggagatgggc 780agccggaccc gcgaccgcct
ggacgaggtg aaggagcagg tggcggaggt gcgcgccaag 840ctggaggagc
aggcccagca gatacgcctg caggccgagg ccttccaggc ccgcctcaag
900agctggttcg agcccctggt ggaagacatg cagcgccagt gggccgggct
ggtggagaag 960gtgcaggctg ccgtgggcac cagcgccgcc cctgtgccca
gcgacaatca ctgaacgccg 1020aagcctgcag ccatgcgacc ccacgccacc
ccgtgcctcc tgcctccgcg cagcctgcag 1080cgggagaccc tgtccccgcc
ccagccgtcc tcctggggtg gaccctagtt taataaagat 1140tcaccaagtt tcacgc
115625515DNAHUMAN 2ggaacttgat gctcagagag gacaagtcat ttgcccaagg
tcacacagct ggcaactggc 60agacgagatt cacgccctgg caatttgact ccagaatcct
aaccttaacc cagaagcacg 120gcttcaagcc ctggaaacca caatacctgt
ggcagccagg gggaggtgct ggaatctcat 180ttcacatgtg gggagggggc
tcctgtgctc aaggtcacaa ccaaagagga agctgtgatt 240aaaacccagg
tcccatttgc aaagcctcga cttttagcag gtgcatcata ctgttcccac
300ccctcccatc ccacttctgt ccagccgcct agccccactt tctttttttt
ctttttttga 360gacagtctcc ctcttgctga ggctggagtg cagtggcgag
atctcggctc actgtaacct 420ccgcctcccg ggttcaagcg attctcctgc
ctcagcctcc caagtagcta ggattacagg 480cgcccgccac cacgcctggc
taacttttgt atttttagta gagatggggt ttcaccatgt 540tggccaggct
ggtctcaaac tcctgacctt aagtgattcg cccactgtgg cctcccaaag
600tgctgggatt acaggcgtga gctaccgccc ccagcccctc ccatcccact
tctgtccagc 660cccctagccc tactttcttt ctgggatcca ggagtccaga
tccccagccc cctctccaga 720ttacattcat ccaggcacag gaaaggacag
ggtcaggaaa ggaggactct gggcggcagc 780ctccacattc cccttccacg
cttggccccc agaatggagg agggtgtctg tattactggg 840cgaggtgtcc
tcccttcctg gggactgtgg ggggtggtca aaagacctct atgccccacc
900tccttcctcc ctctgccctg ctgtgcctgg ggcaggggga gaacagccca
cctcgtgact 960gggctgccca gcccgcccta tccctggggg agggggcggg
acagggggag ccctataatt 1020ggacaagtct gggatccttg agtcctactc
agccccagcg gaggtgaagg acgtccttcc 1080ccaggagccg gtgagaagcg
cagtcggggg cacggggatg agctcagggg cctctagaaa 1140gagctgggac
cctgggaagc cctggcctcc aggtagtctc aggagagcta ctcggggtcg
1200ggcttgggga gaggaggagc gggggtgagg caagcagcag gggactggac
ctgggaaggg 1260ctgggcagca gagacgaccc gacccgctag aaggtggggt
ggggagagca gctggactgg 1320gatgtaagcc atagcaggac tccacgagtt
gtcactatca ttatcgagca cctactgggt 1380gtccccagtg tcctcagatc
tccataactg gggagccagg ggcagcgaca cggtagctag 1440ccgtcgattg
gagaacttta aaatgaggac tgaattagct cataaatgga acacggcgct
1500taactgtgag gttggagctt agaatgtgaa gggagaatga ggaatgcgag
actgggactg 1560agatggaacc ggcggtgggg agggggtggg gggatggaat
ttgaaccccg ggagaggaag 1620atggaatttt ctatggaggc cgacctgggg
atggggagat aagagaagac caggagggag 1680ttaaataggg aatgggttgg
gggcggcttg gtaaatgtgc tgggattagg ctgttgcaga 1740taatgcaaca
aggcttggaa ggctaacctg gggtgaggcc gggttggggg cgctgggggt
1800gggaggagtc ctcactggcg gttgattgac agtttctcct tccccagact
ggccaatcac 1860aggcaggaag atgaaggttc tgtgggctgc gttgctggtc
acattcctgg caggtatggg 1920ggcggggctt gctcggttcc ccccgctcct
ccccctctca tcctcacctc aacctcctgg 1980ccccattcag acagaccctg
ggccccctct tctgaggctt ctgtgctgct tcctggctct 2040gaacagcgat
ttgacgctct ctgggcctcg gtttccccca tccttgagat aggagttaga
2100agttgttttg ttgttgttgt ttgttgttgt tgttttgttt ttttgagatg
aagtctcgct 2160ctgtcgccca ggctggagtg cagtggcggg atctcggctc
actgcaagct ccgcctccca 2220ggtccacgcc attctcctgc ctcagcctcc
caagtagctg ggactacagg cacatgccac 2280cacacccgac taactttttt
gtattttcag tagagacggg gtttcaccat gttggccagg 2340ctggtctgga
actcctgacc tcaggtgatc tgcccgtttc gatctcccaa agtgctggga
2400ttacaggcgt gagccaccgc acctggctgg gagttagagg tttctaatgc
attgcaggca 2460gatagtgaat accagacacg gggcagctgt gatctttatt
ctccatcacc cccacacagc 2520cctgcctggg gcacacaagg acactcaata
catgcttttc cgctgggccg gtggctcacc 2580cctgtaatcc cagcactttg
ggaggccaag gtgggaggat cacttgagcc caggagttca 2640acaccagcct
gggcaacata gtgagaccct gtctctacta aaaatacaaa aattagccag
2700gcatggtgcc acacacctgt gctctcagct actcaggagg ctgaggcagg
aggatcgctt 2760gagcccagaa ggtcaaggtt gcagtgaacc atgttcaggc
cgctgcactc cagcctgggt 2820gacagagcaa gaccctgttt ataaatacat
aatgctttcc aagtgattaa accgactccc 2880ccctcaccct gcccaccatg
gctccaaaga agcatttgtg gagcaccttc tgtgtgcccc 2940taggtagcta
gatgcctgga cggggtcaga aggaccctga cccgaccttg aacttgttcc
3000acacaggatg ccaggccaag gtggagcaag cggtggagac agagccggag
cccgagctgc 3060gccagcagac cgagtggcag agcggccagc gctgggaact
ggcactgggt cgcttttggg 3120attacctgcg ctgggtgcag acactgtctg
agcaggtgca ggaggagctg ctcagctccc 3180aggtcaccca ggaactgagg
tgagtgtccc catcctggcc cttgaccctc ctggtgggcg 3240gctatacctc
cccaggtcca ggtttcattc tgcccctgtc gctaagtctt ggggggcctg
3300ggtctctgct ggttctagct tcctcttccc atttctgact cctggcttta
gctctctgga 3360attctctctc tcagctttgt ctctctctct tcccttctga
ctcagtctct cacactcgtc 3420ctggctctgt ctctgtcctt ccctagctct
tttatataga gacagagaga tggggtctca 3480ctgtgttgcc caggctggtc
ttgaacttct gggctcaagc gatcctcccg cctcggcctc 3540ccaaagtgct
gggattagag gcatgagcac cttgcccggc ctcctagctc cttcttcgtc
3600tctgcctctg ccctctgcat ctgctctctg catctgtctc tgtctccttc
tctcggcctc 3660tgccccgttc cttctctccc tcttgggtct ctctggctca
tccccatctc gcccgcccca 3720tcccagccct tctcccccgc ctccccactg
tgcgacaccc tcccgccctc tcggccgcag 3780ggcgctgatg gacgagacca
tgaaggagtt gaaggcctac aaatcggaac tggaggaaca 3840actgaccccg
gtggcggagg agacgcgggc acggctgtcc aaggagctgc aggcggcgca
3900ggcccggctg ggcgcggaca tggaggacgt gcgcggccgc ctggtgcagt
accgcggcga 3960ggtgcaggcc atgctcggcc agagcaccga ggagctgcgg
gtgcgcctcg cctcccacct 4020gcgcaagctg cgtaagcggc tcctccgcga
tgccgatgac ctgcagaagc gcctggcagt 4080gtaccaggcc ggggcccgcg
agggcgccga gcgcggcctc agcgccatcc gcgagcgcct 4140ggggcccctg
gtggaacagg gccgcgtgcg ggccgccact gtgggctccc tggccggcca
4200gccgctacag gagcgggccc aggcctgggg cgagcggctg cgcgcgcgga
tggaggagat 4260gggcagccgg acccgcgacc gcctggacga ggtgaaggag
caggtggcgg aggtgcgcgc 4320caagctggag gagcaggccc agcagatacg
cctgcaggcc gaggccttcc aggcccgcct 4380caagagctgg ttcgagcccc
tggtggaaga catgcagcgc cagtgggccg ggctggtgga 4440gaaggtgcag
gctgccgtgg gcaccagcgc cgcccctgtg cccagcgaca atcactgaac
4500gccgaagcct gcagccatgc gaccccacgc caccccgtgc ctcctgcctc
cgcgcagcct 4560gcagcgggag accctgtccc cgccccagcc gtcctcctgg
ggtggaccct agtttaataa 4620agattcacca agtttcacgc atctgctggc
ctccccctgt gatttcctct aagccccagc 4680ctcagtttct ctttctgccc
acatactgcc acacaattct cagccccctc ctctccatct 4740gtgtctgtgt
gtatctttct ctctgccctt tttttttttt tagacggagt ctggctctgt
4800cacccaggct agagtgcagt ggcacgatct tggctcactg caacctctgc
ctcttgggtt 4860caagcgattc tgctgcctca gtagctggga ttacaggctc
acaccaccac acccggctaa 4920tttttgtatt tttagtagag acgagctttc
accatgttgg ccaggcaggt ctcaaactcc 4980tgaccaagtg atccacccgc
cggcctccca aagtgctgag attacaggcc tgagccacca 5040tgcccggcct
ctgcccctct ttctttttta gggggcaggg aaaggtctca ccctgtcacc
5100cgccatcaca gctcactgca gcctccacct cctggactca agtgataagt
gatcctcccg 5160cctcagcctt tccagtagct gagactacag gcgcatacca
ctaggattaa tttggggggg 5220ggtggtgtgt gtggagatgg ggtctggctt
tgttggccag gctgatgtgg aattcctggg 5280ctcaagcgat actcccacct
tggcctcctg agtagctgag actactggct agcaccacca 5340cacccagctt
tttattatta tttgtagaga caaggtctca atatgttgcc caggctagtc
5400tcaaacccct ggctcaagag atcctccgcc atcggcctcc caaagtgctg
ggattccagg 5460catgggctcc gagcggcctg cccaacttaa taatattgtt
cctagagttg cactc 551531157DNAHUMAN 3ccccagcgga ggtgaaggac
gtccttcccc aggagccgac tggccaatca caggcaggaa 60gatgaaggtt ctgtgggctg
cgttgctggt cacattcctg gcaggatgcc aggccaaggt 120ggagcaagcg
gtggagacag agccggagcc cgagctgcgc cagcagaccg agtggcagag
180cggccagcgc tgggaactgg cactgggtcg cttttgggat tacctgcgct
gggtgcagac 240actgtctgag caggtgcagg aggagctgct cagctcccaa
gtcacccaag aactgagggc 300gctgatggac gagaccatga aggagttgaa
ggcctacaaa tcggaactgg aggaacaact 360gaccccggta gcggaggaga
cgcgggcacg gctgtccaag gagctgcaga cggcgcaggc 420ccggctgggc
gcggacatgg aggacgtgtg cggccgcctg gtgcagtacc gcggcgaggt
480gcaggccatg ctcggccaga gcaccgagga gctgcgggtg cgcctcgcct
cccacctgcg 540caagctgcgt aagcggctcc tccgcgatcc cgatgacctg
cagaagcgcc tggcagtgta 600ccaggccggg gcccgcgagg gcgccgagcg
cggcctcagc gccatccgcg agcgcctggg 660gcccctggtg gaacagggcc
gcgtgcgggc cgccactgtg ggctccctgg ccggccagcc 720gctacaggag
cgggcccagg cctggggcga gcggctgcgc gcgcggatgg aggagatggg
780cagtcggacc cgcgaccgcc tggacgaggt gaaggagcag gtggcggagg
tgcgcgccaa 840gctggaggag caggcccagc agatacgcct gcaggccgag
gccttccagg cccgcctcaa 900gagctggttc gagcccctgg tggaagacat
gcagcgccag tgggccgggc tggtggagaa 960ggtgcaggct gccgtgggca
ccagcgccgc ccctgtgccc agcgacaatc actgaacgcc 1020gaagcctgca
gccatgcgac cccacgccac cccgtgcctc ctgcctccgc gcagcctgca
1080gcgggagacc ctgtccccgc cccagccgtc ctcctggggt ggaccctagt
ttaataaaga 1140ttcaccaagt ttcacgc 11574317PRTHUMAN 4Met Lys Val Leu
Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys1 5 10 15Gln Ala Lys
Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu 20 25 30Arg Gln
Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala Leu 35 40 45Gly
Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser Glu Gln 50 55
60Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu Leu Arg Ala65
70 75 80Leu Met Asp Glu Thr Met Lys Glu Leu Lys Ala Tyr Lys Ser Glu
Leu 85 90 95Glu Glu Gln Leu Thr Pro Val Ala Glu Glu Thr Arg Ala Arg
Leu Ser 100 105 110Lys Glu Leu Gln Ala Ala Gln Ala Arg Leu Gly Ala
Asp Met Glu Asp 115 120 125Val Cys Gly Arg Leu Val Gln Tyr Arg Gly
Glu Val Gln Ala Met Leu 130 135 140Gly Gln Ser Thr Glu Glu Leu Arg
Val Arg Leu Ala Ser His Leu Arg145 150 155 160Lys Leu Arg Lys Arg
Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg 165 170 175Leu Ala Val
Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu 180 185 190Ser
Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly Arg Val 195 200
205Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg
210 215 220Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala Arg Met Glu Glu
Met Gly225 230 235 240Ser Arg Thr Arg Asp Arg Leu Asp Glu Val Lys
Glu Gln Val Ala Glu 245 250 255Val Arg Ala Lys Leu Glu Glu Gln Ala
Gln Gln Ile Arg Leu Gln Ala 260 265 270Glu Ala Phe Gln Ala Arg Leu
Lys Ser Trp Phe Glu Pro Leu Val Glu 275 280 285Asp Met Gln Arg Gln
Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala 290 295 300Val Gly Thr
Ser Ala Ala Pro Val Pro Ser Asp Asn His305 310 3155317PRTHUMAN 5Met
Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe Leu Ala Gly Cys1 5 10
15Gln Ala Lys Val Glu Gln Ala Val Glu Thr Glu Pro Glu Pro Glu Leu
20 25 30Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln Arg Trp Glu Leu Ala
Leu 35 40 45Gly Arg Phe Trp Asp Tyr Leu Arg Trp Val Gln Thr Leu Ser
Glu Gln 50 55 60Val Gln Glu Glu Leu Leu Ser Ser Gln Val Thr Gln Glu
Leu Arg Ala65 70 75 80Leu Met Asp Glu Thr Met Lys Glu Leu Lys Ala
Tyr Lys Ser Glu Leu 85 90 95Glu Glu Gln Leu Thr Pro Val Ala Glu Glu
Thr Arg Ala Arg Leu Ser 100 105 110Lys Glu Leu Gln Ala Ala Gln Ala
Arg Leu Gly Ala Asp Met Glu Asp 115 120 125Val Arg Gly Arg Leu Val
Gln Tyr Arg Gly Glu Val Gln Ala Met Leu 130 135 140Gly Gln Ser Thr
Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu Arg145 150 155 160Lys
Leu Arg Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln Lys Arg 165 170
175Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu Gly Ala Glu Arg Gly Leu
180 185 190Ser Ala Ile Arg Glu Arg Leu Gly Pro Leu Val Glu Gln Gly
Arg Val 195 200 205Arg Ala Ala Thr Val Gly Ser Leu Ala Gly Gln Pro
Leu Gln Glu Arg 210 215 220Ala Gln Ala Trp Gly Glu Arg Leu Arg Ala
Arg Met Glu Glu Met Gly225 230 235 240Ser Arg Thr Arg Asp Arg Leu
Asp Glu Val Lys Glu Gln Val Ala Glu 245 250 255Val Arg Ala Lys Leu
Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala 260 265 270Glu Ala Phe
Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu Val Glu 275 280 285Asp
Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys Val Gln Ala Ala 290 295
300Val Gly Thr Ser Ala Ala Pro Val Pro Ser Asp Asn His305 310
3156317PRTHUMAN 6Met Lys Val Leu Trp Ala Ala Leu Leu Val Thr Phe
Leu Ala Gly Cys1 5 10 15Gln Ala Lys Val Glu Gln Ala Val Glu Thr Glu
Pro Glu Pro Glu Leu 20 25 30Arg Gln Gln Thr Glu Trp Gln Ser Gly Gln
Arg Trp Glu Leu Ala Leu 35 40 45Gly Arg Phe Trp Asp Tyr Leu Arg Trp
Val Gln Thr Leu Ser Glu Gln 50 55 60Val Gln Glu Glu Leu Leu Ser Ser
Gln Val Thr Gln Glu Leu Arg Ala65 70 75 80Leu Met Asp Glu Thr Met
Lys Glu Leu Lys Ala Tyr Lys Ser Glu Leu 85 90 95Glu Glu Gln Leu Thr
Pro Val Ala Glu Glu Thr Arg Ala Arg Leu Ser 100 105 110Lys Glu Leu
Gln Thr Ala Gln Ala Arg Leu Gly Ala Asp Met Glu Asp 115 120 125Val
Cys Gly Arg Leu Val Gln Tyr Arg Gly Glu Val Gln Ala Met Leu 130 135
140Gly Gln Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu
Arg145 150 155 160Lys Leu Arg Lys Arg Leu Leu Arg Asp Pro Asp Asp
Leu Gln Lys Arg 165 170 175Leu Ala Val Tyr Gln Ala Gly Ala Arg Glu
Gly Ala Glu Arg Gly Leu 180 185 190Ser Ala Ile Arg Glu Arg Leu Gly
Pro Leu Val Glu Gln Gly Arg Val 195 200 205Arg Ala Ala Thr Val Gly
Ser Leu Ala Gly Gln Pro Leu Gln Glu Arg 210 215 220Ala Gln Ala Trp
Gly Glu Arg Leu Arg Ala Arg Met Glu Glu Met Gly225 230 235 240Ser
Arg Thr Arg Asp Arg Leu Asp Glu Val Lys Glu Gln Val Ala Glu 245 250
255Val Arg Ala Lys Leu Glu Glu Gln Ala Gln Gln Ile Arg Leu Gln Ala
260 265 270Glu Ala Phe Gln Ala Arg Leu Lys Ser Trp Phe Glu Pro Leu
Val Glu 275 280 285Asp Met Gln Arg Gln Trp Ala Gly Leu Val Glu Lys
Val Gln Ala Ala 290 295 300Val Gly Thr Ser Ala Ala Pro Val Pro Ser
Asp Asn His305 310 315710412DNAHUMAN 7gtaattgcga gcgagagtga
gtggggccgg gacccgcaga gccgagccga cccttctctc 60ccgggctgcg gcagggcagg
gcggggagct ccgcgcacca acagagccgg ttctcagggc 120gctttgctcc
ttgttttttc cccggttctg ttttctcccc ttctccggaa ggcttgtcaa
180ggggtaggag aaagagacgc aaacacaaaa gtggaaaaca gttaatgacc
agccacggcg 240tccctgctgt gagctctggc cgctgccttc cagggctccc
gagccacacg ctgggggtgc 300tggctgaggg aacatggctt gttggcctca
gctgaggttg ctgctgtgga agaacctcac 360tttcagaaga agacaaacat
gtcagctgct gctggaagtg gcctggcctc tatttatctt 420cctgatcctg
atctctgttc ggctgagcta cccaccctat gaacaacatg aatgccattt
480tccaaataaa gccatgccct ctgcaggaac acttccttgg gttcagggga
ttatctgtaa 540tgccaacaac ccctgtttcc gttacccgac tcctggggag
gctcccggag ttgttggaaa 600ctttaacaaa tccattgtgg ctcgcctgtt
ctcagatgct cggaggcttc ttttatacag 660ccagaaagac accagcatga
aggacatgcg caaagttctg agaacattac agcagatcaa 720gaaatccagc
tcaaacttga agcttcaaga tttcctggtg gacaatgaaa ccttctctgg
780gttcctgtat cacaacctct ctctcccaaa gtctactgtg gacaagatgc
tgagggctga 840tgtcattctc cacaaggtat ttttgcaagg ctaccagtta
catttgacaa gtctgtgcaa 900tggatcaaaa tcagaagaga tgattcaact
tggtgaccaa gaagtttctg agctttgtgg 960cctaccaagg gagaaactgg
ctgcagcaga gcgagtactt cgttccaaca tggacatcct 1020gaagccaatc
ctgagaacac taaactctac atctcccttc ccgagcaagg agctggctga
1080agccacaaaa acattgctgc atagtcttgg gactctggcc caggagctgt
tcagcatgag 1140aagctggagt gacatgcgac aggaggtgat gtttctgacc
aatgtgaaca gctccagctc 1200ctccacccaa atctaccagg ctgtgtctcg
tattgtctgc gggcatcccg agggaggggg 1260gctgaagatc aagtctctca
actggtatga ggacaacaac tacaaagccc
tctttggagg 1320caatggcact gaggaagatg ctgaaacctt ctatgacaac
tctacaactc cttactgcaa 1380tgatttgatg aagaatttgg agtctagtcc
tctttcccgc attatctgga aagctctgaa 1440gccgctgctc gttgggaaga
tcctgtatac acctgacact ccagccacaa ggcaggtcat 1500ggctgaggtg
aacaagacct tccaggaact ggctgtgttc catgatctgg aaggcatgtg
1560ggaggaactc agccccaaga tctggacctt catggagaac agccaagaaa
tggaccttgt 1620ccggatgctg ttggacagca gggacaatga ccacttttgg
gaacagcagt tggatggctt 1680agattggaca gcccaagaca tcgtggcgtt
tttggccaag cacccagagg atgtccagtc 1740cagtaatggt tctgtgtaca
cctggagaga agctttcaac gagactaacc aggcaatccg 1800gaccatatct
cgcttcatgg agtgtgtcaa cctgaacaag ctagaaccca tagcaacaga
1860agtctggctc atcaacaagt ccatggagct gctggatgag aggaagttct
gggctggtat 1920tgtgttcact ggaattactc caggcagcat tgagctgccc
catcatgtca agtacaagat 1980ccgaatggac attgacaatg tggagaggac
aaataaaatc aaggatgggt actgggaccc 2040tggtcctcga gctgacccct
ttgaggacat gcggtacgtc tgggggggct tcgcctactt 2100gcaggatgtg
gtggagcagg caatcatcag ggtgctgacg ggcaccgaga agaaaactgg
2160tgtctatatg caacagatgc cctatccctg ttacgttgat gacatctttc
tgcgggtgat 2220gagccggtca atgcccctct tcatgacgct ggcctggatt
tactcagtgg ctgtgatcat 2280caagggcatc gtgtatgaga aggaggcacg
gctgaaagag accatgcgga tcatgggcct 2340ggacaacagc atcctctggt
ttagctggtt cattagtagc ctcattcctc ttcttgtgag 2400cgctggcctg
ctagtggtca tcctgaagtt aggaaacctg ctgccctaca gtgatcccag
2460cgtggtgttt gtcttcctgt ccgtgtttgc tgtggtgaca atcctgcagt
gcttcctgat 2520tagcacactc ttctccagag ccaacctggc agcagcctgt
gggggcatca tctacttcac 2580gctgtacctg ccctacgtcc tgtgtgtggc
atggcaggac tacgtgggct tcacactcaa 2640gatcttcgct agcctgctgt
ctcctgtggc ttttgggttt ggctgtgagt actttgccct 2700ttttgaggag
cagggcattg gagtgcagtg ggacaacctg tttgagagtc ctgtggagga
2760agatggcttc aatctcacca cttcggtctc catgatgctg tttgacacct
tcctctatgg 2820ggtgatgacc tggtacattg aggctgtctt tccaggccag
tacggaattc ccaggccctg 2880gtattttcct tgcaccaagt cctactggtt
tggcgaggaa agtgatgaga agagccaccc 2940tggttccaac cagaagagaa
tatcagaaat ctgcatggag gaggaaccca cccacttgaa 3000gctgggcgtg
tccattcaga acctggtaaa agtctaccga gatgggatga aggtggctgt
3060cgatggcctg gcactgaatt tttatgaggg ccagatcacc tccttcctgg
gccacaatgg 3120agcggggaag acgaccacca tgtcaatcct gaccgggttg
ttccccccga cctcgggcac 3180cgcctacatc ctgggaaaag acattcgctc
tgagatgagc accatccggc agaacctggg 3240ggtctgtccc cagcataacg
tgctgtttga catgctgact gtcgaagaac acatctggtt 3300ctatgcccgc
ttgaaagggc tctctgagaa gcacgtgaag gcggagatgg agcagatggc
3360cctggatgtt ggtttgccat caagcaagct gaaaagcaaa acaagccagc
tgtcaggtgg 3420aatgcagaga aagctatctg tggccttggc ctttgtcggg
ggatctaagg ttgtcattct 3480ggatgaaccc acagctggtg tggaccctta
ctcccgcagg ggaatatggg agctgctgct 3540gaaataccga caaggccgca
ccattattct ctctacacac cacatggatg aagcggacgt 3600cctgggggac
aggattgcca tcatctccca tgggaagctg tgctgtgtgg gctcctccct
3660gtttctgaag aaccagctgg gaacaggcta ctacctgacc ttggtcaaga
aagatgtgga 3720atcctccctc agttcctgca gaaacagtag tagcactgtg
tcatacctga aaaaggagga 3780cagtgtttct cagagcagtt ctgatgctgg
cctgggcagc gaccatgaga gtgacacgct 3840gaccatcgat gtctctgcta
tctccaacct catcaggaag catgtgtctg aagcccggct 3900ggtggaagac
atagggcatg agctgaccta tgtgctgcca tatgaagctg ctaaggaggg
3960agcctttgtg gaactctttc atgagattga tgaccggctc tcagacctgg
gcatttctag 4020ttatggcatc tcagagacga ccctggaaga aatattcctc
aaggtggccg aagagagtgg 4080ggtggatgct gagacctcag atggtacctt
gccagcaaga cgaaacaggc gggccttcgg 4140ggacaagcag agctgtcttc
gcccgttcac tgaagatgat gctgctgatc caaatgattc 4200tgacatagac
ccagaatcca gagagacaga cttgctcagt gggatggatg gcaaagggtc
4260ctaccaggtg aaaggctgga aacttacaca gcaacagttt gtggcccttt
tgtggaagag 4320actgctaatt gccagacgga gtcggaaagg attttttgct
cagattgtct tgccagctgt 4380gtttgtctgc attgcccttg tgttcagcct
gatcgtgcca ccctttggca agtaccccag 4440cctggaactt cagccctgga
tgtacaacga acagtacaca tttgtcagca atgatgctcc 4500tgaggacacg
ggaaccctgg aactcttaaa cgccctcacc aaagaccctg gcttcgggac
4560ccgctgtatg gaaggaaacc caatcccaga cacgccctgc caggcagggg
aggaagagtg 4620gaccactgcc ccagttcccc agaccatcat ggacctcttc
cagaatggga actggacaat 4680gcagaaccct tcacctgcat gccagtgtag
cagcgacaaa atcaagaaga tgctgcctgt 4740gtgtccccca ggggcagggg
ggctgcctcc tccacaaaga aaacaaaaca ctgcagatat 4800ccttcaggac
ctgacaggaa gaaacatttc ggattatctg gtgaagacgt atgtgcagat
4860catagccaaa agcttaaaga acaagatctg ggtgaatgag tttaggtatg
gcggcttttc 4920cctgggtgtc agtaatactc aagcacttcc tccgagtcaa
gaagttaatg atgccatcaa 4980acaaatgaag aaacacctaa agctggccaa
ggacagttct gcagatcgat ttctcaacag 5040cttgggaaga tttatgacag
gactggacac caaaaataat gtcaaggtgt ggttcaataa 5100caagggctgg
catgcaatca gctctttcct gaatgtcatc aacaatgcca ttctccgggc
5160caacctgcaa aagggagaga accctagcca ttatggaatt actgctttca
atcatcccct 5220gaatctcacc aagcagcagc tctcagaggt ggctctgatg
accacatcag tggatgtcct 5280tgtgtccatc tgtgtcatct ttgcaatgtc
cttcgtccca gccagctttg tcgtattcct 5340gatccaggag cgggtcagca
aagcaaaaca cctgcagttc atcagtggag tgaagcctgt 5400catctactgg
ctctctaatt ttgtctggga tatgtgcaat tacgttgtcc ctgccacact
5460ggtcattatc atcttcatct gcttccagca gaagtcctat gtgtcctcca
ccaatctgcc 5520tgtgctagcc cttctacttt tgctgtatgg gtggtcaatc
acacctctca tgtacccagc 5580ctcctttgtg ttcaagatcc ccagcacagc
ctatgtggtg ctcaccagcg tgaacctctt 5640cattggcatt aatggcagcg
tggccacctt tgtgctggag ctgttcaccg acaataagct 5700gaataatatc
aatgatatcc tgaagtccgt gttcttgatc ttcccacatt tttgcctggg
5760acgagggctc atcgacatgg tgaaaaacca ggcaatggct gatgccctgg
aaaggtttgg 5820ggagaatcgc tttgtgtcac cattatcttg ggacttggtg
ggacgaaacc tcttcgccat 5880ggccgtggaa ggggtggtgt tcttcctcat
tactgttctg atccagtaca gattcttcat 5940caggcccaga cctgtaaatg
caaagctatc tcctctgaat gatgaagatg aagatgtgag 6000gcgggaaaga
cagagaattc ttgatggtgg aggccagaat gacatcttag aaatcaagga
6060gttgacgaag atatatagaa ggaagcggaa gcctgctgtt gacaggattt
gcgtgggcat 6120tcctcctggt gagtgctttg ggctcctggg agttaatggg
gctggaaaat catcaacttt 6180caagatgtta acaggagata ccactgttac
cagaggagat gctttcctta acaaaaatag 6240tatcttatca aacatccatg
aagtacatca gaacatgggc tactgccctc agtttgatgc 6300catcacagag
ctgttgactg ggagagaaca cgtggagttc tttgcccttt tgagaggagt
6360cccagagaaa gaagttggca aggttggtga gtgggcgatt cggaaactgg
gcctcgtgaa 6420gtatggagaa aaatatgctg gtaactatag tggaggcaac
aaacgcaagc tctctacagc 6480catggctttg atcggcgggc ctcctgtggt
gtttctggat gaacccacca caggcatgga 6540tcccaaagcc cggcggttct
tgtggaattg tgccctaagt gttgtcaagg aggggagatc 6600agtagtgctt
acatctcata gtatggaaga atgtgaagct ctttgcacta ggatggcaat
6660catggtcaat ggaaggttca ggtgccttgg cagtgtccag catctaaaaa
ataggtttgg 6720agatggttat acaatagttg tacgaatagc agggtccaac
ccggacctga agcctgtcca 6780ggatttcttt ggacttgcat ttcctggaag
tgttctaaaa gagaaacacc ggaacatgct 6840acaataccag cttccatctt
cattatcttc tctggccagg atattcagca tcctctccca 6900gagcaaaaag
cgactccaca tagaagacta ctctgtttct cagacaacac ttgaccaagt
6960atttgtgaac tttgccaagg accaaagtga tgatgaccac ttaaaagacc
tctcattaca 7020caaaaaccag acagtagtgg acgttgcagt tctcacatct
tttctacagg atgagaaagt 7080gaaagaaagc tatgtatgaa gaatcctgtt
catacggggt ggctgaaagt aaagaggaac 7140tagactttcc tttgcaccat
gtgaagtgtt gtggagaaaa gagccagaag ttgatgtggg 7200aagaagtaaa
ctggatactg tactgatact attcaatgca atgcaattca atgcaatgaa
7260aacaaaattc cattacaggg gcagtgcctt tgtagcctat gtcttgtatg
gctctcaagt 7320gaaagacttg aatttagttt tttacctata cctatgtgaa
actctattat ggaacccaat 7380ggacatatgg gtttgaactc acactttttt
tttttttttt gttcctgtgt attctcattg 7440gggttgcaac aataattcat
caagtaatca tggccagcga ttattgatca aaatcaaaag 7500gtaatgcaca
tcctcattca ctaagccatg ccatgcccag gagactggtt tcccggtgac
7560acatccattg ctggcaatga gtgtgccaga gttattagtg ccaagttttt
cagaaagttt 7620gaagcaccat ggtgtgtcat gctcactttt gtgaaagctg
ctctgctcag agtctatcaa 7680cattgaatat cagttgacag aatggtgcca
tgcgtggcta acatcctgct ttgattccct 7740ctgataagct gttctggtgg
cagtaacatg caacaaaaat gtgggtgtct ccaggcacgg 7800gaaacttggt
tccattgtta tattgtccta tgcttcgagc catgggtcta cagggtcatc
7860cttatgagac tcttaaatat acttagatcc tggtaagagg caaagaatca
acagccaaac 7920tgctggggct gcaagctgct gaagccaggg catgggatta
aagagattgt gcgttcaaac 7980ctagggaagc ctgtgcccat ttgtcctgac
tgtctgctaa catggtacac tgcatctcaa 8040gatgtttatc tgacacaagt
gtattatttc tggctttttg aattaatcta gaaaatgaaa 8100agatggagtt
gtattttgac aaaaatgttt gtacttttta atgttatttg gaattttaag
8160ttctatcagt gacttctgaa tccttagaat ggcctctttg tagaaccctg
tggtatagag 8220gagtatggcc actgccccac tatttttatt ttcttatgta
agtttgcata tcagtcatga 8280ctagtgccta gaaagcaatg tgatggtcag
gatctcatga cattatattt gagtttcttt 8340cagatcattt aggatactct
taatctcact tcatcaatca aatatttttt gagtgtatgc 8400tgtagctgaa
agagtatgta cgtacgtata agactagaga gatattaagt ctcagtacac
8460ttcctgtgcc atgttattca gctcactggt ttacaaatat aggttgtctt
gtggttgtag 8520gagcccactg taacaatact gggcagcctt tttttttttt
tttttaattg caacaatgca 8580aaagccaaga aagtataagg gtcacaagtc
taaacaatga attcttcaac agggaaaaca 8640gctagcttga aaacttgctg
aaaaacacaa cttgtgttta tggcatttag taccttcaaa 8700taattggctt
tgcagatatt ggatacccca ttaaatctga cagtctcaaa tttttcatct
8760cttcaatcac tagtcaagaa aaatataaaa acaacaaata cttccatatg
gagcattttt 8820cagagttttc taacccagtc ttatttttct agtcagtaaa
catttgtaaa aatactgttt 8880cactaatact tactgttaac tgtcttgaga
gaaaagaaaa atatgagaga actattgttt 8940ggggaagttc aagtgatctt
tcaatatcat tactaacttc ttccactttt tccagaattt 9000gaatattaac
gctaaaggtg taagacttca gatttcaaat taatctttct atatttttta
9060aatttacaga atattatata acccactgct gaaaaagaaa aaaatgattg
ttttagaagt 9120taaagtcaat attgatttta aatataagta atgaaggcat
atttccaata actagtgata 9180tggcatcgtt gcattttaca gtatcttcaa
aaatacagaa tttatagaat aatttctcct 9240catttaatat ttttcaaaat
caaagttatg gtttcctcat tttactaaaa tcgtattcta 9300attcttcatt
atagtaaatc tatgagcaac tccttacttc ggttcctctg atttcaaggc
9360catattttaa aaaatcaaaa ggcactgtga actattttga agaaaacaca
acattttaat 9420acagattgaa aggacctctt ctgaagctag aaacaatcta
tagttataca tcttcattaa 9480tactgtgtta ccttttaaaa tagtaatttt
ttacattttc ctgtgtaaac ctaattgtgg 9540tagaaatttt taccaactct
atactcaatc aagcaaaatt tctgtatatt ccctgtggaa 9600tgtacctatg
tgagtttcag aaattctcaa aatacgtgtt caaaaatttc tgcttttgca
9660tctttgggac acctcagaaa acttattaac aactgtgaat atgagaaata
cagaagaaaa 9720taataagccc tctatacata aatgcccagc acaattcatt
gttaaaaaac aaccaaacct 9780cacactactg tatttcatta tctgtactga
aagcaaatgc tttgtgacta ttaaatgttg 9840cacatcattc attcactgta
tagtaatcat tgactaaagc catttgtctg tgttttcttc 9900ttgtggttgt
atatatcagg taaaatattt tccaaagagc catgtgtcat gtaatactga
9960accactttga tattgagaca ttaatttgta cccttgttat tatctactag
taataatgta 10020atactgtaga aatattgctc taattctttt caaaattgtt
gcatccccct tagaatgttt 10080ctatttccat aaggatttag gtatgctatt
atcccttctt ataccctaag atgaagctgt 10140ttttgtgctc tttgttcatc
attggccctc attccaagca ctttacgctg tctgtaatgg 10200gatctatttt
tgcactggaa tatctgagaa ttgcaaaact agacaaaagt ttcacaacag
10260atttctaagt taaatcattt tcattaaaag gaaaaaagaa aaaaaatttt
gtatgtcaat 10320aactttatat gaagtattaa aatgcatatt tctatgttgt
aatataatga gtcacaaaat 10380aaagctgtga cagttctgtt ggtctacaga aa
1041286786DNAHUMAN 8atggcttgtt ggcctcagct gaggttgctg ctgtggaaga
acctcacttt cagaagaaga 60caaacatgtc agctgctgct ggaagtggcc tggcctctat
ttatcttcct gatcctgatc 120tctgttcggc tgagctaccc accctatgaa
caacatgaat gccattttcc aaataaagcc 180atgccctctg caggaacact
tccttgggtt caggggatta tctgtaatgc caacaacccc 240tgtttccgtt
acccgactcc tggggaggct cccggagttg ttggaaactt taacaaatcc
300attgtggctc gcctgttctc agatgctcgg aggcttcttt tatacagcca
gaaagacacc 360agcatgaagg acatgcgcaa agttctgaga acattacagc
agatcaagaa atccagctca 420aacttgaagc ttcaagattt cctggtggac
aatgaaacct tctctgggtt cctgtatcac 480aacctctctc tcccaaagtc
tactgtggac aagatgctga gggctgatgt cattctccac 540aaggtatttt
tgcaaggcta ccagttacat ttgacaagtc tgtgcaatgg atcaaaatca
600gaagagatga ttcaacttgg tgaccaagaa gtttctgagc tttgtggcct
accaagggag 660aaactggctg cagcagagcg agtacttcgt tccaacatgg
acatcctgaa gccaatcctg 720agaacactaa actctacatc tcccttcccg
agcaaggagc tggctgaagc cacaaaaaca 780ttgctgcata gtcttgggac
tctggcccag gagctgttca gcatgagaag ctggagtgac 840atgcgacagg
aggtgatgtt tctgaccaat gtgaacagct ccagctcctc cacccaaatc
900taccaggctg tgtctcgtat tgtctgcggg catcccgagg gaggggggct
gaagatcaag 960tctctcaact ggtatgagga caacaactac aaagccctct
ttggaggcaa tggcactgag 1020gaagatgctg aaaccttcta tgacaactct
acaactcctt actgcaatga tttgatgaag 1080aatttggagt ctagtcctct
ttcccgcatt atctggaaag ctctgaagcc gctgctcgtt 1140gggaagatcc
tgtatacacc tgacactcca gccacaaggc aggtcatggc tgaggtgaac
1200aagaccttcc aggaactggc tgtgttccat gatctggaag gcatgtggga
ggaactcagc 1260cccaagatct ggaccttcat ggagaacagc caagaaatgg
accttgtccg gatgctgttg 1320gacagcaggg acaatgacca cttttgggaa
cagcagttgg atggcttaga ttggacagcc 1380caagacatcg tggcgttttt
ggccaagcac ccagaggatg tccagtccag taatggttct 1440gtgtacacct
ggagagaagc tttcaacgag actaaccagg caatccggac catatctcgc
1500ttcatggagt gtgtcaacct gaacaagcta gaacccatag caacagaagt
ctggctcatc 1560aacaagtcca tggagctgct ggatgagagg aagttctggg
ctggtattgt gttcactgga 1620attactccag gcagcattga gctgccccat
catgtcaagt acaagatccg aatggacatt 1680gacaatgtgg agaggacaaa
taaaatcaag gatgggtact gggaccctgg tcctcgagct 1740gacccctttg
aggacatgcg gtacgtctgg gggggcttcg cctacttgca ggatgtggtg
1800gagcaggcaa tcatcagggt gctgacgggc accgagaaga aaactggtgt
ctatatgcaa 1860cagatgccct atccctgtta cgttgatgac atctttctgc
gggtgatgag ccggtcaatg 1920cccctcttca tgacgctggc ctggatttac
tcagtggctg tgatcatcaa gggcatcgtg 1980tatgagaagg aggcacggct
gaaagagacc atgcggatca tgggcctgga caacagcatc 2040ctctggttta
gctggttcat tagtagcctc attcctcttc ttgtgagcgc tggcctgcta
2100gtggtcatcc tgaagttagg aaacctgctg ccctacagtg atcccagcgt
ggtgtttgtc 2160ttcctgtccg tgtttgctgt ggtgacaatc ctgcagtgct
tcctgattag cacactcttc 2220tccagagcca acctggcagc agcctgtggg
ggcatcatct acttcacgct gtacctgccc 2280tacgtcctgt gtgtggcatg
gcaggactac gtgggcttca cactcaagat cttcgctagc 2340ctgctgtctc
ctgtggcttt tgggtttggc tgtgagtact ttgccctttt tgaggagcag
2400ggcattggag tgcagtggga caacctgttt gagagtcctg tggaggaaga
tggcttcaat 2460ctcaccactt cggtctccat gatgctgttt gacaccttcc
tctatggggt gatgacctgg 2520tacattgagg ctgtctttcc aggccagtac
ggaattccca ggccctggta ttttccttgc 2580accaagtcct actggtttgg
cgaggaaagt gatgagaaga gccaccctgg ttccaaccag 2640aagagaatat
cagaaatctg catggaggag gaacccaccc acttgaagct gggcgtgtcc
2700attcagaacc tggtaaaagt ctaccgagat gggatgaagg tggctgtcga
tggcctggca 2760ctgaattttt atgagggcca gatcacctcc ttcctgggcc
acaatggagc ggggaagacg 2820accaccatgt caatcctgac cgggttgttc
cccccgacct cgggcaccgc ctacatcctg 2880ggaaaagaca ttcgctctga
gatgagcacc atccggcaga acctgggggt ctgtccccag 2940cataacgtgc
tgtttgacat gctgactgtc gaagaacaca tctggttcta tgcccgcttg
3000aaagggctct ctgagaagca cgtgaaggcg gagatggagc agatggccct
ggatgttggt 3060ttgccatcaa gcaagctgaa aagcaaaaca agccagctgt
caggtggaat gcagagaaag 3120ctatctgtgg ccttggcctt tgtcggggga
tctaaggttg tcattctgga tgaacccaca 3180gctggtgtgg acccttactc
ccgcagggga atatgggagc tgctgctgaa ataccgacaa 3240ggccgcacca
ttattctctc tacacaccac atggatgaag cggacgtcct gggggacagg
3300attgccatca tctcccatgg gaagctgtgc tgtgtgggct cctccctgtt
tctgaagaac 3360cagctgggaa caggctacta cctgaccttg gtcaagaaag
atgtggaatc ctccctcagt 3420tcctgcagaa acagtagtag cactgtgtca
tacctgaaaa aggaggacag tgtttctcag 3480agcagttctg atgctggcct
gggcagcgac catgagagtg acacgctgac catcgatgtc 3540tctgctatct
ccaacctcat caggaagcat gtgtctgaag cccggctggt ggaagacata
3600gggcatgagc tgacctatgt gctgccatat gaagctgcta aggagggagc
ctttgtggaa 3660ctctttcatg agattgatga ccggctctca gacctgggca
tttctagtta tggcatctca 3720gagacgaccc tggaagaaat attcctcaag
gtggccgaag agagtggggt ggatgctgag 3780acctcagatg gtaccttgcc
agcaagacga aacaggcggg ccttcgggga caagcagagc 3840tgtcttcgcc
cgttcactga agatgatgct gctgatccaa atgattctga catagaccca
3900gaatccagag agacagactt gctcagtggg atggatggca aagggtccta
ccaggtgaaa 3960ggctggaaac ttacacagca acagtttgtg gcccttttgt
ggaagagact gctaattgcc 4020agacggagtc ggaaaggatt ttttgctcag
attgtcttgc cagctgtgtt tgtctgcatt 4080gcccttgtgt tcagcctgat
cgtgccaccc tttggcaagt accccagcct ggaacttcag 4140ccctggatgt
acaacgaaca gtacacattt gtcagcaatg atgctcctga ggacacggga
4200accctggaac tcttaaacgc cctcaccaaa gaccctggct tcgggacccg
ctgtatggaa 4260ggaaacccaa tcccagacac gccctgccag gcaggggagg
aagagtggac cactgcccca 4320gttccccaga ccatcatgga cctcttccag
aatgggaact ggacaatgca gaacccttca 4380cctgcatgcc agtgtagcag
cgacaaaatc aagaagatgc tgcctgtgtg tcccccaggg 4440gcaggggggc
tgcctcctcc acaaagaaaa caaaacactg cagatatcct tcaggacctg
4500acaggaagaa acatttcgga ttatctggtg aagacgtatg tgcagatcat
agccaaaagc 4560ttaaagaaca agatctgggt gaatgagttt aggtatggcg
gcttttccct gggtgtcagt 4620aatactcaag cacttcctcc gagtcaagaa
gttaatgatg ccatcaaaca aatgaagaaa 4680cacctaaagc tggccaagga
cagttctgca gatcgatttc tcaacagctt gggaagattt 4740atgacaggac
tggacaccaa aaataatgtc aaggtgtggt tcaataacaa gggctggcat
4800gcaatcagct ctttcctgaa tgtcatcaac aatgccattc tccgggccaa
cctgcaaaag 4860ggagagaacc ctagccatta tggaattact gctttcaatc
atcccctgaa tctcaccaag 4920cagcagctct cagaggtggc tctgatgacc
acatcagtgg atgtccttgt gtccatctgt 4980gtcatctttg caatgtcctt
cgtcccagcc agctttgtcg tattcctgat ccaggagcgg 5040gtcagcaaag
caaaacacct gcagttcatc agtggagtga agcctgtcat ctactggctc
5100tctaattttg tctgggatat gtgcaattac gttgtccctg ccacactggt
cattatcatc 5160ttcatctgct tccagcagaa gtcctatgtg tcctccacca
atctgcctgt gctagccctt 5220ctacttttgc tgtatgggtg gtcaatcaca
cctctcatgt acccagcctc ctttgtgttc 5280aagatcccca gcacagccta
tgtggtgctc accagcgtga acctcttcat tggcattaat 5340ggcagcgtgg
ccacctttgt gctggagctg ttcaccgaca ataagctgaa taatatcaat
5400gatatcctga agtccgtgtt cttgatcttc ccacattttt gcctgggacg
agggctcatc 5460gacatggtga aaaaccaggc aatggctgat gccctggaaa
ggtttgggga gaatcgcttt 5520gtgtcaccat tatcttggga cttggtggga
cgaaacctct tcgccatggc cgtggaaggg 5580gtggtgttct tcctcattac
tgttctgatc cagtacagat tcttcatcag gcccagacct 5640gtaaatgcaa
agctatctcc tctgaatgat gaagatgaag atgtgaggcg ggaaagacag
5700agaattcttg atggtggagg ccagaatgac atcttagaaa tcaaggagtt
gacgaagata 5760tatagaagga agcggaagcc tgctgttgac aggatttgcg
tgggcattcc tcctggtgag 5820tgctttgggc tcctgggagt taatggggct
ggaaaatcat caactttcaa gatgttaaca 5880ggagatacca ctgttaccag
aggagatgct ttccttaaca
aaaatagtat cttatcaaac 5940atccatgaag tacatcagaa catgggctac
tgccctcagt ttgatgccat cacagagctg 6000ttgactggga gagaacacgt
ggagttcttt gcccttttga gaggagtccc agagaaagaa 6060gttggcaagg
ttggtgagtg ggcgattcgg aaactgggcc tcgtgaagta tggagaaaaa
6120tatgctggta actatagtgg aggcaacaaa cgcaagctct ctacagccat
ggctttgatc 6180ggcgggcctc ctgtggtgtt tctggatgaa cccaccacag
gcatggatcc caaagcccgg 6240cggttcttgt ggaattgtgc cctaagtgtt
gtcaaggagg ggagatcagt agtgcttaca 6300tctcatagta tggaagagtg
tgaagctctt tgcactagga tggcaatcat ggtcaatgga 6360aggttcaggt
gccttggcag tgtccagcat ctaaaaaata ggtttggaga tggttataca
6420atagttgtac gaatagcagg gtccaacccg gacctgaagc ctgtccagga
tttctttgga 6480cttgcatttc ctggaagtgt tctaaaagag aaacaccgga
acatgctaca ataccagctt 6540ccatcttcat tatcttctct ggccaggata
ttcagcatcc tctcccagag caaaaagcga 6600ctccacatag aagactactc
tgtttctcag acaacacttg accaagtatt tgtgaacttt 6660gccaaggacc
aaagtgatga tgaccactta aaagacctct cattacacaa aaaccagaca
6720gtagtggacg ttgcagttct cacatctttt ctacaggatg agaaagtgaa
agaaagctat 6780gtatga 678692261PRTHUMAN 9Met Ala Cys Trp Pro Gln
Leu Arg Leu Leu Leu Trp Lys Asn Leu Thr1 5 10 15Phe Arg Arg Arg Gln
Thr Cys Gln Leu Leu Leu Glu Val Ala Trp Pro 20 25 30Leu Phe Ile Phe
Leu Ile Leu Ile Ser Val Arg Leu Ser Tyr Pro Pro 35 40 45Tyr Glu Gln
His Glu Cys His Phe Pro Asn Lys Ala Met Pro Ser Ala 50 55 60Gly Thr
Leu Pro Trp Val Gln Gly Ile Ile Cys Asn Ala Asn Asn Pro65 70 75
80Cys Phe Arg Tyr Pro Thr Pro Gly Glu Ala Pro Gly Val Val Gly Asn
85 90 95Phe Asn Lys Ser Ile Val Ala Arg Leu Phe Ser Asp Ala Arg Arg
Leu 100 105 110Leu Leu Tyr Ser Gln Lys Asp Thr Ser Met Lys Asp Met
Arg Lys Val 115 120 125Leu Arg Thr Leu Gln Gln Ile Lys Lys Ser Ser
Ser Asn Leu Lys Leu 130 135 140Gln Asp Phe Leu Val Asp Asn Glu Thr
Phe Ser Gly Phe Leu Tyr His145 150 155 160Asn Leu Ser Leu Pro Lys
Ser Thr Val Asp Lys Met Leu Arg Ala Asp 165 170 175Val Ile Leu His
Lys Val Phe Leu Gln Gly Tyr Gln Leu His Leu Thr 180 185 190Ser Leu
Cys Asn Gly Ser Lys Ser Glu Glu Met Ile Gln Leu Gly Asp 195 200
205Gln Glu Val Ser Glu Leu Cys Gly Leu Pro Arg Glu Lys Leu Ala Ala
210 215 220Ala Glu Arg Val Leu Arg Ser Asn Met Asp Ile Leu Lys Pro
Ile Leu225 230 235 240Arg Thr Leu Asn Ser Thr Ser Pro Phe Pro Ser
Lys Glu Leu Ala Glu 245 250 255Ala Thr Lys Thr Leu Leu His Ser Leu
Gly Thr Leu Ala Gln Glu Leu 260 265 270Phe Ser Met Arg Ser Trp Ser
Asp Met Arg Gln Glu Val Met Phe Leu 275 280 285Thr Asn Val Asn Ser
Ser Ser Ser Ser Thr Gln Ile Tyr Gln Ala Val 290 295 300Ser Arg Ile
Val Cys Gly His Pro Glu Gly Gly Gly Leu Lys Ile Lys305 310 315
320Ser Leu Asn Trp Tyr Glu Asp Asn Asn Tyr Lys Ala Leu Phe Gly Gly
325 330 335Asn Gly Thr Glu Glu Asp Ala Glu Thr Phe Tyr Asp Asn Ser
Thr Thr 340 345 350Pro Tyr Cys Asn Asp Leu Met Lys Asn Leu Glu Ser
Ser Pro Leu Ser 355 360 365Arg Ile Ile Trp Lys Ala Leu Lys Pro Leu
Leu Val Gly Lys Ile Leu 370 375 380Tyr Thr Pro Asp Thr Pro Ala Thr
Arg Gln Val Met Ala Glu Val Asn385 390 395 400Lys Thr Phe Gln Glu
Leu Ala Val Phe His Asp Leu Glu Gly Met Trp 405 410 415Glu Glu Leu
Ser Pro Lys Ile Trp Thr Phe Met Glu Asn Ser Gln Glu 420 425 430Met
Asp Leu Val Arg Met Leu Leu Asp Ser Arg Asp Asn Asp His Phe 435 440
445Trp Glu Gln Gln Leu Asp Gly Leu Asp Trp Thr Ala Gln Asp Ile Val
450 455 460Ala Phe Leu Ala Lys His Pro Glu Asp Val Gln Ser Ser Asn
Gly Ser465 470 475 480Val Tyr Thr Trp Arg Glu Ala Phe Asn Glu Thr
Asn Gln Ala Ile Arg 485 490 495Thr Ile Ser Arg Phe Met Glu Cys Val
Asn Leu Asn Lys Leu Glu Pro 500 505 510Ile Ala Thr Glu Val Trp Leu
Ile Asn Lys Ser Met Glu Leu Leu Asp 515 520 525Glu Arg Lys Phe Trp
Ala Gly Ile Val Phe Thr Gly Ile Thr Pro Gly 530 535 540Ser Ile Glu
Leu Pro His His Val Lys Tyr Lys Ile Arg Met Asp Ile545 550 555
560Asp Asn Val Glu Arg Thr Asn Lys Ile Lys Asp Gly Tyr Trp Asp Pro
565 570 575Gly Pro Arg Ala Asp Pro Phe Glu Asp Met Arg Tyr Val Trp
Gly Gly 580 585 590Phe Ala Tyr Leu Gln Asp Val Val Glu Gln Ala Ile
Ile Arg Val Leu 595 600 605Thr Gly Thr Glu Lys Lys Thr Gly Val Tyr
Met Gln Gln Met Pro Tyr 610 615 620Pro Cys Tyr Val Asp Asp Ile Phe
Leu Arg Val Met Ser Arg Ser Met625 630 635 640Pro Leu Phe Met Thr
Leu Ala Trp Ile Tyr Ser Val Ala Val Ile Ile 645 650 655Lys Gly Ile
Val Tyr Glu Lys Glu Ala Arg Leu Lys Glu Thr Met Arg 660 665 670Ile
Met Gly Leu Asp Asn Ser Ile Leu Trp Phe Ser Trp Phe Ile Ser 675 680
685Ser Leu Ile Pro Leu Leu Val Ser Ala Gly Leu Leu Val Val Ile Leu
690 695 700Lys Leu Gly Asn Leu Leu Pro Tyr Ser Asp Pro Ser Val Val
Phe Val705 710 715 720Phe Leu Ser Val Phe Ala Val Val Thr Ile Leu
Gln Cys Phe Leu Ile 725 730 735Ser Thr Leu Phe Ser Arg Ala Asn Leu
Ala Ala Ala Cys Gly Gly Ile 740 745 750Ile Tyr Phe Thr Leu Tyr Leu
Pro Tyr Val Leu Cys Val Ala Trp Gln 755 760 765Asp Tyr Val Gly Phe
Thr Leu Lys Ile Phe Ala Ser Leu Leu Ser Pro 770 775 780Val Ala Phe
Gly Phe Gly Cys Glu Tyr Phe Ala Leu Phe Glu Glu Gln785 790 795
800Gly Ile Gly Val Gln Trp Asp Asn Leu Phe Glu Ser Pro Val Glu Glu
805 810 815Asp Gly Phe Asn Leu Thr Thr Ser Val Ser Met Met Leu Phe
Asp Thr 820 825 830Phe Leu Tyr Gly Val Met Thr Trp Tyr Ile Glu Ala
Val Phe Pro Gly 835 840 845Gln Tyr Gly Ile Pro Arg Pro Trp Tyr Phe
Pro Cys Thr Lys Ser Tyr 850 855 860Trp Phe Gly Glu Glu Ser Asp Glu
Lys Ser His Pro Gly Ser Asn Gln865 870 875 880Lys Arg Ile Ser Glu
Ile Cys Met Glu Glu Glu Pro Thr His Leu Lys 885 890 895Leu Gly Val
Ser Ile Gln Asn Leu Val Lys Val Tyr Arg Asp Gly Met 900 905 910Lys
Val Ala Val Asp Gly Leu Ala Leu Asn Phe Tyr Glu Gly Gln Ile 915 920
925Thr Ser Phe Leu Gly His Asn Gly Ala Gly Lys Thr Thr Thr Met Ser
930 935 940Ile Leu Thr Gly Leu Phe Pro Pro Thr Ser Gly Thr Ala Tyr
Ile Leu945 950 955 960Gly Lys Asp Ile Arg Ser Glu Met Ser Thr Ile
Arg Gln Asn Leu Gly 965 970 975Val Cys Pro Gln His Asn Val Leu Phe
Asp Met Leu Thr Val Glu Glu 980 985 990His Ile Trp Phe Tyr Ala Arg
Leu Lys Gly Leu Ser Glu Lys His Val 995 1000 1005Lys Ala Glu Met
Glu Gln Met Ala Leu Asp Val Gly Leu Pro Ser 1010 1015 1020Ser Lys
Leu Lys Ser Lys Thr Ser Gln Leu Ser Gly Gly Met Gln 1025 1030
1035Arg Lys Leu Ser Val Ala Leu Ala Phe Val Gly Gly Ser Lys Val
1040 1045 1050Val Ile Leu Asp Glu Pro Thr Ala Gly Val Asp Pro Tyr
Ser Arg 1055 1060 1065Arg Gly Ile Trp Glu Leu Leu Leu Lys Tyr Arg
Gln Gly Arg Thr 1070 1075 1080Ile Ile Leu Ser Thr His His Met Asp
Glu Ala Asp Val Leu Gly 1085 1090 1095Asp Arg Ile Ala Ile Ile Ser
His Gly Lys Leu Cys Cys Val Gly 1100 1105 1110Ser Ser Leu Phe Leu
Lys Asn Gln Leu Gly Thr Gly Tyr Tyr Leu 1115 1120 1125Thr Leu Val
Lys Lys Asp Val Glu Ser Ser Leu Ser Ser Cys Arg 1130 1135 1140Asn
Ser Ser Ser Thr Val Ser Tyr Leu Lys Lys Glu Asp Ser Val 1145 1150
1155Ser Gln Ser Ser Ser Asp Ala Gly Leu Gly Ser Asp His Glu Ser
1160 1165 1170Asp Thr Leu Thr Ile Asp Val Ser Ala Ile Ser Asn Leu
Ile Arg 1175 1180 1185Lys His Val Ser Glu Ala Arg Leu Val Glu Asp
Ile Gly His Glu 1190 1195 1200Leu Thr Tyr Val Leu Pro Tyr Glu Ala
Ala Lys Glu Gly Ala Phe 1205 1210 1215Val Glu Leu Phe His Glu Ile
Asp Asp Arg Leu Ser Asp Leu Gly 1220 1225 1230Ile Ser Ser Tyr Gly
Ile Ser Glu Thr Thr Leu Glu Glu Ile Phe 1235 1240 1245Leu Lys Val
Ala Glu Glu Ser Gly Val Asp Ala Glu Thr Ser Asp 1250 1255 1260Gly
Thr Leu Pro Ala Arg Arg Asn Arg Arg Ala Phe Gly Asp Lys 1265 1270
1275Gln Ser Cys Leu Arg Pro Phe Thr Glu Asp Asp Ala Ala Asp Pro
1280 1285 1290Asn Asp Ser Asp Ile Asp Pro Glu Ser Arg Glu Thr Asp
Leu Leu 1295 1300 1305Ser Gly Met Asp Gly Lys Gly Ser Tyr Gln Val
Lys Gly Trp Lys 1310 1315 1320Leu Thr Gln Gln Gln Phe Val Ala Leu
Leu Trp Lys Arg Leu Leu 1325 1330 1335Ile Ala Arg Arg Ser Arg Lys
Gly Phe Phe Ala Gln Ile Val Leu 1340 1345 1350Pro Ala Val Phe Val
Cys Ile Ala Leu Val Phe Ser Leu Ile Val 1355 1360 1365Pro Pro Phe
Gly Lys Tyr Pro Ser Leu Glu Leu Gln Pro Trp Met 1370 1375 1380Tyr
Asn Glu Gln Tyr Thr Phe Val Ser Asn Asp Ala Pro Glu Asp 1385 1390
1395Thr Gly Thr Leu Glu Leu Leu Asn Ala Leu Thr Lys Asp Pro Gly
1400 1405 1410Phe Gly Thr Arg Cys Met Glu Gly Asn Pro Ile Pro Asp
Thr Pro 1415 1420 1425Cys Gln Ala Gly Glu Glu Glu Trp Thr Thr Ala
Pro Val Pro Gln 1430 1435 1440Thr Ile Met Asp Leu Phe Gln Asn Gly
Asn Trp Thr Met Gln Asn 1445 1450 1455Pro Ser Pro Ala Cys Gln Cys
Ser Ser Asp Lys Ile Lys Lys Met 1460 1465 1470Leu Pro Val Cys Pro
Pro Gly Ala Gly Gly Leu Pro Pro Pro Gln 1475 1480 1485Arg Lys Gln
Asn Thr Ala Asp Ile Leu Gln Asp Leu Thr Gly Arg 1490 1495 1500Asn
Ile Ser Asp Tyr Leu Val Lys Thr Tyr Val Gln Ile Ile Ala 1505 1510
1515Lys Ser Leu Lys Asn Lys Ile Trp Val Asn Glu Phe Arg Tyr Gly
1520 1525 1530Gly Phe Ser Leu Gly Val Ser Asn Thr Gln Ala Leu Pro
Pro Ser 1535 1540 1545Gln Glu Val Asn Asp Ala Ile Lys Gln Met Lys
Lys His Leu Lys 1550 1555 1560Leu Ala Lys Asp Ser Ser Ala Asp Arg
Phe Leu Asn Ser Leu Gly 1565 1570 1575Arg Phe Met Thr Gly Leu Asp
Thr Lys Asn Asn Val Lys Val Trp 1580 1585 1590Phe Asn Asn Lys Gly
Trp His Ala Ile Ser Ser Phe Leu Asn Val 1595 1600 1605Ile Asn Asn
Ala Ile Leu Arg Ala Asn Leu Gln Lys Gly Glu Asn 1610 1615 1620Pro
Ser His Tyr Gly Ile Thr Ala Phe Asn His Pro Leu Asn Leu 1625 1630
1635Thr Lys Gln Gln Leu Ser Glu Val Ala Leu Met Thr Thr Ser Val
1640 1645 1650Asp Val Leu Val Ser Ile Cys Val Ile Phe Ala Met Ser
Phe Val 1655 1660 1665Pro Ala Ser Phe Val Val Phe Leu Ile Gln Glu
Arg Val Ser Lys 1670 1675 1680Ala Lys His Leu Gln Phe Ile Ser Gly
Val Lys Pro Val Ile Tyr 1685 1690 1695Trp Leu Ser Asn Phe Val Trp
Asp Met Cys Asn Tyr Val Val Pro 1700 1705 1710Ala Thr Leu Val Ile
Ile Ile Phe Ile Cys Phe Gln Gln Lys Ser 1715 1720 1725Tyr Val Ser
Ser Thr Asn Leu Pro Val Leu Ala Leu Leu Leu Leu 1730 1735 1740Leu
Tyr Gly Trp Ser Ile Thr Pro Leu Met Tyr Pro Ala Ser Phe 1745 1750
1755Val Phe Lys Ile Pro Ser Thr Ala Tyr Val Val Leu Thr Ser Val
1760 1765 1770Asn Leu Phe Ile Gly Ile Asn Gly Ser Val Ala Thr Phe
Val Leu 1775 1780 1785Glu Leu Phe Thr Asp Asn Lys Leu Asn Asn Ile
Asn Asp Ile Leu 1790 1795 1800Lys Ser Val Phe Leu Ile Phe Pro His
Phe Cys Leu Gly Arg Gly 1805 1810 1815Leu Ile Asp Met Val Lys Asn
Gln Ala Met Ala Asp Ala Leu Glu 1820 1825 1830Arg Phe Gly Glu Asn
Arg Phe Val Ser Pro Leu Ser Trp Asp Leu 1835 1840 1845Val Gly Arg
Asn Leu Phe Ala Met Ala Val Glu Gly Val Val Phe 1850 1855 1860Phe
Leu Ile Thr Val Leu Ile Gln Tyr Arg Phe Phe Ile Arg Pro 1865 1870
1875Arg Pro Val Asn Ala Lys Leu Ser Pro Leu Asn Asp Glu Asp Glu
1880 1885 1890Asp Val Arg Arg Glu Arg Gln Arg Ile Leu Asp Gly Gly
Gly Gln 1895 1900 1905Asn Asp Ile Leu Glu Ile Lys Glu Leu Thr Lys
Ile Tyr Arg Arg 1910 1915 1920Lys Arg Lys Pro Ala Val Asp Arg Ile
Cys Val Gly Ile Pro Pro 1925 1930 1935Gly Glu Cys Phe Gly Leu Leu
Gly Val Asn Gly Ala Gly Lys Ser 1940 1945 1950Ser Thr Phe Lys Met
Leu Thr Gly Asp Thr Thr Val Thr Arg Gly 1955 1960 1965Asp Ala Phe
Leu Asn Lys Asn Ser Ile Leu Ser Asn Ile His Glu 1970 1975 1980Val
His Gln Asn Met Gly Tyr Cys Pro Gln Phe Asp Ala Ile Thr 1985 1990
1995Glu Leu Leu Thr Gly Arg Glu His Val Glu Phe Phe Ala Leu Leu
2000 2005 2010Arg Gly Val Pro Glu Lys Glu Val Gly Lys Val Gly Glu
Trp Ala 2015 2020 2025Ile Arg Lys Leu Gly Leu Val Lys Tyr Gly Glu
Lys Tyr Ala Gly 2030 2035 2040Asn Tyr Ser Gly Gly Asn Lys Arg Lys
Leu Ser Thr Ala Met Ala 2045 2050 2055Leu Ile Gly Gly Pro Pro Val
Val Phe Leu Asp Glu Pro Thr Thr 2060 2065 2070Gly Met Asp Pro Lys
Ala Arg Arg Phe Leu Trp Asn Cys Ala Leu 2075 2080 2085Ser Val Val
Lys Glu Gly Arg Ser Val Val Leu Thr Ser His Ser 2090 2095 2100Met
Glu Glu Cys Glu Ala Leu Cys Thr Arg Met Ala Ile Met Val 2105 2110
2115Asn Gly Arg Phe Arg Cys Leu Gly Ser Val Gln His Leu Lys Asn
2120 2125 2130Arg Phe Gly Asp Gly Tyr Thr Ile Val Val Arg Ile Ala
Gly Ser 2135 2140 2145Asn Pro Asp Leu Lys Pro Val Gln Asp Phe Phe
Gly Leu Ala Phe 2150 2155 2160Pro Gly Ser Val Leu Lys Glu Lys His
Arg Asn Met Leu Gln Tyr 2165 2170 2175Gln Leu Pro Ser Ser Leu Ser
Ser Leu Ala Arg Ile Phe Ser Ile 2180 2185 2190Leu Ser Gln Ser Lys
Lys Arg Leu His Ile Glu Asp Tyr Ser Val 2195 2200 2205Ser Gln Thr
Thr Leu Asp Gln Val Phe Val Asn Phe Ala Lys Asp 2210 2215 2220Gln
Ser Asp Asp Asp His Leu Lys Asp Leu Ser Leu His Lys Asn 2225 2230
2235Gln Thr Val Val Asp Val Ala Val Leu Thr Ser Phe Leu Gln Asp
2240 2245 2250Glu Lys Val Lys Glu Ser Tyr Val 2255
2260102261PRTHUMAN 10Met Ala Cys Trp Pro Gln Leu Arg Leu Leu Leu
Trp Lys Asn Leu Thr1 5 10 15Phe Arg Arg Arg Gln Thr Cys Gln Leu Leu
Leu Glu Val Ala Trp Pro 20 25 30Leu Phe Ile Phe Leu Ile Leu Ile
Ser Val Arg Leu Ser Tyr Pro Pro 35 40 45Tyr Glu Gln His Glu Cys His
Phe Pro Asn Lys Ala Met Pro Ser Ala 50 55 60Gly Thr Leu Pro Trp Val
Gln Gly Ile Ile Cys Asn Ala Asn Asn Pro65 70 75 80Cys Phe Arg Tyr
Pro Thr Pro Gly Glu Ala Pro Gly Val Val Gly Asn 85 90 95Phe Asn Lys
Ser Ile Val Ala Arg Leu Phe Ser Asp Ala Arg Arg Leu 100 105 110Leu
Leu Tyr Ser Gln Lys Asp Thr Ser Met Lys Asp Met Arg Lys Val 115 120
125Leu Arg Thr Leu Gln Gln Ile Lys Lys Ser Ser Ser Asn Leu Lys Leu
130 135 140Gln Asp Phe Leu Val Asp Asn Glu Thr Phe Ser Gly Phe Leu
Tyr His145 150 155 160Asn Leu Ser Leu Pro Lys Ser Thr Val Asp Lys
Met Leu Arg Ala Asp 165 170 175Val Ile Leu His Lys Val Phe Leu Gln
Gly Tyr Gln Leu His Leu Thr 180 185 190Ser Leu Cys Asn Gly Ser Lys
Ser Glu Glu Met Ile Gln Leu Gly Asp 195 200 205Gln Glu Val Ser Glu
Leu Cys Gly Leu Pro Arg Glu Lys Leu Ala Ala 210 215 220Ala Glu Arg
Val Leu Arg Ser Asn Met Asp Ile Leu Lys Pro Ile Leu225 230 235
240Arg Thr Leu Asn Ser Thr Ser Pro Phe Pro Ser Lys Glu Leu Ala Glu
245 250 255Ala Thr Lys Thr Leu Leu His Ser Leu Gly Thr Leu Ala Gln
Glu Leu 260 265 270Phe Ser Met Arg Ser Trp Ser Asp Met Arg Gln Glu
Val Met Phe Leu 275 280 285Thr Asn Val Asn Ser Ser Ser Ser Ser Thr
Gln Ile Tyr Gln Ala Val 290 295 300Ser Arg Ile Val Cys Gly His Pro
Glu Gly Gly Gly Leu Lys Ile Lys305 310 315 320Ser Leu Asn Trp Tyr
Glu Asp Asn Asn Tyr Lys Ala Leu Phe Gly Gly 325 330 335Asn Gly Thr
Glu Glu Asp Ala Glu Thr Phe Tyr Asp Asn Ser Thr Thr 340 345 350Pro
Tyr Cys Asn Asp Leu Met Lys Asn Leu Glu Ser Ser Pro Leu Ser 355 360
365Arg Ile Ile Trp Lys Ala Leu Lys Pro Leu Leu Val Gly Lys Ile Leu
370 375 380Tyr Thr Pro Asp Thr Pro Ala Thr Arg Gln Val Met Ala Glu
Val Asn385 390 395 400Lys Thr Phe Gln Glu Leu Ala Val Phe His Asp
Leu Glu Gly Met Trp 405 410 415Glu Glu Leu Ser Pro Lys Ile Trp Thr
Phe Met Glu Asn Ser Gln Glu 420 425 430Met Asp Leu Val Arg Met Leu
Leu Asp Ser Arg Asp Asn Asp His Phe 435 440 445Trp Glu Gln Gln Leu
Asp Gly Leu Asp Trp Thr Ala Gln Asp Ile Val 450 455 460Ala Phe Leu
Ala Lys His Pro Glu Asp Val Gln Ser Ser Asn Gly Ser465 470 475
480Val Tyr Thr Trp Arg Glu Ala Phe Asn Glu Thr Asn Gln Ala Ile Arg
485 490 495Thr Ile Ser Arg Phe Met Glu Cys Val Asn Leu Asn Lys Leu
Glu Pro 500 505 510Ile Ala Thr Glu Val Trp Leu Ile Asn Lys Ser Met
Glu Leu Leu Asp 515 520 525Glu Arg Lys Phe Trp Ala Gly Ile Val Phe
Thr Gly Ile Thr Pro Gly 530 535 540Ser Ile Glu Leu Pro His His Val
Lys Tyr Lys Ile Arg Met Asp Ile545 550 555 560Asp Asn Val Glu Arg
Thr Asn Lys Ile Lys Asp Gly Tyr Trp Asp Pro 565 570 575Gly Pro Arg
Ala Asp Pro Phe Glu Asp Met Arg Tyr Val Trp Gly Gly 580 585 590Phe
Ala Tyr Leu Gln Asp Val Val Glu Gln Ala Ile Ile Arg Val Leu 595 600
605Thr Gly Thr Glu Lys Lys Thr Gly Val Tyr Met Gln Gln Met Pro Tyr
610 615 620Pro Cys Tyr Val Asp Asp Ile Phe Leu Arg Val Met Ser Arg
Ser Met625 630 635 640Pro Leu Phe Met Thr Leu Ala Trp Ile Tyr Ser
Val Ala Val Ile Ile 645 650 655Lys Gly Ile Val Tyr Glu Lys Glu Ala
Arg Leu Lys Glu Thr Met Arg 660 665 670Ile Met Gly Leu Asp Asn Ser
Ile Leu Trp Phe Ser Trp Phe Ile Ser 675 680 685Ser Leu Ile Pro Leu
Leu Val Ser Ala Gly Leu Leu Val Val Ile Leu 690 695 700Lys Leu Gly
Asn Leu Leu Pro Tyr Ser Asp Pro Ser Val Val Phe Val705 710 715
720Phe Leu Ser Val Phe Ala Val Val Thr Ile Leu Gln Cys Phe Leu Ile
725 730 735Ser Thr Leu Phe Ser Arg Ala Asn Leu Ala Ala Ala Cys Gly
Gly Ile 740 745 750Ile Tyr Phe Thr Leu Tyr Leu Pro Tyr Val Leu Cys
Val Ala Trp Gln 755 760 765Asp Tyr Val Gly Phe Thr Leu Lys Ile Phe
Ala Ser Leu Leu Ser Pro 770 775 780Val Ala Phe Gly Phe Gly Cys Glu
Tyr Phe Ala Leu Phe Glu Glu Gln785 790 795 800Gly Ile Gly Val Gln
Trp Asp Asn Leu Phe Glu Ser Pro Val Glu Glu 805 810 815Asp Gly Phe
Asn Leu Thr Thr Ser Val Ser Met Met Leu Phe Asp Thr 820 825 830Phe
Leu Tyr Gly Val Met Thr Trp Tyr Ile Glu Ala Val Phe Pro Gly 835 840
845Gln Tyr Gly Ile Pro Arg Pro Trp Tyr Phe Pro Cys Thr Lys Ser Tyr
850 855 860Trp Phe Gly Glu Glu Ser Asp Glu Lys Ser His Pro Gly Ser
Asn Gln865 870 875 880Lys Arg Ile Ser Glu Ile Cys Met Glu Glu Glu
Pro Thr His Leu Lys 885 890 895Leu Gly Val Ser Ile Gln Asn Leu Val
Lys Val Tyr Arg Asp Gly Met 900 905 910Lys Val Ala Val Asp Gly Leu
Ala Leu Asn Phe Tyr Glu Gly Gln Ile 915 920 925Thr Ser Phe Leu Gly
His Asn Gly Ala Gly Lys Thr Thr Thr Met Ser 930 935 940Ile Leu Thr
Gly Leu Phe Pro Pro Thr Ser Gly Thr Ala Tyr Ile Leu945 950 955
960Gly Lys Asp Ile Arg Ser Glu Met Ser Thr Ile Arg Gln Asn Leu Gly
965 970 975Val Cys Pro Gln His Asn Val Leu Phe Asp Met Leu Thr Val
Glu Glu 980 985 990His Ile Trp Phe Tyr Ala Arg Leu Lys Gly Leu Ser
Glu Lys His Val 995 1000 1005Lys Ala Glu Met Glu Gln Met Ala Leu
Asp Val Gly Leu Pro Ser 1010 1015 1020Ser Lys Leu Lys Ser Lys Thr
Ser Gln Leu Ser Gly Gly Met Gln 1025 1030 1035Arg Lys Leu Ser Val
Ala Leu Ala Phe Val Gly Gly Ser Lys Val 1040 1045 1050Val Ile Leu
Asp Glu Pro Thr Ala Gly Val Asp Pro Tyr Ser Arg 1055 1060 1065Arg
Gly Ile Trp Glu Leu Leu Leu Lys Tyr Arg Gln Gly Arg Thr 1070 1075
1080Ile Ile Leu Ser Thr His His Met Asp Glu Ala Asp Val Leu Gly
1085 1090 1095Asp Arg Ile Ala Ile Ile Ser His Gly Lys Leu Cys Cys
Val Gly 1100 1105 1110Ser Ser Leu Phe Leu Lys Asn Gln Leu Gly Thr
Gly Tyr Tyr Leu 1115 1120 1125Thr Leu Val Lys Lys Asp Val Glu Ser
Ser Leu Ser Ser Cys Arg 1130 1135 1140Asn Ser Ser Ser Thr Val Ser
Tyr Leu Lys Lys Glu Asp Ser Val 1145 1150 1155Ser Gln Ser Ser Ser
Asp Ala Gly Leu Gly Ser Asp His Glu Ser 1160 1165 1170Asp Thr Leu
Thr Ile Asp Val Ser Ala Ile Ser Asn Leu Ile Arg 1175 1180 1185Lys
His Val Ser Glu Ala Arg Leu Val Glu Asp Ile Gly His Glu 1190 1195
1200Leu Thr Tyr Val Leu Pro Tyr Glu Ala Ala Lys Glu Gly Ala Phe
1205 1210 1215Val Glu Leu Phe His Glu Ile Asp Asp Arg Leu Ser Asp
Leu Gly 1220 1225 1230Ile Ser Ser Tyr Gly Ile Ser Glu Thr Thr Leu
Glu Glu Ile Phe 1235 1240 1245Leu Lys Val Ala Glu Glu Ser Gly Val
Asp Ala Glu Thr Ser Asp 1250 1255 1260Gly Thr Leu Pro Ala Arg Arg
Asn Arg Arg Ala Phe Gly Asp Lys 1265 1270 1275Gln Ser Cys Leu Arg
Pro Phe Thr Glu Asp Asp Ala Ala Asp Pro 1280 1285 1290Asn Asp Ser
Asp Ile Asp Pro Glu Ser Arg Glu Thr Asp Leu Leu 1295 1300 1305Ser
Gly Met Asp Gly Lys Gly Ser Tyr Gln Val Lys Gly Trp Lys 1310 1315
1320Leu Thr Gln Gln Gln Phe Val Ala Leu Leu Trp Lys Arg Leu Leu
1325 1330 1335Ile Ala Arg Arg Ser Arg Lys Gly Phe Phe Ala Gln Ile
Val Leu 1340 1345 1350Pro Ala Val Phe Val Cys Ile Ala Leu Val Phe
Ser Leu Ile Val 1355 1360 1365Pro Pro Phe Gly Lys Tyr Pro Ser Leu
Glu Leu Gln Pro Trp Met 1370 1375 1380Tyr Asn Glu Gln Tyr Thr Phe
Val Ser Asn Asp Ala Pro Glu Asp 1385 1390 1395Thr Gly Thr Leu Glu
Leu Leu Asn Ala Leu Thr Lys Asp Pro Gly 1400 1405 1410Phe Gly Thr
Arg Cys Met Glu Gly Asn Pro Ile Pro Asp Thr Pro 1415 1420 1425Cys
Gln Ala Gly Glu Glu Glu Trp Thr Thr Ala Pro Val Pro Gln 1430 1435
1440Thr Ile Met Asp Leu Phe Gln Asn Gly Asn Trp Thr Met Gln Asn
1445 1450 1455Pro Ser Pro Ala Cys Gln Cys Ser Ser Asp Lys Ile Lys
Lys Met 1460 1465 1470Leu Pro Val Cys Pro Pro Gly Ala Gly Gly Leu
Pro Pro Pro Gln 1475 1480 1485Arg Lys Gln Asn Thr Ala Asp Ile Leu
Gln Asp Leu Thr Gly Arg 1490 1495 1500Asn Ile Ser Asp Tyr Leu Val
Lys Thr Tyr Val Gln Ile Ile Ala 1505 1510 1515Lys Ser Leu Lys Asn
Lys Ile Trp Val Asn Glu Phe Arg Tyr Gly 1520 1525 1530Gly Phe Ser
Leu Gly Val Ser Asn Thr Gln Ala Leu Pro Pro Ser 1535 1540 1545Gln
Glu Val Asn Asp Ala Ile Lys Gln Met Lys Lys His Leu Lys 1550 1555
1560Leu Ala Lys Asp Ser Ser Ala Asp Arg Phe Leu Asn Ser Leu Gly
1565 1570 1575Arg Phe Met Thr Gly Leu Asp Thr Lys Asn Asn Val Lys
Val Trp 1580 1585 1590Phe Asn Asn Lys Gly Trp His Ala Ile Ser Ser
Phe Leu Asn Val 1595 1600 1605Ile Asn Asn Ala Ile Leu Arg Ala Asn
Leu Gln Lys Gly Glu Asn 1610 1615 1620Pro Ser His Tyr Gly Ile Thr
Ala Phe Asn His Pro Leu Asn Leu 1625 1630 1635Thr Lys Gln Gln Leu
Ser Glu Val Ala Leu Met Thr Thr Ser Val 1640 1645 1650Asp Val Leu
Val Ser Ile Cys Val Ile Phe Ala Met Ser Phe Val 1655 1660 1665Pro
Ala Ser Phe Val Val Phe Leu Ile Gln Glu Arg Val Ser Lys 1670 1675
1680Ala Lys His Leu Gln Phe Ile Ser Gly Val Lys Pro Val Ile Tyr
1685 1690 1695Trp Leu Ser Asn Phe Val Trp Asp Met Cys Asn Tyr Val
Val Pro 1700 1705 1710Ala Thr Leu Val Ile Ile Ile Phe Ile Cys Phe
Gln Gln Lys Ser 1715 1720 1725Tyr Val Ser Ser Thr Asn Leu Pro Val
Leu Ala Leu Leu Leu Leu 1730 1735 1740Leu Tyr Gly Trp Ser Ile Thr
Pro Leu Met Tyr Pro Ala Ser Phe 1745 1750 1755Val Phe Lys Ile Pro
Ser Thr Ala Tyr Val Val Leu Thr Ser Val 1760 1765 1770Asn Leu Phe
Ile Gly Ile Asn Gly Ser Val Ala Thr Phe Val Leu 1775 1780 1785Glu
Leu Phe Thr Asp Asn Lys Leu Asn Asn Ile Asn Asp Ile Leu 1790 1795
1800Lys Ser Val Phe Leu Ile Phe Pro His Phe Cys Leu Gly Arg Gly
1805 1810 1815Leu Ile Asp Met Val Lys Asn Gln Ala Met Ala Asp Ala
Leu Glu 1820 1825 1830Arg Phe Gly Glu Asn Arg Phe Val Ser Pro Leu
Ser Trp Asp Leu 1835 1840 1845Val Gly Arg Asn Leu Phe Ala Met Ala
Val Glu Gly Val Val Phe 1850 1855 1860Phe Leu Ile Thr Val Leu Ile
Gln Tyr Arg Phe Phe Ile Arg Pro 1865 1870 1875Arg Pro Val Asn Ala
Lys Leu Ser Pro Leu Asn Asp Glu Asp Glu 1880 1885 1890Asp Val Arg
Arg Glu Arg Gln Arg Ile Leu Asp Gly Gly Gly Gln 1895 1900 1905Asn
Asp Ile Leu Glu Ile Lys Glu Leu Thr Lys Ile Tyr Arg Arg 1910 1915
1920Lys Arg Lys Pro Ala Val Asp Arg Ile Cys Val Gly Ile Pro Pro
1925 1930 1935Gly Glu Cys Phe Gly Leu Leu Gly Val Asn Gly Ala Gly
Lys Ser 1940 1945 1950Ser Thr Phe Lys Met Leu Thr Gly Asp Thr Thr
Val Thr Arg Gly 1955 1960 1965Asp Ala Phe Leu Asn Lys Asn Ser Ile
Leu Ser Asn Ile His Glu 1970 1975 1980Val His Gln Asn Met Gly Tyr
Cys Pro Gln Phe Asp Ala Ile Thr 1985 1990 1995Glu Leu Leu Thr Gly
Arg Glu His Val Glu Phe Phe Ala Leu Leu 2000 2005 2010Arg Gly Val
Pro Glu Lys Glu Val Gly Lys Val Gly Glu Trp Ala 2015 2020 2025Ile
Arg Lys Leu Gly Leu Val Lys Tyr Gly Glu Lys Tyr Ala Gly 2030 2035
2040Asn Tyr Ser Gly Gly Asn Lys Arg Lys Leu Ser Thr Ala Met Ala
2045 2050 2055Leu Ile Gly Gly Pro Pro Val Val Phe Leu Asp Glu Pro
Thr Thr 2060 2065 2070Gly Met Asp Pro Lys Ala Arg Arg Phe Leu Trp
Asn Cys Ala Leu 2075 2080 2085Ser Val Val Lys Glu Gly Arg Ser Val
Val Leu Thr Ser His Ser 2090 2095 2100Met Glu Glu Cys Glu Ala Leu
Cys Thr Arg Met Ala Ile Met Val 2105 2110 2115Asn Gly Arg Phe Arg
Cys Leu Gly Ser Val Gln His Leu Lys Asn 2120 2125 2130Arg Phe Gly
Asp Gly Tyr Thr Ile Val Val Arg Ile Ala Gly Ser 2135 2140 2145Asn
Pro Asp Leu Lys Pro Val Gln Asp Phe Phe Gly Leu Ala Phe 2150 2155
2160Pro Gly Ser Val Leu Lys Glu Lys His Arg Asn Met Leu Gln Tyr
2165 2170 2175Gln Leu Pro Ser Ser Leu Ser Ser Leu Ala Arg Ile Phe
Ser Ile 2180 2185 2190Leu Ser Gln Ser Lys Lys Arg Leu His Ile Glu
Asp Tyr Ser Val 2195 2200 2205Ser Gln Thr Thr Leu Asp Gln Val Phe
Val Asn Phe Ala Lys Asp 2210 2215 2220Gln Ser Asp Asp Asp His Leu
Lys Asp Leu Ser Leu His Lys Asn 2225 2230 2235Gln Thr Val Val Asp
Val Ala Val Leu Thr Ser Phe Leu Gln Asp 2240 2245 2250Glu Lys Val
Lys Glu Ser Tyr Val 2255 2260112566DNAHUMAN 11cgtcgccgtc cccgtctcct
gccaggcgcg gagccctgcg agccgcgggt gggccccagg 60cgcgcagaca tgggctgctc
cgccaaagcg cgctgggctg ccggggcgct gggcgtcgcg 120gggctactgt
gcgctgtgct gggcgctgtc atgatcgtga tggtgccgtc gctcatcaag
180cagcaggtcc ttaagaacgt gcgcatcgac cccagtagcc tgtccttcaa
catgtggaag 240gagatcccta tccccttcta tctctccgtc tacttctttg
acgtcatgaa ccccagcgag 300atcctgaagg gcgagaagcc gcaggtgcgg
gagcgcgggc cctacgtgta cagggagtcc 360aggcacaaaa gcaacatcac
cttcaacaac aacgacaccg tgtccttcct cgagtaccgc 420accttccagt
tccagccctc caagtcccac ggctcggaga gcgactacat cgtcatgccc
480aacatcctgg tcttgggtgc ggcggtgatg atggagaata agcccatgac
cctgaagctc 540atcatgacct tggcattcac caccctcggc gaacgtgcct
tcatgaaccg cactgtgggt 600gagatcatgt ggggctacaa ggaccccctt
gtgaatctca tcaacaagta ctttccaggc 660atgttcccct tcaaggacaa
gttcggatta tttgctgagc tcaacaactc cgactctggg 720ctcttcacgg
tgttcacggg ggtccagaac atcagcagga tccacctcgt ggacaagtgg
780aacgggctga gcaaggttga cttctggcat tccgatcagt gcaacatgat
caatggaact 840tctgggcaaa tgtggccgcc cttcatgact cctgagtcct
cgctggagtt ctacagcccg 900gaggcctgcc gatccatgaa gctaatgtac
aaggagtcag gggtgtttga aggcatcccc 960acctatcgct tcgtggctcc
caaaaccctg tttgccaacg ggtccatcta cccacccaac 1020gaaggcttct
gcccgtgcct ggagtctgga attcagaacg tcagcacctg caggttcagt
1080gcccccttgt ttctctccca tcctcacttc ctcaacgccg acccggttct
ggcagaagcg 1140gtgactggcc tgcaccctaa ccaggaggca cactccttgt
tcctggacat ccacccggtc 1200acgggaatcc ccatgaactg ctctgtgaaa
ctgcagctga gcctctacat gaaatctgtc 1260gcaggcattg gacaaactgg
gaagattgag cctgtggtcc tgccgctgct ctggtttgca 1320gagagcgggg
ccatggaggg ggagactctt cacacattct acactcagct ggtgttgatg
1380cccaaggtga
tgcactatgc ccagtacgtc ctcctggcgc tgggctgcgt cctgctgctg
1440gtccctgtca tctgccaaat ccggagccaa gagaaatgct atttattttg
gagtagtagt 1500aaaaagggct caaaggataa ggaggccatt caggcctatt
ctgaatccct gatgacatca 1560gctcccaagg gctctgtgct gcaggaagca
aaactgtagg gtcctgagga caccgtgagc 1620cagccaggcc tggccgctgg
gcctgaccgg ccccccagcc cctacacccc gcttctcccg 1680gactctccca
gcagacagcc ccccagcccc acagcctgag cctcccagct gccatgtgcc
1740tgttgcacac ctgcacacac gccctggcac acatacacac atgcgtgcag
gcttgtgcag 1800acactcaggg atggagctgc tgctgaaggg acttgtaggg
agaggctcgt caacaagcac 1860tgttctggaa ccttctctcc acgtggccca
caggctgacc acaggggctg tgggtcctgc 1920gtccccttcc tcgggtgagc
ctggcctgtc ccgttcagcc gttgggccag gcttcctccc 1980ctccaaggtg
aaacactgca gtcccggtgt ggtggctccc catgcaggac gggccaggct
2040gggagtgccg ccttcctgtg ccaaattcag tggggactca gtgcccaggc
cctggcacga 2100gctttggcct tggtctacct gccaggccag gcaaagcgcc
tttacacagg cctcggaaaa 2160caatggagtg agcacaagat gccctgtgca
gctgcccgag ggtctccgcc caccccggcc 2220ggactttgat ccccccgaag
tcttcacagg cactgcatcg ggttgtctgg cgcccttttc 2280ctccagccta
aactgacatc atcctatgga ctgagccggc cactctctgg ccgaagtggc
2340gcaggctgtg cccccgagct gcccccaccc cctcacaggg tccctcagat
tataggtgcc 2400caggctgagg tgaagaggcc tgggggccct gccttccggg
cgctcctgga ccctggggca 2460aacctgtgac ccttttctac tggaatagaa
atgagtttta tcatctttga aaaataattc 2520actcttgaag taataaacgt
ttaaaaaaat ggaaaaaaaa aaaaaa 256612509PRTHUMAN 12Met Gly Cys Ser
Ala Lys Ala Arg Trp Ala Ala Gly Ala Leu Gly Val1 5 10 15Ala Gly Leu
Leu Cys Ala Val Leu Gly Ala Val Met Ile Val Met Val 20 25 30Pro Ser
Leu Ile Lys Gln Gln Val Leu Lys Asn Val Arg Ile Asp Pro 35 40 45Ser
Ser Leu Ser Phe Asn Met Trp Lys Glu Ile Pro Ile Pro Phe Tyr 50 55
60Leu Ser Val Tyr Phe Phe Asp Val Met Asn Pro Ser Glu Ile Leu Lys65
70 75 80Gly Glu Lys Pro Gln Val Arg Glu Arg Gly Pro Tyr Val Tyr Arg
Glu 85 90 95Ser Arg His Lys Ser Asn Ile Thr Phe Asn Asn Asn Asp Thr
Val Ser 100 105 110Phe Leu Glu Tyr Arg Thr Phe Gln Phe Gln Pro Ser
Lys Ser His Gly 115 120 125Ser Glu Ser Asp Tyr Ile Val Met Pro Asn
Ile Leu Val Leu Gly Ala 130 135 140Ala Val Met Met Glu Asn Lys Pro
Met Thr Leu Lys Leu Ile Met Thr145 150 155 160Leu Ala Phe Thr Thr
Leu Gly Glu Arg Ala Phe Met Asn Arg Thr Val 165 170 175Gly Glu Ile
Met Trp Gly Tyr Lys Asp Pro Leu Val Asn Leu Ile Asn 180 185 190Lys
Tyr Phe Pro Gly Met Phe Pro Phe Lys Asp Lys Phe Gly Leu Phe 195 200
205Ala Glu Leu Asn Asn Ser Asp Ser Gly Leu Phe Thr Val Phe Thr Gly
210 215 220Val Gln Asn Ile Ser Arg Ile His Leu Val Asp Lys Trp Asn
Gly Leu225 230 235 240Ser Lys Val Asp Phe Trp His Ser Asp Gln Cys
Asn Met Ile Asn Gly 245 250 255Thr Ser Gly Gln Met Trp Pro Pro Phe
Met Thr Pro Glu Ser Ser Leu 260 265 270Glu Phe Tyr Ser Pro Glu Ala
Cys Arg Ser Met Lys Leu Met Tyr Lys 275 280 285Glu Ser Gly Val Phe
Glu Gly Ile Pro Thr Tyr Arg Phe Val Ala Pro 290 295 300Lys Thr Leu
Phe Ala Asn Gly Ser Ile Tyr Pro Pro Asn Glu Gly Phe305 310 315
320Cys Pro Cys Leu Glu Ser Gly Ile Gln Asn Val Ser Thr Cys Arg Phe
325 330 335Ser Ala Pro Leu Phe Leu Ser His Pro His Phe Leu Asn Ala
Asp Pro 340 345 350Val Leu Ala Glu Ala Val Thr Gly Leu His Pro Asn
Gln Glu Ala His 355 360 365Ser Leu Phe Leu Asp Ile His Pro Val Thr
Gly Ile Pro Met Asn Cys 370 375 380Ser Val Lys Leu Gln Leu Ser Leu
Tyr Met Lys Ser Val Ala Gly Ile385 390 395 400Gly Gln Thr Gly Lys
Ile Glu Pro Val Val Leu Pro Leu Leu Trp Phe 405 410 415Ala Glu Ser
Gly Ala Met Glu Gly Glu Thr Leu His Thr Phe Tyr Thr 420 425 430Gln
Leu Val Leu Met Pro Lys Val Met His Tyr Ala Gln Tyr Val Leu 435 440
445Leu Ala Leu Gly Cys Val Leu Leu Leu Val Pro Val Ile Cys Gln Ile
450 455 460Arg Ser Gln Glu Lys Cys Tyr Leu Phe Trp Ser Ser Ser Lys
Lys Gly465 470 475 480Ser Lys Asp Lys Glu Ala Ile Gln Ala Tyr Ser
Glu Ser Leu Met Thr 485 490 495Ser Ala Pro Lys Gly Ser Val Leu Gln
Glu Ala Lys Leu 500 5051325DNAARTIFICIAL SEQUENCEPrimer
13taagcttggc acggctgtcc aagga 251426DNAARTIFICIAL SEQUENCEPrimer
14acagaattcg ccccggcctg gtacac 261518PRTARTIFICIAL
SEQUENCESynthetic Peptide 15Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val
Ala Glu Lys Leu Lys Glu1 5 10 15Ala Phe1618PRTARTIFICIAL
SEQUENCESynthetic Peptide 16Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val
Ala Glu Lys Phe Lys Glu1 5 10 15Ala Phe1767PRTARTIFICIAL
SEQUENCESynthetic Peptide 17Leu Ser Pro Leu Gly Glu Glu Met Arg Asp
Arg Ala Arg Ala His Val1 5 10 15Asp Ala Leu Arg Thr His Leu Ala Pro
Tyr Ser Asp Glu Leu Arg Gln 20 25 30Pro Leu Ala Ala Arg Leu Glu Ala
Leu Lys Glu Asn Gly Gly Ala Arg 35 40 45Leu Ala Glu Tyr His Ala Lys
Ala Thr Glu His Leu Ser Thr Leu Ser 50 55 60Glu Lys Ala65
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