U.S. patent application number 12/860387 was filed with the patent office on 2011-03-31 for cardiovascular safety assay.
This patent application is currently assigned to JANSSEN PHARMACEUTICA, N.V.. Invention is credited to GODELIEVE IRMA CHRISTINE MARIA HEYLEN, CORNELUS GERARDUS MARIA JANSSEN, JURZAK MIREK, HENRICUS PETRUS MARTINUS MARIA VAN ASSOUW.
Application Number | 20110076696 12/860387 |
Document ID | / |
Family ID | 8180635 |
Filed Date | 2011-03-31 |
United States Patent
Application |
20110076696 |
Kind Code |
A1 |
HEYLEN; GODELIEVE IRMA CHRISTINE
MARIA ; et al. |
March 31, 2011 |
CARDIOVASCULAR SAFETY ASSAY
Abstract
The present invention provides assays and kits for the screening
of test compounds for their capability to induce cardiotoxicity in
a subject. Said assays and kits are based on the finding that the
interaction of astemizole with the HERG potassium channel can be
exploited to predict cardiotoxicity of compounds during the
development of new therapeutics and other agents.
Inventors: |
HEYLEN; GODELIEVE IRMA CHRISTINE
MARIA; (WESTMALLE, BE) ; JANSSEN; CORNELUS GERARDUS
MARIA; (VOSSELAAR, BE) ; MIREK; JURZAK;
(FRANKFIRT, DE) ; VAN ASSOUW; HENRICUS PETRUS MARTINUS
MARIA; (JE OIRSCHOT, NL) |
Assignee: |
JANSSEN PHARMACEUTICA, N.V.
BEERSE
BE
|
Family ID: |
8180635 |
Appl. No.: |
12/860387 |
Filed: |
August 20, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11593399 |
Nov 6, 2006 |
7820453 |
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12860387 |
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10483617 |
Jan 13, 2004 |
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PCT/EP2002/007364 |
Jul 2, 2002 |
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11593399 |
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Current U.S.
Class: |
435/7.21 ;
436/501; 436/504 |
Current CPC
Class: |
G01N 2800/32 20130101;
G01N 33/60 20130101; G01N 33/6887 20130101; G01N 2500/02 20130101;
G01N 33/6893 20130101; G01N 33/5014 20130101 |
Class at
Publication: |
435/7.21 ;
436/504; 436/501 |
International
Class: |
G01N 33/567 20060101
G01N033/567; G01N 33/566 20060101 G01N033/566 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 13, 2001 |
EP |
01202689 |
Claims
1-36. (canceled)
37. A kit comprising: a) a source containing a HERG polypeptide or
functional fragment thereof; b) radiolabeled astemizole of formula
III: ##STR00004##
38. A kit according to claim 37 wherein the source containing said
HERG polypeptide or functional equivalent thereof is selected from:
i) an isolated and purified nucleic acid which encodes a HERG
polypeptide having an amino acid sequence that is at least 80%
identical to that of SEQ ID NO: 2 or a functional fragment thereof;
ii) an isolated and purified nucleic acid which encodes a HERG
polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or a
functional fragment thereof; iii) cells expressing on the surface
thereof a HERG polypeptide having an amino acid sequence that is at
least 80% identical to that of SEQ ID NO:2 or a functional fragment
thereof; or iv) membrane preparations of cells expressing on the
surface thereof a HERG polypeptide having an amino acid sequence
that is at least 80% identical to that of SEQ ID NO: 2 or a
functional fragment thereof.
39. A kit according to claim 37 wherein the source containing HERG
is an isolated and purified HERG polypeptide having an amino acid
sequence that is at least 80% identical to that of SEQ ID NO: 2 or
a functional fragment thereof, bound to a solid support.
40. A kit according to claim 39 wherein the solid support is a
fluorescer comprising solid support.
41. A kit according to claim 39 wherein the source containing HERG
consists of membrane preparations of cells expressing on the
surface thereof HERG polypeptide channels encoded by the nucleic
acid sequence consisting of SEQ ID NO: 2.
42. A kit according to claim 41 wherein the cells are HEK293
cells.
43. A kit according to claim 42 optionally comprising means to
remove the excess of unbound labeled reference compound from the
incubation mixture.
44. A kit according to claim 42 wherein the separating means
consist of GF/B filtration.
45. An assay for screening test compounds, comprising: a)
incubating an isolated and purified polynucleotide which encodes
HERG comprising the nucleic acid sequence of SEQ ID NO: 2 or a
functional fragment thereof with: i) radiolabeled astemizole of
formula III ##STR00005## ii) the test compound; and b) measuring
the effect.
46. An assay for screening test compounds, comprising: a)
incubating an isolated and purified polynucleotide which encodes a
HERG polypeptide having an amino sequence that is at least 80%
identical to that of SEQ ID NO: 2; said nucleic acid sequence
comprising the nucleic acid sequence of SEQ ID NO: 1 or a
functional fragment thereof with: i) radiolabeled astemizole of
formula III ##STR00006## ii) the test compound; and b) measuring
the effect.
47. An assay for screening test compounds, comprising: a)
incubating membrane preparations of cells expressing on the surface
thereof a HERG polypeptide encoded by nucleic acid sequence
consisting of SEQ ID NO: 1 with: i) radiolabeled astemizole of
formula III ##STR00007## ii) the test compound; and b) measuring
the effect.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/593,399, filed Nov. 6, 2006, which is a divisional of U.S.
application Ser. No. 10/483,617, filed Jan. 13, 2004, now
abandoned, which is a 371 of PCT/EP2002/07364, filed Jul. 2, 2002,
which claims priority to European Patent Application No. 01202689,
filed Jul. 13, 2001, all of which are incorporated by reference in
their entirety.
FIELD OF INVENTION
[0002] The present invention relates to the field of cardiovascular
safety assays and provides assays and kits for the screening of
test compounds for their capability to induce cardiotoxicity in a
subject. Said assays and kits are based on the finding that the
interaction of astemizole with the HERG potassium channel can be
exploited to predict potential cardiotoxicity of compounds during
the development of new therapeutics and other agents. The present
invention finds particularly advantageous use in high throughput
screening of chemical compound libraries.
BACKGROUND OF THE INVENTION
[0003] Evidence has accrued that several drugs may prolong cardiac
repolarisation (hence, "measured as" the QT interval of the surface
electrocardiogram) to such a degree that potentially
life-threatening ventricular arrhythmias e.g. torsades de pointes
(TdP) may occur, especially in case of overdosage or
pharmacokinetic interaction.
[0004] The number of drugs reported to induce QT interval
prolongation with or without TdP continues to increase (W.
Haverkamp et al. (2000) Cardiovascular Research 47, 219-233). As
many as 50 clinically available or still investigational
non-cardiovascular drugs and cardiovascular non-anti-arrhythmic
drugs have been implicated. A number of drugs, both old and new,
have either been withdrawn from the market or have had their sale
restricted. Of concern is the interval, usually measured in years,
from the marketing of these drugs to initial recognition of their
association with QT interval prolongation and/or TdP. It would
therefore be beneficial to investigate any new chemical entity for
this potential side effect before its first use in man at an early
stage of the development of new therapeutics and/or other
agents.
[0005] A key component in the present development of new
therapeutic agents consists of High Throughput Screening (HTS) of
chemical compound libraries. Pharmaceutical companies have
established large collections of structurally distinct compounds,
which act as the starting point for drug target lead identification
programs. A typical corporate compound collection now comprises
between 100,000 and 1,000,000 discrete chemical entities. While a
few years ago a throughput of a few thousand compounds a day and
per assay was considered to be sufficient, pharmaceutical companies
nowadays aim at ultra high throughput screening techniques with
several hundreds of thousands of compounds tested per week. In a
typical HTS related screen format, assays are performed in
multi-well microplates, such as 96, 384 or 1536 well plates. The
use of these plates facilitates automation such as the use of
automated reagent dispensers and robotic pipetting instrumentation.
Further, to reduce the cycle time, the costs and the resources for
biochemicals such as recombinant proteins, HTS related screens are
preferably performed at room temperature with a single measurement
for each of the compounds tested in the assay.
[0006] A decisive criterion in the lead evaluation process will be
an early recognition of their potential association with QT
prolongation and/or TdP. However, there are currently no reliable,
fast, easy screening methods available to assess cardiotoxicity,
which can cope with the number of compounds identified in the
currently deployed HTS techniques. It is an object of this
invention to solve this problem in the art by providing assays and
kits which are based on the finding that the interaction of
astemizole with the HERG potassium channel can be exploited to
predict cardiotoxicity of compounds during the development of new
therapeutics and other agents.
[0007] The currently available in vitro models comprise
heterologous expression systems, disaggregated cells, isolated
tissues and the isolated intact (Langendorf-perfused) heart. In all
models the effect of potassium current blockade is assessed by
measurement of either ionic currents using two-electrode voltage
clamp recordings (Dascal N. (1987) Crit. Rev. Biochem 22, 341-356)
or patch-clamp recordings (Zhou Z. et al., (1998) Biophysical
Journal 74, 230-241), of membrane potentials using microelectrodes
or confocal microscopy (Dall'Asta V. et al. (1997) Exp. Cell
Research 231, 260-268). None of the aforementioned methods can be
used in an HTS screen in view of the complexity of the experimental
procedures, the slow cycling times, the nature of the source
materials (i.e. isolated tissues and disaggregated cells thereof)
and the reliability of the test results.
[0008] The present inventors surprisingly found that a binding
assay using labeled astemizole as a specific ligand for the HERG
channel can be used to predict the potential association of
compounds with QT prolongation and/or TdP. This binding assay
solves the aforementioned problems and can be deployed in an HTS
related screen format.
[0009] A similar assay has been described by Chadwick C. et al.
(Chadwick C. et al., (1993) Circulation Research 72, 707-714)
wherein [.sup.3H]-dofetilide has been identified as a specific
radioligand for the cardiac delayed rectifier K.sup.+ channel. This
article further demonstrates a good correlation between dofetilide
displacement and potassium channel blocking activity of a number of
antiarrhythmic compounds. This binding assay facilitates the
characterization of drug-channel interactions at the molecular
level.
[0010] In this assay labeled dofetilide has been prepared from the
dibromo precursor by .sup.3H-exchange yielding the incorporation of
two .sup.3H-labels per molecule. There is a direct correlation
between the number of .sup.3H-labels per molecule and the
sensitivity of the binding assay. The present invention provides an
improved binding assay over the above, as the use of a
desmethylastemizole precursor in a reaction with
[3.sup.3H]-methyliodide resulted in the incorporation of three
.sup.3H-labels per molecule astemizole. The specific activity of
the thus obtained radioligand is 1.5-2 times higher than the
specific activity of [.sup.3H]-dofetilide.
[0011] Further, the dofetilide assay could not be used in an HTS
related screen format since the ventricular myocytes isolated from
adult male guinea pigs had to be used within 6 hours of isolation.
In addition only 36% of the isolated cells were viable and could be
used in the binding assay. In order to be used in an HTS related
screen, the starting material should be readily available and in
sufficient amounts. The present invention solves this problem as
membrane preparations of HEK293 cells, stably expressing the HERG
potassium channel, are used. Said cells can be maintained in
culture in sufficient amount avoiding the need and supply of animal
models and as cell membranes are used in the binding assay, the
latter can be stored in binding assay ready aliquots at -80.degree.
C. for later use. A further drawback of the dofetilide binding
assay described by Chadwick et al. has to do with the incubation
protocol. As viable myocytes are used, the incubation has to be
performed at the physiological temperature of 34.degree. C. The
latter undoubtedly increases the costs, possible cycle time and
complexity of the assay if to be performed in an HTS related screen
format. The present invention solves this problem as it was
surprisingly demonstrated that the membrane preparations could be
incubated at room temperature. Especially in light of a study by
Zhoe Z. et al. Zhou Z. et al., (1998) Biophysical Journal 74,
230-241) which concluded that the kinetic properties measured for
HERG are markedly dependent on temperature and that differences
observed in several reports, are diminished when studies are
performed at physiological temperatures, i.e. 35.degree. C.
[0012] This and other aspects of the invention will be described
herein below.
SUMMARY OF THE INVENTION
[0013] The present invention provides an assay for screening test
compounds for their capability to induce cardiotoxicity in a
subject, the method comprising incubating a source containing HERG
or a fragment thereof with a reference compound and the test
compound, for a time sufficient to allow binding of the reference
compound and of the test compound with the HERG polypeptide channel
and measuring the effect of the test compound on the amount of
reference compound bound to HERG.
[0014] In a preferred embodiment of this invention, the assay
consists of incubating membrane preparations of cells, preferably
HEK293 cells, expressing on the surface thereof the HERG
polypeptide channel comprising the amino acid sequence of SEQ ID
NO:2 or a fragment thereof; with a labeled reference compound.
Wherein said labeled reference compound is a drug capable to induce
cardiac arrhythmia in a subject, preferably said labeled reference
compound is [.sup.3H]-astemizole. Incubating the above together
with the test compound and measure the effect of the test compound
on the amount of reference compound bound to the HERG polypeptide
channel. In a further embodiment the means of measurement consist
of separating means to remove the excess of unbound labeled
reference compound from the incubation mixture and of means for
detection of the labeled reference compound wherein the latter
preferably consists of radiolabeled measurement using scintillation
counting.
[0015] The invention further provides a high-throughput assay for
screening compounds for their capability to induce cardiotoxicity
in a subject, the assay comprising; [0016] a) contacting membrane
preparations of cells expressing on the surface thereof HERG
polypeptide channels having an amino acid sequence that is at least
80% identical to that of SEQ ID NO:2 or fragments thereof, with a
labeled reference compound for a time sufficient to allow binding
of the reference compound with the HERG polypeptide channel; [0017]
b) contacting membrane preparations of cells expressing on the
surface thereof HERG polypeptide channels having an amino acid
sequence that is at least 80% identical to that of SEQ ID NO:2 or
fragments thereof, with the labeled reference compound of step a)
together with the test compound for a time sufficient to allow
binding of the reference compound and of the test compound with the
HERG polypeptide channel; [0018] c) measuring the amount of labeled
reference compound bound to the HERG channel in step a); [0019] d)
measuring the amount of labeled reference compound bound to the
HERG channel in step b); and [0020] e) compare the amount of
labeled reference compound bound to the HERG channel measured in
step a) with the amount of labeled reference compound bound to the
HERG polypeptide channel measured in step b). In a preferred
embodiment of the high-throughput screening assay, the membrane
preparations are derived from cells, preferably HEK293 cells,
expressing on the surface thereof HERG polypeptide channels encoded
by the amino acid sequence consisting of SEQ ID NO:2. In a further
embodiment of the high-throughput screening assay the labeled
reference compound is astemizole, preferably
[.sup.3H]-astemizole.
[0021] The present invention also encompasses kits for screening
compounds for their capability to induce cardiotoxicity in a
subject as well as the use of reagents, including polynucleotides,
polypeptides and suitable reference compounds in the assays of the
present invention.
BRIEF DESCRIPTION OF THE DRAWING
[0022] FIG. 1A shows the saturation binding of [.sup.3H]-astemizole
to cell membrane preparations of HEK293 cells stably transfected
with the HERG channel cDNA. TB represents Total Binding measured,
NSB represents Non Specific Binding measured and SB represents
Specif Binding measured.
[0023] FIG. 1B shows the Scatchard plot for the saturation binding
experiments. From the fitted line a K.sub.D of 3.07.+-.2.26 nM
(n=11) could be determined with a B.sub.max (Maximal Binding) of
3260.+-.900 fmol/mg protein (n=11).
[0024] FIG. 2 shows the binding affinities of 42 reference
compounds compared to the electrophysiological patch clamp data. A
Spearman rank correlation coefficient of 0.87 could be
obtained.
DETAILED DESCRIPTION
[0025] The present invention relates to the field of cardiovascular
safety assays and provides assays and kits for the screening of
test compounds for their capability to induce cardiotoxicity in a
subject. Said assays and kits are based on the finding that the
interaction of astemizole with the HERG potassium channel can be
exploited to predict cardiotoxicity of compounds during the
development of new therapeutics and other agents. The present
invention finds particularly advantageous use in high throughput
screening of chemical compound libraries.
[0026] In one embodiment of the present invention, the assay for
screening test compounds comprises: a) incubating a source
containing HERG or a fragment thereof with i) a reference compound,
ii) the test compound; and b) measuring the effect of the test
compound on the amount of reference compound bound to HERG.
[0027] In a specific embodiment of the present invention the assays
are used to assess the capability of the test compounds to induce
cardiac arrhythmia in a subject.
[0028] As used herein the term "test compound" refers to a
chemically defined molecule whose cardiac arrhythmia inducing
capability is assessed in an assay according to the invention. Test
compounds include, but are not limited to, drugs, ligands (natural
or synthetic), polypeptides, peptides, peptide mimics,
polysaccharides, saccharides, glycoproteins, nucleic acids,
polynucleotides and small organic molecules. In one embodiment test
compounds comprise an existing library of compounds. In another
embodiment, test compounds comprise a novel library of
compounds.
[0029] As used herein the term "reference compound" refers to a
drug capable to induce cardiotoxicity in a subject. Reference
compounds include, but are not limited to, astemizole, terfenadine,
erythromycin, sparfloxain, probucol, terodiline and sertindole.
[0030] As used herein the term "HERG" refers to the Human
Ether-a-go-go-Related Gene channel. It is a delayed rectifier
potassium channel that plays a role in the control of action
potential repolarization in many cell types. HERG was originally
cloned from human hippocampus and it is strongly expressed in the
heart. The HERG polypeptides according to the invention include
isolated and purified proteins having an amino acid sequence that
is at least 80% identical to that of SEQ ID NO:2 or a fragment
thereof. In a further embodiment the HERG polypeptide channel
according to the invention has an amino acid sequence comprising
the amino acid sequence of SEQ ID NO:2. In a preferred embodiment
the HERG polypeptide according to the invention consists of SEQ ID
NO:2.
[0031] Variants of the defined sequence and fragments also form
part of the invention. Variants include those that vary from the
parent sequence by conservative amino acid changes, wherein
"conservative amino acid changes" refers to the replacement of one
or more amino acid residue(s) in the parent sequence without
affecting the biological activity of the parent molecule based on
the art recognized substitutability of certain amino acids (See
e.g. M. Dayhoff, In Atlas of Protein Sequence and Structure, Vol.
5, Supp. 3, pgs 345-352, 1987). Further variants are variants in
which several, 5-10, 1-5, or 1-2 amino acids are substituted,
deleted or added in any combination.
[0032] Methods for comparing the identity and similarity of two or
more sequences are well known in the art. Thus for instance,
programs available in the Winconsin Sequence Analysis Package,
version 9.1 (Devreux J. et al, Nucleic Acid Res., 12, 387-395,
1984), for example the programs BESTFIT and GAP, may be used to
determine the % identity between two polynucleotides and the %
identity and the % similarity between two polypeptide sequences.
BESTFIT uses the "local homology" algorithm of Smith and Waterman
(J. Mol. Biol., 147, 195-197, 1981) and finds the best single
region of similarity between two sequences. BESTFIT is more suited
to compare two polynucleotide or two polypeptide sequences that are
dissimilar in length, the program assuming that the shorter
sequence represents a portion of the longer. In comparison, GAP
aligns two sequences, finding a "maximum similarity", according to
the algorithm of Neddleman and Wunsch (J. Mol. Biol., 48, 443-453,
1970). GAP is more suited to compare sequences that are
approximately the same length and an alignment is expected over the
entire length. Preferably, the parameters "Gap Weight" and "Length
Weight" used in each program are 50 and 3 for polynucleotide
sequences, and 12 and 4 for polypeptide sequences, respectively.
Preferably, % identities and similarities are determined when the
two sequences being compared are optimally aligned. Other programs
for determining identity and/or similarity between sequences are
also known in the art, for instance the BLAST family of programs
(Altschul S F et al, Nucleic Acids Res., 25:3389-3402, 1997).
[0033] Those skilled in the art will recognize that the
polypeptides according to the invention, i.e. the HERG polypeptide
channel, could be obtained by a plurality of recombinant DNA
techniques including, for example, hybridization, polymerase chain
reaction (PCR) amplification, or de novo DNA synthesis (See e.g.,
T. Maniatis et al. Molecular Cloning: A Laboratory Manual, 2d Ed.
Chap. 14 (1989)). Thus, in a further embodiment the present
invention provides the use of the isolated and purified
polynucleotides encoding the HERG polypeptide or a fragment
thereof, in an assay or kit according to the invention. In another
embodiment the present invention provides the use of the isolated
and purified polynucleotide encoding the HERG polypeptide channel
or a fragment thereof comprising the polynucleotide sequence of SEQ
ID NO:1. In a preferred embodiment the present invention provides
the use of the isolated and purified polynucleotide encoding the
HERG polypeptide channel consisting of the polynucleotide sequence
of SEQ ID NO:1.
[0034] The term "fragments thereof" describes a piece, or
sub-region of protein or polynucleotide molecule whose sequence is
disclosed herein, such that said fragment comprises 5 or more amino
acids that are contiguous in the parent protein, or such that said
fragment comprises 15 or more nucleic acids that are contiguous in
the parent polynucleotide. The term "fragments thereof" is intended
to include "functional fragments" wherein the isolated fragment,
piece or sub-region comprises a functionally distinct region such
as an active site, a binding site or a phosphorylation site of the
receptor protein. Functional fragments may be produced by cloning
technology, or as the natural products of alternative splicing
techniques.
[0035] As used herein, "isolated" refers to the fact that the
polynucleotides, proteins and polypeptides, or respective fragments
thereof in question, have been removed from its in vivo environment
so that it can be manipulated by the skilled artisan, such as but
not limited to sequencing, restriction digestion, site-directed
mutagenesis, and subcloning into expression vectors for a nucleic
acid fragment as well as obtaining the protein or protein fragments
in quantities that afford the opportunity to generate polyclonal
antibodies, monoclonal antibodies, amino acid sequencing, and
peptide digestion. Therefore, the nucleic acids as described herein
can be present in whole cells or in cell lysates or in a partially,
substantially or wholly purified form.
[0036] A polypeptide is considered "purified" when it is purified
away from environmental contaminants. Thus a polypeptide isolated
from cells is considered to be substantially purified when purified
from cellular components by standard methods while a chemically
synthesized polypeptide sequence is considered to be substantially
purified when purified from its chemical precursors. A
"substantially pure" protein or nucleic acid will typically
comprise at least 85% of a sample with greater percentages being
preferred. One method for determining the purity of a protein or
nucleic acid molecule, is by electrophoresing a preparation in a
matrix such as polyacrylamide or agarose. Purity is evidenced by
the appearance of a single band after staining. Other methods for
assessing purity include chromatography and analytical
centrifugation.
[0037] The term "time sufficient to allow binding" as used herein
refers to the time needed to generate a detectable amount of
labeled reference compound bound to the HERG polypeptide channel.
The time needed to generate this detectable amount will vary
depending on the assay system. One of skill in the art will know
the amount of time sufficient to generate a detectable amount of
labeled reference compound bound to the HERG polypeptide channel
based upon the assay system.
Assays
[0038] Assays of the present invention can be designed in many
formats generally known in the art of screening compounds for
binding polypeptide channels.
[0039] The assays of the present invention advantageously exploit
the fact that the interaction of astemizole with the HERG potassium
channel can be exploited to predict cardiotoxicity of compounds
during the development of new therapeutics and other agents.
Therefore, the present invention provides an assay for screening
test compounds, the assay comprising a) incubating a source
containing HERG or a fragment thereof with; i) a reference
compound, ii) the test compound; and b) measuring the effect of the
test compound on the amount of reference compound bound to
HERG.
[0040] In a first embodiment of this invention the source
containing HERG is an isolated and purified protein which encodes
HERG having an amino acid sequence that is at least 80% identical
to that of SEQ ID NO:2 or a fragment thereof.
[0041] In a second embodiment of this invention the source
containing HERG is an isolated and purified protein which encodes
HERG comprising the amino acid sequence of SEQ ID NO: 2 or a
fragment thereof.
[0042] In a further embodiment of this invention the source
containing HERG are cells expressing on the surface thereof the
HERG polypeptide channel or a fragment thereof.
[0043] In another embodiment of this invention the source
containing HERG are membrane preparations of cells expressing on
the surface thereof the HERG polypeptide channel or a fragment
thereof.
[0044] In an alternative embodiment of this invention, the
reference compound is a compound capable to induce cardiotoxicity
in a subject, preferably selected from the group consisting of
astemizole, terfenadine, erythromycin, sparfloxain, probucol,
terodiline and sertindole. In a preferred embodiment the reference
compound is astemizole. It is a further object of this invention to
provide assays wherein the reference compound is labeled,
preferably radiolabeled.
[0045] In a preferred embodiment, the assay for screening test
compounds for their capability to induce cardiotoxicity in a
subject consists of a) incubating membrane preparations of cells
expressing on the surface thereof HERG polypeptide channels encoded
by the amino acid sequence consisting of SEQ ID NO:2 with i)
[.sup.3H]-astemizole, ii) the compound to be tested; and measuring
the effect of the test compound on the amount of reference compound
bound to HERG. The label of the reference compound is used to
measure this effect wherein said label can be measured using
amongst others scintillation counting.
[0046] A specific embodiment of the assays according to the
invention, consists of an high-throughput assay for screening test
compounds, the assay comprising: a) contacting membrane
preparations of cells expressing on the surface thereof HERG
polypeptide channels having an amino acid sequence that is at least
80% identical to that of SEQ ID NO:2 or fragments thereof, with a
labeled reference compound for a time sufficient to allow binding
of the reference compound with the HERG polypeptide channel; b)
contacting membrane preparations of cells expressing on the surface
thereof. HERG polypeptide channels having an amino acid sequence
that is at least 80% identical to that of SEQ ID NO:2 or fragments
thereof, with the labeled reference compound of step a) together
with the test compound for a time sufficient to allow binding of
the reference compound and of the test compound with the HERG
polypeptide channel; c) measuring the amount of labeled reference
compound bound to the HERG channel in step a); d) measuring the
amount of labeled reference compound bound to the HERG channel in
step b); and e) compare the amount of labeled reference compound
bound to the HERG channel measured in step a) with the amount of
labeled reference compound bound to the HERG polypeptide channel
measured in step b).
[0047] In a further embodiment the membrane preparations of the
high-throughput screening assay consist of membrane preparations of
cells expressing on the surface thereof the HERG polypeptide
channel comprising the amino acid sequence of SEQ ID NO:2 or
fragments thereof.
[0048] In a preferred embodiment of this invention the membrane
preparations of the high-throughput screening assay consist of
membrane preparations of cells, preferably HEK 293 cells,
expressing on the surface thereof HERG polypeptide channels
consisting of the amino acid sequence of SEQ ID NO:2.
[0049] In a further preferred embodiment, the labeled reference
compound in the high-throughput screening assay consists of
[.sup.3H]-labeled astemizole. Said label can be measured using
amongst others scintillation counting.
[0050] In another specific embodiment the present invention
provides a high-throughput proximity detection assay for screening
test compounds the assay comprising: [0051] i) HERG labeled with a
first label capable of participating in a proximity detection
assay; [0052] ii) a reference compound labeled with a second label
capable of participating in a proximity detection assay; [0053]
iii) contacting HERG of step i) and a reference compound of step
ii) together with a test compound for a time sufficient to allow
binding of the reference compound and of the test compound to HERG;
and [0054] iv) detect an interaction between HERG of step i) and a
reference compound of step ii) by means of proximity of the first
label with the second label when HERG and the reference compound
interact. The proximity of the first label to the second label,
brought about by the interaction of HERG and the reference compound
results in the production of a detectable signal. This may be
achieved by e.g. a scintillation proximity assay (SPA) system, in
which one of the labels is a radiolabel suitable for use in SPA and
the other label is a fluorescer comprised in a solid phase. The
detectable signal is light energy emitted when the labeled HERG
protein interacts with the labeled reference compound, bringing the
radiolabel sufficiently close to the fluorescer. Scintillation
proximity assay technology is described in U.S. Pat. No.
4,568,649.
[0055] Alternatively, the detectable signal may be a change in an
existing signal output, eg. fluorescence. Fluorescence resonance
energy transfer (FRET) is a method which works on this principle
and is described by Tsien R. et al. (Tsien R. et al. (1993) Trends
Cell Biol. 3: 242-245). It employs two different fluorescent
molecules, a donor and an acceptor, such that when these are in
sufficient proximity to one another the fluorescence of the donor
molecule is absorbed by the acceptor molecule and light of another
wavelength is emitted. Thus, when there is an interaction between
two molecules such as HERG and a reference compound, each of which
is labeled with one of these fluorescent molecules, a detectable
signal is produced.
[0056] By such proximity assays as are described above, the
screening assay according to the invention may be performed in a
single step, i.e. without the need of a separation step to remove
the excess of labeled reference compound from the incubation
mixture using separation means such as filtration.
[0057] In a preferred embodiment of the high-throughput proximity
detection assay, HERG is labeled with the fluorescer comprised in a
solid phase, such as coated scintillation proximity assay beads and
the reference compound is labeled with the radiolabel preferably
the reference compound is radiolabeled astemizole of formula
(III).
##STR00001##
[0058] It will be readily appreciated by the skilled artisan that
the binding of astemizole with HERG may also be used in a method
for the structure-based or rational design of compound which
affects the aforementioned binding, by: [0059] a) probing the
structure of the binding site of the HERG polypeptide channel with
astemizole; [0060] b) identifying contacting atoms in the binding
site of the HERG polypeptide channel that interact with astemizole
during binding; [0061] c) design test compounds that interact with
the atoms identified in (b) to affect the HERG-astemizole
interaction; and [0062] d) contact said designed test compound with
a source containing HERG or a fragment thereof, to measure the
capability of said compound to affect the amount of labeled
astemizole bound to HERG. It will be further appreciated that this
will normally be an iterative process.
Kits
[0063] The present invention also provides kits that can be used in
the above assays. In one embodiment the kit comprises a) a source
containing HERG; b) a reference compound.
[0064] In a first embodiment the kit comprises a source containing
HERG selected from: i) an isolated and purified protein which
encodes HERG having an amino acid sequence that is at least 80%
identical to that of SEQ ID NO:2 or a fragment thereof; ii) an
isolated and purified protein which encodes HERG comprising the
amino acid sequence of SEQ ID NO:2 or a fragment thereof; iii)
cells expressing on the surface thereof the HERG polypeptide
channel or a fragment thereof; or iv) membrane preparations of
cells expressing on the surface thereof the HERG polypeptide
channel or a fragment thereof.
[0065] In a further embodiment the kit comprises a reference
compound is selected from the group consisting of astemizole,
terfenadine, erythromycin, sparfloxain, probucol, terodiline and
sertindole. In a preferred embodiment the reference compound is
astemizole. It is a further object of this invention to provide
kits wherein the reference compound is labeled, preferably
radiolabeled.
[0066] In a specific embodiment the isolated and purified HERG
polypeptide channel is bound to a solid support, preferably to a
fluorescer comprising solid support such as coated scintillation
proximity beads.
[0067] Thus, in a specific embodiment the kit comprises a) an
isolated and purified HERG polypeptide channel or a fragment
thereof, bound to a solid support; and b) a labeled reference
compound. Preferably this specific embodiment consists of a kit
comprising a) an isolated and purified HERG polypeptide channel
consisting of the amino acid sequence of SEQ ID NO:2, bound to
fluorescer comprising solid support; and b) a radiolabeled
reference compound, preferably [.sup.3H]-labeled astemizole.
[0068] In another specific embodiment the kit comprises a) membrane
preparations of cells, preferably HEK293 cells, expressing on the
surface thereof the HERG polypeptide channel consisting of the
amino acid sequence of SEQ ID NO:2; b) [.sup.3H]-labeled
astemizole; and c) means for measurement of the amount of labeled
reference compound bound to HERG.
[0069] The means of measurement consist of separating means to
remove the excess of unbound labeled reference compound from the
incubation mixture and of means for detection of the labeled
reference compound. The person skilled in the art will know the
separating means available for removing the excess of unbound
labeled reference compound from the incubation mixture. Said
separating means include, but are not limited to, magnetic beads,
centrifugation techniques and filtration techniques. The means for
detecting the labeled reference compound will be depended on the
labeled used. Said labels may be fluorescent or radiolabels. The
skilled man will know the detection means available depending on
the label used.
[0070] In a specific embodiment the separating means consists of
GF/B filtration (Whatman Inc, Clifton, N.J.). In another specific
embodiment the detection means consists of scintillation counting
in a TOPCOUNT.TM. (Packard, Meriden, Conn.).
[0071] In a further embodiment the kits of the invention further
comprise instructions and/or multiple well plates for performing
the assay.
[0072] This invention will be better understood by reference to the
Experimental Details that follow, but those skilled in the art will
readily appreciate that these are only illustrative of the
invention as described more fully in the claims that follow
thereafter. Additionally, throughout this application, various
publications are cited. The disclosure of these publications is
hereby incorporated by reference into this application to describe
more fully the state of the art to which this invention
pertains.
Example 1
DNA Constructs and Stable Transfection of HEK293 Cells
[0073] HERG cDNA (Genbank Accession number: U04270 (SEQ ID NO:1))
was subcloned into bamHI/EcoRI sites of the pcDNA3 vector
(Invitrogen). This vector contains a CMV promotor and a SV40
promotor, which drive the expression of the inserted cDNA (HERG)
and neomycin resistance gene, respectively. The HEK293 cells were
transfected with this construct by a calcium phosphate precipitate
method (Gibco) or a lipofectamine method (Gibco). After selection
in 800 .mu.g/ml geneticin (G418; Gibco) for 15-20 days, single
colonies were picked with cloning cylinders and tested for HERG
current. The stably transfected cells were cultured in minimum
essential medium (MEM) supplemented with 10% fetal bovine serum and
400 .mu.g/ml geneticin.
[0074] For electrophysiological study, the cells were harvested
from the culture dish by trypsinization, washed twice with standard
MEM medium and seeded on small petri-dishes coated with
poly-L-lysine. Experiments were performed on the cells 1-2 days
after plating.
Example 2
Membrane Preparations of HEK293 Cells Stably Transfected with the
HERG Potassium Channel
[0075] HEK293 cells stably transfected with the HERG channel cDNA,
were grown in DMEM culture medium enriched with 10% fetal calf
serum and antibiotics. Collected cells were homogenized in Tris-HCl
50 mM pH 7.4 using an Ultraturrax homogenizer and the homogenate
was centrifuged for 10 min at 23,500.times.g in a SORVALL.TM.
centrifuge. The cell membranes were washed once by
re-homogenization and re-centrifugation. The membranes were
suspended in Tris-HCl 50 mM pH 7.4, aliquoted and stored at -80
C.
Example 3
Radiolabeling of Astemizole
##STR00002##
[0077] A solution of 4.6 g of astemizole (I) (10 mmol) in a 48%
aqueous hydrobromic acid solution (80 ml) was stirred and refluxed
for 2 hours. The reaction mixture was allowed to cool to room
temperature and the formed precipitate was filtered and washed with
water. The solids were dissolved in a mixture of
N,N-dimethylformamide (20 ml) and water (20 ml) and the mixture was
made alkaline by introducing slowly and with stirring a
concentrated aqueous ammoniumhydroxide solution. Then water (100
ml) was added and the mixture was stirred for 1 h. The precipitate
was filtered off and dried to the air for 18 h to yield
desmethylastemizole (II).
[0078] From this amount a fraction was taken and thoroughly
purified in portions via preparative HPLC on a Hypersyl ODS (5
.mu.m) bonded phase stainless steel column (7.1 mm ID.times.300 mm)
to yield astemizole free desmethylastemizole. Detection took place
at 282 nm and elution was performed isocratically with
acetonitrile-water-diisopropylamine (56:44:0.2, v/v) at a flow rate
of 4.0 ml/min.
##STR00003##
[0079] A fraction of the HPLC purified desmethylastemizole (II)
(26.7 mg, 60 .mu.mol) was dissolved in N,N-dimethylformamide (1.0
ml). To this solution 1N aqueous sodium hydroxide solution (60
.mu.l, 60 mop was added. The mixture was stirred for 25 minutes at
room temperature and added dropwise to a precooled solution
(-78.degree. C.) of [3.sup.3H]-methyliodide (370 MBq) in toluene.
The reaction mixture was vortexed and then left without cooling for
3 hours. The toluene was evaporated from the reaction mixture on a
waterbath of 40.degree. C. at aspirator pressure and the residue
was purified in portions via preparative HPLC as described above.
The product containing fractions were combined and depleted to 70
ml with methanol to give [.sup.3H]-astemizole (III) with a total
radioactivity of 198 MBq and a specific activity of 3.14 TBq/mmol
(85 Ci/mmol).
Example 4
Radioligand Binding Assay
[0080] Membranes were thawed and re-homogenized in incubation
buffer (Hepes 10 nM pH 7.4, 40 mM KCl, 20 mM KH.sub.2PO.sub.4, 5 mM
MgCl.sub.2, 0.5 mM KHCO.sub.3, 10 mM glucose, 50 mM glutamate, 20
mM aspartate, 14 mM heptanoic acid, 1 mM EGTA, 0.1% BSA) and 20-100
.mu.g protein was incubated with [.sup.3H]-astemizole for 60 min at
25.degree. C. with or without competitor followed by rapid
filtration over GF/B filter using a Filtermate 196 harvester
(Packard, Meriden, Conn.). Filters were rinsed extensively with
ice-cold rinse-buffer (Tris-HCl 25 mM pH 7.4, 130 mM NaCl, 5.5 mM
KCl, 5 mM glucose, 0.8 mM MgCl.sub.2, 50 .mu.M CaCl.sub.2, 0.1%
BSA). Filter bound radioactivity was determined by scintillation
counting in a TOPCOUNT.TM. (Packard, Meriden, Conn.) and results
were expressed as counts per minute (cpm).
[0081] Initially, various parameters including buffer, radioligand
and compound to determined non-specific binding, were investigated
in order to select the optimal conditions.
[0082] In a saturation binding experiment, increasing
concentrations of [.sup.3H]-astemizole were incubated with
membranes, re-suspended in buffer. Non-specific binding was
measured in the presence of 10 .mu.M R66204 (FIG. 1).
[0083] The effect of BSA and/or cyclodextrine present in the
incubation buffer, and of various ways of compound addition prior
to the experiment, was investigated by comparing the binding
affinities of 22 reference compounds to the electrophysiology data.
Compounds were dissolved in DMSO and further diluted in the same
solvent using a MULTIPROBEII.TM. pipetting station (Packard,
Meriden, Conn.). The final DMSO concentration in all experiments
was 1%. From this analysis it appears that compounds can be added
directly from the DMSO stock solution. Attempts to increase the
solubility of the compounds by addition of BSA and/or cyclodextrin
did not improve the correlation significantly.
Example 5
Whole-Cell Voltage Clamp Technique (Patch Clamp)
[0084] Solutions: The bath solution contained (in mM) 150 NaCl, 4
KCl, 5 glucose, 10 HEPES, 1.8 CaCl.sub.2 and 1 MgCl.sub.2 (pH 7.4
with NaOH). The pipette solution contained (in mM) 120 KCl, 5 EGTA,
10 HEPES, 4 MgATP, 0.5 CaCl.sub.2 and 2 MgCl.sub.2 (pH 7.2 with
KOH). Compounds were dissolved in DMSO to obtain a stock solution
of 10.sup.-2 M or 10.sup.-1 M. Control (=bath solution+DMSO) and
test solutions (=bath solution+DMSO+compound to be tested)
contained 0.3%, 0.1% or 0.03% DMSO. Test and control solutions were
applied to the cell under study using an Y-tube system, allowing to
rapidly change solutions (less than 0.5 s) in the vicinity of the
cell under study. Electrophysiological measurements: A Petri dish
containing attached HEK293 cells expressing HERG was fixed on the
stage of a Patch Clamp Tower. An inverted microscope was used to
observe the cells. The Petri dish was constantly perfused with the
bath solution at room temperature.
[0085] Patch pipettes were pulled from borosilicate glass
capillaries using a horizontal Flaming/Brown micropipette puller
without further fire-polishing. The microelectrodes used had an
input resistance between 1.5 and 3 M.OMEGA. when filled with the
pipette solution.
[0086] The membrane current of the cells was measured at distinct
membrane potentials with the patch clamp technique by means of an
EPC-9 patch clamp amplifier. Data were acquired and analysed using
the programs Pulse and Pulsefit (HEKA), DataAccess (Bruxton) and
Igor (Wavemetrics). The current signals were low-pass filtered and
subsequently digitised. The liquid junction potential was
electronically corrected, before establishing the seal. After
disruption of the membrane, the cell capacitance and the series
resistance were compensated using the circuit of the EPC-9 patch
clamp amplifier.
[0087] The holding potential was -80 mV. The HERG current
(K.sup.+-selective outward current) was determined as the maximal
tail current at -40 mV after a 2 second depolarization to +60 mV.
Pulse cycling rate was 15 s. Before each test pulse a short pulse
(0.5 s) from the holding potential to -60 mV was given to determine
leak current. After establishing whole-cell configuration a 5
minute equilibration period allowed for internal perfusion of the
cell with the pipette solution. Thereafter test pulses were given
for 5 minutes to quantify the HERG current under control
conditions. While continuing the pulse protocol, perfusion was
switched from control solution to drug-containing solution. The
effect of the drug was measured after 5 minutes of drug
application. One to three concentrations of the drug were tested
per cell (applied cumulatively).
Parameter analysis of the measurements: The HERG current was
determined as the maximal tail current at -40 mV after a 2 second
depolarization to +60 mV, starting from a holding potential of -80
mV.
[0088] During the initial 5 minutes measured in the presence of the
control solution, the amplitude of the HERG-mediated membrane
K.sup.+ current gradually decreased with time (run-down). In order
to quantify accurately the extent of block by the compounds, this
continuous run-down of the K.sup.+ current has to be taken into
account. Therefore the time course of the K.sup.+ current (measured
at -40 mV) was fitted exponentially to the initial period of 5
minutes in control solution and extrapolated for the remainder of
the experiment. These extrapolations give the estimated amplitude
of the current if no drug would have been given. To determine the
extent of block by the compounds, the ratio of the measured current
was calculated by dividing each measured current amplitude by the
value of the fitted current at the same point in time.
Example 6
Pharmacological Evaluation of the Binding Assay
[0089] For the pharmacological evaluation of the binding assay, 322
compounds were tested at 8 concentrations, for their ability to
inhibit [.sup.3H]-astemizole binding to the HERG channel and
pIC.sub.50-values were calculated by non-linear regression
analysis. If pIC.sub.50 values were available, the rank order
(Spearman) of the potencies for binding and patch clamp was
compared.
[0090] If in the patch clamp assay, compounds only have been tested
at <4 concentrations, a score was assigned to both binding- and
patch clamp data according to the following criteria: [0091] score
1: pIC50<6 or % blockade<50% at 10.sup.-6 M or higher [0092]
score 2: pIC50 between 6-8 or % blockade<50% between 10.sup.-6
and 10.sup.-8 M [0093] score 3: pIC50>8 or % blockade>50% at
10.sup.-8 M or lower
[0094] The rank order of potencies of 42 reference compounds to
displace the [.sup.3H]-astemizole binding from the HERG channel,
correlates well with the electrophysiological data for the
functional blockade of the rapid activating delayed rectifier
K.sup.+ current (r.sub.SP=0.87) (FIG. 2).
[0095] For 94% of the compounds tested, the binding data correlate
with the patch clamp data. In 2% of the cases the binding assay
scored higher than the patch clamp assay, for the remaining 4% it
is the other way around, i.e. the patch clamp assay scores higher
than the binding assay.
[0096] In view of this good correlation between binding data and
electrophysiological data it may be concluded that the radioligand
binding assay can be used as a primary screening tool for the
prediction of potential cardiovascular side-effects.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 2 <210> SEQ ID NO 1 <211> LENGTH: 4070 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (184)..(3663)
<300> PUBLICATION INFORMATION: <301> AUTHORS: Warmke,
J. W. <302> TITLE: Human putative potassium channel subunit
(h-erg) mRNA, complete cds. <308> DATABASE ACCESSION NUMBER:
GenBank / U04270 <309> DATABASE ENTRY DATE: 1993-12-09
<313> RELEVANT RESIDUES IN SEQ ID NO: 1 TO 4070 <400>
SEQUENCE: 1 acgcggcctg ctcaggcctc cagcggccgg tcggagggga ggcgggaggc
gagcgaggac 60 ccgcgcccgc agtccagtct gtgcgcgccc gtgctcgctt
ggcgcggtgc gggaccagcg 120 ccggccaccc gaagcctagt gcgtcgccgg
gtgggtgggc ccgcccggcg ccatgggctc 180 agg atg ccg gtg cgg agg ggc
cac gtc gcg ccg cag aac acc ttc ctg 228 Met Pro Val Arg Arg Gly His
Val Ala Pro Gln Asn Thr Phe Leu 1 5 10 15 gac acc atc atc cgc aag
ttt gag ggc cag agc cgt aag ttc atc atc 276 Asp Thr Ile Ile Arg Lys
Phe Glu Gly Gln Ser Arg Lys Phe Ile Ile 20 25 30 gcc aac gct cgg
gtg gag aac tgc gcc gtc atc tac tgc aac gac ggc 324 Ala Asn Ala Arg
Val Glu Asn Cys Ala Val Ile Tyr Cys Asn Asp Gly 35 40 45 ttc tgc
gag ctg tgc ggc tac tcg cgg gcc gag gtg atg cag cga ccc 372 Phe Cys
Glu Leu Cys Gly Tyr Ser Arg Ala Glu Val Met Gln Arg Pro 50 55 60
tgc acc tgc gac ttc ctg cac ggg ccg cgc acg cag cgc cgc gct gcc 420
Cys Thr Cys Asp Phe Leu His Gly Pro Arg Thr Gln Arg Arg Ala Ala 65
70 75 gcg cag atc gcg cag gca ctg ctg ggc gcc gag gag cgc aaa gtg
gaa 468 Ala Gln Ile Ala Gln Ala Leu Leu Gly Ala Glu Glu Arg Lys Val
Glu 80 85 90 95 atc gcc ttc tac cgg aaa gat ggg agc tgc ttc cta tgt
ctg gtg gat 516 Ile Ala Phe Tyr Arg Lys Asp Gly Ser Cys Phe Leu Cys
Leu Val Asp 100 105 110 gtg gtg ccc gtg aag aac gag gat ggg gct gtc
atc atg ttc atc ctc 564 Val Val Pro Val Lys Asn Glu Asp Gly Ala Val
Ile Met Phe Ile Leu 115 120 125 aat ttc gag gtg gtg atg gag aag gac
atg gtg ggg tcc ccg gct cat 612 Asn Phe Glu Val Val Met Glu Lys Asp
Met Val Gly Ser Pro Ala His 130 135 140 gac acc aac cac cgg ggc ccc
ccc acc agc tgg ctg gcc cca ggc cgc 660 Asp Thr Asn His Arg Gly Pro
Pro Thr Ser Trp Leu Ala Pro Gly Arg 145 150 155 gcc aag acc ttc cgc
ctg aag ctg ccc gcg ctg ctg gcg ctg acg gcc 708 Ala Lys Thr Phe Arg
Leu Lys Leu Pro Ala Leu Leu Ala Leu Thr Ala 160 165 170 175 cgg gag
tcg tcg gtg cgg tcg ggc ggc gcg ggc ggc gcg ggc gcc ccg 756 Arg Glu
Ser Ser Val Arg Ser Gly Gly Ala Gly Gly Ala Gly Ala Pro 180 185 190
ggg gcc gtg gtg gtg gac gtg gac ctg acg ccc gcg gca ccc agc agc 804
Gly Ala Val Val Val Asp Val Asp Leu Thr Pro Ala Ala Pro Ser Ser 195
200 205 gag tcg ctg gcc ctg gac gaa gtg aca gcc atg gac aac cac gtg
gca 852 Glu Ser Leu Ala Leu Asp Glu Val Thr Ala Met Asp Asn His Val
Ala 210 215 220 ggg ctc ggg ccc gcg gag gag cgg cgt gcg ctg gtg ggt
ccc ggc tct 900 Gly Leu Gly Pro Ala Glu Glu Arg Arg Ala Leu Val Gly
Pro Gly Ser 225 230 235 ccg ccc cgc agc gcg ccc ggc cag ctc cca tcg
ccc cgg gcg cac agc 948 Pro Pro Arg Ser Ala Pro Gly Gln Leu Pro Ser
Pro Arg Ala His Ser 240 245 250 255 ctc aac ccc gac gcc tcg ggc tcc
agc tgc agc ctg gcc cgg acg cgc 996 Leu Asn Pro Asp Ala Ser Gly Ser
Ser Cys Ser Leu Ala Arg Thr Arg 260 265 270 tcc cga gaa agc tgc gcc
agc gtg cgc cgc gcc tcg tcg gcc gac gac 1044 Ser Arg Glu Ser Cys
Ala Ser Val Arg Arg Ala Ser Ser Ala Asp Asp 275 280 285 atc gag gcc
atg cgc gcc ggg gtg ctg ccc ccg cca ccg cgc cac gcc 1092 Ile Glu
Ala Met Arg Ala Gly Val Leu Pro Pro Pro Pro Arg His Ala 290 295 300
agc acc ggg gcc atg cac cca ctg cgc agc ggc ttg ctc aac tcc acc
1140 Ser Thr Gly Ala Met His Pro Leu Arg Ser Gly Leu Leu Asn Ser
Thr 305 310 315 tcg gac tcc gac ctc gtg cgc tac cgc acc att agc aag
att ccc caa 1188 Ser Asp Ser Asp Leu Val Arg Tyr Arg Thr Ile Ser
Lys Ile Pro Gln 320 325 330 335 atc acc ctc aac ttt gtg gac ctc aag
ggc gac ccc ttc ttg gct tcg 1236 Ile Thr Leu Asn Phe Val Asp Leu
Lys Gly Asp Pro Phe Leu Ala Ser 340 345 350 ccc acc agt gac cgt gag
atc ata gca cct aag ata aag gag cga acc 1284 Pro Thr Ser Asp Arg
Glu Ile Ile Ala Pro Lys Ile Lys Glu Arg Thr 355 360 365 cac aat gtc
act gag aag gtc acc cag gtc ctg tcc ctg ggc gcc gac 1332 His Asn
Val Thr Glu Lys Val Thr Gln Val Leu Ser Leu Gly Ala Asp 370 375 380
gtg ctg cct gag tac aag ctg cag gca ccg cgc atc cac cgc tgg acc
1380 Val Leu Pro Glu Tyr Lys Leu Gln Ala Pro Arg Ile His Arg Trp
Thr 385 390 395 atc ctg cat tac agc ccc ttc aag gcc gtg tgg gac tgg
ctc atc ctg 1428 Ile Leu His Tyr Ser Pro Phe Lys Ala Val Trp Asp
Trp Leu Ile Leu 400 405 410 415 ctg ctg gtc atc tac acg gct gtc ttc
aca ccc tac tcg gct gcc ttc 1476 Leu Leu Val Ile Tyr Thr Ala Val
Phe Thr Pro Tyr Ser Ala Ala Phe 420 425 430 ctg ctg aag gag acg gaa
gaa ggc ccg cct gct acc gag tgt ggc tac 1524 Leu Leu Lys Glu Thr
Glu Glu Gly Pro Pro Ala Thr Glu Cys Gly Tyr 435 440 445 gcc tgc cag
ccg ctg gct gtg gtg gac ctc atc gtg gac atc atg ttc 1572 Ala Cys
Gln Pro Leu Ala Val Val Asp Leu Ile Val Asp Ile Met Phe 450 455 460
att gtg gac atc ctc atc aac ttc cgc acc acc tac gtc aat gcc aac
1620 Ile Val Asp Ile Leu Ile Asn Phe Arg Thr Thr Tyr Val Asn Ala
Asn 465 470 475 gag gag gtg gtc agc cac ccc ggc cgc atc gcc gtc cac
tac ttc aag 1668 Glu Glu Val Val Ser His Pro Gly Arg Ile Ala Val
His Tyr Phe Lys 480 485 490 495 ggc tgg ttc ctc atc gac atg gtg gcc
gcc atc ccc ttc gac ctg ctc 1716 Gly Trp Phe Leu Ile Asp Met Val
Ala Ala Ile Pro Phe Asp Leu Leu 500 505 510 atc ttc ggc tct ggc tct
gag gag ctg atc ggg ctg ctg aag act gcg 1764 Ile Phe Gly Ser Gly
Ser Glu Glu Leu Ile Gly Leu Leu Lys Thr Ala 515 520 525 cgg ctg ctg
cgg ctg gtg cgc gtg gcg cgg aag ctg gat cgc tac tca 1812 Arg Leu
Leu Arg Leu Val Arg Val Ala Arg Lys Leu Asp Arg Tyr Ser 530 535 540
gag tac ggc gcg gcc gtg ctg ttc ttg ctc atg tgc acc ttt gcg ctc
1860 Glu Tyr Gly Ala Ala Val Leu Phe Leu Leu Met Cys Thr Phe Ala
Leu 545 550 555 atc gcg cac tgg cta gcc tgc atc tgg tac gcc atc ggc
aac atg gag 1908 Ile Ala His Trp Leu Ala Cys Ile Trp Tyr Ala Ile
Gly Asn Met Glu 560 565 570 575 cag cca cac atg gac tca cgc atc ggc
tgg ctg cac aac ctg ggc gac 1956 Gln Pro His Met Asp Ser Arg Ile
Gly Trp Leu His Asn Leu Gly Asp 580 585 590 cag ata ggc aaa ccc tac
aac agc agc ggc ctg ggc ggc ccc tcc atc 2004 Gln Ile Gly Lys Pro
Tyr Asn Ser Ser Gly Leu Gly Gly Pro Ser Ile 595 600 605 aag gac aag
tat gtg acg gcg ctc tac ttc acc ttc agc agc ctc acc 2052 Lys Asp
Lys Tyr Val Thr Ala Leu Tyr Phe Thr Phe Ser Ser Leu Thr 610 615 620
agt gtg ggc ttc ggc aac gtc tct ccc aac acc aac tca gag aag atc
2100 Ser Val Gly Phe Gly Asn Val Ser Pro Asn Thr Asn Ser Glu Lys
Ile 625 630 635 ttc tcc atc tgc gtc atg ctc att ggc tcc ctc atg tat
gct agc atc 2148 Phe Ser Ile Cys Val Met Leu Ile Gly Ser Leu Met
Tyr Ala Ser Ile 640 645 650 655 ttc ggc aac gtg tcg gcc atc atc cag
cgg ctg tac tcg ggc aca gcc 2196 Phe Gly Asn Val Ser Ala Ile Ile
Gln Arg Leu Tyr Ser Gly Thr Ala 660 665 670 cgc tac cac aca cag atg
ctg cgg gtg cgg gag ttc atc cgc ttc cac 2244 Arg Tyr His Thr Gln
Met Leu Arg Val Arg Glu Phe Ile Arg Phe His 675 680 685 cag atc ccc
aat ccc ctg cgc cag cgc ctc gag gag tac ttc cag cac 2292 Gln Ile
Pro Asn Pro Leu Arg Gln Arg Leu Glu Glu Tyr Phe Gln His 690 695 700
gcc tgg tcc tac acc aac ggc atc gac atg aac gcg gtg ctg aag ggc
2340 Ala Trp Ser Tyr Thr Asn Gly Ile Asp Met Asn Ala Val Leu Lys
Gly 705 710 715 ttc cct gag tgc ctg cag gct gac atc tgc ctg cac ctg
aac cgc tca 2388 Phe Pro Glu Cys Leu Gln Ala Asp Ile Cys Leu His
Leu Asn Arg Ser 720 725 730 735 ctg ctg cag cac tgc aaa ccc ttc cga
ggg gcc acc aag ggc tgc ctt 2436 Leu Leu Gln His Cys Lys Pro Phe
Arg Gly Ala Thr Lys Gly Cys Leu 740 745 750 cgg gcc ctg gcc atg aag
ttc aag acc aca cat gca ccg cca ggg gac 2484 Arg Ala Leu Ala Met
Lys Phe Lys Thr Thr His Ala Pro Pro Gly Asp 755 760 765 aca ctg gtg
cat gct ggg gac ctg ctc acc gcc ctg tac ttc atc tcc 2532 Thr Leu
Val His Ala Gly Asp Leu Leu Thr Ala Leu Tyr Phe Ile Ser 770 775 780
cgg ggc tcc atc gag atc ctg cgg ggc gac gtc gtc gtg gcc atc ctg
2580 Arg Gly Ser Ile Glu Ile Leu Arg Gly Asp Val Val Val Ala Ile
Leu 785 790 795 ggg aag aat gac atc ttt ggg gag cct ctg aac ctg tat
gca agg cct 2628 Gly Lys Asn Asp Ile Phe Gly Glu Pro Leu Asn Leu
Tyr Ala Arg Pro 800 805 810 815 ggc aag tcg aac ggg gat gtg cgg gcc
ctc acc tac tgt gac cta cac 2676 Gly Lys Ser Asn Gly Asp Val Arg
Ala Leu Thr Tyr Cys Asp Leu His 820 825 830 aag atc cat cgg gac gac
ctg ctg gag gtg ctg gac atg tac cct gag 2724 Lys Ile His Arg Asp
Asp Leu Leu Glu Val Leu Asp Met Tyr Pro Glu 835 840 845 ttc tcc gac
cac ttc tgg tcc agc ctg gag atc acc ttc aac ctg cga 2772 Phe Ser
Asp His Phe Trp Ser Ser Leu Glu Ile Thr Phe Asn Leu Arg 850 855 860
gat acc aac atg atc ccg ggc tcc ccc ggc agt acg gag tta gag ggt
2820 Asp Thr Asn Met Ile Pro Gly Ser Pro Gly Ser Thr Glu Leu Glu
Gly 865 870 875 ggc ttc agt cgg caa cgc aag cgc aag ttg tcc ttc cgc
agg cgc acg 2868 Gly Phe Ser Arg Gln Arg Lys Arg Lys Leu Ser Phe
Arg Arg Arg Thr 880 885 890 895 gac aag gac acg gag cag cca ggg gag
gtg tcg gcc ttg ggg ccg ggc 2916 Asp Lys Asp Thr Glu Gln Pro Gly
Glu Val Ser Ala Leu Gly Pro Gly 900 905 910 cgg gcg ggg gca ggg ccg
agt agc cgg ggc cgg ccg ggg ggg ccg tgg 2964 Arg Ala Gly Ala Gly
Pro Ser Ser Arg Gly Arg Pro Gly Gly Pro Trp 915 920 925 ggg gag agc
ccg tcc agt ggc ccc tcc agc cct gag agc agt gag gat 3012 Gly Glu
Ser Pro Ser Ser Gly Pro Ser Ser Pro Glu Ser Ser Glu Asp 930 935 940
gag ggc cca ggc cgc agc tcc agc ccc ctc cgc ctg gtg ccc ttc tcc
3060 Glu Gly Pro Gly Arg Ser Ser Ser Pro Leu Arg Leu Val Pro Phe
Ser 945 950 955 agc ccc agg ccc ccc gga gag ccg ccg ggt ggg gag ccc
ctg atg gag 3108 Ser Pro Arg Pro Pro Gly Glu Pro Pro Gly Gly Glu
Pro Leu Met Glu 960 965 970 975 gac tgc gag aag agc agc gac act tgc
aac ccc ctg tca ggc gcc ttc 3156 Asp Cys Glu Lys Ser Ser Asp Thr
Cys Asn Pro Leu Ser Gly Ala Phe 980 985 990 tca gga gtg tcc aac att
ttc agc ttc tgg ggg gac agt cgg ggc cgc 3204 Ser Gly Val Ser Asn
Ile Phe Ser Phe Trp Gly Asp Ser Arg Gly Arg 995 1000 1005 cag tac
cag gag ctc cct cga tgc ccc gcc ccc acc ccc agc ctc ctc 3252 Gln
Tyr Gln Glu Leu Pro Arg Cys Pro Ala Pro Thr Pro Ser Leu Leu 1010
1015 1020 aac atc ccc ctc tcc agc ccg ggt cgg cgg ccc cgg ggc gac
gtg gag 3300 Asn Ile Pro Leu Ser Ser Pro Gly Arg Arg Pro Arg Gly
Asp Val Glu 1025 1030 1035 agc agg ctg gat gcc ctc cag cgc cag ctc
aac agg ctg gag acc cgg 3348 Ser Arg Leu Asp Ala Leu Gln Arg Gln
Leu Asn Arg Leu Glu Thr Arg 1040 1045 1050 1055 ctg agt gca gac atg
gcc act gtc ctg cag ctg cta cag agg cag atg 3396 Leu Ser Ala Asp
Met Ala Thr Val Leu Gln Leu Leu Gln Arg Gln Met 1060 1065 1070 acg
ctg gtc ccg ccc gcc tac agt gct gtg acc acc ccg ggg cct ggc 3444
Thr Leu Val Pro Pro Ala Tyr Ser Ala Val Thr Thr Pro Gly Pro Gly
1075 1080 1085 ccc act tcc aca tcc ccg ctg ttg ccc gtc agc ccc ctc
ccc acc ctc 3492 Pro Thr Ser Thr Ser Pro Leu Leu Pro Val Ser Pro
Leu Pro Thr Leu 1090 1095 1100 acc ttg gac tcg ctt tct cag gtt tcc
cag ttc atg gcg tgt gag gag 3540 Thr Leu Asp Ser Leu Ser Gln Val
Ser Gln Phe Met Ala Cys Glu Glu 1105 1110 1115 ctg ccc ccg ggg gcc
cca gag ctt ccc caa gaa ggc ccc aca cga cgc 3588 Leu Pro Pro Gly
Ala Pro Glu Leu Pro Gln Glu Gly Pro Thr Arg Arg 1120 1125 1130 1135
ctc tcc cta ccg ggc cag ctg ggg gcc ctc acc tcc cag ccc ctg cac
3636 Leu Ser Leu Pro Gly Gln Leu Gly Ala Leu Thr Ser Gln Pro Leu
His 1140 1145 1150 aga cac ggc tcg gac ccg ggc agt tag tggggctgcc
cagtgtggac 3683 Arg His Gly Ser Asp Pro Gly Ser 1155 1160
acgtggctca cccagggatc aaggcgctgc tgggccgctc cccttggagg ccctgctcag
3743 gaggccctga ccgtggaagg ggagaggaac tcgaaagcac agctcctccc
ccagcccttg 3803 ggaccatctt ctcctgcagt cccctgggcc ccagtgagag
gggcaggggc agggccggca 3863 gtaggtgggg cctgtggtcc ccccactgcc
ctgagggcat tagctggtct aactgcccgg 3923 aggcacccgg ccctgggcct
taggcacctc aaggactttt ctgctattta ctgctcttat 3983 tgttaaggat
aataattaag gatcatatga ataattaatg aagatgctga tgactatgaa 4043
taataaataa ttatcctgag gagaaaa 4070 <210> SEQ ID NO 2
<211> LENGTH: 1159 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met Pro Val Arg Arg
Gly His Val Ala Pro Gln Asn Thr Phe Leu Asp 1 5 10 15 Thr Ile Ile
Arg Lys Phe Glu Gly Gln Ser Arg Lys Phe Ile Ile Ala 20 25 30 Asn
Ala Arg Val Glu Asn Cys Ala Val Ile Tyr Cys Asn Asp Gly Phe 35 40
45 Cys Glu Leu Cys Gly Tyr Ser Arg Ala Glu Val Met Gln Arg Pro Cys
50 55 60 Thr Cys Asp Phe Leu His Gly Pro Arg Thr Gln Arg Arg Ala
Ala Ala 65 70 75 80 Gln Ile Ala Gln Ala Leu Leu Gly Ala Glu Glu Arg
Lys Val Glu Ile 85 90 95 Ala Phe Tyr Arg Lys Asp Gly Ser Cys Phe
Leu Cys Leu Val Asp Val 100 105 110 Val Pro Val Lys Asn Glu Asp Gly
Ala Val Ile Met Phe Ile Leu Asn 115 120 125 Phe Glu Val Val Met Glu
Lys Asp Met Val Gly Ser Pro Ala His Asp 130 135 140 Thr Asn His Arg
Gly Pro Pro Thr Ser Trp Leu Ala Pro Gly Arg Ala 145 150 155 160 Lys
Thr Phe Arg Leu Lys Leu Pro Ala Leu Leu Ala Leu Thr Ala Arg 165 170
175 Glu Ser Ser Val Arg Ser Gly Gly Ala Gly Gly Ala Gly Ala Pro Gly
180 185 190 Ala Val Val Val Asp Val Asp Leu Thr Pro Ala Ala Pro Ser
Ser Glu 195 200 205 Ser Leu Ala Leu Asp Glu Val Thr Ala Met Asp Asn
His Val Ala Gly 210 215 220 Leu Gly Pro Ala Glu Glu Arg Arg Ala Leu
Val Gly Pro Gly Ser Pro 225 230 235 240 Pro Arg Ser Ala Pro Gly Gln
Leu Pro Ser Pro Arg Ala His Ser Leu 245 250 255 Asn Pro Asp Ala Ser
Gly Ser Ser Cys Ser Leu Ala Arg Thr Arg Ser 260 265 270 Arg Glu Ser
Cys Ala Ser Val Arg Arg Ala Ser Ser Ala Asp Asp Ile 275 280 285 Glu
Ala Met Arg Ala Gly Val Leu Pro Pro Pro Pro Arg His Ala Ser 290 295
300 Thr Gly Ala Met His Pro Leu Arg Ser Gly Leu Leu Asn Ser Thr Ser
305 310 315 320 Asp Ser Asp Leu Val Arg Tyr Arg Thr Ile Ser Lys Ile
Pro Gln Ile 325 330 335 Thr Leu Asn Phe Val Asp Leu Lys Gly Asp Pro
Phe Leu Ala Ser Pro 340 345 350 Thr Ser Asp Arg Glu Ile Ile Ala Pro
Lys Ile Lys Glu Arg Thr His 355 360 365 Asn Val Thr Glu Lys Val Thr
Gln Val Leu Ser Leu Gly Ala Asp Val 370 375 380 Leu Pro Glu Tyr Lys
Leu Gln Ala Pro Arg Ile His Arg Trp Thr Ile 385 390 395 400 Leu His
Tyr Ser Pro Phe Lys Ala Val Trp Asp Trp Leu Ile Leu Leu 405 410 415
Leu Val Ile Tyr Thr Ala Val Phe Thr Pro Tyr Ser Ala Ala Phe Leu 420
425 430 Leu Lys Glu Thr Glu Glu Gly Pro Pro Ala Thr Glu Cys Gly Tyr
Ala 435 440 445 Cys Gln Pro Leu Ala Val Val Asp Leu Ile Val Asp Ile
Met Phe Ile 450 455 460 Val Asp Ile Leu Ile Asn Phe Arg Thr Thr Tyr
Val Asn Ala Asn Glu 465 470 475 480 Glu Val Val Ser His Pro Gly Arg
Ile Ala Val His Tyr Phe Lys Gly 485 490 495 Trp Phe Leu Ile Asp Met
Val Ala Ala Ile Pro Phe Asp Leu Leu Ile 500 505 510 Phe Gly Ser Gly
Ser Glu Glu Leu Ile Gly Leu Leu Lys Thr Ala Arg 515 520 525 Leu Leu
Arg Leu Val Arg Val Ala Arg Lys Leu Asp Arg Tyr Ser Glu 530 535 540
Tyr Gly Ala Ala Val Leu Phe Leu Leu Met Cys Thr Phe Ala Leu Ile 545
550 555 560 Ala His Trp Leu Ala Cys Ile Trp Tyr Ala Ile Gly Asn Met
Glu Gln 565 570 575 Pro His Met Asp Ser Arg Ile Gly Trp Leu His Asn
Leu Gly Asp Gln 580 585 590 Ile Gly Lys Pro Tyr Asn Ser Ser Gly Leu
Gly Gly Pro Ser Ile Lys 595 600 605 Asp Lys Tyr Val Thr Ala Leu Tyr
Phe Thr Phe Ser Ser Leu Thr Ser 610 615 620 Val Gly Phe Gly Asn Val
Ser Pro Asn Thr Asn Ser Glu Lys Ile Phe 625 630 635 640 Ser Ile Cys
Val Met Leu Ile Gly Ser Leu Met Tyr Ala Ser Ile Phe 645 650 655 Gly
Asn Val Ser Ala Ile Ile Gln Arg Leu Tyr Ser Gly Thr Ala Arg 660 665
670 Tyr His Thr Gln Met Leu Arg Val Arg Glu Phe Ile Arg Phe His Gln
675 680 685 Ile Pro Asn Pro Leu Arg Gln Arg Leu Glu Glu Tyr Phe Gln
His Ala 690 695 700 Trp Ser Tyr Thr Asn Gly Ile Asp Met Asn Ala Val
Leu Lys Gly Phe 705 710 715 720 Pro Glu Cys Leu Gln Ala Asp Ile Cys
Leu His Leu Asn Arg Ser Leu 725 730 735 Leu Gln His Cys Lys Pro Phe
Arg Gly Ala Thr Lys Gly Cys Leu Arg 740 745 750 Ala Leu Ala Met Lys
Phe Lys Thr Thr His Ala Pro Pro Gly Asp Thr 755 760 765 Leu Val His
Ala Gly Asp Leu Leu Thr Ala Leu Tyr Phe Ile Ser Arg 770 775 780 Gly
Ser Ile Glu Ile Leu Arg Gly Asp Val Val Val Ala Ile Leu Gly 785 790
795 800 Lys Asn Asp Ile Phe Gly Glu Pro Leu Asn Leu Tyr Ala Arg Pro
Gly 805 810 815 Lys Ser Asn Gly Asp Val Arg Ala Leu Thr Tyr Cys Asp
Leu His Lys 820 825 830 Ile His Arg Asp Asp Leu Leu Glu Val Leu Asp
Met Tyr Pro Glu Phe 835 840 845 Ser Asp His Phe Trp Ser Ser Leu Glu
Ile Thr Phe Asn Leu Arg Asp 850 855 860 Thr Asn Met Ile Pro Gly Ser
Pro Gly Ser Thr Glu Leu Glu Gly Gly 865 870 875 880 Phe Ser Arg Gln
Arg Lys Arg Lys Leu Ser Phe Arg Arg Arg Thr Asp 885 890 895 Lys Asp
Thr Glu Gln Pro Gly Glu Val Ser Ala Leu Gly Pro Gly Arg 900 905 910
Ala Gly Ala Gly Pro Ser Ser Arg Gly Arg Pro Gly Gly Pro Trp Gly 915
920 925 Glu Ser Pro Ser Ser Gly Pro Ser Ser Pro Glu Ser Ser Glu Asp
Glu 930 935 940 Gly Pro Gly Arg Ser Ser Ser Pro Leu Arg Leu Val Pro
Phe Ser Ser 945 950 955 960 Pro Arg Pro Pro Gly Glu Pro Pro Gly Gly
Glu Pro Leu Met Glu Asp 965 970 975 Cys Glu Lys Ser Ser Asp Thr Cys
Asn Pro Leu Ser Gly Ala Phe Ser 980 985 990 Gly Val Ser Asn Ile Phe
Ser Phe Trp Gly Asp Ser Arg Gly Arg Gln 995 1000 1005 Tyr Gln Glu
Leu Pro Arg Cys Pro Ala Pro Thr Pro Ser Leu Leu Asn 1010 1015 1020
Ile Pro Leu Ser Ser Pro Gly Arg Arg Pro Arg Gly Asp Val Glu Ser
1025 1030 1035 1040 Arg Leu Asp Ala Leu Gln Arg Gln Leu Asn Arg Leu
Glu Thr Arg Leu 1045 1050 1055 Ser Ala Asp Met Ala Thr Val Leu Gln
Leu Leu Gln Arg Gln Met Thr 1060 1065 1070 Leu Val Pro Pro Ala Tyr
Ser Ala Val Thr Thr Pro Gly Pro Gly Pro 1075 1080 1085 Thr Ser Thr
Ser Pro Leu Leu Pro Val Ser Pro Leu Pro Thr Leu Thr 1090 1095 1100
Leu Asp Ser Leu Ser Gln Val Ser Gln Phe Met Ala Cys Glu Glu Leu
1105 1110 1115 1120 Pro Pro Gly Ala Pro Glu Leu Pro Gln Glu Gly Pro
Thr Arg Arg Leu 1125 1130 1135 Ser Leu Pro Gly Gln Leu Gly Ala Leu
Thr Ser Gln Pro Leu His Arg 1140 1145 1150 His Gly Ser Asp Pro Gly
Ser 1155
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 2 <210>
SEQ ID NO 1 <211> LENGTH: 4070 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (184)..(3663) <300>
PUBLICATION INFORMATION: <301> AUTHORS: Warmke, J. W.
<302> TITLE: Human putative potassium channel subunit (h-erg)
mRNA, complete cds. <308> DATABASE ACCESSION NUMBER: GenBank
/ U04270 <309> DATABASE ENTRY DATE: 1993-12-09 <313>
RELEVANT RESIDUES IN SEQ ID NO: 1 TO 4070 <400> SEQUENCE: 1
acgcggcctg ctcaggcctc cagcggccgg tcggagggga ggcgggaggc gagcgaggac
60 ccgcgcccgc agtccagtct gtgcgcgccc gtgctcgctt ggcgcggtgc
gggaccagcg 120 ccggccaccc gaagcctagt gcgtcgccgg gtgggtgggc
ccgcccggcg ccatgggctc 180 agg atg ccg gtg cgg agg ggc cac gtc gcg
ccg cag aac acc ttc ctg 228 Met Pro Val Arg Arg Gly His Val Ala Pro
Gln Asn Thr Phe Leu 1 5 10 15 gac acc atc atc cgc aag ttt gag ggc
cag agc cgt aag ttc atc atc 276 Asp Thr Ile Ile Arg Lys Phe Glu Gly
Gln Ser Arg Lys Phe Ile Ile 20 25 30 gcc aac gct cgg gtg gag aac
tgc gcc gtc atc tac tgc aac gac ggc 324 Ala Asn Ala Arg Val Glu Asn
Cys Ala Val Ile Tyr Cys Asn Asp Gly 35 40 45 ttc tgc gag ctg tgc
ggc tac tcg cgg gcc gag gtg atg cag cga ccc 372 Phe Cys Glu Leu Cys
Gly Tyr Ser Arg Ala Glu Val Met Gln Arg Pro 50 55 60 tgc acc tgc
gac ttc ctg cac ggg ccg cgc acg cag cgc cgc gct gcc 420 Cys Thr Cys
Asp Phe Leu His Gly Pro Arg Thr Gln Arg Arg Ala Ala 65 70 75 gcg
cag atc gcg cag gca ctg ctg ggc gcc gag gag cgc aaa gtg gaa 468 Ala
Gln Ile Ala Gln Ala Leu Leu Gly Ala Glu Glu Arg Lys Val Glu 80 85
90 95 atc gcc ttc tac cgg aaa gat ggg agc tgc ttc cta tgt ctg gtg
gat 516 Ile Ala Phe Tyr Arg Lys Asp Gly Ser Cys Phe Leu Cys Leu Val
Asp 100 105 110 gtg gtg ccc gtg aag aac gag gat ggg gct gtc atc atg
ttc atc ctc 564 Val Val Pro Val Lys Asn Glu Asp Gly Ala Val Ile Met
Phe Ile Leu 115 120 125 aat ttc gag gtg gtg atg gag aag gac atg gtg
ggg tcc ccg gct cat 612 Asn Phe Glu Val Val Met Glu Lys Asp Met Val
Gly Ser Pro Ala His 130 135 140 gac acc aac cac cgg ggc ccc ccc acc
agc tgg ctg gcc cca ggc cgc 660 Asp Thr Asn His Arg Gly Pro Pro Thr
Ser Trp Leu Ala Pro Gly Arg 145 150 155 gcc aag acc ttc cgc ctg aag
ctg ccc gcg ctg ctg gcg ctg acg gcc 708 Ala Lys Thr Phe Arg Leu Lys
Leu Pro Ala Leu Leu Ala Leu Thr Ala 160 165 170 175 cgg gag tcg tcg
gtg cgg tcg ggc ggc gcg ggc ggc gcg ggc gcc ccg 756 Arg Glu Ser Ser
Val Arg Ser Gly Gly Ala Gly Gly Ala Gly Ala Pro 180 185 190 ggg gcc
gtg gtg gtg gac gtg gac ctg acg ccc gcg gca ccc agc agc 804 Gly Ala
Val Val Val Asp Val Asp Leu Thr Pro Ala Ala Pro Ser Ser 195 200 205
gag tcg ctg gcc ctg gac gaa gtg aca gcc atg gac aac cac gtg gca 852
Glu Ser Leu Ala Leu Asp Glu Val Thr Ala Met Asp Asn His Val Ala 210
215 220 ggg ctc ggg ccc gcg gag gag cgg cgt gcg ctg gtg ggt ccc ggc
tct 900 Gly Leu Gly Pro Ala Glu Glu Arg Arg Ala Leu Val Gly Pro Gly
Ser 225 230 235 ccg ccc cgc agc gcg ccc ggc cag ctc cca tcg ccc cgg
gcg cac agc 948 Pro Pro Arg Ser Ala Pro Gly Gln Leu Pro Ser Pro Arg
Ala His Ser 240 245 250 255 ctc aac ccc gac gcc tcg ggc tcc agc tgc
agc ctg gcc cgg acg cgc 996 Leu Asn Pro Asp Ala Ser Gly Ser Ser Cys
Ser Leu Ala Arg Thr Arg 260 265 270 tcc cga gaa agc tgc gcc agc gtg
cgc cgc gcc tcg tcg gcc gac gac 1044 Ser Arg Glu Ser Cys Ala Ser
Val Arg Arg Ala Ser Ser Ala Asp Asp 275 280 285 atc gag gcc atg cgc
gcc ggg gtg ctg ccc ccg cca ccg cgc cac gcc 1092 Ile Glu Ala Met
Arg Ala Gly Val Leu Pro Pro Pro Pro Arg His Ala 290 295 300 agc acc
ggg gcc atg cac cca ctg cgc agc ggc ttg ctc aac tcc acc 1140 Ser
Thr Gly Ala Met His Pro Leu Arg Ser Gly Leu Leu Asn Ser Thr 305 310
315 tcg gac tcc gac ctc gtg cgc tac cgc acc att agc aag att ccc caa
1188 Ser Asp Ser Asp Leu Val Arg Tyr Arg Thr Ile Ser Lys Ile Pro
Gln 320 325 330 335 atc acc ctc aac ttt gtg gac ctc aag ggc gac ccc
ttc ttg gct tcg 1236 Ile Thr Leu Asn Phe Val Asp Leu Lys Gly Asp
Pro Phe Leu Ala Ser 340 345 350 ccc acc agt gac cgt gag atc ata gca
cct aag ata aag gag cga acc 1284 Pro Thr Ser Asp Arg Glu Ile Ile
Ala Pro Lys Ile Lys Glu Arg Thr 355 360 365 cac aat gtc act gag aag
gtc acc cag gtc ctg tcc ctg ggc gcc gac 1332 His Asn Val Thr Glu
Lys Val Thr Gln Val Leu Ser Leu Gly Ala Asp 370 375 380 gtg ctg cct
gag tac aag ctg cag gca ccg cgc atc cac cgc tgg acc 1380 Val Leu
Pro Glu Tyr Lys Leu Gln Ala Pro Arg Ile His Arg Trp Thr 385 390 395
atc ctg cat tac agc ccc ttc aag gcc gtg tgg gac tgg ctc atc ctg
1428 Ile Leu His Tyr Ser Pro Phe Lys Ala Val Trp Asp Trp Leu Ile
Leu 400 405 410 415 ctg ctg gtc atc tac acg gct gtc ttc aca ccc tac
tcg gct gcc ttc 1476 Leu Leu Val Ile Tyr Thr Ala Val Phe Thr Pro
Tyr Ser Ala Ala Phe 420 425 430 ctg ctg aag gag acg gaa gaa ggc ccg
cct gct acc gag tgt ggc tac 1524 Leu Leu Lys Glu Thr Glu Glu Gly
Pro Pro Ala Thr Glu Cys Gly Tyr 435 440 445 gcc tgc cag ccg ctg gct
gtg gtg gac ctc atc gtg gac atc atg ttc 1572 Ala Cys Gln Pro Leu
Ala Val Val Asp Leu Ile Val Asp Ile Met Phe 450 455 460 att gtg gac
atc ctc atc aac ttc cgc acc acc tac gtc aat gcc aac 1620 Ile Val
Asp Ile Leu Ile Asn Phe Arg Thr Thr Tyr Val Asn Ala Asn 465 470 475
gag gag gtg gtc agc cac ccc ggc cgc atc gcc gtc cac tac ttc aag
1668 Glu Glu Val Val Ser His Pro Gly Arg Ile Ala Val His Tyr Phe
Lys 480 485 490 495 ggc tgg ttc ctc atc gac atg gtg gcc gcc atc ccc
ttc gac ctg ctc 1716 Gly Trp Phe Leu Ile Asp Met Val Ala Ala Ile
Pro Phe Asp Leu Leu 500 505 510 atc ttc ggc tct ggc tct gag gag ctg
atc ggg ctg ctg aag act gcg 1764 Ile Phe Gly Ser Gly Ser Glu Glu
Leu Ile Gly Leu Leu Lys Thr Ala 515 520 525 cgg ctg ctg cgg ctg gtg
cgc gtg gcg cgg aag ctg gat cgc tac tca 1812 Arg Leu Leu Arg Leu
Val Arg Val Ala Arg Lys Leu Asp Arg Tyr Ser 530 535 540 gag tac ggc
gcg gcc gtg ctg ttc ttg ctc atg tgc acc ttt gcg ctc 1860 Glu Tyr
Gly Ala Ala Val Leu Phe Leu Leu Met Cys Thr Phe Ala Leu 545 550 555
atc gcg cac tgg cta gcc tgc atc tgg tac gcc atc ggc aac atg gag
1908 Ile Ala His Trp Leu Ala Cys Ile Trp Tyr Ala Ile Gly Asn Met
Glu 560 565 570 575 cag cca cac atg gac tca cgc atc ggc tgg ctg cac
aac ctg ggc gac 1956 Gln Pro His Met Asp Ser Arg Ile Gly Trp Leu
His Asn Leu Gly Asp 580 585 590 cag ata ggc aaa ccc tac aac agc agc
ggc ctg ggc ggc ccc tcc atc 2004 Gln Ile Gly Lys Pro Tyr Asn Ser
Ser Gly Leu Gly Gly Pro Ser Ile 595 600 605 aag gac aag tat gtg acg
gcg ctc tac ttc acc ttc agc agc ctc acc 2052 Lys Asp Lys Tyr Val
Thr Ala Leu Tyr Phe Thr Phe Ser Ser Leu Thr 610 615 620 agt gtg ggc
ttc ggc aac gtc tct ccc aac acc aac tca gag aag atc 2100 Ser Val
Gly Phe Gly Asn Val Ser Pro Asn Thr Asn Ser Glu Lys Ile 625 630 635
ttc tcc atc tgc gtc atg ctc att ggc tcc ctc atg tat gct agc atc
2148 Phe Ser Ile Cys Val Met Leu Ile Gly Ser Leu Met Tyr Ala Ser
Ile 640 645 650 655 ttc ggc aac gtg tcg gcc atc atc cag cgg ctg tac
tcg ggc aca gcc 2196 Phe Gly Asn Val Ser Ala Ile Ile Gln Arg Leu
Tyr Ser Gly Thr Ala 660 665 670 cgc tac cac aca cag atg ctg cgg gtg
cgg gag ttc atc cgc ttc cac 2244 Arg Tyr His Thr Gln Met Leu Arg
Val Arg Glu Phe Ile Arg Phe His 675 680 685 cag atc ccc aat ccc ctg
cgc cag cgc ctc gag gag tac ttc cag cac 2292 Gln Ile Pro Asn Pro
Leu Arg Gln Arg Leu Glu Glu Tyr Phe Gln His 690 695 700 gcc tgg tcc
tac acc aac ggc atc gac atg aac gcg gtg ctg aag ggc 2340 Ala Trp
Ser Tyr Thr Asn Gly Ile Asp Met Asn Ala Val Leu Lys Gly 705 710 715
ttc cct gag tgc ctg cag gct gac atc tgc ctg cac ctg aac cgc tca
2388 Phe Pro Glu Cys Leu Gln Ala Asp Ile Cys Leu His Leu Asn Arg
Ser 720 725 730 735 ctg ctg cag cac tgc aaa ccc ttc cga ggg gcc acc
aag ggc tgc ctt 2436 Leu Leu Gln His Cys Lys Pro Phe Arg Gly Ala
Thr Lys Gly Cys Leu 740 745 750 cgg gcc ctg gcc atg aag ttc aag acc
aca cat gca ccg cca ggg gac 2484 Arg Ala Leu Ala Met Lys Phe Lys
Thr Thr His Ala Pro Pro Gly Asp 755 760 765 aca ctg gtg cat gct ggg
gac ctg ctc acc gcc ctg tac ttc atc tcc 2532 Thr Leu Val His Ala
Gly Asp Leu Leu Thr Ala Leu Tyr Phe Ile Ser 770 775 780 cgg ggc tcc
atc gag atc ctg cgg ggc gac gtc gtc gtg gcc atc ctg 2580 Arg Gly
Ser Ile Glu Ile Leu Arg Gly Asp Val Val Val Ala Ile Leu 785 790 795
ggg aag aat gac atc ttt ggg gag cct ctg aac ctg tat gca agg cct
2628 Gly Lys Asn Asp Ile Phe Gly Glu Pro Leu Asn Leu Tyr Ala Arg
Pro 800 805 810 815 ggc aag tcg aac ggg gat gtg cgg gcc ctc acc tac
tgt gac cta cac 2676 Gly Lys Ser Asn Gly Asp Val Arg Ala Leu Thr
Tyr Cys Asp Leu His 820 825 830 aag atc cat cgg gac gac ctg ctg gag
gtg ctg gac atg tac cct gag 2724 Lys Ile His Arg Asp Asp Leu Leu
Glu Val Leu Asp Met Tyr Pro Glu 835 840 845 ttc tcc gac cac ttc tgg
tcc agc ctg gag atc acc ttc aac ctg cga 2772 Phe Ser Asp His Phe
Trp Ser Ser Leu Glu Ile Thr Phe Asn Leu Arg 850 855 860 gat acc aac
atg atc ccg ggc tcc ccc ggc agt acg gag tta gag ggt 2820 Asp Thr
Asn Met Ile Pro Gly Ser Pro Gly Ser Thr Glu Leu Glu Gly 865 870 875
ggc ttc agt cgg caa cgc aag cgc aag ttg tcc ttc cgc agg cgc acg
2868
Gly Phe Ser Arg Gln Arg Lys Arg Lys Leu Ser Phe Arg Arg Arg Thr 880
885 890 895 gac aag gac acg gag cag cca ggg gag gtg tcg gcc ttg ggg
ccg ggc 2916 Asp Lys Asp Thr Glu Gln Pro Gly Glu Val Ser Ala Leu
Gly Pro Gly 900 905 910 cgg gcg ggg gca ggg ccg agt agc cgg ggc cgg
ccg ggg ggg ccg tgg 2964 Arg Ala Gly Ala Gly Pro Ser Ser Arg Gly
Arg Pro Gly Gly Pro Trp 915 920 925 ggg gag agc ccg tcc agt ggc ccc
tcc agc cct gag agc agt gag gat 3012 Gly Glu Ser Pro Ser Ser Gly
Pro Ser Ser Pro Glu Ser Ser Glu Asp 930 935 940 gag ggc cca ggc cgc
agc tcc agc ccc ctc cgc ctg gtg ccc ttc tcc 3060 Glu Gly Pro Gly
Arg Ser Ser Ser Pro Leu Arg Leu Val Pro Phe Ser 945 950 955 agc ccc
agg ccc ccc gga gag ccg ccg ggt ggg gag ccc ctg atg gag 3108 Ser
Pro Arg Pro Pro Gly Glu Pro Pro Gly Gly Glu Pro Leu Met Glu 960 965
970 975 gac tgc gag aag agc agc gac act tgc aac ccc ctg tca ggc gcc
ttc 3156 Asp Cys Glu Lys Ser Ser Asp Thr Cys Asn Pro Leu Ser Gly
Ala Phe 980 985 990 tca gga gtg tcc aac att ttc agc ttc tgg ggg gac
agt cgg ggc cgc 3204 Ser Gly Val Ser Asn Ile Phe Ser Phe Trp Gly
Asp Ser Arg Gly Arg 995 1000 1005 cag tac cag gag ctc cct cga tgc
ccc gcc ccc acc ccc agc ctc ctc 3252 Gln Tyr Gln Glu Leu Pro Arg
Cys Pro Ala Pro Thr Pro Ser Leu Leu 1010 1015 1020 aac atc ccc ctc
tcc agc ccg ggt cgg cgg ccc cgg ggc gac gtg gag 3300 Asn Ile Pro
Leu Ser Ser Pro Gly Arg Arg Pro Arg Gly Asp Val Glu 1025 1030 1035
agc agg ctg gat gcc ctc cag cgc cag ctc aac agg ctg gag acc cgg
3348 Ser Arg Leu Asp Ala Leu Gln Arg Gln Leu Asn Arg Leu Glu Thr
Arg 1040 1045 1050 1055 ctg agt gca gac atg gcc act gtc ctg cag ctg
cta cag agg cag atg 3396 Leu Ser Ala Asp Met Ala Thr Val Leu Gln
Leu Leu Gln Arg Gln Met 1060 1065 1070 acg ctg gtc ccg ccc gcc tac
agt gct gtg acc acc ccg ggg cct ggc 3444 Thr Leu Val Pro Pro Ala
Tyr Ser Ala Val Thr Thr Pro Gly Pro Gly 1075 1080 1085 ccc act tcc
aca tcc ccg ctg ttg ccc gtc agc ccc ctc ccc acc ctc 3492 Pro Thr
Ser Thr Ser Pro Leu Leu Pro Val Ser Pro Leu Pro Thr Leu 1090 1095
1100 acc ttg gac tcg ctt tct cag gtt tcc cag ttc atg gcg tgt gag
gag 3540 Thr Leu Asp Ser Leu Ser Gln Val Ser Gln Phe Met Ala Cys
Glu Glu 1105 1110 1115 ctg ccc ccg ggg gcc cca gag ctt ccc caa gaa
ggc ccc aca cga cgc 3588 Leu Pro Pro Gly Ala Pro Glu Leu Pro Gln
Glu Gly Pro Thr Arg Arg 1120 1125 1130 1135 ctc tcc cta ccg ggc cag
ctg ggg gcc ctc acc tcc cag ccc ctg cac 3636 Leu Ser Leu Pro Gly
Gln Leu Gly Ala Leu Thr Ser Gln Pro Leu His 1140 1145 1150 aga cac
ggc tcg gac ccg ggc agt tag tggggctgcc cagtgtggac 3683 Arg His Gly
Ser Asp Pro Gly Ser 1155 1160 acgtggctca cccagggatc aaggcgctgc
tgggccgctc cccttggagg ccctgctcag 3743 gaggccctga ccgtggaagg
ggagaggaac tcgaaagcac agctcctccc ccagcccttg 3803 ggaccatctt
ctcctgcagt cccctgggcc ccagtgagag gggcaggggc agggccggca 3863
gtaggtgggg cctgtggtcc ccccactgcc ctgagggcat tagctggtct aactgcccgg
3923 aggcacccgg ccctgggcct taggcacctc aaggactttt ctgctattta
ctgctcttat 3983 tgttaaggat aataattaag gatcatatga ataattaatg
aagatgctga tgactatgaa 4043 taataaataa ttatcctgag gagaaaa 4070
<210> SEQ ID NO 2 <211> LENGTH: 1159 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Met
Pro Val Arg Arg Gly His Val Ala Pro Gln Asn Thr Phe Leu Asp 1 5 10
15 Thr Ile Ile Arg Lys Phe Glu Gly Gln Ser Arg Lys Phe Ile Ile Ala
20 25 30 Asn Ala Arg Val Glu Asn Cys Ala Val Ile Tyr Cys Asn Asp
Gly Phe 35 40 45 Cys Glu Leu Cys Gly Tyr Ser Arg Ala Glu Val Met
Gln Arg Pro Cys 50 55 60 Thr Cys Asp Phe Leu His Gly Pro Arg Thr
Gln Arg Arg Ala Ala Ala 65 70 75 80 Gln Ile Ala Gln Ala Leu Leu Gly
Ala Glu Glu Arg Lys Val Glu Ile 85 90 95 Ala Phe Tyr Arg Lys Asp
Gly Ser Cys Phe Leu Cys Leu Val Asp Val 100 105 110 Val Pro Val Lys
Asn Glu Asp Gly Ala Val Ile Met Phe Ile Leu Asn 115 120 125 Phe Glu
Val Val Met Glu Lys Asp Met Val Gly Ser Pro Ala His Asp 130 135 140
Thr Asn His Arg Gly Pro Pro Thr Ser Trp Leu Ala Pro Gly Arg Ala 145
150 155 160 Lys Thr Phe Arg Leu Lys Leu Pro Ala Leu Leu Ala Leu Thr
Ala Arg 165 170 175 Glu Ser Ser Val Arg Ser Gly Gly Ala Gly Gly Ala
Gly Ala Pro Gly 180 185 190 Ala Val Val Val Asp Val Asp Leu Thr Pro
Ala Ala Pro Ser Ser Glu 195 200 205 Ser Leu Ala Leu Asp Glu Val Thr
Ala Met Asp Asn His Val Ala Gly 210 215 220 Leu Gly Pro Ala Glu Glu
Arg Arg Ala Leu Val Gly Pro Gly Ser Pro 225 230 235 240 Pro Arg Ser
Ala Pro Gly Gln Leu Pro Ser Pro Arg Ala His Ser Leu 245 250 255 Asn
Pro Asp Ala Ser Gly Ser Ser Cys Ser Leu Ala Arg Thr Arg Ser 260 265
270 Arg Glu Ser Cys Ala Ser Val Arg Arg Ala Ser Ser Ala Asp Asp Ile
275 280 285 Glu Ala Met Arg Ala Gly Val Leu Pro Pro Pro Pro Arg His
Ala Ser 290 295 300 Thr Gly Ala Met His Pro Leu Arg Ser Gly Leu Leu
Asn Ser Thr Ser 305 310 315 320 Asp Ser Asp Leu Val Arg Tyr Arg Thr
Ile Ser Lys Ile Pro Gln Ile 325 330 335 Thr Leu Asn Phe Val Asp Leu
Lys Gly Asp Pro Phe Leu Ala Ser Pro 340 345 350 Thr Ser Asp Arg Glu
Ile Ile Ala Pro Lys Ile Lys Glu Arg Thr His 355 360 365 Asn Val Thr
Glu Lys Val Thr Gln Val Leu Ser Leu Gly Ala Asp Val 370 375 380 Leu
Pro Glu Tyr Lys Leu Gln Ala Pro Arg Ile His Arg Trp Thr Ile 385 390
395 400 Leu His Tyr Ser Pro Phe Lys Ala Val Trp Asp Trp Leu Ile Leu
Leu 405 410 415 Leu Val Ile Tyr Thr Ala Val Phe Thr Pro Tyr Ser Ala
Ala Phe Leu 420 425 430 Leu Lys Glu Thr Glu Glu Gly Pro Pro Ala Thr
Glu Cys Gly Tyr Ala 435 440 445 Cys Gln Pro Leu Ala Val Val Asp Leu
Ile Val Asp Ile Met Phe Ile 450 455 460 Val Asp Ile Leu Ile Asn Phe
Arg Thr Thr Tyr Val Asn Ala Asn Glu 465 470 475 480 Glu Val Val Ser
His Pro Gly Arg Ile Ala Val His Tyr Phe Lys Gly 485 490 495 Trp Phe
Leu Ile Asp Met Val Ala Ala Ile Pro Phe Asp Leu Leu Ile 500 505 510
Phe Gly Ser Gly Ser Glu Glu Leu Ile Gly Leu Leu Lys Thr Ala Arg 515
520 525 Leu Leu Arg Leu Val Arg Val Ala Arg Lys Leu Asp Arg Tyr Ser
Glu 530 535 540 Tyr Gly Ala Ala Val Leu Phe Leu Leu Met Cys Thr Phe
Ala Leu Ile 545 550 555 560 Ala His Trp Leu Ala Cys Ile Trp Tyr Ala
Ile Gly Asn Met Glu Gln 565 570 575 Pro His Met Asp Ser Arg Ile Gly
Trp Leu His Asn Leu Gly Asp Gln 580 585 590 Ile Gly Lys Pro Tyr Asn
Ser Ser Gly Leu Gly Gly Pro Ser Ile Lys 595 600 605 Asp Lys Tyr Val
Thr Ala Leu Tyr Phe Thr Phe Ser Ser Leu Thr Ser 610 615 620 Val Gly
Phe Gly Asn Val Ser Pro Asn Thr Asn Ser Glu Lys Ile Phe 625 630 635
640 Ser Ile Cys Val Met Leu Ile Gly Ser Leu Met Tyr Ala Ser Ile Phe
645 650 655 Gly Asn Val Ser Ala Ile Ile Gln Arg Leu Tyr Ser Gly Thr
Ala Arg 660 665 670 Tyr His Thr Gln Met Leu Arg Val Arg Glu Phe Ile
Arg Phe His Gln 675 680 685 Ile Pro Asn Pro Leu Arg Gln Arg Leu Glu
Glu Tyr Phe Gln His Ala 690 695 700 Trp Ser Tyr Thr Asn Gly Ile Asp
Met Asn Ala Val Leu Lys Gly Phe 705 710 715 720 Pro Glu Cys Leu Gln
Ala Asp Ile Cys Leu His Leu Asn Arg Ser Leu 725 730 735 Leu Gln His
Cys Lys Pro Phe Arg Gly Ala Thr Lys Gly Cys Leu Arg 740 745 750 Ala
Leu Ala Met Lys Phe Lys Thr Thr His Ala Pro Pro Gly Asp Thr 755 760
765 Leu Val His Ala Gly Asp Leu Leu Thr Ala Leu Tyr Phe Ile Ser Arg
770 775 780 Gly Ser Ile Glu Ile Leu Arg Gly Asp Val Val Val Ala Ile
Leu Gly 785 790 795 800 Lys Asn Asp Ile Phe Gly Glu Pro Leu Asn Leu
Tyr Ala Arg Pro Gly 805 810 815 Lys Ser Asn Gly Asp Val Arg Ala Leu
Thr Tyr Cys Asp Leu His Lys 820 825 830 Ile His Arg Asp Asp Leu Leu
Glu Val Leu Asp Met Tyr Pro Glu Phe 835 840 845
Ser Asp His Phe Trp Ser Ser Leu Glu Ile Thr Phe Asn Leu Arg Asp 850
855 860 Thr Asn Met Ile Pro Gly Ser Pro Gly Ser Thr Glu Leu Glu Gly
Gly 865 870 875 880 Phe Ser Arg Gln Arg Lys Arg Lys Leu Ser Phe Arg
Arg Arg Thr Asp 885 890 895 Lys Asp Thr Glu Gln Pro Gly Glu Val Ser
Ala Leu Gly Pro Gly Arg 900 905 910 Ala Gly Ala Gly Pro Ser Ser Arg
Gly Arg Pro Gly Gly Pro Trp Gly 915 920 925 Glu Ser Pro Ser Ser Gly
Pro Ser Ser Pro Glu Ser Ser Glu Asp Glu 930 935 940 Gly Pro Gly Arg
Ser Ser Ser Pro Leu Arg Leu Val Pro Phe Ser Ser 945 950 955 960 Pro
Arg Pro Pro Gly Glu Pro Pro Gly Gly Glu Pro Leu Met Glu Asp 965 970
975 Cys Glu Lys Ser Ser Asp Thr Cys Asn Pro Leu Ser Gly Ala Phe Ser
980 985 990 Gly Val Ser Asn Ile Phe Ser Phe Trp Gly Asp Ser Arg Gly
Arg Gln 995 1000 1005 Tyr Gln Glu Leu Pro Arg Cys Pro Ala Pro Thr
Pro Ser Leu Leu Asn 1010 1015 1020 Ile Pro Leu Ser Ser Pro Gly Arg
Arg Pro Arg Gly Asp Val Glu Ser 1025 1030 1035 1040 Arg Leu Asp Ala
Leu Gln Arg Gln Leu Asn Arg Leu Glu Thr Arg Leu 1045 1050 1055 Ser
Ala Asp Met Ala Thr Val Leu Gln Leu Leu Gln Arg Gln Met Thr 1060
1065 1070 Leu Val Pro Pro Ala Tyr Ser Ala Val Thr Thr Pro Gly Pro
Gly Pro 1075 1080 1085 Thr Ser Thr Ser Pro Leu Leu Pro Val Ser Pro
Leu Pro Thr Leu Thr 1090 1095 1100 Leu Asp Ser Leu Ser Gln Val Ser
Gln Phe Met Ala Cys Glu Glu Leu 1105 1110 1115 1120 Pro Pro Gly Ala
Pro Glu Leu Pro Gln Glu Gly Pro Thr Arg Arg Leu 1125 1130 1135 Ser
Leu Pro Gly Gln Leu Gly Ala Leu Thr Ser Gln Pro Leu His Arg 1140
1145 1150 His Gly Ser Asp Pro Gly Ser 1155
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