U.S. patent application number 13/008330 was filed with the patent office on 2011-09-29 for human salty taste receptor and methods of modulating salty taste perception.
This patent application is currently assigned to MONELL CHEMICAL SENSES CENTER. Invention is credited to Joseph G. Brand, Taufiqul Huque.
Application Number | 20110236313 13/008330 |
Document ID | / |
Family ID | 39325121 |
Filed Date | 2011-09-29 |
United States Patent
Application |
20110236313 |
Kind Code |
A1 |
Brand; Joseph G. ; et
al. |
September 29, 2011 |
HUMAN SALTY TASTE RECEPTOR AND METHODS OF MODULATING SALTY TASTE
PERCEPTION
Abstract
Methods for identifying modulators of the epithelial sodium ion
channel and for identifying modulators of salty taste perception
are described. Also featured are isolated human salty taste
receptors, artificial lipid bilayers comprising an epithelial
sodium ion channels, and kits for practicing the claimed
methods.
Inventors: |
Brand; Joseph G.; (Wayne,
PA) ; Huque; Taufiqul; (Philadelphia, PA) |
Assignee: |
MONELL CHEMICAL SENSES
CENTER
Philadelphia
PA
|
Family ID: |
39325121 |
Appl. No.: |
13/008330 |
Filed: |
January 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11875200 |
Oct 19, 2007 |
7892756 |
|
|
13008330 |
|
|
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60853290 |
Oct 19, 2006 |
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Current U.S.
Class: |
424/9.1 ;
426/649; 435/29; 506/7; 530/327; 544/407; 564/235 |
Current CPC
Class: |
G01N 33/566 20130101;
G01N 33/6872 20130101; C07K 14/705 20130101; G01N 33/554 20130101;
G01N 33/84 20130101; A61P 3/12 20180101 |
Class at
Publication: |
424/9.1 ; 506/7;
530/327; 544/407; 564/235; 435/29; 426/649 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C40B 30/00 20060101 C40B030/00; C07K 7/06 20060101
C07K007/06; C07D 241/26 20060101 C07D241/26; C07C 279/26 20060101
C07C279/26; C12Q 1/02 20060101 C12Q001/02; A23L 1/237 20060101
A23L001/237 |
Claims
1. A method for identifying modulators of epithelial sodium ion
channels, comprising: assembling at least one epithelial sodium ion
channel in an artificial lipid membrane, wherein the epithelial
sodium ion channel comprises at least three subunits, wherein the
subunits are independently an alpha subunit, a beta subunit, a
gamma subunit, a delta subunit or an epsilon subunit; contacting
the ion channel with a test compound in the presence of sodium or
lithium; and determining a modulation of the biological activity of
the epithelial sodium ion channel in the presence of the test
compound relative to the biological activity of the epithelial
sodium ion channel in the absence of the test compound.
2. The method of claim 1 wherein the epithelial ion channel
comprises at least one delta subunit, at least one beta subunit,
and at least one gamma subunit.
3. The method of claim 1, wherein the epithelial ion channel
comprises at least one alpha subunit, at least one beta subunit, at
least one delta, and at least one gamma subunit.
4. The method of claim 3, further comprising at least one epsilon
subunit.
5. The method of claim 1, further comprising contacting the
epithelial sodium ion channel with an epithelial sodium ion channel
antagonist.
6. The method of claim 1, wherein the artificial lipid membrane is
a micelle, liposome, or lipid bilayer.
7. The method of claim 1, wherein at least two subunits of an
epithelial sodium ion channel are present in the lipid membrane at
differing ratios relative to each other.
8. The method of claim 1, wherein the epithelial sodium ion channel
comprises at least one biological activity of a functional human
salty taste receptor.
9. A method according to claim 1, adapted for high throughput
screening.
10. An artificial lipid membrane comprising at least one type of
phospholipid and an epithelial sodium ion channel or specific
ratios of epithelial sodium ion channel subunits, wherein the
subunits are independently an alpha subunit, a beta subunit, a
gamma subunit, a delta subunit, or an epsilon subunit.
11. The artificial lipid membrane of claim 10, wherein the membrane
is a micelle, liposome, or lipid bilayer.
12. The artificial lipid membrane of claim 10, comprising at least
one epithelial sodium ion channel comprising at least one alpha
subunit, at least one beta subunit, at least one delta subunit, and
at least one gamma subunit.
13. The artificial lipid membrane of claim 12, further comprising
at least one epsilon subunit.
14. The artificial lipid membrane of claim 10, comprising at least
one epithelial sodium channel comprising at least one delta
subunit, at least one beta subunit, and at least one gamma
subunit.
15. A method for preparing the artificial lipid membrane of claim
10, comprising: admixing a micelle or liposome comprising at least
one phospholipid with an epithelial sodium ion channel or specific
ratios of epithelial sodium ion channel subunits, wherein the
epithelial sodium ion channel or epithelial sodium ion channel
subunits are dissolved in a suitable aqueous buffer comprising at
least one surfactant; incubating said micelle or liposome with the
epithelial sodium ion channel or epithelial sodium ion channel
subunit for a sufficient amount of time; and removing the at least
one surfactant.
16. The method of claim 15, further comprising reconstituting the
proteo-liposome into a planar lipid bilayer.
17. A method for identifying modulators of salty taste perception,
comprising: assembling at least one epithelial sodium ion channel
in an artificial lipid membrane, wherein the epithelial sodium ion
channel comprises at least one beta subunit, at least one gamma
subunit, and at least one delta subunit; contacting the ion channel
with a test compound in the presence of sodium or lithium;
determining a modulation of the biological activity of the
epithelial sodium ion channel in the presence of the test compound
relative to the biological activity of the epithelial sodium ion
channel in the absence of the test compound; and administering the
test compound to a subject and determining a modulation of salty
taste perception in the subject relative to the level of salty
taste perception in the subject in the absence of the test
compound.
18. The method of claim 17 wherein the delta subunit comprises the
amino acid sequence of SEQ ID NO:12.
19. The method of claim 17 further comprising the step of screening
positive candidate compounds in a cell-based assay for epithelial
sodium channel activity.
20. An isolated human salty taste receptor comprising at least one
beta polypeptide subunit, at least one gamma polypeptide subunit
and at least one delta polypeptide subunit, wherein said delta
polypeptide subunit comprises the amino acid sequence of SEQ ID
NO:12.
21. The isolated human salty taste receptor of claim 20 wherein
said delta polypeptide subunit has the amino acid sequence of SEQ
ID NO:9.
22. A kit for identifying modulators of the human salty taste
receptor comprising: at least one phospholipid; substantially
purified epithelial sodium ion channel subunits comprising delta
subunits, beta subunits, and gamma subunits; optionally comprising
an epithelial sodium ion channel modulator, sodium or lithium; and
instructions for using the kit in a method for identifying
modulators of the human salty taste receptor.
23. The kit of claim 22 wherein the subunits are admixed in known
ratios in a single container.
24. The kit of claim 22 wherein at least two subunits are present
at differing ratios relative to each other.
25. The kit of claim 22, wherein the modulator is amiloride,
phenamil, benzamil, chlorhexidine or a source of guanidinium ion or
an organic polyamine.
26. A compound identified by the method of claim 1, wherein the
compound activates the epithelial sodium ion channel and induces a
salty taste perception when administered to the mouth of a subject,
provided that the compound is not sodium or lithium.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/875,200, filed Oct. 19, 2007, which claims benefit to U.S.
Provisional Application No. 60/853,290 filed Oct. 19, 2006, the
entire contents of which are incorporated by reference herein, in
their entirety and for all purposes.
FIELD
[0002] The invention relates generally to the field of cell
biology. More specifically, the invention relates to sodium ion
channels and their role in the recognition of salty taste in
humans.
BACKGROUND
[0003] Various publications, including patents, published
applications, technical articles and scholarly articles are cited
throughout the specification. Each of these cited publications is
incorporated by reference herein, in its entirety and for all
purposes.
[0004] Sodium plays an important role in the body's metabolism,
including, among other things, electrical impulse transmission and
fluid and electrolyte homeostasis. In addition, sodium contributes
to the development and stability of flavor in the various foods
ingested by animals, particularly by humans. The sodium ion can
inhibit the bitter taste of some stimuli, thereby modifying the
taste of food. This inhibitory effect of sodium on bitter taste
does not depend on the saltiness of the compound containing the
sodium ion, but rather depends on the concentration of the sodium
ion.
[0005] Excess intake of sodium, however, has been implicated in
various disease states, including gastric cancer and hypertension.
Hypertension is a major risk factor for heart disease, stroke, and
kidney disease. Because of the potential negative health effects of
excess sodium consumption, the United States FDA recommends that
adults limit their intake to less than 2400 milligrams of sodium
per day. Nevertheless, Americans generally far exceed this
recommended allowance. As such, various medical and scientific
groups have recommended drastic reductions in sodium intake.
[0006] To further the goal of reduced sodium intake, numerous salty
taste mimics and salty taste enhancers have been developed. In
general, such mimics have not proven commercially viable as they
lack the clean saltiness of sodium chloride, and most do not affect
food flavor as sodium salt does.
[0007] The dearth of mimics of salty taste, commonly known as salt
substitutes, reflects the extreme structural specificity of the
taste receptor. As far is known, only sodium chloride (NaCl) and
lithium chloride (LiCl) impart a true salty taste. Both heavier
anions paired with Na and Li, and heavier cations paired with Cl
tend to be bitter. The cation specificity suggests an ion channel,
while the chloride effects suggests paracellular shunts. In
addition, the concentration at which NaCl imparts a salty taste is
above 50 mM, a concentration on the higher end of receptor
processes. These two observations--the specificity for Na and Li,
and the effective concentration range--are believed to be the key
to discovering the mechanism of salty taste in humans.
[0008] Over the past two decades, numerous studies, both
qualitative and quantitative, of salt-induced changes in neural
activity in the presence or absence of specific inhibitors and
enhancers have led to the supposition that an epithelial sodium
channel (ENaC) acts as the primary receptor for saltiness (Brand et
al. (1985) Brain Res. 334:207-14; Feigin et al. (1994) Am. J.
Physiol. 266(Cell Physiol):C1165-72; and, Brelin et al. (2006) Adv.
Otorhinolaryngol. 63:152-90). While the ENaC serves as the salt
receptor for many experimental animals (Halpern, BP (1998)
Neurosci. Biobehav. Rev. 23(1):5-47), no conclusive evidence has
emerged that the same holds true for human beings. Notably, the
inability of amiloride to inhibit sodium-induced salty taste
response in humans suggests that ENaCs are not involved in human
salty taste recognition, at least to the extent observed in other
animals.
[0009] Because of this discrepancy between human and animal models,
the transduction mechanisms underlying the perception of salty
taste in humans remain under investigation. Sufficient activation
of the nerve eventually evokes the sensation of saltiness in the
higher cortical areas (Schoenfeld, Mass. et al. (2004)
Neuroscience. 127:347-53).
[0010] Because of the robust response shown to amiloride by taste
cells of many rodents, the ENaCs in these cells are assumed to be
located primarily at the apical membrane, above the level of the
tight junctions. This location makes them susceptible to the action
of drugs such as amiloride. It is assumed that amiloride cannot
pass the tight junctions. Augmenting the direct mechanism at the
apical membrane is a paracellular shunt pathway into the
basolateral area of taste buds below the tight junction level
(Mierson, S et al. (1996) J. Neurophysiol. 76:1297-309). Since
sodium can pass the tight junctions, the paracellular mechanism
should result in an amiloride insensitive salty taste response. The
human salty response may be amiloride-insensitive because the vast
majority of taste cell ENaCs are located below these tight
junctions. Other mechanisms for salt perception may exist. These
could be entirely different from the ENaC, or an alternative
manifestation of the ENaC due to sodium load or hormonal influences
on ENaC expression or composition.
[0011] ENaCs comprise a family of cation channel proteins mediating
sodium permeation in epithelia (Mano, I et al. (1999) Bioessays
21:568-78). Expression cloning originally demonstrated that there
are three homologous genes, each encoding one of the three subunits
of the channel--i.e., alpha (.alpha.), beta (.beta.) and gamma
(.gamma.) (Canessa, C M et al. (1994) Nature 367:463-7).
Co-expression of all three subunits is essential for maximal
Na+channel activity, although the alpha subunit by itself produces
a small current. A fourth subunit, delta (6) was later cloned and
shown to be similar to the alpha subunit both structurally and
functionally, albeit with a 30-fold lower affinity for amiloride
(Waldmann et al. (1995) J. Biol. Chem. 270:27411-4). This lower
amiloride sensitivity is assumed to be reflected in a motif called
the PreMR2 sequence. The transmembrane topology of the ENaCs
comprises two hydrophobic transmembrane domains flanking a long
extracellular loop, with intracellular amino and carboxyl termini.
The subunit stoichiometry of the ENaCs may be species-specific and
tissue-specific, since there is evidence for an
.alpha.2.beta..gamma. configuration in rats (Firsov et al. (1998)
EMBO J. 17:344-52) and an (.alpha.)1.beta.(1).gamma.(1) arrangement
in humans (Staruschenko, A (2005) Biophys. J. 88:3966-75).
[0012] For improved health and wellness, there is a need to
diminish sodium intake. This need must be balanced with the desire
for the taste of sodium, and the ability of sodium to impart
improved flavor in food. One attractive means to diminish dietary
sodium without sacrificing sodium flavor is to use modulators of
salty taste. Thus, there is a need to establish the definitive
receptor for salty taste perception and for a means to identify
modulators of salty taste perception.
SUMMARY
[0013] The invention provides an isolated human salty taste
receptor comprising at least one beta polypeptide subunit, at least
one gamma polypeptide subunit, and at least one delta polypeptide
subunit wherein said delta polypeptide subunit comprises the amino
acid sequence of SEQ ID NO:12. In some aspects, the delta
polypeptide subunit has the amino acid sequence of SEQ ID NO:9.
Also provided is an isolated human salty taste receptors comprising
at least one alpha polypeptide subunit, at least one beta
polypeptide subunit, at least one delta polypeptide subunit, and at
least one gamma polypeptide subunit.
[0014] The invention also provides a method for identifying
modulators of epithelial sodium ion channels. Such methods include
assembling at least one epithelial sodium ion channel in a lipid
membrane (wherein the epithelial sodium ion channel comprises at
least three types of subunits, which are independently an alpha
subunit, a beta subunit, a gamma subunit, a delta subunit, and an
epsilon subunit); contacting the ion channel with a test compound
in the presence of sodium ions or lithium ions; and determining a
modulation of the biological activity of the epithelial sodium ion
channel in the presence of the test compound relative to the
biological activity of the epithelial sodium ion channel in the
absence of the test compound. The lipid membrane is preferably an
artificial membrane.
[0015] In some aspects, the epithelial ion channel comprises one
alpha subunit, one beta subunit, and one gamma subunit. In other
aspects, the epithelial ion channel comprises one alpha subunit,
one beta subunit, one gamma subunit, and one epsilon subunit. In
other aspects, the epithelial ion channel comprises two alpha
subunits, one beta subunit, and one gamma subunit. In further
aspects the epithelial ion channel comprises three alpha subunits,
three beta subunits, and three gamma subunits. Additional aspects
include those wherein the epithelial ion channel comprises one
delta subunit, one beta subunit, and one gamma subunit. In other
aspects, the epithelial ion channel comprises two delta subunits,
one beta subunit, and one gamma subunit. In still further aspects,
the epithelial ion channel comprises two delta subunits, two beta
subunits, and two gamma subunits. In still further aspects, the
epithelial ion channel comprises three delta subunits, three beta
subunits, and three gamma subunits.
[0016] In the method for identifying modulators of epithelial
sodium channels, the method may further include contacting the
epithelial sodium ion channel with an epithelial sodium ion channel
antagonist, such as, but not limited to chlorhexidine, amiloride,
phenamil, benzamil or a homolog, analog, or derivative thereof.
[0017] In the method for identifying modulators of epithelial
sodium channels, suitable lipid components for the membrane include
at least one of phosphatidylcholine, phoshpatidylethanolamine,
phostphatidylserine, phosphatidylglyine, phosphatidylinositol,
sphingomyelin, cholesterol, cardiolipin, or a homolog, analog, or
derivative thereof. As such the lipids may be organized as a
micelle, liposome, or lipid bilayer.
[0018] In some aspects of the method for identifying modulators of
epithelial sodium channels, at least two subunits of an epithelial
sodium ion channel are present in the lipid membrane at differing
ratios relative to each other.
[0019] In the step for determining a modulation of the biological
activity of the epithelial sodium ion channel, any suitable means
known in the art may be used, such as, but not limited to, voltage
clamping, and/or measurement of an indicator dye. The method may be
adapted for high throughput screening.
[0020] The method for identifying modulators of epithelial sodium
channels thus provides compounds identified by the method that act
as modulators of the epithelial sodium channels. These compounds
may be formulated into compositions by admixing the compounds with
a pharmaceutically acceptable carrier.
[0021] In a specific aspect, the invention provides a method for
identifying modulators of the human salty taste receptor
comprising: assembling at least one salty taste receptor in a lipid
membrane, wherein the salty taste receptor comprises at least one
beta subunit, at least one gamma subunit, and at least one delta
subunit; contacting the ion channel with a test compound in the
presence of sodium ions or lithium ions; and determining a
modulation of the biological activity of the salty taste receptor
in the presence of the test compound relative to the biological
activity of the salty taste receptor in the absence of the test
compound.
[0022] In some aspects, the human salty taste receptor comprises
one alpha subunit, one beta subunit, and one gamma subunit. In
other aspects, the salty taste receptor comprises one alpha
subunit, one beta subunit, one gamma subunit, and one epsilon
subunit. In other aspects, the salty taste receptor comprises two
alpha subunits, one beta subunit, and one gamma subunit. In further
aspects the salty taste receptor comprises three alpha subunits,
three beta subunits, and three gamma subunits. Additional aspects
include those wherein the salty taste receptor comprises one delta
subunit, one beta subunit, and one gamma subunit. In other aspects,
the salty taste receptor comprises two delta subunits, one beta
subunit, and one gamma subunit. In still further aspects, the salty
taste receptor comprises two delta subunits, two beta subunits, and
two gamma subunits. In still further aspects, the salty taste
receptor comprises three delta subunits, three beta subunits, and
three gamma subunits.
[0023] In the method for identifying modulators of the human salty
taste receptor, the delta subunit preferably comprises the amino
acid sequence of SEQ ID NO:12. In some aspects, the delta receptor
comprises the amino acid sequence of SEQ ID NO:9. In the method for
identifying modulators of the human salty taste receptor, the
method may further comprise contacting the epithelial sodium ion
channel with an epithelial sodium ion channel antagonist, such as,
but not limited to, chlorhexidine, amiloride, phenamil, benzamil or
a homolog, analog, or derivative thereof.
[0024] In the method for identifying modulators of the human salty
taste receptor, the lipid membrane may comprise at least one of
phosphatidylcholine, phoshpatidylethanolamine, phostphatidylserine,
phosphatidylglyine, phosphatidylinositol, sphingomyelin,
cholesterol, cardiolipin, or a homolog, analog, or derivative
thereof. The lipids may be organized as a liposome or lipid
bilayer.
[0025] In some aspects of the method for identifying modulators of
the human salty taste receptor, at least two subunits of an
epithelial sodium ion channel are present in the lipid membrane at
differing ratios relative to each other. The channels in the
membrane preferably comprise at least one biological activity of a
functional human salty taste receptor.
[0026] In the step for determining a modulation of the biological
activity of the salty taste receptor, any suitable means known in
the art may be used, such as, but not limited to voltage clamping,
and/or measurement of an indicator dye. The method may be adapted
for high throughput screening.
[0027] Compounds that modulate human salty taste perception are
identified by the method of the invention and may include, for
example, salty taste mimics, enhancers, modifiers, and inhibitors.
The invention thus provides modulators of human salty taste
perception which may further be used in compositions by admixing
the compounds with a pharmaceutically acceptable carrier, or foods
and beverages to modulate the salty taste perception of the food or
beverage.
[0028] The invention also provides an artificial lipid membrane
comprising at least one type of phospholipid and an epithelial
sodium ion channel or specific ratios of epithelial sodium ion
channel subunits wherein the subunits are selected from the group
consisting of alpha subunits, beta subunits, gamma subunits, delta
subunits, and epsilon subunits.
[0029] The artificial lipid membrane may comprise at least one
phospholipid including phosphatidylcholine,
phoshpatidylethanolamine, phostphatidylserine, phosphatidylglyine,
phosphatidylinositol, sphingomyelin, cholesterol, cardiolipin, or a
homolog, analog, or derivative thereof. The lipid membrane may be
organized, for example, as a liposome or lipid bilayer.
[0030] In some aspects, the artificial lipid membrane comprises at
least one epithelial ion channel comprising one alpha subunit, one
beta subunit, and one gamma subunit. In other aspects, the
epithelial ion channel comprises one alpha subunit, one beta
subunit, one gamma subunit, and one epsilon subunit. In other
aspects, the epithelial ion channel comprises two alpha subunits,
one beta subunit, and one gamma subunit. In further aspects the
epithelial ion channel comprises three alpha subunits, three beta
subunits, and three gamma subunits. Additional aspects include
those wherein the epithelial ion channel comprises one delta
subunit, one beta subunit, and one gamma subunit. In other aspects,
the epithelial ion channel comprises two delta subunits, one beta
subunit, and one gamma subunit. In still further aspects, the
epithelial ion channel comprises two delta subunits, two beta
subunits, and two gamma subunits. In still further aspects, the
epithelial ion channel comprises three delta subunits, three beta
subunits, and three gamma subunits.
[0031] The method also provides a method for preparing such
artificial lipid membrane comprising admixing a liposome comprising
at least one phospholipid with an epithelial sodium ion channel or
specific ratios of epithelial sodium ion channel subunits wherein
the epithelial sodium ion channel or epithelial sodium ion channel
subunits are dissolved in a suitable aqueous buffer comprising at
least one detergent, incubating the liposome with the epithelial
sodium ion channel or epithelial sodium ion channel subunit for a
sufficient amount of time, and removing the at least one
detergent.
[0032] The method of preparing the artificial lipid membrane may
further comprise reconstituting the proteo-liposome into a planar
lipid bilayer.
[0033] The invention further provides a method for identifying
modulators of salty taste perception comprising: assembling at
least one epithelial sodium ion channel in a lipid membrane,
wherein the epithelial sodium ion channel comprises at least one of
an alpha subunit, a beta subunit, a gamma subunit, a delta subunit,
or an epsilon subunit; contacting the ion channel with a test
compound in the presence of sodium or lithium; determining a
modulation of the biological activity of the epithelial sodium ion
channel in the presence of the test compound relative to the
biological activity of the epithelial sodium ion channel in the
absence of the test compound; and administering the test compound
to a subject and determining a modulation of salty taste perception
in the subject relative to the level of salty taste perception in
the subject in the absence of the test compound. Preferably, the
epithelial sodium ion channel comprises at least one beta subunit,
at least one gamma subunit, and at least one delta subunit.
[0034] In some aspects, the epithelial ion channel comprises one
alpha subunit, one beta subunit, and one gamma subunit. In other
aspects, the epithelial ion channel comprises one alpha subunit,
one beta subunit, one gamma subunit, and one epsilon subunit. In
other aspects, the epithelial ion channel comprises two alpha
subunits, one beta subunit, and one gamma subunit. In further
aspects the epithelial ion channel comprises three alpha subunits,
three beta subunits, and three gamma subunits. Additional aspects
include those wherein the epithelial ion channel comprises one
delta subunit, one beta subunit, and one gamma subunit. In other
aspects, the epithelial ion channel comprises two delta subunits,
one beta subunit, and one gamma subunit. In still further aspects,
the epithelial ion channel comprises two delta subunits, two beta
subunits, and two gamma subunits. In still further aspects, the
epithelial ion channel comprises three delta subunits, three beta
subunits, and three gamma subunits.
[0035] In some aspects, the delta subunit comprises the amino acid
sequence of SEQ ID NO:12. In some aspects, the delta subunit
comprises the amino acid sequence of SEQ ID NO:9.
[0036] In some aspects, the subject is a human.
[0037] The method permits identification of a compound that reacts
in vitro with the human salty taste receptor and which is perceived
by subjects as salty. The invention thus provides such compounds
which may be used in compositions by admixing the compounds with a
pharmaceutically acceptable carrier, or foods or beverages to
modulate the salty taste perception of the food or beverage.
Preferably, the compounds allow perception of salty taste, but
which have a reduced effect on blood pressure as compared to salt
and which have no untoward effect on the subject.
[0038] In some aspects, the compounds can be additionally screened
by cell based assays for epithelial sodium channel activity.
[0039] The invention also provides kits for identifying modulators
of the human salty taste receptor comprising at least one form of
phospholipid; substantially purified epithelial sodium ion channel
subunits comprising alpha subunits, delta subunits, beta subunits,
gamma subunits, or epsilon subunits; and optionally comprising an
epithelial sodium ion channel modulator, sodium or lithium, and
instructions for using the kit in a method for identifying
modulators of the human salty taste receptor.
[0040] The instructions may provide, for example, directions to
admix the subunits in specific ratios to achieve various forms of
the epithelial sodium ion channel of interest. In some aspects, at
least two subunits are added to be present at differing ratios
relative to each other.
[0041] The kit may contain a modulator such as, but not limited to
amiloride, phenamil, benzamil, chlorhexidine, or a source of
guanidinium ion.
[0042] The invention also provides a method of modulating salty
taste perception (either by stimulating salty taste perception or
inhibiting salty taste perception) comprising contacting a human
salty taste receptor with a compound that stimulates salty taste
perception wherein the salty taste receptor comprises at least one
beta polypeptide subunit, at least one gamma polypeptide subunit,
and at least one delta polypeptide subunit wherein said delta
polypeptide subunit comprises the amino acid sequence of SEQ ID
NO:12, and wherein said compound specifically interacts with said
delta subunit.
[0043] In some aspects, the human salty taste receptor comprises
one alpha subunit, one beta subunit, and one gamma subunit. In
other aspects, the salty taste receptor comprises one alpha
subunit, one beta subunit, one gamma subunit, and one epsilon
subunit. In other aspects, the salty taste receptor comprises two
alpha subunits, one beta subunit, and one gamma subunit. In further
aspects the salty taste receptor comprises three alpha subunits,
three beta subunits, and three gamma subunits. Additional aspects
include those wherein the salty taste receptor comprises one delta
subunit, one beta subunit, and one gamma subunit. In other aspects,
the salty taste receptor comprises two delta subunits, one beta
subunit, and one gamma subunit. In still further aspects, the salty
taste receptor comprises two delta subunits, two beta subunits, and
two gamma subunits. In still further aspects, the salty taste
receptor comprises three delta subunits, three beta subunits, and
three gamma subunits.
[0044] In some aspects the compound specifically interacts a
portion of the delta subunit containing the amino acid sequence of
SEQ ID NO:12. In some aspects, the compound binds to the portion of
the delta subunit containing the amino acid sequence of SEQ ID
NO:12.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 shows a human taste bud stained by an ATPase
histochemical procedure.
[0046] FIG. 2 shows antibody detection of the second messenger
enzyme, phospholipase Cbeta2 (PLCbeta2) using an
immunohistochemical procedure on human taste cells. Panel A shows
the subset of cells labeled by the antibody. Panel B is a contrast
image of the taste bud and of the surrounding fungiform
papillae.
[0047] FIGS. 3 (a, b, c , d) shows an alignment of ENaC delta
subunit sequenced from cDNA of ten individuals (labeled DENACA,
DENACD, DENACE, DENACG, DENACH, DENACI, DENACJ, DENACT, DENACTV and
DENACW, respectively), as compared with the GeneBank sequence of
the top row (DENACGB). DENACA, DENACD, DENACE, DENACG, DENACH,
DENACI, DENACJ, DENACT, DENACTV and DENACW correspond to SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ
ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID
NO:26, respectively.
[0048] FIG. 4 shows amiloride inhibition of ENaC of different
composition. ENaC composed of human delta, beta, gamma was less
sensitive to amiloride than that composed of human alpha, beta,
gamma.
[0049] FIG. 5 shows immuno-labeling of a subset of cells in a human
taste bud.
[0050] FIG. 6 shows the capture of an isolated human taste bud cell
by a micro-pipette from an aqueous suspension. The cell thus
captured is placed in an RNA-preserving medium for further
study.
[0051] FIG. 7 shows an early quantitative RT-PCR of a single cell
tracing the amplification of partial transcripts of the ENaC
subunits, alpha, beta, gamma, and delta. The result suggests a cell
containing equal copies of delta, beta, and gamma, with the alpha
transcript showing as a genomic control.
[0052] FIG. 8 (a, b, c , d) shows an alignment of ENaC gamma
subunit sequenced from cDNA of ten individuals (labeled GENACA,
GENACB, GENACD, GENACE, GENACG, GENACH, GENACJ, GENACT, GENACV, and
GENACW, respectively) compared with the GeneBank sequence of the
top row (GENACGB). GENACA, GENACB, GENACD, GENACE, GENACG, GENACH,
GENACJ, GENACT, GENACV, and GENACW correspond to SEQ ID NO:27, SEQ
ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36,
respectively.
[0053] FIG. 9 shows single channel recording of the activity of the
catfish putative taste receptor for L-arginine in planar lipid
bilayers. Proteoliposomes containing purified receptor protein from
catfish taste epithelium are fused to planar lipid bilayers.
Control records (trace shown in part A) were obtained after
addition of proteolipisomes to the membrane bathing solution before
addition of L-Arg. The addition of 10 .mu.M L-Arg to the cis-side
of the bilayer evoked regular periodic channel activity (trace
shown in panel B, including the inset that shows the current record
at an expanded scale). After several minutes of single channel
recording, 100 .mu.M D-Arg was added to the cis-side (trace shown
in panel C) and activity ceased. Transmembrane potential was -100
mV. Traces shown in all panels are continuous records of that
specific condition.
DETAILED DESCRIPTION
[0054] It is to be understood that this invention is not limited to
particular methods, reagents, compounds, compositions, or
biological systems, which can, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular aspects only, and is not intended to be
limiting.
[0055] Various terms relating to the methods and other aspects of
the present invention are used throughout the specification and
claims. Such terms are to be given their ordinary meaning in the
art unless otherwise indicated. Other specifically defined terms
are to be construed in a manner consistent with the definition
provided herein.
[0056] As used in this specification and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the content clearly dictates otherwise. Thus, for example,
reference to "a cell" includes a combination of two or more cells,
and the like.
[0057] The term "about" as used herein when referring to a
measurable value such as an amount, a temporal duration, and the
like, is meant to encompass variations of .+-.20% or .+-.10%, more
preferably .+-.5%, even more preferably .+-.1%, and still more
preferably .+-.0.1% from the specified value, as such variations
are appropriate to perform the disclosed methods.
[0058] As used herein the "Epithelial Sodium Channel" or, as
abbreviated, "ENaC," refers to a multisubunit protein that is
responsible for flow of or transport of sodium ions across specific
epithelium or cell membranes. ENaCs are generally composed of
multiple subunits, generally .alpha., .beta., .gamma. subunits.
There are also .delta. and .epsilon. subunits which may be in some
ENaCs in specific tissues. The "salty taste receptor" as discovered
herein, is a species of ENaC that is localized in taste cells and
in one aspect is composed of .beta., .gamma., and .delta.
subunits.
[0059] As used herein, "test compound" refers to any purified
molecule, substantially purified molecule, molecules that are one
or more components of a mixture of compounds, or a mixture of a
compound with any other material that can be analyzed using the
methods of the present invention. Test compounds can be organic or
inorganic chemicals, or biomolecules, and all fragments, analogs,
homologs, conjugates, and derivatives thereof. Biomolecules include
proteins, polypeptides, nucleic acids, lipids, monosaccharides,
polysaccharides, and all fragments, analogs, homologs, conjugates,
and derivatives thereof. Test compounds can be of natural or
synthetic origin, and can be isolated or purified from their
naturally occurring sources, or can be synthesized de novo. Test
compounds can be defined in terms of structure or composition, or
can be undefined. The compound can be an isolated product of
unknown structure, a mixture of several known products, or an
undefined composition comprising one or more compounds. Examples of
undefined compositions include cell and tissue extracts, growth
medium in which prokaryotic, eukaryotic, and archaebacterial cells
have been cultured, fermentation broths, protein expression
libraries, and the like.
[0060] As used herein, the terms "modulate" means any change,
increase, or decrease in the amount, quality, or effect of a
particular activity or protein. "Modulators" refer to any
inhibitory or activating molecules identified using in vitro and in
vivo assays for, e.g., agonists, antagonists, and their homologs,
including fragments, variants, and mimetics, as defined herein,
that exert substantially the same biological activity as the
molecule "Inhibitors" or "antagonists" are modulating compounds
that reduce, decrease, block, prevent, delay activation,
inactivate, desensitize, or downregulate the biological activity or
expression of a molecule or pathway of interest. "Inducers,"
"activators," or "agonists" are modulating compounds that increase,
induce, stimulate, open, activate, facilitate, enhance activation,
sensitize, or upregulate a molecule or pathway of interest. In some
preferred aspects of the invention, the level of inhibition or
upregulation of the expression or biological activity of a molecule
or pathway of interest refers to a decrease (inhibition or
downregulation) or increase (upregulation) of greater than from
about 50% to about 99%, and more specifically, about 50%, 51%, 52%,
53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69% 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. The inhibition or
upregulation may be direct, i.e., operate on the molecule or
pathway of interest itself, or indirect, i.e., operate on a
molecule or pathway that affects the molecule or pathway of
interest.
[0061] "Pharmaceutically acceptable carrier" refers to a medium
that does not interfere with the effectiveness of the biological
activity of the active ingredient(s) of a composition, and is not
toxic to the subject to which it is administered.
[0062] "Ct" or "threshold cycle" refers to the PCR cycle in which a
noticeable increase in reporter fluorescence above a baseline
signal is initially detected.
[0063] ".DELTA.Ct" refers to the difference between the Ct of a
sample assay and the Ct of a control sample. Thus,
.DELTA.Ct=Ct(target)-Ct(control).
[0064] ".DELTA..DELTA.Ct" refers to the difference between the
average .DELTA.Ct value of a target sample and the average
.DELTA.Ct for a corresponding calibrator sample. Thus,
.DELTA..DELTA.Ct(test sample)=Avg.DELTA.Ct(test
sample)-Avg.DELTA.Ct(calibrator sample).
[0065] "Biological activity" as used herein refers to a measurable
function of an ENaC, including but not limited to, maintenance of a
sodium gradient across the membrane, changes in ion flux, changes
in membrane potential, current amplitude, voltage gating,
sensitivity to chlorhexidine, amiloride, or its analogs,
stimulation by bretylium, novobiocin, or guanidinium ions, binding
to subunit-specific monoclonal antibodies, and the like.
[0066] The present invention is based on the discovery that the
human salty taste receptor is an epithelial sodium ion channel. It
is thus an object of the present invention to use the precise molar
ratios of the ENaC subunits and to reconstitute the ENaCs in a
lipid bilayer in order to identify compounds that modulate the
biological activity of the ENaCs. In particular it is an object of
the present invention to use the precise molar ratios of the salty
taste receptor subunits to reconstitute the salty taste receptor in
the a lipid bilayer in order to identify compounds that modulate
the biological activity of the salty taste receptor and to identify
compounds that modulate salty taste perception in human beings.
Without intending to be limited to any particular theory or
mechanism of action, it is believed that a passive influx of sodium
ions through epithelial sodium channels in certain taste receptor
cells causes a change in intracellular ion balance leading to a
depolarization, ultimately resulting in neurotransmitter release,
which in turn produces a perception of salty taste.
[0067] In one aspect, the invention provides assays to identify
compounds that bind and/or modulate the human salty taste receptor.
The methods comprise assembling at least one epithelial sodium ion
channel in a lipid membrane, wherein the epithelial sodium ion
channel comprises an alpha, beta, gamma, or delta subunit,
contacting the at least one ion channel with a test compound in the
presence of sodium or lithium, and determining a modulation of the
biological activity of the at least one epithelial sodium ion
channel in the presence of the test compound relative to the
biological activity of the at least one subunit in the absence of
the test compound.
[0068] Where the biological activity of the sample containing the
test compound is higher than the activity in the sample lacking the
test compound, the compound is an agonist. If the activity of the
sample containing the test compound is lower than the activity in
the sample lacking the test compound, the compound is an
antagonist.
[0069] Epithelial sodium ion channels are heteromultimeric
complexes that are comprised of different subunits. Various
subunits of ENaC have been identified, and include, without
limitation, the alpha subunit, the beta subunit, the gamma subunit,
the delta subunit, and the epsilon subunit. The ENaC subunits may
derived from any species, however, mammalian ENaC subunits are
preferred and the most preferred species is human. Examples of
nucleic acid sequences encoding human ENaC subunits and the deduced
amino acid sequences are provided herein. Other subunits with amino
acid sequences that are substantially homologous or which represent
isoforms of the subunit proteins may be used in practicing the
invention. Amino acid sequences that are "substantially homologous"
are at least protein sequences that are from about 80% to about
100% identical to the sequence provided herein for the subunit
sequence. More preferably, the sequences are about 85% to about
100% identical. Most preferably, the sequences are about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
reference sequence provided herein for the subunit.
[0070] Representative nucleotide sequences encoding human alpha
subunit, human beta subunit, human gamma subunit, and human delta
subunit are provided as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and
SEQ ID NO:7, respectively. The deduced amino acid sequences for
human alpha subunit, human beta subunit, human gamma subunit, and
human delta subunit are provided as SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:6, and SEQ ID NO:8, respectively. In a preferred aspect for
the salty taste receptor, the delta subunit comprises a cysteine at
position 532 with respect to SEQ ID NO:8. The delta receptor with
Cys.sub.532 is shown in SEQ ID NO:9. Such substitution may arise
due to a alteration in the triplet codon from tac to tgc (with
respect to that shown in SEQ ID NO:7) and results in a change from
tyrosine (Tyr) to cysteine (Cys).
[0071] In some aspects, the ENaC is comprised of at least one alpha
subunit, at least one beta subunit, and at least one gamma subunit
(e.g., (.alpha.)1(.beta.)1(.gamma.)1). In other aspects, the ENaC
is comprised of at least one alpha subunit, at least one beta
subunit, at least one gamma subunit, and at least one delta
subunit. In other aspects, the ENaC is composed of two alpha
subunits, one beta subunit, one gamma subunit
(.alpha.2.beta..gamma.). In other aspects, the ENaC is composed of
three alpha subunits, three beta subunits, and three gamma subunits
((.alpha.)3(.beta.)3(.gamma.)3). In another aspect, the ENaC
comprises an epsilon subunit and at least one other subunit such as
an alpha subunit, beta subunit, delta subunit, gamma subunit, or
combinations thereof. In still other aspects the ENaC comprises a
plurality of beta subunits. In a preferred aspect, the ENaC is
comprised of at least one beta subunit, at least one gamma subunit,
and at least one delta subunit (the salty taste receptor). The most
preferred aspect is an ENaC comprising at least one beta, at least
one gamma, and at least one delta subunit (e.g.,
(.beta.)1(.gamma.)1)(.delta.)1 in which the delta subunit contains
Cys.sub.532.
[0072] The various subunits can be present in the ENaC in different
ratios relative to other subunits. The observed variation may
relate to which tissue the particular ENaC of interest is expressed
in. For example, but not by way of limitation, an ENaC can be
comprised of two alpha, one beta, and one gamma subunit. Thus, in
certain aspects of the invention, the ENaC assembled into a lipid
membrane is comprised of at least two subunits that are present in
different ratios relative to the other subunits. In other aspects,
the ENaC is comprised of at least two subunits that are present in
the same ratio relative to the other subunits. The ratios of the
ENaC subunits may also vary depending on the tissue in which the
ENaCs of interest are expressed. Further, there may be important
sequence variability in the form of each subunit expressed in
various tissues. For example, but not by way of limitation, the
delta subunit of ENaC expressed in the salty taste receptor
preferably has a cysteine in the putative amiloride binding site of
delta at position 532 of SEQ ID NO:8 (which encodes human delta
from kidney). Human kidney delta has a tyrosine at this position.
Thus, when expressing a human salty taste receptor, it is preferred
to use a delta with the putative amiloride binding site of
MGSLCSLWFGA (SEQ ID NO:12) which includes CYS.sub.532. As this
motif is at least a putative site for amiloride binding, other
compounds that modulate the human salty taste receptor may also
bind to this site.
[0073] In certain aspects of the invention, the lipid membranes
produced with the ENaC subunits in them contain ENaC subunits that
form the salty taste receptor. These salty taste receptors include
at least one beta, at least one gamma, and at least one delta
subunit. In preferred aspects, the delta subunit comprises
Cys.sub.532. In other aspects, the ENaC contains subunits selected
from alpha, beta, gamma, delta, and epsilon. In some aspects, the
ENaC is composed of at least one alpha, at least one beta, and at
least one gamma. In other aspects, the ENaC comprises at least one
epsilon subunit.
[0074] The ENaC or the various subunits that are to be assembled
into the lipid membrane can be obtained from any source suitable in
the art. For example, an ENaC or any subunit thereof can be freshly
isolated from any cell that expresses and ENaC, including cell
lines and stable cell lines. For example, but not by way of
limitation, ENaC are expressed in neural tissue, the pancreas,
testes, ovaries, tongue, colon, kidneys, lungs, sweat glands, and
the like. In some aspects, the ENaC for salty taste perception is
isolated from the papillae of the tongue. In other aspects, an ENaC
or any subunit thereof can be recombinantly expressed, purified and
used to reconstitute a lipid membrane to form functional ENaCs.
[0075] In certain aspects, each subunit of the ENaC is separately
expressed in a recombinant expression system such as, but not
limited to bacterial cells, Spodoptera frugiperda cells, mammalian
cells, and frog oocytes. The expressed protein is purified by
standard biochemical means as is well-known in the art.
Alternatively, expressed protein may be immunopurified using
immobilized antibodies that specifically bind the ENaC subunits.
Methods for purifying proteins by immunoaffinity (using antibodies
that specifically bind the subunit or ENaC of interest). In other
aspects, the ENaC subunits are expressed as a fusion protein with a
polypeptide that allows for rapid purification and subsequent
cleavage from the expressed protein. Such purification systems
include, but are not limited to the pGEX system
(glutathione-S-transferase fusion proteins) and multi-histidine
fusion proteins (for nickel binding affinity purification). These
and other types of purification are described in numerous
references and are well-known to those of skill in the art. In
certain preferred aspects, the ENaC subunits are expressed
simultaneously using a baculovirus system and Spodoptera frugiperda
cells and membrane fractions are prepared as described in Rao, U.S.
et al. (2002) "Activation of Large Conductance Sodium Channels Upon
Expression of Amiloride-Sensitive Sodium Channel in SF9 Insect
Cells" J. Biol. Chem. 277(7):4900-4905.
[0076] In certain aspects, the subunits of the ENaC are
substantially purified prior to incorporation into the membrane. As
used herein, "substantially purified" refers to subunits that are
at least 80% free of contaminating material (e.g., proteins,
polysaccharides, and lipids) derived from the cells from which they
are obtained. Preferably, the subunits are at least about 85% free
of contaminating material. More preferably, the subunits are at
least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
more free of contaminating material.
[0077] To recreate a particular ENaC which may be present in a
tissue in the artificial membranes of the invention, the ratio of
the subunits present in the ENaC may be determined by quantitative
PCR. As the ratio of protein subunits of a multimeric receptor
often correlates to the amount of mRNA produced in a cell for the
given receptor, quantitative PCR can provide an efficient means of
determining the ratio of mRNA present. Protocols for performing
quantitative PCR are well known in the art. Further, given the
sequences of the ENaCs provided herein and the knowledge in the art
and software available for selecting PCR oligonucleotide primers
that can specifically and reliably amplify messages for particular
genes, one of skill in the art may easily and routinely perform
quantitative PCR on tissue samples and determine the identity and
ratio of the subunits that form a particular ENaC. Assays for
determining the relative amounts of mRNA are well known in the art.
Once the ratio of mRNA is determined, one may extrapolate the
amount of protein of each subunit that must be added to the
membrane to provide the appropriate stoichiometric amounts of
protein to form biologically active ENaCs.
[0078] The concentration of affinity-purified protein can be
determined by measuring the total nitrogen content of the protein
eluate and comparing the nitrogen content with the total protein
content of the eluate. Nitrogen content can be determined by any
means suitable in the art, such as the well-known Kjeldahl Nitrogen
Method. Protein concentration can be determined, for example, by
spectrophotometry whereby a protein sample is analyzed for its
absorption of light at 280 nm to derive an absorption coefficient.
Any means known in the art for assessing concentration and/or
purity of protein may be used.
[0079] The invention thus provides artificial membrane systems
containing substantially purified ENaC protein subunits that
assemble into functional ENaCs. Specifically, the invention
provides artificial membrane systems containing substantially
purified human salty taste receptor. These membrane systems permit
analysis of ENaCs, including, but not limited to the salty taste
receptor apart from contaminating proteins such as endogenous
ENaCs. The invention permits the assembly of ENaCs in which the
subunits are added at known ratios to permit the assembly of
precise ratios of selected subunits. The lipid membrane can
comprise any combination of lipids. Non-limiting examples of
suitable lipids include phosphatidylcholine,
phoshpatidylethanolamine, phostphatidylserine, phosphatidylglyine,
phosphatidylinositol, sphingomyelin, cholesterol, cardiolipin, or a
homolog, analog, or derivative thereof. Phospholipids are
preferred, and can be obtained from any source suitable in the art.
For example, the phospholipids can be extracted from a cell, or can
be synthetic phospholipids, which are commercially available.
[0080] The lipid membrane can be in any conformation or phase,
including without limitation, liposomes, a lipid bilayer, or the
hexagonal phase. Liposomes and lipid bilayers are particularly
preferred.
[0081] The effect of the test compound on the biological activity
of the ENaC an be determined by any means suitable in the art. The
test compound can be assessed at multiple concentrations. In some
aspects, the test compound is assessed for its ability to modulate
at least one biological activity of the ENaC. In preferred aspects,
the ENaC is the salty taste receptor.
[0082] The biological activity of the ENaC can be determined by
measuring the current of ENaC assembled in the lipid membrane.
Voltage clamping is one preferable technique to measure ENaC
current. Voltage clamp techniques are well known in the art.
(Nagel, G et al. (2005) J. Physiol. 564(Pt 3):671-82; Staruschenko,
A et al. (2004) J. Biol. Chem. 279:27729-34; Tong, Q et al. (2004)
J. Biol. Chem. 279:22654-63; Sheng, S et al. (2000) J. Biol. Chem.
275:8572-81). The following parameters can be measured using a
voltage clamp: single channel conductance, channel open time,
voltage dependence, blockade induced by application of a particular
compound, and activation induced by application of a particular
compound. Other suitable techniques for measuring the biological
activity of ENaCs include flux assays, patch clamping,
voltage-sensitive dyes, and ion-sensitive dyes. Preferably, ENaC
activity is measured by membrane electrophysiology or by assessing
the change in fluorescence of a membrane potential dye in response
to sodium or lithium, or analogs thereof (e.g., isotopes). All such
assays are well known in the art. (Gill, S et al. (2003) Assay Drug
Dev. Technol. 1:709-17, flux assay; Caldwell, R A et al. (2005) Am.
J. Physiol. Lung Cell Mol. Physiol. 288:L813-9, patch clamp). A
variety of voltage sensitive dyes are commercially available,
including without limitation styryl dyes, oxonol dyes, and
merocyanine-rhodanine dyes. Selection of the appropriate voltage
sensitive dye is within the relevant skill in the art. Similarly, a
variety of ion sensitive dyes are commercially available, including
single excitation dyes, dual excitation ratiometric dyes, and dual
emission ratiometric dyes.
[0083] The salty taste receptor is responsive to sodium and lithium
ions. However, unlike other ENaCs, the human salty taste receptor
is not sensitive to amiloride. Thus, amiloride should not inhibit
or stimulate the salty taste receptor ENaC. Conversely,
chlorhexidine acts as an inhibitor of the salty taste response in
humans, and may be used in assays to identify salty taste
modifiers. One may assess specificity of stimulation of the salty
taste receptor with test compounds by showing that the effect is
inhibited by chlorhexidine. Moreover, test compounds that can
overcome the effect of chlorhexidine (and stimulate the salty taste
receptors in the membrane systems of the invention) are strong
salty taste enhancers. Basic compounds containing guanidinium ions
as well as certain amines act as salty taste enhancers. These
include guanidine, arginine, and homoarginine. Both L- and
D-arginine are equally effective. While not wishing to be bound by
any particular theory of operation, this lack of enantiomeric
specificity suggests that the primary enhancing effect derives from
a compact, basic moiety, in this case the guanidinium ion. Thus,
salty taste receptors in the membrane system of the invention may
be stimulated by contacting them with a source of guanidinium ions.
It may be assumed that these enhancing compounds interact directly
with the human salty taste ion channel, since most sodium channel
blockers and enhancers contain guanidinium groups that interact
with acidic moieties inside the channel pore lumen. Thus, the
molecular mechanisms of human salty taste share selected functional
features in common with known sodium channels but also have unique
pharmacological attributes.
[0084] Amiloride and amiloride derivatives (e.g., phenamil,
benzamil and the like) may be useful in assessing other ENaCs, such
as those containing an alpha subunit. Amiloride and derivatives may
also be used in assays to inhibit the background (endogenous ENaCs)
if the purity of the subunit preparations is low such that host
cell ENaCs are contaminating the preparation. Thus, in some aspects
of the invention, the methods further comprise contacting the ENaC
with a sodium ion channel antagonist. Such antagonists are
well-known to those of skill in the art. Preferably, the antagonist
is amiloride, chlorhexidine, or homologs, analogs, or derivatives
thereof.
[0085] The invention also includes within its scope high-throughput
screening assays to identify compounds that modulate the biological
activity of the salty taste receptor. High-throughput screening
assays permit screening of large numbers of test compounds in an
efficient manner. For example, but not by way of limitation, lipid
membranes comprising an assembled ENaC can be dispersed throughout
a multi-well plate such as a 96-well microtiter plate. Each well of
the microtiter plate can be used to run a separate assay against a
candidate modulator. A microtiter plate permits screening of
multiple concentrations of a test compound, multiple test
compounds, alone or in combination with other test compounds, and
multiple ENaCs, including ENaCs with varying ratios of subdomains
as described and exemplified herein under identical assay
conditions. In other aspects of the invention, planar lipid
bilayers containing the ENaC of interest is contacted with a test
compound and a measurement is taken. The solution on one or both
sides of the planar bilayer is changed and the bilayer is contacted
with a second test compound. This may be continuously used as a
high-throughput assay. The assays may take place in the presence of
additional agonists or antagonists. Data obtained for the test
compounds are compared with measurements taken in the presence of
known agonists or antagonists and/or to control samples (such as a
non-stimulatory/non-inhibitory medium).
[0086] Serial assays may be performed to narrow down the pool of
test compounds that act as salty taste modifiers. For example, the
in vitro assays of the invention may be combined with cell-based
assays as a secondary or confirming screening step. Such assays
have been described, for example, in published U.S. Patent
Application 2005/0059094 to Servant et al.
[0087] An additional aspect of the invention features methods for
identifying compounds that modulate salty taste perception in a
subject by a combination of in vitro and in vivo screening assays.
In one aspect, a test compound is first screened in vitro to
determine its modulatory effect on an epithelial sodium ion
channel, and then screened further in vivo to determine if the
compound can modulate, preferably enhance, salty taste perception
in a subject.
[0088] In one aspect, the in vitro screening assay comprises
identifying modulators of the human salty taste receptor comprising
contacting a test compound with at least one ENaC and determining a
decrease in the biological activity of the ENaC in the presence of
the test compound relative to the biological activity of the ENaC
in the absence of the test compound. This aspect can be practiced
according to the details described herein. In one aspect, the in
vivo screening assay comprises identifying compounds that enhance
salty taste perception in a subject comprising administering a test
compound to the subject and determining whether salty taste
perception is enhanced in the subject relative to the level of
salty taste perception by the subject in the absence of the test
compound.
[0089] For in vivo screening, subjects can be recruited via an
Institutional Review Board-approved method such as general
advertisement in print media. Prior to entering the study, each
subject provides informed consent. The participants can be
requested to refrain from eating, drinking or chewing gum for at
least one hour prior to testing. Subjects can be paid to
participate in the study.
[0090] Experimental solutions containing a candidate test compound
to be administered to study subjects can be presented in the form
of a binary mixture such as the compound and an inorganic salt such
as NaCl. Preliminary experiments can be carried out to establish an
appropriate concentration range for the test compound and inorganic
salts. For example, four concentrations of the test compound are
used with four concentrations of the inorganic salts. Aqueous
solutions can be prepared to encompass all possible combinations of
the concentrations of the test compound with the inorganic
salts.
[0091] To assess the salty taste amplifying properties of a given
stimulus, any means suitable in the art can be used. One
non-limiting example of such means is the method of magnitude
estimation. Magnitude estimation measures ratings of the perceived
intensities of saltiness from a sample. Subjects participating in
saltiness assessments can be instructed to rate the saltiness or
relative saltiness of a solution. Each solution can be sampled by
the subject once, twice, three times, or more.
[0092] Prior to sampling a test solution, subjects can be
instructed to rinse their mouth. For example, subjects can be
instructed to rinse with and expectorate water four times,
preferably within a short duration of time such as period of
approximately two minutes. Test samples and inorganic salt
solutions can then be administered to the subjects, preferably in
random order, and without replacement. For example, solutions can
be prepared in polystyrene medicine cups (Dynarex, N.Y.) in 10 ml
aliquots, and administered to the subjects. The subject can be
instructed to rate the relative saltiness of the solution, and the
relative saltiness ratings for each solution can be arithmetically
averaged to yield single ratings of saltiness.
[0093] Magnitude estimation may not reveal differences due to
variations in subject number use. To eliminate the variance
produced by idiosyncratic number usage in the magnitude estimation
task, the saltiness ratings can be standardized to the grand
arithmetic mean of the saltiness ratings of NaCl alone in water
(averaged across all NaCl concentrations). Each subject's mean
saltiness rating can be divided into the grand saltiness mean, and
the quotient can be used as the multiplicative standardization
factor for that individual's saltiness rating. This procedure
equates mean saltiness ratings of NaCl in water across
subjects.
[0094] Analysis of variance (ANOVA) can be conducted on the
standardized repeated measures data from the magnitude estimation,
and post-hoc pairwise comparisons can be conducted with Tukey's
honest significant difference (HSD) analysis.
[0095] An alternative to magnitude estimation is a forced-ranking
procedure, wherein a series of two-alternative forced-choice
pairings are used to rank the saltiness of aqueous solutions of
NaCl in the presence or absence of a test compound putative
enhancer. In this procedure, subjects can be instructed to taste
half of the first solution (for example, 5 ml or 10 ml solution) of
the first pair of samples for three seconds and expectorate.
Subjects then rinse twice and taste half of the second sample,
expectorate, rinse twice and taste the remainder of the two
solutions using the same procedure. After tasting both solutions
twice, subjects can be asked to indicate which solution they
thought was saltier. If they report that neither solution seemed
saltier, subjects can be asked to guess (forced-choice). The
procedure can repeated for all samples.
[0096] The saltiness rankings can be calculated based on the number
of times a particular solution is chosen as being saltier than all
other solutions using the Friedman analysis of pairwise rankings.
The Tukey HSD can be calculated to determine if the differences
between individual rankings are significant.
[0097] Compounds identified by any of the foregoing inventive
screening methods are contemplated to be within the scope of this
invention. Such compounds are preferably agonists of ENaCs, more
preferably agonists of the human salty taste receptor, and even
more preferably are enhancers of human salty taste receptors. Such
compounds may be formulated as a nutraceutical or pharmaceutical
composition by admixing such compound in an amount effective to
enhance salty taste perception in the subject to which it is
administered and a pharmaceutically or nutraceutically acceptable
carrier, as described herein.
[0098] It is an object of the invention to use the assays to
identify compounds that are perceived as salty, as well as to
identify compounds that enhance salty taste (such that a reduced
amount of sodium or lithium is perceived as a higher concentration
of sodium or lithium). The invention enables the screening of
libraries of compounds including natural or synthetic molecules
including, but not limited to proteins, peptides, oligonucleotides,
polynucleotides, polysaccharides, lipids, small organic molecules,
and the like, for their ability to act as salt substitutes, salty
taste enhancers, or salty taste inhibitors. The invention includes
salt substitutes, salty taste enhancers, and salty taste inhibitors
identified by the methods of the invention.
[0099] Also featured in accordance with the present invention are
artificial lipid membranes and methods for preparing the same. The
artificial lipid membranes comprise at least one lipid and an
assembled ENaC or at least one subunit of an ENaC. In preferred
aspects, the ENaC is a salty taste receptor. The lipid membrane can
be comprised of any suitable lipid, and are preferably comprised of
phospholipids. Suitable lipids include, without limitation,
phosphatidylcholine, phoshpatidylethanolamine, phostphatidylserine,
phosphatidylglyine, phosphatidylinositol, sphingomyelin,
cholesterol, cardiolipin, or a homolog, analog, or derivative
thereof, and these can be obtained from any source suitable in the
art. The lipid membrane can be in any conformation, and preferably
is a liposome or lipid bilayer.
[0100] In one aspect, an artificial lipid membrane is prepared by
admixing a liposome that comprises at least one phospholipid with
an ENaC or a particular subunit or subunits of an ENaC that has
dissolved in a suitable aqueous buffer. The aqueous buffer
comprises at least one detergent. Suitable detergents are well
known in the art, and include without limitation, Tween, Triton,
CHAPS, sodium cholate, and octyl-glucoside. After mixing the
phospholipids and ENaC or subunits thereof, the mixture is allowed
to incubate for several minutes, preferably at least about 20
minutes, to permit assembly of the ENaC into a lipid membrane.
Following the incubation, the detergent is removed according to any
means suitable in the art, such as those described and exemplified
herein. Other methods known in the art of preparing lipids and
liposomes containing proteins may be used to produce the lipids and
liposomes containing the ENaC subunits.
[0101] In some aspects, the ENaC is assembled into a liposome. The
liposome can be converted into a planar lipid bilayer by use of
techniques that are well known and routine in the art, including
those that are described and exemplified herein. In some aspects,
the liposomes contain a substance other than found in the
surrounding milieu. For example, but not by way of limitation, the
liposomes may contain a fluorescent voltage-sensitive or membrane
potential dye that is responsive to sodium or lithium, to indicate
a change in sodium content as a marker of sodium flow upon
stimulation with a test compound.
[0102] The invention also features kits for identifying modulators
of the human salty taste receptor. The kits comprise at least one
phospholipid, an isolated epithelial sodium ion channel subunit(s),
and optionally a source of sodium and/or lithium ions, and
instructions for using the kit in a method for identifying
modulators of the human salty taste receptor. In some aspects, the
kits optionally comprise an epithelial sodium ion channel
antagonist and/or agonist.
[0103] The invention provides a method for modulating salty taste
perception in a subject by contacting a salty taste receptor with a
compound that specifically interacts with the putative
amiloride-sensitive region of the delta subunit that contains
Cys.sub.532. In human subjects, this delta subunit comprises the
amino acid sequence of SEQ ID NO:12. The modulators may enhance or
inhibit salty taste perception by either stimulating the receptor
or blocking the receptor. The compounds may interact with the delta
receptor by binding to the receptor, preferably in the putative
amiloride sensitive region having the amino acid sequence of SEQ ID
NO:12.
[0104] Using computer programs for rational-based drug design that
are available in the art, molecular modeling may be performed based
on the primary amino acid sequence data available herein and
knowledge in the art as to tertiary structure of ion channels to
provide a three dimensional model of the human salty taste
receptor. Such modeling permits the rational selection of candidate
compounds that will interact with specific modulatory sites
including, as example, the putative amiloride binding site of the
delta subunit, motif SEQ ID NO:12. These compounds, or classes of
compounds, will act as salty taste modifiers. Compounds that
interact with these regions (e.g., delta subunit SEQ ID NO:12) are
useful as modifiers of salty taste perception. Thus, the data
presented herein provide a structural-functional relationship
between the subunits comprising the salty taste receptor and the
areas of the subunits that are likely involved in salty taste
perception.
[0105] The following actual and prophetic examples are provided to
describe the invention in more detail. They are intended to
illustrate, not to limit the invention.
Example 1
[0106] Procedure for Obtaining Human Fungiform Papillae and Taste
Cells. Human fungiform papillae containing taste buds are routinely
obtained from the anterior dorsal surface of the tongues of
volunteers by a minor surgical biopsy procedure carried out under
local anesthesia. The general procedure is described in Spielman,
AI et al. Collection of taste tissue from mammals. Experimental
Cell Biology of Taste and Olfaction. Spielman A I and Brand J G
eds. CRC Press, Boca Raton, Fla., pp 25-32. Volunteers give
informed consent. This procedure has been reviewed and approved by
an Institutional Review Board. The excised papillae can be
subsequently used either for RNA extraction, immunohistochemistry
or in situ hybridization, or in a procedure that results in a
suspension of isolated taste cells.
[0107] RNA extraction, histochemistry an in situ analysis. When
used for total RNA extraction, papillae are immediately subjected
to a standard extraction procedure using TRIzol.TM. reagent
(Invitrogen, Carlsbad, Calif.). The RNA extract is treated with
DNase to remove most genomic DNA. Any DNA remaining could otherwise
yield false positive results in subsequent steps where the use of
intron-spanning primers is not possible. Genomic material, however,
is useful in quantitative reverse trancriptase polymerase chain
reaction (QRT-PCR) because the single copy of the genomic DNA
signals the point of highest sensitivity of the PCR, and provides
thereby a convenient end-point for the procedure. Reverse
transcription is then performed on the RNA to yield a DNA copy of
the RNA, known as complementary DNA or cDNA. This cDNA will used as
the substrate in the polymerase chain reaction.
[0108] Because the fungiform papillae RNA and subsequent cDNA are
generally of high quality, the entire coding sequence or open
reading frame (ORF) of the protein under study can be immediately
amplified. The oligo-nucleotide primers used to effect this
amplification are designed based on the published sequence of the
same or similar protein annotated in GenBank. The PCR reaction
products can be analyzed by agarose gel electrophoresis. This
procedure is often used to obtain the entire coding sequence of a
gene known to be expressed in taste bud cells, the full sequence of
which cannot be obtained readily from single cell analysis.
[0109] The excised papillae may also be used for general or
immunohistochemical, or in situ hybridization analysis. Various
techniques and procedures are available and can be used to fix and
protect the tissue. As an example, FIG. 1 shows a human taste bud
stained by an ATPase histochemical procedure. FIG. 2 shows antibody
detection of the second messenger enzyme, phospholipase Cbeta2
(PLCbeta2) using an immunohistochemical procedure on human taste
cells. The procedure is as follows: Histological sections (8 to 10
microns) of fungiform papillae were washed three times in
1.times.PBS for 10 minutes., placed in blocking buffer at room
temperature for 4-18 hours. Blocking buffer was removed and primary
antibody (rabbit anti-PLCbeta2) was added in three concentrations
(1:50, 1:100, and 1:200 in buffer). The primary antibody solution
was removed and the slides were washed three times in PBS. The
first wash drained immediately while the subsequent washes were
incubated for 10-20 minutes each. Excess fluid was removed and a
the secondary antibody solution (CY3-labeled goat anti-rabbit,
1:1000) was added to the sections and the slides were incubated at
room temperature for 45-120 minutes. The slides were washed three
times in PBS. The first wash drained immediately while the
subsequent washes were incubated for 10-20 minutes each. The excess
fluid was drained, but slides were allowed to remain wet.
Coverslips were placed on the slides and the slides were examined
under a fluorescence microscope.
[0110] RT-PCR for identifying ENaC subunits and sequencing the
same. Extraction of total RNA from biopsied fungiform papillae is
carried out as described above, without DNase treatment, followed
by synthesis of first-strand cDNA. Amplification of ENaC subunits
(no more than 500 bp in size) can be performed with the PCR Core
System I reagent kit (Promega Corp., Madison Wis.) using primers as
above.
[0111] If a product of apparently the correct size is obtained,
this product is excised from the gel and purified. The product is
then ligated into a plasmid vector to yield a recombinant plasmid
which has the gene for the coding sequence of the protein (e.g.,
ENaC .delta.) inserted into it. The recombinant plasmid is used to
transform bacterial cells which, when provided with an appropriate
growth medium, produce large amounts of plasmid. Purification of
the bacterial culture yields the recombinant plasmid in a pure
form, which enables one to get the sequence of the protein gene
from human fungiform papillae. Finally, a bioinformatic analysis of
the sequence, using the BLAST program confirms that the correct
sequence has indeed been obtained.
[0112] Using this procedure, evidence was found for transcripts of
four ENaC subunit types in human fungiform papillae. These subunits
are the alpha, beta, gamma, and delta ENaC subunits. The complete
ORF of the alpha subunit was rarely observed, but the complete ORF
of the other subunits was nearly always observed. Surprisingly, it
was discovered that the delta subunit of ENaC is present in human
fungiform papillae.
Example 2
Identification of the Human Salty Taste Receptor and the Importance
of the Delta Subunit
[0113] In accordance with the present invention, the delta subunit
of the ENaC in the fungiform (taste) papillae of humans. The clones
in which the subunit was detected were from pooled cDNA from 3
individuals who agreed to undergo a biopsy procedure to remove
several fungiform papillae from the anterior dorsal surface of the
tongue.
[0114] Characteristics of the delta subunit. The delta subunit of
the epithelial sodium channel was detected in the fungiform
papillae from thirteen individuals by RT-PCR. The detected
fragments were amplified by PCR and subcloned. The polynucleotide
encoding delta subunit from these thirteen individuals was then
fully sequenced. It was determined that the human delta subunit
from fungiform papillae differed from human delta subunit cloned
from kidney in the putative amiloride binding site. The putative
amiloride binding site contains a tyrosine at amino acid 532 in
delta subunit from kidney (SEQ ID NO:8), but amino acid 532 in
delta from fungiform papillae was cysteines in each of the thirteen
samples sequenced (SEQ ID NO:9):
TABLE-US-00001 TABLE 1 Sequence of putative amiloride Source of
delta binding site Kidney delta: MGSLYSLWFGA (SEQ ID NO: 11) Taste
delta# 1: MGSLCSLWFGA (SEQ ID NO: 12) Taste delta# 2: MGSLCSLWFGA
(SEQ ID NO: 12) Taste delta# 3: MGSLCSLWFGA (SEQ ID NO: 12)
[0115] FIG. 3 shows an amino acid sequence alignment of 11 delta
subunits, where the first sequence is the GenBank sequence with the
other 10 sequences being from 10 different individuals. At position
180, a possible polymorphism (R to P) is indicated. Other positions
indicating possible polymorphisms are with positions 278 (F to I),
355 (S to R), 389 (E to Q), and 566 (R to H). Position 566 has also
been implicated in amiloride binding. Without intending to be
limited to any particular theory or mechanism of action, the
polymorphisms may play a role in sensitivity to salty stimuli or
may play a role in sensitivity to taste modulators.
[0116] The Y to C change at position 532 is significant as it may
help explain why rat salty taste receptors are apparently amiloride
sensitive while human salty taste receptors are not. As the rat's
delta ENaC subunit is a psuedogene, it is not expressed. It is
believed that the amiloride-sensitive alpha subunit functions as
part of the salty receptor in rat. Although this substitution does
not significantly alter the receptor sensitivity and specificity,
the pharmacology of the channel is altered.
[0117] While the delta subunit is amiloride sensitive, it is less
so than the alpha (FIG. 4). Thus, if the human salty taste receptor
ENaC contained the usual form of delta, it too should show
amiloride sensitivity. However, the putative amiloride binding site
in delta from human taste cells contains a non-conservative
substitution and may therefore have a different sensitivity to
amiloride than delta subunit in kidney. Without intending to be
limited to any particular theory or mechanism of action, it delta
shows less sensitivity, this observation potentially can be
interpreted to mean that delta is in the human salty taste
receptor, particularly because amiloride cannot cross tight
junctions. Because of the differences between rat and human, the
rat is probably not a good model for salty taste perception in
humans.
[0118] Cellular specificity of the human fungiform delta ENaC
subunit. A human taste bud is shown in FIG. 1, wherein an 8 micron
section of a human fungiform papilla is stained by an ATPase
histochemical procedure. The question now became whether some, all,
or none of these taste cells expressed delta ENaC. To view only
those cells expressing the delta subunit, an antibody to the delta
form of human ENaC was raised in rabbits. A representative
photograph is shown in FIG. 5. The slide shows tissue specific
labeling on a subset of cells within a human taste bud. The
implication is that the human salty taste receptor is an ENaC
composed of a multimer of delta, beta, and gamma subunits or of
subunits alpha, delta, beta, and gamma. This specificity of delta
in the taste cells accompanied by a notable dearth of full-length
alpha in these same buds, suggested that the human salty taste
receptor is a delta-containing ENaC and not simply an alpha, beta,
gamma ENaC as suggested by others.
[0119] Isolation of human taste bud cells. A suspension of single
isolated taste bud cells was prepared from human fungiform papillae
by incubation of biopsied papillae in a collagenase-based enzymatic
procedure, followed by washing of the papillae to effectively
removed enzyme, then trituration of same through a glass pipette.
The resulting suspension was enriched for cells of the taste bud.
Individual cells were captured using a glass micropipette (See FIG.
6) and individually placed into a tube containing 2 to 10 .mu.A of
RNAlater.
[0120] The delta subunit is located in taste bud cells. cDNA was
derived from 7 human fungiform taste cells that were individually
isolated and captured, as described above, and then pooled. A
product of the correct size (.about.500 bp) was noted and its
identity as a human ENaC delta subunit was confirmed by sequencing.
Using this PCR procedure of identifying overlapping segments of the
ORF of delta ENaC, the complete ORF of taste cell delta ENaC was
obtained.
[0121] Single cell RT-PCR using nested primers was also performed,
and revealed that two out of twelve human taste bud cells tested
provided strong evidence for expression of delta, beta, and gamma
subunits, without expressing full-length alpha (data not shown).
One early single cell Q-RT-PCR revealed no message for the alpha
subunit but approximately equal numbers of message copies for
delta, gamma and beta (FIG. 7).
[0122] Using calcium imaging on a preparation of isolated taste
cells, it is possible to identify those individual cells that are
activated by sodium chloride. These cells are captured and their
contents analyzed by Q-SC-RT-PCR. In a group of 30 salt sensitive
cells, the primary expressed subunit was determined to be delta.
Eight expressed delta, beta, and gamma, while 7 expressed delta,
alpha, beta, and gamma.
[0123] An alignment of the amino acid sequences of the 10 gamma
subunits sequenced from taste cells as compared to the GenBank
sequence for gamma is shown in FIG. 8.
[0124] Having identified the salty receptor as delta, beta, gamma
or delta, alpha, beta, gamma, each subunit will be expressed and
reconstituted into lipid bilayers for analyses, as provided by the
examples below.
Example 3
Preparation of Liposomes and Artificial Lipid Bilayers
[0125] This example demonstrates the techniques that are readily
practiced to solubilize an abundant receptor from its membrane
milieu, purify the receptor, and reconstitute the receptor in an
artificial lipid membrane such as a lipid bilayer. Such membranes
serve as an artificial biological membrane in which the receptor
resumes its native conformation and can be studied in detail and in
isolation, e.g., without interference from other proteins or the
metabolic whimsy of a living cell.
[0126] This example, in part, describes the extraction,
purification, and membrane-reconstitution of a taste receptor for
L-arginine (L-arg) from catfish. The methods, which are published
and described in Grosvenor, W et al. (2004) BMC Neuroscience 5:25,
can be readily adapted for reconstitution of ENaC, ENaC subunits,
and salty taste receptors in lipid membranes as described below.
The catfish has served as a model for taste receptor studies
because the receptors are very sensitive to certain amino acids.
One such amino acid is L-arg. Like the ENaC, the taste receptor for
L-arg in catfish is an ion channel. Parallel approaches are
utilized in solubilizing the L-arg and ENaC-type receptors. The
receptors differ primarily by origin--the L-arg receptor is
purified from catfish taste tissue and the ENaC subunits are
synthesized by a heterologous cell culture expression system.
[0127] Liposome generation. Liposomes are used to carry the
solubilized receptor to the bilayer construct. The major challenge
to studying a membrane-soluble protein is developing a procedure to
move the protein from its native membrane or synthesis end point to
an artificial lipid bilayer. Solubilization usually uses detergent
and this detergent must be removed to avoid damage to the bilayer.
The liposome performs this transfer by taking up the protein from
the detergent system and giving it up to the bilayer.
[0128] Liposomes are prepared by adding 5 mg of the mixture of
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine:1,2-dioleoyl-sn-glycero-3-p-
hosphocholine (DOPE:DOPC) in a 2:1 ratio in 0.5 ml of chloroform to
a round bottom flask. The flask is rotated for 30-40 min at
4.degree. C. After evaporation of the chloroform, a thin layer of
lipid is formed to which 2 ml of buffer solution (300 mM NaCl, 50
mM Tris, pH=7) is added. After addition of the buffer, the flask is
bath-sonicated 3 times with 3-5 min pulses to induce liposome
formation. Alternatively, the probe sonicator can be pulsed for
only 30-40 sec.
[0129] Dissolution of the L-ArgR into liposomes and pharmacology of
L-ArgR in a lipid membrane. An amount (0.01 to 0.5 .mu.g) of the
L-ArgR dissolved in 100 mM NaCl/50 mM Tris, pH=7.1 containing one
of several detergents such as Tween, CHAPS, Triton, Sodium Cholate,
or/and Octyl-glucoside is added to the liposomes. The ratio of
protein to lipid (mass:mass) for measuring channel activity as
single cannel events is 1:2000-5000. The ratio for measuring
macroscopic properties is in the range of 1:50-100. The
protein-lipid mixture is incubated for 20-30 mins.
[0130] The properties of the L-ArgR as measured in the bilayer are
very similar to those observed through taste nerve recordings from
the animal, when the L-ArgR is in its native state. For example,
the taste nerve recordings indicate that the sensitivity of the
native receptor is in the tenths of uM of L-arg, while D-arg
inhibits the receptor. FIG. 9 shows that when the L-ArgR is
reconstituted into a bilayer, the same properties are seen. This
demonstrates that a receptor like the L-ArgR is more directly and
more readily studied when in the bilayer than when in the native
state where its activity must be inferred from secondary neural
recordings.
[0131] Reconstitution of the ENaC protein into liposomes. An amount
(0.01 to 0.5 .mu.g) of the sodium channel of interest, including
ENaC.alpha., ENaC.delta., or specific ratio mixtures derived from
the ENaC subunits, .alpha., .beta., .gamma., .delta., dissolved in
100 mM NaCl/50 mM Tris, pH=7.1 containing one of several detergents
such as Tween, CHAPS, Triton, Sodium Cholate, or/and
Octyl-glucoside will be added to the liposomes. The ratio of
protein to lipid (mass:mass) for measuring channel activity as
single cannel events is 1:2000-5000. The ratio for measuring
macroscopic properties is in the range of 1:50-100. The
protein-lipid mixture will be incubated for 20-30 mins. This
procedure will be followed because the ENaC protein, being a
membrane channel, needs to remain in solution while it is
reconstituted into liposomes.
[0132] Reconstituted detergent-free proteo-liposomes containing one
or more of the ENaCs can be prepared at least two ways. In one
method, they can be formed through centrifugation of the
protein:lipid mixture through gel filtration columns. These gel
columns are prepared from Sephadex G-50 (fine), swollen overnight,
and poured into 5-ml disposable columns (1.5-ml bed volume).
Columns are pre-spun in a centrifuge at .about.1,000.times.g. The
protein:lipid mixture is loaded on the top of the column, and
proteo-liposomes free of detergent can be recovered by spinning the
columns at 700 g for 1 min. Alternatively, detergent can be removed
by dialysis. For dialysis, the protein:lipid mixture is loaded into
a cassette dialysis unit and the mixture dialyzed overnight against
2000 ml of a Tris/NaCl/sucrose (detergent-free) buffer at 4.degree.
C. Phospholipid vesicles containing the protein are expected to
form spontaneously as the concentration of detergent decreases
during the dialysis.
[0133] Reconstitution of the channel proteins from proteo-liposomes
into a planar lipid bilayer. The planar lipid bilayer is formed on
an aperture between two aqueous compartments, which for operational
purposes are called cis and trans compartments. The voltage
generator will be connected to the cis compartment, with an Ag/AgCl
electrode, to control membrane potential. The trans compartment
(virtual ground) will be connected to the input of the
current-measuring amplifier through a second Ag/AgCl electrode.
[0134] Forming the bilayer. A 4:1 mixture of DOPE:DOPC will be
dissolved in 25 .mu.l of n-decane (concentration ranging from 15 to
25 mg/ml). This mixture will be kept at room temperature and
prepared each day that the experiment is performed. Electrode
compartments will be filled with 3 M KCl and the Ag/AgCl electrodes
will be placed in the compartment. The cis and trans compartments
will be filled with the recording bath solution (100 mM NaCl, 10
Tris, pH 7) and agar bridges will be placed between them and the
electrode compartments. To form the bilayer, a droplet of the lipid
mixture will be spread onto the hole from the cis side.
[0135] The lipids can be determined to be completely formed around
the hole when the resistance increased and the signal is not
saturating. To verify that an organized bilayer has formed, the
voltage pulses across the bilayer can be applied and "capacitive
currents" can be measured. For a hole of 100 .mu.m, the capacitance
is expected to be of the order of 50-100 pF. The electrical
resistance of the bilayer is expected to be higher than 109
Ohm.
[0136] Reconstitution. After the bilayer has formed, 10-15 .mu.l of
the proteo-liposomes will be added to the cis-side of the bilayer
under constant stirring. When channel subunits are incorporated
into the bilayer, the currents are expected to change in steps.
Macroscopic current will be measured when many channels are
incorporated.
[0137] Liposome fusion with the bilayer happens spontaneously, and
currents will be able to be recorded within about 5 to 30 minutes.
In some cases, it may be necessary to facilitate the liposome
fusion by: creating a concentration gradient across the liposome by
adding the liposomes formed previously in 300 mM NaCl to a bilayer
bathing solution containing 100 mM NaCl, or by creating a
concentration gradient across the lipid bilayer by adding 100 mM
NaCl to the cis side and 10 mM NaCl to the trans side, or by
changing bilayer and/or liposome lipid composition by the addition
of negatively charged lipid such as DOPS to the bilayer.
Example 4
Expression of Varying Ratios of ENaC Receptor Subdomains In Lipid
Bilayers
[0138] This is a prophetic example. The ENaC is a heteromultimeric
complex generally comprised of three subunits: either of subunits
.alpha., .beta., and .gamma. (in most tissues as .alpha.2
.beta..gamma. complex) or subunits .delta., .beta., and .gamma..
These subunits can assemble in varying ratios, often dictated by
the tissue source. Varying the relative ratios of the subunits
confers unique kinetics and pharmacology upon these channels.
Without intending to be limited to any particular theory or
mechanism of action, it is believed that the .delta. subunit
replaces the .alpha. subunit in many tissues, and that such a
substitution may modify particularly the pharmacology of the
channel.
[0139] Single cell quantitative PCR with specific reference to
estimation of the ratios of ENaC subunits. While there is no
guarantee of a one-to-one correspondence between amount of message
and amount of protein, Q-PCR is one tool available for estimation
of ENaC ratio. Assuming a salty taste cell is active, it is likely
to have a number of copies of a particular subunit. It is likely
that the ratio of message copies will be at least approximately
that of the protein products. Quantitative single cell PCR can be
used to gain a semi-quantitative picture of the relative abundance
of message for any proteins of interest. The procedure, although
theoretically straight forward, presents a number of challenging
obstacles. With taste cells, for example, RNA quality can be
problematic because the time-consuming procedure currently used to
obtain isolated cells (see above) is conducive to destruction of
RNA. To be confident in the experimental technique, the following
procedure can be carried out: (1) Design several unique primer
pairs for each gene of interest, using only those that have almost
identical efficiency under the same PCR conditions for every gene
of interest. (2) Construct a primer set (mixture of primer pairs)
from the appropriate pairs above that registers as a blank when
used in a water control PCR reaction. (3) Collect individual cells
(as above) into an RNase-free environment, lyse the cell and
reverse transcribe the single cell mRNA content using a
commercially available kit. (4) Run a limited (10-25) number of
cycles of the first stage PCR with the primer set and condition
above, so that all of the reactions are in a linear amplification
phase. (5) Dilute the above reaction (100.times.-1000.times.), and
use an aliquot as template along with a single pair of primers from
the set above and perform the second stage of PCR (in
duplicate/triplicate) using a Q-PCR machine. (6) A relative
quantification method is used for data analysis. Normalization is
based on amplification of a genomic DNA that is not
translated/transcribed of which there is, by definition, one copy
of the gene. The differences in gene expression can be determined
by comparing ratio (.DELTA.Ct between target gene and genomic
reference sequence in sample) differences (.DELTA..DELTA.Cts, the
differences of .DELTA.Cts between two samples).
[0140] A taste bud cell containing message for human ENaC subunits
.delta., .beta., and .gamma., but no message for .alpha. was
apparent. The Q-PCR trace of this analysis is shown in FIG. 7. From
this single cell, it can be concluded that the ENAC of that cell is
of multimeric structure, .delta.1 .beta.1 .gamma.1. However, as
these traces are not normalized, the definitive structure may have
a different stoichiometric ratio. The best evidence, however,
suggests that the human salty taste receptor is composed of
.delta.1 .beta.1 .gamma.1.
[0141] Once sequence confirmation is obtained, the recombinant
plasmid can be used as the substrate in a process known as in vitro
protein expression (IVPE). This procedure, be it either cell driven
or a cell-free system, allows generation of large amounts (mM) of
desired protein, in this case, ENaC subunits, .delta., .alpha.,
.beta., and .gamma.. A Western blot can be used to confirm the
identity of the manufactured protein. Analysis of the reaction
mixture using an antibody to the protein (a Western blot) is used
to confirm that the desired protein has indeed been obtained.
[0142] The desired protein can be isolated and purified.
Purification of the protein by affinity chromatography involves
chemically linking an antibody to the protein with a column matrix
such as Sephadex. Passing the IVPE reaction mixture through the
column results in binding of the protein to its antibody on the
column. Elution of the column with an appropriate reagent yields
the enriched protein. The protein eluate can be quantified by
measuring total nitrogen, as in the Kjeldahl procedure. This
measure of total nitrogen content is then compared to the protein's
absorption at 280 nm to calculate an absorption coefficient. From
this point, absorption at 280 nm becomes a convenient and accurate
measure of protein concentration.
[0143] Knowing the actual concentration of each subunit of the ENaC
allows the combination of these subunits in specific ratios, these
having been estimated by the Q-PCR of single cells. As these
proteins are membrane associated, they will require some amount of
detergent to remain soluble. While their being soluble is required
for combining them in specific ratios, detergent will destroy the
lipid bilayer into which they need to be reconstituted to measure
activity. Thus, reconstitution of the lipid bilayer with the
isolated proteins requires that any detergent be removed. Detergent
can be removed by any means suitable in the art, such as dialysis
as described herein. Reconstitution of isolated proteins into lipid
membranes has been described (Grosvenor, W et al. (2004) BMC
Neurosci. 5:2202-5), and summarized in the examples above. Because
the subunits for human ENaC are synthesized, an advantage is gained
as careful control over the composition an ratios of any putative
salt taste receptor subunits can be exerted.
[0144] The present invention is not limited to the embodiments
described and exemplified above, but is capable of variation and
modification within the scope of the appended claims.
SEQUENCE LISTING
TABLE-US-00002 [0145] SEQ ID NO: 1 nt for human alpha atggagggga
acaagctgga ggagcaggac tctagccctc cacagtccac tccagggctc atgaagggga
acaagcgtga ggagcagggg ctgggccccg aacctgcggc gccccagcag cccacggcgg
aggaggaggc cctgatcgag ttccaccgct cctaccgaga gctcttcgag ttcttctgca
acaacaccac catccacggc gccatccgcc tggtgtgctc ccagcacaac cgcatgaaga
cggccttctg ggcagtgctg tggctctgca cctttggcat gatgtactgg caattcggcc
tgcttttcgg agagtacttc agctaccccg tcagcctcaa catcaacctc aactcggaca
agctcgtctt ccccgcagtg accatctgca ccctcaatcc ctacaggtac ccggaaatta
aagaggagct ggaggagctg gaccgcatca cagagcagac gctctttgac ctgtacaaat
acagctcctt caccactctc gtggccggct cccgcagccg tcgcgacctg cgggggactc
tgccgcaccc cttgcagcgc ctgagggtcc cgcccccgcc tcacggggcc cgtcgagccc
gtagcgtggc ctccagcttg cgggacaaca acccccaggt ggactggaag gactggaaga
tcggcttcca gctgtgcaac cagaacaaat cggactgctt ctaccagaca tactcatcag
gggtggatgc ggtgagggag tggtaccgct tccactacat caacatcctg tcgaggctgc
cagagactct gccatccctg gaggaggaca cgctgggcaa cttcatcttc gcctgccgct
tcaaccaggt ctcctgcaac caggcgaatt actctcactt ccaccacccg atgtatggaa
actgctatac tttcaatgac aagaacaact ccaacctctg gatgtcttcc atgcctggaa
tcaacaacgg tctgtccctg atgctgcgcg cagagcagaa tgacttcatt cccctgctgt
ccacagtgac tggggcccgg gtaatggtgc acgggcagga tgaacctgcc tttatggatg
atggtggctt taacttgcgg cctggcgtgg agacctccat cagcatgagg aaggaaaccc
tggacagact tgggggcgat tatggcgact gcaccaagaa tggcagtgat gttcctgttg
agaaccttta cccttcaaag tacacacagc aggtgtgtat tcactcctgc ttccaggaga
gcatgatcaa ggagtgtggc tgtgcctaca tcttctatcc gcggccccag aacgtggagt
actgtgacta cagaaagcac agttcctggg ggtactgcta ctataagctc caggttgact
tctcctcaga ccacctgggc tgtttcacca agtgccggaa gccatgcagc gtgaccagct
accagctctc tgctggttac tcacgatggc cctcggtgac atcccaggaa tgggtcttcc
agatgctatc gcgacagaac aattacaccg tcaacaacaa gagaaatgga gtggccaaag
tcaacatctt cttcaaggag ctgaactaca aaaccaattc tgagtctccc tctgtcacga
tggtcaccct cctgtccaac ctgggcagcc agtggagcct gtggttcggc tcctcggtgt
tgtctgtggt ggagatggct gagctcgtct ttgacctgct ggtcatcatg ttcctcatgc
tgctccgaag gttccgaagc cgatactggt ctccaggccg agggggcagg ggtgctcagg
aggtagcctc caccctggca tcctcccctc cttcccactt ctgcccccac cccatgtctc
tgtccttgtc ccagccaggc cctgctccct ctccagcctt gacagcccct ccccctgcct
atgccaccct gggcccccgc ccatctccag ggggctctgc aggggccagt tcctccacct
gtcctctggg ggggccctga SEQ ID NO: 2 pro for human alpha MEGNKLEEQD
SSPPQSTPGL MKGNKREEQG LGPEPAAPQQ PTAEEEALIE FHRSYRELFE FFCNNTTIHG
AIRLVCSQHN RMKTAFWAVL WLCTFGMMYW QFGLLFGEYF SYPVSLNINL NSDKLVFPAV
TICTLNPYRY PEIKEELEEL DRITEQTLFD LYKYSSFTTL VAGSRSRRDL RGTLPHPLQR
LRVPPPPHGA RRARSVASSL RDNNPQVDWK DWKIGFQLCN QNKSDCFYQT YSSGVDAVRE
WYRFHYINIL SRLPETLPSL EEDTLGNFIF ACRFNQVSCN QANYSHFHHP MYGNCYTFND
KNNSNLWMSS MPGINNGLSL MLRAEQNDFI PLLSTVTGAR VMVHGQDEPA FMDDGGFNLR
PGVETSISMR KETLDRLGGD YGDCTKNGSD VPVENLYPSK YTQQVCIHSC FQESMIKECG
CAYIFYPRPQ NVEYCDYRKH SSWGYCYYKL QVDFSSDHLG CFTKCRKPCS VTSYQLSAGY
SRWPSVTSQE WVFQMLSRQN NYTVNNKRNG VAKVNIFFKE LNYKTNSESP SVTMVTLLSN
LGSQWSLWFG SSVLSVVEMA ELVFDLLVIM FLMLLRRFRS RWPSPGRGGR GAQEVASTLA
SSPPSHFCPH PMSLSLSQPG PAPSPALTAP PPAYATLGPR PSPGGSAGAS SSTCPLGGP
SEQ ID NO: 3 nt for human beta atgcacgtga agaagtacct gctgaagggc
ctgcatcggc tgcagaaggg ccccggctac acgtacaagg agctgctggt gtggtactgc
gacaacacca acacccacgg ccccaagcgc atcatctgtg aggggcccaa gaagaaagcc
atgtggttcc tgctcaccct gctcttcgcc gccctcgtct gctggcagtg gggcatcttc
atcaggacct acttgagctg ggaggtcagc gtctccctct ccgtaggctt caagaccatg
gacttccccg ccgtcaccat ctgcaatgct agccccttca agtattccaa aatcaagcat
ttgctgaagg acctggatga gctgatggaa gctgtcctgg agagaatcct ggctcctgag
ctaagccatg ccaatgccac caggaacctg aacttctcca tctggaacca cacacccctg
gtccttattg atgaacggaa cccccaccac cccatggtcc ttgatctctt tggagacaac
cacaatggct taacaagcag ctcagcatca gaaaagatct gtaatgccca cgggtgcaaa
atggccatga gactatgtag cctcaacagg acccagtgta ccttccggaa cttcaccagt
gctacccagg cattgacaga gtggtacatc ctgcaggcca ccaacatctt tgcacaggtg
ccacagcagg agctagtaga gatgagctac cccggcgagc agatgatcct ggcctgccta
ttcggagctg agccctgcaa ctaccggaac ttcacgtcca tcttctaccc tcactatggc
aactgttaca tcttcaactg gggcatgaca gagaaggcac ttccttcggc caaccctgga
actgaattcg gcctgaagtt gatcctggac ataggccagg aagactacgt ccccttcctt
gcgtccacgg ccggggtcag gctgatgctt cacgagcaga ggtcataccc cttcatcaga
gatgagggca tctacGccat gtcggggaca gagacgtcca tcggggtact cgtggacaag
cttcagcgca tgggggagcc ctacagcccg tgcaccgtga atggttctga ggtccccgtc
caaaacttct acagtgacta caacacgacc tactccatcc aggcctgtct tcgctcctgc
ttccaagacc acatgatccg taactgcaac tgtggccact acctgtaccc actGccccgt
ggggagaaat actgcaacaa ccgggacttc ccagactggg cccattgcta ctcagatcta
cagatgagcg tggcgcagag agagacctgc attggcatgt gcaaggagtc ctgcaatgac
acccagtaca agatgaccat ctccatggct gactggcctt ctgaggcctc cgaggactgg
attttccacg tcttgtctca ggagcgggac caaagcacca atatcaccct gagcaggaag
ggaattgtca agctcaacat ctacttccaa gaatttaact atcgcaccat tgaagaatca
gcagccaata acatcgtctg gctgctctcg aatctgggtg gccagtttgg cttctggatg
gggggctctg tgctgtgcct catcgagttt ggggagatca tcatcgactt tgtgtggatc
accatcatca agctggtggc cttggccaag agcctacggc agcggcgagc ccaagccagc
tacgctggcc caccgcccac cgtggccgag ctggtggagg cccacaccaa ctttggcttc
cagcctgaca cggccccccg cagccccaac actgggccct accccagtga gcaggccctg
cccatcccag gcaccccgcc ccccaactat gactccctgc gtctgcagcc gctggacgtc
atcgagtctg acagtgaggg tgatgccatc taa SEQ ID NO: 4 pro for human
beta MHVKKYLLKG LHRLQKGPGY TYKELLVWYC DNTNTHGPKR IICEGPKKKA
MWFLLTLLFA ALVCWQWGIF IRTYLSWEVS VSLSVGFKTM DFPAVTICNA SPFKYSKIKH
LLKDLDELME AVLERILAPE LSHANATRNL NFSIWNHTPL VLIDERNPHH PMVLDLFGDN
HNGLTSSSAS EKICNAHGCK MAMRLCSLNR
TQCTFRNFTS ATQALTEWYI LQATNIFAQV PQQELVEMSY PGEQMILACL FGAEPCNYRN
FTSIFYPHYG NCYIFNWGMT EKALPSANPG TEFGLKLILD IGQEDYVPFL ASTAGVRLML
HEQRSYPFIR DEGIYAMSGT ETSIGVLVDK LQRMGEPYSP CTVNGSEVPV QNFYSDYNTT
YSIQACLRSC FQDHMIRNCN CGHYLYPLPR GEKYCNNRDF PDWAHCYSDL QMSVAQRETC
IGMCKESCND TQYKMTISMA DWPSEASEDW IFHVLSQERD QSTNITLSRK GIVKLNIYFQ
EFNYRTIEES AANNIVWLLS NLGGQFGFWM GGSVLCLIEF GEIIIDFVWI TIIKLVALAK
SLRQRRAQAS YAGPPPTVAE LVEAHTNFGF QPDTAPRSPN TGPYPSEQAL PIPGTPPPNY
DSLRLQPLDV IESDSEGDAI SEQ ID NO: 5 nt for human taste gamma
atggcacccg gagagaagat caaagccaaa atcaagaaga atctgcccgt gacgggccct
caggcgccga ccattaaaga gctgatgcgg tggtactgcc tcaacaccaa cacccatggc
tgtcgccgca tcgtggtgtc ccgcggccgt ctgcgccgcc tcctctggat cgggttcaca
ctgactgccg tggccctcat cctctggcag tgcgccctcc tcgtcttctc cttctatact
gtctcagttt ccatcaaagt ccacttccgg aagctggatt ttcctgcagt caccatctgc
aacatcaacc cctacaagta cagcaccgtt cgccaccttc tagctgactt ggaacaggag
accagagagg ccctgaagtc cctgtatggc tttccagagt cccggaagcg ccgagaggcg
gagtcctgga actccgtctc agagggaaag cagcctagat tctcccaccg gattccgctg
ctgatctttg atcaggatga gaagggcaag gccagggact tcttcacagg gaggaagcgg
aaagtcggcg gtagcatcat tcacaaggct tcaaatgtca tgcacatcga gtccaagcaa
gtggtgggat tccaactgtg ctcaaatgac acctccgact gtgccaccta caccttcagc
tcgggaatca atgccattca ggagtggtat aagctacact acatgaacat catggcacag
gtgcctctgg agaagaaaat caacatgagc tattctgctg aggagctgct ggtgacctgc
ttctttgatg gagtgtcctg tgatgccagg aatttcacgc ttttccacca cccgatgcat
gggaattgct atactttcaa caacagagaa aatgagacca ttctcagcac ctccatgggg
ggcagcgaat atgggctgca agtcattttg tacataaacg aagaggaata caacccattc
ctcgtgtcct ccactggagc taaggtgatc atccatcggc aggatgagta tcccttcgtc
gaagatgtgg gaacagagat tgagacagca atggtcacct ctataggaat gcacctgaca
gagtccttca agctgagtga gccctacagt cagtgcacgg aggacgggag tgacgtgcca
atcaggaaca tctacaacgc tgcctactcg ctccagatct gccttcattc atgcttccag
acaaagatgg tggagaaatg tgggtgtgcc cagtacagcc agcctctacc tcctgcagcc
aactactgca actaccagca gcaccccaac tggatgtatt gttactacca actgcatcga
gcctttgtcc aggaagagct gggctgccag tctgtgtgca aggaagcctg cagctttaaa
gagtggacac taaccacaag cctggcacaa tggccatctg tggtttcgga gaagtggttg
ctgcctgttc tcacttggga ccaaggccgg caagtaaaca aaaagctcaa caagacagac
ttggccaaac tcttgatatt ctacaaagac ctgaaccaga gatccatcat ggagagccca
gccaacagta ttgagatgct tctgtccaac ttcggtggcc agctgggcct gtggatgagc
tgctctgttg tctgcgtcat cgagatcatc gaggtcttct tcattgactt cttctctatc
attgcccgcc gccagtggca gaaagccaag gagtggtggg cctggaaaca ggctccccca
tgtccagaag ctccccgtag cccacagggc caggacaatc cagccctgga tatagacgat
gacctaccca ctttcaactc tgctttgcac ctgcctccag ccctaggaac ccaagtgccc
ggcacaccgc cccccaaata caataccttg cgcttggaga gggccttttc caaccagctc
acagataccc agatgctaga tgagctctga SEQ ID NO: 6 pro for human taste
gamma MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR
LRRLLWIGFT LTAVALILWQ CALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV
RHLLADLEQE TREALKSLYG FPESRKRREA ESWNSVSEGK QPRFSHRIPL LIFDQDEKGK
ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSND TSDCATYTFS SGINAIQEWY
KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMH GNCYTFNNRE
NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETA
MVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA
QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ
WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN
FGGQLGLWMS CSVVCVIEII EVFFIDFFSI IARRQWQKAK EWWAWKQAPP CPEAPRSPQG
QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTL RLERAFSNQL TDTQMLDEL
SEQ ID NO: 7 not for human kidney delta atggctgagc accgaagcat
ggacgggaga atggaagcag ccacacgggg gggctctcac ctccaggctg cagcccagac
gccccccagg ccggggccac catcagcacc accaccacca cccaaggagg ggcaccagga
ggggctggtg gagctgcccg cctcgttccg ggagctgctc accttcttct gcaccaatgc
caccatccac ggcgccatcc gcctggtctg ctcccgcggg aaccgcctca agacgacgtc
ctgggggctg ctgtccctgg gagccctggt cgcgctctgc tggcagctgg ggctcctctt
tgagcgtcac tggcaccgcc cggtcctcat ggccgtctct gtgcactcgg agcgcaagct
gctcccgctg gtcaccctgt gtgacgggaa cccacgtcgg ccgagtccgg tcctccgcca
tctggagctg ctggacgagt ttgccaggga gaacattgac tccctgtaca acgtcaacct
cagcaaaggc agagccgccc tctccgccac tgtcccccgc cacgagcccc ccttccacct
ggaccgggag atccgtctgc agaggctgag ccactcgggc agccgggtca gagtggggtt
cagactgtgc aacagcacgg gcggcgactg cttttaccga ggctacacgt caggcgtggc
ggctgtccag gactggtacc acttccacta tgtggatatc ctggccctgc tgcccgcggc
atgggaggac agccacggga gccaggacgg ccacttcgtc ctctcctgca gttacgatgg
cctggactgc caggcccgac agttccggac cttccaccac cccacctacg gcagctgcta
cacggtcgat ggcgtctgga cagctcagcg ccccggcatc acccacggag tcggcctggt
cctcagggtt gagcagcagc ctcacctccc tctgctgtcc acgctggccg gcatcagggt
catggttcac ggccgtaacc acacgccctt cctggggcac cacagcttca gcgtccggcc
agggacggag gccaccatca gcatccgaga ggacgaggtg caccggctcg ggagccccta
cggccactgc accgccggcg gggaaggcgt ggaggtggag ctgctacaca acacctccta
caccaggcag gcctgcctgg tgtcctgctt ccagcagctg atggtggaga cctgctcctg
tggctactac ctccaccctc tgccggcggg ggctgagtac tgcagctctg cccggcaccc
tgcctgggga cactgcttct accgcctcta ccaggacctg gagacccacc ggctcccctg
tacctcccgc tgccccaggc cctgcaggga gtctgcattc aagctctcca ctgggacctc
caggtggcct tccgccaagt cagctggatg gactctggcc acgctaggtg aacaggggct
gccgcatcag agccacagac agaggagcag cctggccaaa atcaacatcg tctaccagga
gctcaactac cgctcagtgg aggaggcgcc cgtgtactcg gtgccgcagc tgctctccgc
catgggcagc ctctacagcc tgtggtttgg ggcctccgtc ctctccctcc tggagctcct
ggagctgctg ctcgatgctt ctgccctcac cctggtgcta ggcggccgcc ggctccgcag
ggcgtggttc tcctggccca gagccagccc tgcctcaggg gcgtccagca tcaagccaga
ggccagtcag atgcccccgc ctgcaggcgg cacgtcagat gacccggagc ccagcgggcc
tcatctccca cgggtgatgc
ttccaggggt tctggcggga gtctcagccg aagagagctg ggctgggccc cagccccttg
agactctgga cacctga Note: tac->tgc provides Tyr->Cys at 532
SEQ ID NO: 8 pro for human kidney delta (with Y.sub.532) MAEHRSMDGR
MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIH
GAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL
VTLCDGNPRR PSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE
IRLQRLSHSG SRVRVGFRLC NSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED
SHGSQDGHFV LSCSYDGLDC QARQFRTFHH PTYGSCYTVD GVWTAQRPGI THGVGLVLRV
EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTE ATISIREDEV HRLGSPYGHC
TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEY CSSARHPAWG
HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQ
SHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LYSLWFGASV LSLLELLELL
LDASALTLVL GGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP
RVMLPGVLAG VSAEESWAGP QPLETLDT SEQ ID NO: 9 pro for human brain
delta (with C.sub.532) MAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP
PKEGHQEGLV ELPASFRELL TFFCTNATIH GAIRLVCSRG NRLKTTSWGL LSLGALVALC
WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRR PSPVLRHLEL LDEFARENID
SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLC NSTGGDCFYR
GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHH
PTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH
HSFSVRPGTE ATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL
MVETCSCGYY LHPLPAGAEY CSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF
KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQ SHRQRSSLAK INIVYQELNY RSVEEAPVYS
VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVL GGRRLRRAWF SWPRASPASG
ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGP QPLETLDT SEQ
ID NO: 10 motif for human kidney alpha MGSQWSLWFGA SEQ ID NO: 11
motif for human kidney delta MGSLYSLWFGA SEQ ID NO: 12 motif for
human taste bud delta MGSLCSLWFGA SEQ ID NO: 13 pro for human taste
gamma (Gamma A) MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG
CRRIVVSRGR LRRLLWIGFT LTAVALILWQ CALLVFSFYT VSVSIKVHFR KLDFPAVTIC
NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREA ESWNSVSEGK QPRFSHRIPP
LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSND TSDCATYTFS
LGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMH
GNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV
EDVGTEIETA MVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ
TKMVEKCGCA QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSLK
EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD LNQRSIMESP
ANSIEMLLSN FGGQLGLWMS CSVVCVVEII EVFFIDFFSI IARRQWQKAK EWWAWKQAPP
CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTL RLERAFSNQL
TDTQMLDEL SEQ ID NO: 14 pro for human taste gamma (Gamma B)
MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT
LTAVALILWQ CALLVFSFYA VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE
TREALKSLYG FPESRKRREA ESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR
KVGGSIIHKA SNVMHIESKQ VVGFQLCSND TSDCATYTFS SGINAIQEWY KLHYMNIMAQ
VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMH GNCYTFNNRE NETILSTSMG
GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETA MVTSIGMHLT
ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAA
NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL
LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS
CSVVCVIEII EVFFIDFFSI IARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD
DLPTFNSALH LPPALGTQVP GTPPPKYNTL RLERAFSNSL TDTQMLDEL SEQ ID NO: 15
pro for human taste gamma (Gamma C) MAPGEKIKAK IKKNLPVTGP
QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQ CALLVFSFYT
VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREA
ESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ
VVGFQLCSND TSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC
FFDGVSCDAR NFTLFHHPMH GNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF
LVSSTGAKVI IHRQDEYPFV EDVGTEIETA MVTSIGMHLT ESFKLSEPYS QCTEDGSDVP
IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR
AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD
LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSI
IARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD GLPTFNSALH LPPALGTQVP
GTPPPKYNTL RLERAFSNQL TDTQMLDEL SEQ ID NO: 16 pro for human lung
gamma (X87160) MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG
CRRIVVSRGR LRRLLWIGFT LTAVALILWQ CALLVFSFYT VSVSIKVHFR KLDFPAVTIC
NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREA ESWNSVSEGK QPRFSHRIPL
LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSND TSDCATYTFS
SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMH
GNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPSV
EDVGTEIETT MVTSIGMHLT ESFKLSEPSS QCTEGGSDVP IRNIYNAAYS LQICLHSCFQ
TKMVEKCGCA QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACRFK
EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD LNQRSIMESP
ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSI IARRQWQKAK EWWAWKQAPP
CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTL RLERAFSNQL
TDTQMLDEL SEQ ID NO: 17 pro for human delta subject DENACA
MAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL
TFFCTNATIH GAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS
VHSERKLLPL VTLCDGNPRR PSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR
HEPPFHLDRE IRLQRLSHSG SRVRVGFRLC NSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI
LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTIHH PTYGSCYTVD GVWTAQRPGI
THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTE
ATIRIREDEV
HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEY
CSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA
TLGEQGLPHQ SHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV
LSLLELLELL LDASALTLVL GGRRLHRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD
DPEPSGPHLP RVMLPGVLAG VSAEESWAGP QPLETLDT SEQ ID NO: 18 pro for
human delta subject DENACD MAEHRSMDGR MEAATRGGSH LQAAAQTPPR
PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIH GAIRLVCSRG NRLKTTSWGL
LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRR PSPVLRHLEL
LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLC
NSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC
QARQFRTFHH PTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH
GRNHTPFLGH HSFSVRPGTE ATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ
ACLVSCFQQL MVETCSCGYY LHPLPAGAEY CSSARHPAWG HCFYRLYQDL ETHRLPCTSR
CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQ SHRQRSSLAK INIVYQELNY
RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVL GGRRLRRAWF
SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGP
QPLETLDT SEQ ID NO: 19 pro for human delta subject DENAE MAEHRSMDGR
MEAATRGGPH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIH
GAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL
VTLCDGNPRR PSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPP HEPPFHLDRE
IRLQSLSHSG SRVRVGFRLC NSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED
SHGSQDGHFV LSCSYDGLDC QARQFRTFHH PTYGSCYTVD GVWTAQRPGI THGVGLVLRV
EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTE ATISIREDEV HRLGSPYGHC
TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEY CSSARHPAWG
HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQ
SHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL
LDASALTLVL GGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP
RVMLPGVLAG VSAEESWAGP QPLETLDK SEQ ID NO: 20 pro for human delta
subject DENACG MAEHRSMDGR MEAATRGGPH LQAAAQTPPR PGPPSAPPPP
PKEGHQEGLV ELPASFRELL TFFCTNATIH GAIRLVCSRG NRLKTTSWGL LSLGALVALC
WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRR PSPVLRHLEL LDEFARENID
SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLC NSTGGDCFYR
GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHH
PTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH
HSFSVRPGTE ATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL
MVETCSCGYY LHPLPAGAEY CSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF
KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQ SHRQRSSLAK INIVYQGLNY RSVEEAPVYS
VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVL GGRRLRRAWF SWPRASPASG
ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGP QPLETLDT SEQ
ID NO: 21 pro for human delta subject DENACH MAEHRSMDGR MEAATRGGSH
LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIH GTIRLVCSRG
NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRR
PSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPP HEPPFHLDRE IRLQRLSHSG
SRVRVGFRLC NSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV
LSCSYDGLDC QARQFRTFHH PTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS
TLAGIRVMVH GRNHTPFLGH HSFSVRPGTE ATISIREDEV HRLGSPYGHC TAGGEGVEVQ
LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEY CSSARHPAWG HCFYRLYRDL
ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQ SHRQRSSLAK
INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVL
GGRRLRRAWF SWPRASPASG ASSIKPEAGQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG
VSAEESWAGP QPLETLDT SEQ ID NO: 22 pro for human delta subject
DENACI MAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV
ELPASFRELL TFFCTNATIH GAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH
WHRPVLMAVS VHSERKLLPL VTLCDGNPRR PSPVLRHLEL LDEFARENID SLYNVNLSKG
RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLC NSTGGDCFYR GYTSGVAAVQ
DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHH PTYGSCYTVD
GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTE
ATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY
LHPLPAGAEY CSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP
SAKSAGWTLA TLGEQGLPHQ GHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS
LCSLWFGASV LSLLELLELL LDASALTLVL GGRRLRRAWF SWPRASPASG ASSIKPEASQ
MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGP QPLETLDT SEQ ID NO: 23
pro for human delta subject DENACJ MAEHRSMDGR MEAATRGGSH LQAAAQTPPR
PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIH GAIRLVCSRG NRLKTTSWGL
LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRR PSPVLRHLEL
LDEFARENID SLYNVNLSKG RAALSATVPP HEPPFHLDRE IRLQRLSHSG SRVRVGFRLC
NSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC
QARQFRTFHH PTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH
GRNHTPFLGH HSFSVRPGTE ATISIREDEV HRLGSPYGHC TAGGEGVEVQ PLHNTSYTRQ
ACLVSCFQQL MVETCSCGYY LHPLPAGAEY CSSARHPAWG HCFYRLYQDL ETHRLPCTSR
CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQ SHRQRSSLAK INIVYQELNY
RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVL GGRRLRRAWF
SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGP
QPLETLDT SEQ ID NO: 24 pro for human delta subject DENACT
MAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL
TFFCTNATIH GAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS
VHSERKLLPL VTLCDGNPRR PSPVLRHLEL LDEFARENID SLYSVNLSKG RAALSATVPR
HEPPFHLDRE IRLQRLSHSG SRVRVGFRLC NSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI
LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHH PTYGSCYTVD GVWTAQRPGI
THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTE ATISIREDEV
HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ PCLVSCFQQL MVETCSCGYY LHPLPAGAEY
CSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA
TLGEQGLPHQ SHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV
LSLLELLELL LDASALTLVL GGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD
DPEPSGPHLP RVMLPGVLAG VSAEESWAGP QPLETLDT
SEQ ID NO: 25 pro for human delta subject DENACV MAEHRSMDGR
MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIH
GAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL
VTLCDGNPRR PSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE
IRLQRLSHSG SRVRVGFRLC NSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED
SHGSQDGHFV LSCSYDGLDC QARQFRTFHH PTYGSCYTVD GVWTAQRPGI THGVGLVLRV
EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTE ATISIREDEV HRLGSPYGHC
TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEY CGSARHPAWG
HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQ
SHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL
LDASALTLVL GGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP
RVMLPGVLAG VSAEESWAGP QPLETLDT SEQ ID NO: 26 pro for human delta
subject DENACW MAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP
PKEGHQEGLV ELPASFRELL TFFCTNATIH GAIRLVCSRG NRLKTTSWGL LSLGALVALC
WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRR PSPVLRHLEL LDEFARENID
SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLC NSTGGDCFYR
GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTIHH
PTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH
HSFSVRPGTE ATIRIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL
MVETCSCGYY LHPLPAGAEY CSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF
KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQ SHRQRSSLAK INIVYQELNY RSVEEAPVYS
VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVL GGRRLHRAWF SWPRASPASG
ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGP QPLETLDT SEQ
ID NO: 27 pro for human gamma subject GENACA MAPGEKIKAK IKKNLPVTGP
QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQ CALLVFSFYT
VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREA
ESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ
VVGFQLCSND TSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC
FFDGVSCDAR NFTLFHHPMH GNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF
LVSSTGAKVI IHRQDEYPFV EDVGTEIETA MVTSIGMHLT ESFKLSEPYS QCTEDGSDVP
IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR
AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD
LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSI
IARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP
GTPPPKYNTL RLERAFSNQL TDTQMLDEL SEQ ID NO: 28 pro for human gamma
subject GENACB MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG
CRRIVVSRGR LRRLLWIGFT LTAVALILWQ CALLVFSFYT VSVSIKVNFR KLDFPAVTIC
NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREA ESWNSVSEGK QPRFSHRIPL
LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSND TSDCATYTFS
SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMH
GNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV
EDVGTEIETA MVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ
TKMVEKCGCA QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK
EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD LNQRSIMESP
ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSI IARRQWQKAK EWWAWKQAPP
CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTL RLERAFSNQL
TDTQMLDEL SEQ ID NO: 29 pro for human gamma subject GENACD
MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT
LTAVALILWQ CALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE
TREALKSLYG FPESRKRREA ESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR
KVGGSIIHKA SNVMHIESKQ VVGFQLCSND TSDCATYTFS SGINAIQEWY KLHYMNIMAQ
VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMH GNCYTFNNRE NETILSTSMG
GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETA MVTSIGMHLT
ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAA
NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL
LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS
CSVVCVIEII EVFFIDFFSI IARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD
DLPTFNSALH LPPALGTQVP GTPPPKYNTL RLERAFSNQL TDTQMLDEL SEQ ID NO: 30
pro for human gamma subject GENACE MAPGEKIKAK IKKNLPVTGP QAPTIKELMR
WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQ CALLVFSFYT VSVSIKVHFR
KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREA ESWNSVSEGK
QPRFSHRIPL LIFDQDEKGE ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSND
TSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR
NFTLFHHPMH GNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI
IHRQDEYPFV EDVGTEIETA MVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS
LQICLHSCFQ TKMVEKCGCA QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ
SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD
LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSI IARRQWQKAK
EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP STPPPKYNTL
RLERAFSNQL TDTQMLDEL SEQ ID NO: 31 pro for human gamma subject
GENACG MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR
LRRLLWIGFT LTAVALILWQ CALLVFSFYA VSVSIKVHFR KLDFPAVTIC NINPYKYSTV
RHLLADLEQE TREALKSLYG FPESRKRREA ESWNSVSEGK QPRFSHRIPL LIFDQDEKGK
ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSND TSDCATYTFS SGINAIQEWY
KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMH GNCYTFNNRE
NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETA
MVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA
QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ
WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN
FGGQLGLWMS CSVVCVIEII EVFFIDFFSI TARRQWQKAK EWWAWKQAPP CPEAPRSPQG
QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTL RLERAFSNSL TDTQMLDEL
SEQ ID NO: 32 pro for human gamma subject GENACH
MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT
LTAVALILWQ CALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE
TREALKSLYG FPESRKRREA ESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR
KVGGSIIHKA SNVMHIESKQ VVGFQLCSND TSDCATYTFS SGINAIQEWY KLHYMNIMAQ
VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMH GNCYTFNNRE NETILSTSMG
GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETA MVTSIGMHLT
ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAA
NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL
LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS
CSVVCVIEII EVFFIDFFSI IARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD
GLPTFNSALH LPPALGTQVP GTPPPKYNTL RLERAFSNQL TDTQMLDEL SEQ ID NO: 33
pro for human gamma subject GENACJ MAPGEKIKAK IKKNLPVTGP QAPTIKELMR
WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQ CALLVFSFYT VSVSIKVHFR
KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREA ESWNSVSEGK
QPRFSHRIPP LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSND
TSDCATYTFS LGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR
NFTLFHHPMH GNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI
IHRQDEYPFV EDVGTEIETA MVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS
LQICLHSCFQ TKMVEKCGCA QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ
SVCKEACSLK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD
LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSI TARRQWQKAK
EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTL
RLERAFSNQL TDTQMLDEL SEQ ID NO: 34 pro for human gamma subject
GENACT MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR
LRRLLWIGFT LTAVALILWQ CALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV
RHLLADLEQE TREALKSLYG FPESRKRREA ESWNSVSEGK QPRFSHRIPL LIFDQDEKGK
ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSND TSDCATYTFS SGINAIQEWY
KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMH GNCYTFNNRE
NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETA
MVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA
QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ
WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN
FGGQLGLWMS CSVVCVIEII EVFFIDFFSI IARRQWQKAK EWWAWKQAPP CPEAPRSPQG
QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTL RLERAFSNQL TDTQMLDEL
SEQ ID NO: 35 pro for human gamma subject GENACV MAPGEKIKAK
IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQ
CALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG
FPESRKRREA ESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA
SNVMHIESKQ VVGFQLCSND TSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS
YSAEELLVTC FFDGVSCDAR NFTLFHHPMH GNCYTFNNRE NETILSTSMG GSEYGLQVIL
YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETA MVTSIGMHLT ESFKLSEPYS
QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAA NYCNYQQHPN
WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGR
QVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII
EVFFIDFFSI IARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH
LPPALGTQVP GTPPPKYNTL RLERAFSNQL TDTQMLDEL SEQ ID NO: 36 pro for
human gamma subject GENACW MAPGEKIKAK IKKNLPVTGP QAPTIKELMR
WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILRQ CALLVFSFYT VSVSIKVHFR
KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREA ESWNSVSEGK
QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSND
TSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR
NFTLFHHPMH GNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI
IHRQDEYPFV EDVGTEIETA MVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS
LQICLHSCFQ TKMVEKCGCA QYSQPLPPAA NYCNYQQHPN WMYCYYQLHR AFVQEELGCQ
SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGR QVNKKLNKTD LAKLLIFYKD
LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSI IARRQWQKAK
EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTL
RLERAFSNQL TDTQMLDEL
Sequence CWU 1
1
3812010DNAHomo sapiens 1atggagggga acaagctgga ggagcaggac tctagccctc
cacagtccac tccagggctc 60atgaagggga acaagcgtga ggagcagggg ctgggccccg
aacctgcggc gccccagcag 120cccacggcgg aggaggaggc cctgatcgag
ttccaccgct cctaccgaga gctcttcgag 180ttcttctgca acaacaccac
catccacggc gccatccgcc tggtgtgctc ccagcacaac 240cgcatgaaga
cggccttctg ggcagtgctg tggctctgca cctttggcat gatgtactgg
300caattcggcc tgcttttcgg agagtacttc agctaccccg tcagcctcaa
catcaacctc 360aactcggaca agctcgtctt ccccgcagtg accatctgca
ccctcaatcc ctacaggtac 420ccggaaatta aagaggagct ggaggagctg
gaccgcatca cagagcagac gctctttgac 480ctgtacaaat acagctcctt
caccactctc gtggccggct cccgcagccg tcgcgacctg 540cgggggactc
tgccgcaccc cttgcagcgc ctgagggtcc cgcccccgcc tcacggggcc
600cgtcgagccc gtagcgtggc ctccagcttg cgggacaaca acccccaggt
ggactggaag 660gactggaaga tcggcttcca gctgtgcaac cagaacaaat
cggactgctt ctaccagaca 720tactcatcag gggtggatgc ggtgagggag
tggtaccgct tccactacat caacatcctg 780tcgaggctgc cagagactct
gccatccctg gaggaggaca cgctgggcaa cttcatcttc 840gcctgccgct
tcaaccaggt ctcctgcaac caggcgaatt actctcactt ccaccacccg
900atgtatggaa actgctatac tttcaatgac aagaacaact ccaacctctg
gatgtcttcc 960atgcctggaa tcaacaacgg tctgtccctg atgctgcgcg
cagagcagaa tgacttcatt 1020cccctgctgt ccacagtgac tggggcccgg
gtaatggtgc acgggcagga tgaacctgcc 1080tttatggatg atggtggctt
taacttgcgg cctggcgtgg agacctccat cagcatgagg 1140aaggaaaccc
tggacagact tgggggcgat tatggcgact gcaccaagaa tggcagtgat
1200gttcctgttg agaaccttta cccttcaaag tacacacagc aggtgtgtat
tcactcctgc 1260ttccaggaga gcatgatcaa ggagtgtggc tgtgcctaca
tcttctatcc gcggccccag 1320aacgtggagt actgtgacta cagaaagcac
agttcctggg ggtactgcta ctataagctc 1380caggttgact tctcctcaga
ccacctgggc tgtttcacca agtgccggaa gccatgcagc 1440gtgaccagct
accagctctc tgctggttac tcacgatggc cctcggtgac atcccaggaa
1500tgggtcttcc agatgctatc gcgacagaac aattacaccg tcaacaacaa
gagaaatgga 1560gtggccaaag tcaacatctt cttcaaggag ctgaactaca
aaaccaattc tgagtctccc 1620tctgtcacga tggtcaccct cctgtccaac
ctgggcagcc agtggagcct gtggttcggc 1680tcctcggtgt tgtctgtggt
ggagatggct gagctcgtct ttgacctgct ggtcatcatg 1740ttcctcatgc
tgctccgaag gttccgaagc cgatactggt ctccaggccg agggggcagg
1800ggtgctcagg aggtagcctc caccctggca tcctcccctc cttcccactt
ctgcccccac 1860cccatgtctc tgtccttgtc ccagccaggc cctgctccct
ctccagcctt gacagcccct 1920ccccctgcct atgccaccct gggcccccgc
ccatctccag ggggctctgc aggggccagt 1980tcctccacct gtcctctggg
ggggccctga 20102669PRTHomo Sapiens 2Met Glu Gly Asn Lys Leu Glu Glu
Gln Asp Ser Ser Pro Pro Gln Ser1 5 10 15Thr Pro Gly Leu Met Lys Gly
Asn Lys Arg Glu Glu Gln Gly Leu Gly 20 25 30Pro Glu Pro Ala Ala Pro
Gln Gln Pro Thr Ala Glu Glu Glu Ala Leu 35 40 45Ile Glu Phe His Arg
Ser Tyr Arg Glu Leu Phe Glu Phe Phe Cys Asn 50 55 60Asn Thr Thr Ile
His Gly Ala Ile Arg Leu Val Cys Ser Gln His Asn65 70 75 80Arg Met
Lys Thr Ala Phe Trp Ala Val Leu Trp Leu Cys Thr Phe Gly 85 90 95Met
Met Tyr Trp Gln Phe Gly Leu Leu Phe Gly Glu Tyr Phe Ser Tyr 100 105
110Pro Val Ser Leu Asn Ile Asn Leu Asn Ser Asp Lys Leu Val Phe Pro
115 120 125Ala Val Thr Ile Cys Thr Leu Asn Pro Tyr Arg Tyr Pro Glu
Ile Lys 130 135 140Glu Glu Leu Glu Glu Leu Asp Arg Ile Thr Glu Gln
Thr Leu Phe Asp145 150 155 160Leu Tyr Lys Tyr Ser Ser Phe Thr Thr
Leu Val Ala Gly Ser Arg Ser 165 170 175Arg Arg Asp Leu Arg Gly Thr
Leu Pro His Pro Leu Gln Arg Leu Arg 180 185 190Val Pro Pro Pro Pro
His Gly Ala Arg Arg Ala Arg Ser Val Ala Ser 195 200 205Ser Leu Arg
Asp Asn Asn Pro Gln Val Asp Trp Lys Asp Trp Lys Ile 210 215 220Gly
Phe Gln Leu Cys Asn Gln Asn Lys Ser Asp Cys Phe Tyr Gln Thr225 230
235 240Tyr Ser Ser Gly Val Asp Ala Val Arg Glu Trp Tyr Arg Phe His
Tyr 245 250 255Ile Asn Ile Leu Ser Arg Leu Pro Glu Thr Leu Pro Ser
Leu Glu Glu 260 265 270Asp Thr Leu Gly Asn Phe Ile Phe Ala Cys Arg
Phe Asn Gln Val Ser 275 280 285Cys Asn Gln Ala Asn Tyr Ser His Phe
His His Pro Met Tyr Gly Asn 290 295 300Cys Tyr Thr Phe Asn Asp Lys
Asn Asn Ser Asn Leu Trp Met Ser Ser305 310 315 320Met Pro Gly Ile
Asn Asn Gly Leu Ser Leu Met Leu Arg Ala Glu Gln 325 330 335Asn Asp
Phe Ile Pro Leu Leu Ser Thr Val Thr Gly Ala Arg Val Met 340 345
350Val His Gly Gln Asp Glu Pro Ala Phe Met Asp Asp Gly Gly Phe Asn
355 360 365Leu Arg Pro Gly Val Glu Thr Ser Ile Ser Met Arg Lys Glu
Thr Leu 370 375 380Asp Arg Leu Gly Gly Asp Tyr Gly Asp Cys Thr Lys
Asn Gly Ser Asp385 390 395 400Val Pro Val Glu Asn Leu Tyr Pro Ser
Lys Tyr Thr Gln Gln Val Cys 405 410 415Ile His Ser Cys Phe Gln Glu
Ser Met Ile Lys Glu Cys Gly Cys Ala 420 425 430Tyr Ile Phe Tyr Pro
Arg Pro Gln Asn Val Glu Tyr Cys Asp Tyr Arg 435 440 445Lys His Ser
Ser Trp Gly Tyr Cys Tyr Tyr Lys Leu Gln Val Asp Phe 450 455 460Ser
Ser Asp His Leu Gly Cys Phe Thr Lys Cys Arg Lys Pro Cys Ser465 470
475 480Val Thr Ser Tyr Gln Leu Ser Ala Gly Tyr Ser Arg Trp Pro Ser
Val 485 490 495Thr Ser Gln Glu Trp Val Phe Gln Met Leu Ser Arg Gln
Asn Asn Tyr 500 505 510Thr Val Asn Asn Lys Arg Asn Gly Val Ala Lys
Val Asn Ile Phe Phe 515 520 525Lys Glu Leu Asn Tyr Lys Thr Asn Ser
Glu Ser Pro Ser Val Thr Met 530 535 540Val Thr Leu Leu Ser Asn Leu
Gly Ser Gln Trp Ser Leu Trp Phe Gly545 550 555 560Ser Ser Val Leu
Ser Val Val Glu Met Ala Glu Leu Val Phe Asp Leu 565 570 575Leu Val
Ile Met Phe Leu Met Leu Leu Arg Arg Phe Arg Ser Arg Trp 580 585
590Pro Ser Pro Gly Arg Gly Gly Arg Gly Ala Gln Glu Val Ala Ser Thr
595 600 605Leu Ala Ser Ser Pro Pro Ser His Phe Cys Pro His Pro Met
Ser Leu 610 615 620Ser Leu Ser Gln Pro Gly Pro Ala Pro Ser Pro Ala
Leu Thr Ala Pro625 630 635 640Pro Pro Ala Tyr Ala Thr Leu Gly Pro
Arg Pro Ser Pro Gly Gly Ser 645 650 655Ala Gly Ala Ser Ser Ser Thr
Cys Pro Leu Gly Gly Pro 660 66531923DNAHomo Sapiens 3atgcacgtga
agaagtacct gctgaagggc ctgcatcggc tgcagaaggg ccccggctac 60acgtacaagg
agctgctggt gtggtactgc gacaacacca acacccacgg ccccaagcgc
120atcatctgtg aggggcccaa gaagaaagcc atgtggttcc tgctcaccct
gctcttcgcc 180gccctcgtct gctggcagtg gggcatcttc atcaggacct
acttgagctg ggaggtcagc 240gtctccctct ccgtaggctt caagaccatg
gacttccccg ccgtcaccat ctgcaatgct 300agccccttca agtattccaa
aatcaagcat ttgctgaagg acctggatga gctgatggaa 360gctgtcctgg
agagaatcct ggctcctgag ctaagccatg ccaatgccac caggaacctg
420aacttctcca tctggaacca cacacccctg gtccttattg atgaacggaa
cccccaccac 480cccatggtcc ttgatctctt tggagacaac cacaatggct
taacaagcag ctcagcatca 540gaaaagatct gtaatgccca cgggtgcaaa
atggccatga gactatgtag cctcaacagg 600acccagtgta ccttccggaa
cttcaccagt gctacccagg cattgacaga gtggtacatc 660ctgcaggcca
ccaacatctt tgcacaggtg ccacagcagg agctagtaga gatgagctac
720cccggcgagc agatgatcct ggcctgccta ttcggagctg agccctgcaa
ctaccggaac 780ttcacgtcca tcttctaccc tcactatggc aactgttaca
tcttcaactg gggcatgaca 840gagaaggcac ttccttcggc caaccctgga
actgaattcg gcctgaagtt gatcctggac 900ataggccagg aagactacgt
ccccttcctt gcgtccacgg ccggggtcag gctgatgctt 960cacgagcaga
ggtcataccc cttcatcaga gatgagggca tctacgccat gtcggggaca
1020gagacgtcca tcggggtact cgtggacaag cttcagcgca tgggggagcc
ctacagcccg 1080tgcaccgtga atggttctga ggtccccgtc caaaacttct
acagtgacta caacacgacc 1140tactccatcc aggcctgtct tcgctcctgc
ttccaagacc acatgatccg taactgcaac 1200tgtggccact acctgtaccc
actgccccgt ggggagaaat actgcaacaa ccgggacttc 1260ccagactggg
cccattgcta ctcagatcta cagatgagcg tggcgcagag agagacctgc
1320attggcatgt gcaaggagtc ctgcaatgac acccagtaca agatgaccat
ctccatggct 1380gactggcctt ctgaggcctc cgaggactgg attttccacg
tcttgtctca ggagcgggac 1440caaagcacca atatcaccct gagcaggaag
ggaattgtca agctcaacat ctacttccaa 1500gaatttaact atcgcaccat
tgaagaatca gcagccaata acatcgtctg gctgctctcg 1560aatctgggtg
gccagtttgg cttctggatg gggggctctg tgctgtgcct catcgagttt
1620ggggagatca tcatcgactt tgtgtggatc accatcatca agctggtggc
cttggccaag 1680agcctacggc agcggcgagc ccaagccagc tacgctggcc
caccgcccac cgtggccgag 1740ctggtggagg cccacaccaa ctttggcttc
cagcctgaca cggccccccg cagccccaac 1800actgggccct accccagtga
gcaggccctg cccatcccag gcaccccgcc ccccaactat 1860gactccctgc
gtctgcagcc gctggacgtc atcgagtctg acagtgaggg tgatgccatc 1920taa
19234640PRTHomo Sapiens 4Met His Val Lys Lys Tyr Leu Leu Lys Gly
Leu His Arg Leu Gln Lys1 5 10 15Gly Pro Gly Tyr Thr Tyr Lys Glu Leu
Leu Val Trp Tyr Cys Asp Asn 20 25 30Thr Asn Thr His Gly Pro Lys Arg
Ile Ile Cys Glu Gly Pro Lys Lys 35 40 45Lys Ala Met Trp Phe Leu Leu
Thr Leu Leu Phe Ala Ala Leu Val Cys 50 55 60Trp Gln Trp Gly Ile Phe
Ile Arg Thr Tyr Leu Ser Trp Glu Val Ser65 70 75 80Val Ser Leu Ser
Val Gly Phe Lys Thr Met Asp Phe Pro Ala Val Thr 85 90 95Ile Cys Asn
Ala Ser Pro Phe Lys Tyr Ser Lys Ile Lys His Leu Leu 100 105 110Lys
Asp Leu Asp Glu Leu Met Glu Ala Val Leu Glu Arg Ile Leu Ala 115 120
125Pro Glu Leu Ser His Ala Asn Ala Thr Arg Asn Leu Asn Phe Ser Ile
130 135 140Trp Asn His Thr Pro Leu Val Leu Ile Asp Glu Arg Asn Pro
His His145 150 155 160Pro Met Val Leu Asp Leu Phe Gly Asp Asn His
Asn Gly Leu Thr Ser 165 170 175Ser Ser Ala Ser Glu Lys Ile Cys Asn
Ala His Gly Cys Lys Met Ala 180 185 190Met Arg Leu Cys Ser Leu Asn
Arg Thr Gln Cys Thr Phe Arg Asn Phe 195 200 205Thr Ser Ala Thr Gln
Ala Leu Thr Glu Trp Tyr Ile Leu Gln Ala Thr 210 215 220Asn Ile Phe
Ala Gln Val Pro Gln Gln Glu Leu Val Glu Met Ser Tyr225 230 235
240Pro Gly Glu Gln Met Ile Leu Ala Cys Leu Phe Gly Ala Glu Pro Cys
245 250 255Asn Tyr Arg Asn Phe Thr Ser Ile Phe Tyr Pro His Tyr Gly
Asn Cys 260 265 270Tyr Ile Phe Asn Trp Gly Met Thr Glu Lys Ala Leu
Pro Ser Ala Asn 275 280 285Pro Gly Thr Glu Phe Gly Leu Lys Leu Ile
Leu Asp Ile Gly Gln Glu 290 295 300Asp Tyr Val Pro Phe Leu Ala Ser
Thr Ala Gly Val Arg Leu Met Leu305 310 315 320His Glu Gln Arg Ser
Tyr Pro Phe Ile Arg Asp Glu Gly Ile Tyr Ala 325 330 335Met Ser Gly
Thr Glu Thr Ser Ile Gly Val Leu Val Asp Lys Leu Gln 340 345 350Arg
Met Gly Glu Pro Tyr Ser Pro Cys Thr Val Asn Gly Ser Glu Val 355 360
365Pro Val Gln Asn Phe Tyr Ser Asp Tyr Asn Thr Thr Tyr Ser Ile Gln
370 375 380Ala Cys Leu Arg Ser Cys Phe Gln Asp His Met Ile Arg Asn
Cys Asn385 390 395 400Cys Gly His Tyr Leu Tyr Pro Leu Pro Arg Gly
Glu Lys Tyr Cys Asn 405 410 415Asn Arg Asp Phe Pro Asp Trp Ala His
Cys Tyr Ser Asp Leu Gln Met 420 425 430Ser Val Ala Gln Arg Glu Thr
Cys Ile Gly Met Cys Lys Glu Ser Cys 435 440 445Asn Asp Thr Gln Tyr
Lys Met Thr Ile Ser Met Ala Asp Trp Pro Ser 450 455 460Glu Ala Ser
Glu Asp Trp Ile Phe His Val Leu Ser Gln Glu Arg Asp465 470 475
480Gln Ser Thr Asn Ile Thr Leu Ser Arg Lys Gly Ile Val Lys Leu Asn
485 490 495Ile Tyr Phe Gln Glu Phe Asn Tyr Arg Thr Ile Glu Glu Ser
Ala Ala 500 505 510Asn Asn Ile Val Trp Leu Leu Ser Asn Leu Gly Gly
Gln Phe Gly Phe 515 520 525Trp Met Gly Gly Ser Val Leu Cys Leu Ile
Glu Phe Gly Glu Ile Ile 530 535 540Ile Asp Phe Val Trp Ile Thr Ile
Ile Lys Leu Val Ala Leu Ala Lys545 550 555 560Ser Leu Arg Gln Arg
Arg Ala Gln Ala Ser Tyr Ala Gly Pro Pro Pro 565 570 575Thr Val Ala
Glu Leu Val Glu Ala His Thr Asn Phe Gly Phe Gln Pro 580 585 590Asp
Thr Ala Pro Arg Ser Pro Asn Thr Gly Pro Tyr Pro Ser Glu Gln 595 600
605Ala Leu Pro Ile Pro Gly Thr Pro Pro Pro Asn Tyr Asp Ser Leu Arg
610 615 620Leu Gln Pro Leu Asp Val Ile Glu Ser Asp Ser Glu Gly Asp
Ala Ile625 630 635 64051950DNAHomo Sapiens 5atggcacccg gagagaagat
caaagccaaa atcaagaaga atctgcccgt gacgggccct 60caggcgccga ccattaaaga
gctgatgcgg tggtactgcc tcaacaccaa cacccatggc 120tgtcgccgca
tcgtggtgtc ccgcggccgt ctgcgccgcc tcctctggat cgggttcaca
180ctgactgccg tggccctcat cctctggcag tgcgccctcc tcgtcttctc
cttctatact 240gtctcagttt ccatcaaagt ccacttccgg aagctggatt
ttcctgcagt caccatctgc 300aacatcaacc cctacaagta cagcaccgtt
cgccaccttc tagctgactt ggaacaggag 360accagagagg ccctgaagtc
cctgtatggc tttccagagt cccggaagcg ccgagaggcg 420gagtcctgga
actccgtctc agagggaaag cagcctagat tctcccaccg gattccgctg
480ctgatctttg atcaggatga gaagggcaag gccagggact tcttcacagg
gaggaagcgg 540aaagtcggcg gtagcatcat tcacaaggct tcaaatgtca
tgcacatcga gtccaagcaa 600gtggtgggat tccaactgtg ctcaaatgac
acctccgact gtgccaccta caccttcagc 660tcgggaatca atgccattca
ggagtggtat aagctacact acatgaacat catggcacag 720gtgcctctgg
agaagaaaat caacatgagc tattctgctg aggagctgct ggtgacctgc
780ttctttgatg gagtgtcctg tgatgccagg aatttcacgc ttttccacca
cccgatgcat 840gggaattgct atactttcaa caacagagaa aatgagacca
ttctcagcac ctccatgggg 900ggcagcgaat atgggctgca agtcattttg
tacataaacg aagaggaata caacccattc 960ctcgtgtcct ccactggagc
taaggtgatc atccatcggc aggatgagta tcccttcgtc 1020gaagatgtgg
gaacagagat tgagacagca atggtcacct ctataggaat gcacctgaca
1080gagtccttca agctgagtga gccctacagt cagtgcacgg aggacgggag
tgacgtgcca 1140atcaggaaca tctacaacgc tgcctactcg ctccagatct
gccttcattc atgcttccag 1200acaaagatgg tggagaaatg tgggtgtgcc
cagtacagcc agcctctacc tcctgcagcc 1260aactactgca actaccagca
gcaccccaac tggatgtatt gttactacca actgcatcga 1320gcctttgtcc
aggaagagct gggctgccag tctgtgtgca aggaagcctg cagctttaaa
1380gagtggacac taaccacaag cctggcacaa tggccatctg tggtttcgga
gaagtggttg 1440ctgcctgttc tcacttggga ccaaggccgg caagtaaaca
aaaagctcaa caagacagac 1500ttggccaaac tcttgatatt ctacaaagac
ctgaaccaga gatccatcat ggagagccca 1560gccaacagta ttgagatgct
tctgtccaac ttcggtggcc agctgggcct gtggatgagc 1620tgctctgttg
tctgcgtcat cgagatcatc gaggtcttct tcattgactt cttctctatc
1680attgcccgcc gccagtggca gaaagccaag gagtggtggg cctggaaaca
ggctccccca 1740tgtccagaag ctccccgtag cccacagggc caggacaatc
cagccctgga tatagacgat 1800gacctaccca ctttcaactc tgctttgcac
ctgcctccag ccctaggaac ccaagtgccc 1860ggcacaccgc cccccaaata
caataccttg cgcttggaga gggccttttc caaccagctc 1920acagataccc
agatgctaga tgagctctga 19506649PRTHomo Sapiens 6Met Ala Pro Gly Glu
Lys Ile Lys Ala Lys Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro
Gln Ala Pro Thr Ile Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn
Thr Asn Thr His Gly Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg
Leu Arg Arg Leu Leu Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala
Leu Ile Leu Trp Gln Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75
80Val Ser Val Ser Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala
85 90 95Val Thr Ile Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg
His 100 105 110Leu Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu
Lys Ser Leu 115 120 125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu
Ala Glu Ser Trp Asn 130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg
Phe Ser His Arg Ile Pro Leu145 150 155 160Leu Ile
Phe Asp Gln Asp Glu Lys Gly Lys Ala Arg Asp Phe Phe Thr 165 170
175Gly Arg Lys Arg Lys Val Gly Gly Ser Ile Ile His Lys Ala Ser Asn
180 185 190Val Met His Ile Glu Ser Lys Gln Val Val Gly Phe Gln Leu
Cys Ser 195 200 205Asn Asp Thr Ser Asp Cys Ala Thr Tyr Thr Phe Ser
Ser Gly Ile Asn 210 215 220Ala Ile Gln Glu Trp Tyr Lys Leu His Tyr
Met Asn Ile Met Ala Gln225 230 235 240Val Pro Leu Glu Lys Lys Ile
Asn Met Ser Tyr Ser Ala Glu Glu Leu 245 250 255Leu Val Thr Cys Phe
Phe Asp Gly Val Ser Cys Asp Ala Arg Asn Phe 260 265 270Thr Leu Phe
His His Pro Met His Gly Asn Cys Tyr Thr Phe Asn Asn 275 280 285Arg
Glu Asn Glu Thr Ile Leu Ser Thr Ser Met Gly Gly Ser Glu Tyr 290 295
300Gly Leu Gln Val Ile Leu Tyr Ile Asn Glu Glu Glu Tyr Asn Pro
Phe305 310 315 320Leu Val Ser Ser Thr Gly Ala Lys Val Ile Ile His
Arg Gln Asp Glu 325 330 335Tyr Pro Phe Val Glu Asp Val Gly Thr Glu
Ile Glu Thr Ala Met Val 340 345 350Thr Ser Ile Gly Met His Leu Thr
Glu Ser Phe Lys Leu Ser Glu Pro 355 360 365Tyr Ser Gln Cys Thr Glu
Asp Gly Ser Asp Val Pro Ile Arg Asn Ile 370 375 380Tyr Asn Ala Ala
Tyr Ser Leu Gln Ile Cys Leu His Ser Cys Phe Gln385 390 395 400Thr
Lys Met Val Glu Lys Cys Gly Cys Ala Gln Tyr Ser Gln Pro Leu 405 410
415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr Gln Gln His Pro Asn Trp Met
420 425 430Tyr Cys Tyr Tyr Gln Leu His Arg Ala Phe Val Gln Glu Glu
Leu Gly 435 440 445Cys Gln Ser Val Cys Lys Glu Ala Cys Ser Phe Lys
Glu Trp Thr Leu 450 455 460Thr Thr Ser Leu Ala Gln Trp Pro Ser Val
Val Ser Glu Lys Trp Leu465 470 475 480Leu Pro Val Leu Thr Trp Asp
Gln Gly Arg Gln Val Asn Lys Lys Leu 485 490 495Asn Lys Thr Asp Leu
Ala Lys Leu Leu Ile Phe Tyr Lys Asp Leu Asn 500 505 510Gln Arg Ser
Ile Met Glu Ser Pro Ala Asn Ser Ile Glu Met Leu Leu 515 520 525Ser
Asn Phe Gly Gly Gln Leu Gly Leu Trp Met Ser Cys Ser Val Val 530 535
540Cys Val Ile Glu Ile Ile Glu Val Phe Phe Ile Asp Phe Phe Ser
Ile545 550 555 560Ile Ala Arg Arg Gln Trp Gln Lys Ala Lys Glu Trp
Trp Ala Trp Lys 565 570 575Gln Ala Pro Pro Cys Pro Glu Ala Pro Arg
Ser Pro Gln Gly Gln Asp 580 585 590Asn Pro Ala Leu Asp Ile Asp Asp
Asp Leu Pro Thr Phe Asn Ser Ala 595 600 605Leu His Leu Pro Pro Ala
Leu Gly Thr Gln Val Pro Gly Thr Pro Pro 610 615 620Pro Lys Tyr Asn
Thr Leu Arg Leu Glu Arg Ala Phe Ser Asn Gln Leu625 630 635 640Thr
Asp Thr Gln Met Leu Asp Glu Leu 64571917DNAHomo Sapiens 7atggctgagc
accgaagcat ggacgggaga atggaagcag ccacacgggg gggctctcac 60ctccaggctg
cagcccagac gccccccagg ccggggccac catcagcacc accaccacca
120cccaaggagg ggcaccagga ggggctggtg gagctgcccg cctcgttccg
ggagctgctc 180accttcttct gcaccaatgc caccatccac ggcgccatcc
gcctggtctg ctcccgcggg 240aaccgcctca agacgacgtc ctgggggctg
ctgtccctgg gagccctggt cgcgctctgc 300tggcagctgg ggctcctctt
tgagcgtcac tggcaccgcc cggtcctcat ggccgtctct 360gtgcactcgg
agcgcaagct gctcccgctg gtcaccctgt gtgacgggaa cccacgtcgg
420ccgagtccgg tcctccgcca tctggagctg ctggacgagt ttgccaggga
gaacattgac 480tccctgtaca acgtcaacct cagcaaaggc agagccgccc
tctccgccac tgtcccccgc 540cacgagcccc ccttccacct ggaccgggag
atccgtctgc agaggctgag ccactcgggc 600agccgggtca gagtggggtt
cagactgtgc aacagcacgg gcggcgactg cttttaccga 660ggctacacgt
caggcgtggc ggctgtccag gactggtacc acttccacta tgtggatatc
720ctggccctgc tgcccgcggc atgggaggac agccacggga gccaggacgg
ccacttcgtc 780ctctcctgca gttacgatgg cctggactgc caggcccgac
agttccggac cttccaccac 840cccacctacg gcagctgcta cacggtcgat
ggcgtctgga cagctcagcg ccccggcatc 900acccacggag tcggcctggt
cctcagggtt gagcagcagc ctcacctccc tctgctgtcc 960acgctggccg
gcatcagggt catggttcac ggccgtaacc acacgccctt cctggggcac
1020cacagcttca gcgtccggcc agggacggag gccaccatca gcatccgaga
ggacgaggtg 1080caccggctcg ggagccccta cggccactgc accgccggcg
gggaaggcgt ggaggtggag 1140ctgctacaca acacctccta caccaggcag
gcctgcctgg tgtcctgctt ccagcagctg 1200atggtggaga cctgctcctg
tggctactac ctccaccctc tgccggcggg ggctgagtac 1260tgcagctctg
cccggcaccc tgcctgggga cactgcttct accgcctcta ccaggacctg
1320gagacccacc ggctcccctg tacctcccgc tgccccaggc cctgcaggga
gtctgcattc 1380aagctctcca ctgggacctc caggtggcct tccgccaagt
cagctggatg gactctggcc 1440acgctaggtg aacaggggct gccgcatcag
agccacagac agaggagcag cctggccaaa 1500atcaacatcg tctaccagga
gctcaactac cgctcagtgg aggaggcgcc cgtgtactcg 1560gtgccgcagc
tgctctccgc catgggcagc ctctacagcc tgtggtttgg ggcctccgtc
1620ctctccctcc tggagctcct ggagctgctg ctcgatgctt ctgccctcac
cctggtgcta 1680ggcggccgcc ggctccgcag ggcgtggttc tcctggccca
gagccagccc tgcctcaggg 1740gcgtccagca tcaagccaga ggccagtcag
atgcccccgc ctgcaggcgg cacgtcagat 1800gacccggagc ccagcgggcc
tcatctccca cgggtgatgc ttccaggggt tctggcggga 1860gtctcagccg
aagagagctg ggctgggccc cagccccttg agactctgga cacctga 19178638PRTHomo
Sapiens 8Met Ala Glu His Arg Ser Met Asp Gly Arg Met Glu Ala Ala
Thr Arg1 5 10 15Gly Gly Ser His Leu Gln Ala Ala Ala Gln Thr Pro Pro
Arg Pro Gly 20 25 30Pro Pro Ser Ala Pro Pro Pro Pro Pro Lys Glu Gly
His Gln Glu Gly 35 40 45Leu Val Glu Leu Pro Ala Ser Phe Arg Glu Leu
Leu Thr Phe Phe Cys 50 55 60Thr Asn Ala Thr Ile His Gly Ala Ile Arg
Leu Val Cys Ser Arg Gly65 70 75 80Asn Arg Leu Lys Thr Thr Ser Trp
Gly Leu Leu Ser Leu Gly Ala Leu 85 90 95Val Ala Leu Cys Trp Gln Leu
Gly Leu Leu Phe Glu Arg His Trp His 100 105 110Arg Pro Val Leu Met
Ala Val Ser Val His Ser Glu Arg Lys Leu Leu 115 120 125Pro Leu Val
Thr Leu Cys Asp Gly Asn Pro Arg Arg Pro Ser Pro Val 130 135 140Leu
Arg His Leu Glu Leu Leu Asp Glu Phe Ala Arg Glu Asn Ile Asp145 150
155 160Ser Leu Tyr Asn Val Asn Leu Ser Lys Gly Arg Ala Ala Leu Ser
Ala 165 170 175Thr Val Pro Arg His Glu Pro Pro Phe His Leu Asp Arg
Glu Ile Arg 180 185 190Leu Gln Arg Leu Ser His Ser Gly Ser Arg Val
Arg Val Gly Phe Arg 195 200 205Leu Cys Asn Ser Thr Gly Gly Asp Cys
Phe Tyr Arg Gly Tyr Thr Ser 210 215 220Gly Val Ala Ala Val Gln Asp
Trp Tyr His Phe His Tyr Val Asp Ile225 230 235 240Leu Ala Leu Leu
Pro Ala Ala Trp Glu Asp Ser His Gly Ser Gln Asp 245 250 255Gly His
Phe Val Leu Ser Cys Ser Tyr Asp Gly Leu Asp Cys Gln Ala 260 265
270Arg Gln Phe Arg Thr Phe His His Pro Thr Tyr Gly Ser Cys Tyr Thr
275 280 285Val Asp Gly Val Trp Thr Ala Gln Arg Pro Gly Ile Thr His
Gly Val 290 295 300Gly Leu Val Leu Arg Val Glu Gln Gln Pro His Leu
Pro Leu Leu Ser305 310 315 320Thr Leu Ala Gly Ile Arg Val Met Val
His Gly Arg Asn His Thr Pro 325 330 335Phe Leu Gly His His Ser Phe
Ser Val Arg Pro Gly Thr Glu Ala Thr 340 345 350Ile Ser Ile Arg Glu
Asp Glu Val His Arg Leu Gly Ser Pro Tyr Gly 355 360 365His Cys Thr
Ala Gly Gly Glu Gly Val Glu Val Glu Leu Leu His Asn 370 375 380Thr
Ser Tyr Thr Arg Gln Ala Cys Leu Val Ser Cys Phe Gln Gln Leu385 390
395 400Met Val Glu Thr Cys Ser Cys Gly Tyr Tyr Leu His Pro Leu Pro
Ala 405 410 415Gly Ala Glu Tyr Cys Ser Ser Ala Arg His Pro Ala Trp
Gly His Cys 420 425 430Phe Tyr Arg Leu Tyr Gln Asp Leu Glu Thr His
Arg Leu Pro Cys Thr 435 440 445Ser Arg Cys Pro Arg Pro Cys Arg Glu
Ser Ala Phe Lys Leu Ser Thr 450 455 460Gly Thr Ser Arg Trp Pro Ser
Ala Lys Ser Ala Gly Trp Thr Leu Ala465 470 475 480Thr Leu Gly Glu
Gln Gly Leu Pro His Gln Ser His Arg Gln Arg Ser 485 490 495Ser Leu
Ala Lys Ile Asn Ile Val Tyr Gln Glu Leu Asn Tyr Arg Ser 500 505
510Val Glu Glu Ala Pro Val Tyr Ser Val Pro Gln Leu Leu Ser Ala Met
515 520 525Gly Ser Leu Tyr Ser Leu Trp Phe Gly Ala Ser Val Leu Ser
Leu Leu 530 535 540Glu Leu Leu Glu Leu Leu Leu Asp Ala Ser Ala Leu
Thr Leu Val Leu545 550 555 560Gly Gly Arg Arg Leu Arg Arg Ala Trp
Phe Ser Trp Pro Arg Ala Ser 565 570 575Pro Ala Ser Gly Ala Ser Ser
Ile Lys Pro Glu Ala Ser Gln Met Pro 580 585 590Pro Pro Ala Gly Gly
Thr Ser Asp Asp Pro Glu Pro Ser Gly Pro His 595 600 605Leu Pro Arg
Val Met Leu Pro Gly Val Leu Ala Gly Val Ser Ala Glu 610 615 620Glu
Ser Trp Ala Gly Pro Gln Pro Leu Glu Thr Leu Asp Thr625 630
6359638PRTHomo Sapiens 9Met Ala Glu His Arg Ser Met Asp Gly Arg Met
Glu Ala Ala Thr Arg1 5 10 15Gly Gly Ser His Leu Gln Ala Ala Ala Gln
Thr Pro Pro Arg Pro Gly 20 25 30Pro Pro Ser Ala Pro Pro Pro Pro Pro
Lys Glu Gly His Gln Glu Gly 35 40 45Leu Val Glu Leu Pro Ala Ser Phe
Arg Glu Leu Leu Thr Phe Phe Cys 50 55 60Thr Asn Ala Thr Ile His Gly
Ala Ile Arg Leu Val Cys Ser Arg Gly65 70 75 80Asn Arg Leu Lys Thr
Thr Ser Trp Gly Leu Leu Ser Leu Gly Ala Leu 85 90 95Val Ala Leu Cys
Trp Gln Leu Gly Leu Leu Phe Glu Arg His Trp His 100 105 110Arg Pro
Val Leu Met Ala Val Ser Val His Ser Glu Arg Lys Leu Leu 115 120
125Pro Leu Val Thr Leu Cys Asp Gly Asn Pro Arg Arg Pro Ser Pro Val
130 135 140Leu Arg His Leu Glu Leu Leu Asp Glu Phe Ala Arg Glu Asn
Ile Asp145 150 155 160Ser Leu Tyr Asn Val Asn Leu Ser Lys Gly Arg
Ala Ala Leu Ser Ala 165 170 175Thr Val Pro Arg His Glu Pro Pro Phe
His Leu Asp Arg Glu Ile Arg 180 185 190Leu Gln Arg Leu Ser His Ser
Gly Ser Arg Val Arg Val Gly Phe Arg 195 200 205Leu Cys Asn Ser Thr
Gly Gly Asp Cys Phe Tyr Arg Gly Tyr Thr Ser 210 215 220Gly Val Ala
Ala Val Gln Asp Trp Tyr His Phe His Tyr Val Asp Ile225 230 235
240Leu Ala Leu Leu Pro Ala Ala Trp Glu Asp Ser His Gly Ser Gln Asp
245 250 255Gly His Phe Val Leu Ser Cys Ser Tyr Asp Gly Leu Asp Cys
Gln Ala 260 265 270Arg Gln Phe Arg Thr Phe His His Pro Thr Tyr Gly
Ser Cys Tyr Thr 275 280 285Val Asp Gly Val Trp Thr Ala Gln Arg Pro
Gly Ile Thr His Gly Val 290 295 300Gly Leu Val Leu Arg Val Glu Gln
Gln Pro His Leu Pro Leu Leu Ser305 310 315 320Thr Leu Ala Gly Ile
Arg Val Met Val His Gly Arg Asn His Thr Pro 325 330 335Phe Leu Gly
His His Ser Phe Ser Val Arg Pro Gly Thr Glu Ala Thr 340 345 350Ile
Ser Ile Arg Glu Asp Glu Val His Arg Leu Gly Ser Pro Tyr Gly 355 360
365His Cys Thr Ala Gly Gly Glu Gly Val Glu Val Glu Leu Leu His Asn
370 375 380Thr Ser Tyr Thr Arg Gln Ala Cys Leu Val Ser Cys Phe Gln
Gln Leu385 390 395 400Met Val Glu Thr Cys Ser Cys Gly Tyr Tyr Leu
His Pro Leu Pro Ala 405 410 415Gly Ala Glu Tyr Cys Ser Ser Ala Arg
His Pro Ala Trp Gly His Cys 420 425 430Phe Tyr Arg Leu Tyr Gln Asp
Leu Glu Thr His Arg Leu Pro Cys Thr 435 440 445Ser Arg Cys Pro Arg
Pro Cys Arg Glu Ser Ala Phe Lys Leu Ser Thr 450 455 460Gly Thr Ser
Arg Trp Pro Ser Ala Lys Ser Ala Gly Trp Thr Leu Ala465 470 475
480Thr Leu Gly Glu Gln Gly Leu Pro His Gln Ser His Arg Gln Arg Ser
485 490 495Ser Leu Ala Lys Ile Asn Ile Val Tyr Gln Glu Leu Asn Tyr
Arg Ser 500 505 510Val Glu Glu Ala Pro Val Tyr Ser Val Pro Gln Leu
Leu Ser Ala Met 515 520 525Gly Ser Leu Cys Ser Leu Trp Phe Gly Ala
Ser Val Leu Ser Leu Leu 530 535 540Glu Leu Leu Glu Leu Leu Leu Asp
Ala Ser Ala Leu Thr Leu Val Leu545 550 555 560Gly Gly Arg Arg Leu
Arg Arg Ala Trp Phe Ser Trp Pro Arg Ala Ser 565 570 575Pro Ala Ser
Gly Ala Ser Ser Ile Lys Pro Glu Ala Ser Gln Met Pro 580 585 590Pro
Pro Ala Gly Gly Thr Ser Asp Asp Pro Glu Pro Ser Gly Pro His 595 600
605Leu Pro Arg Val Met Leu Pro Gly Val Leu Ala Gly Val Ser Ala Glu
610 615 620Glu Ser Trp Ala Gly Pro Gln Pro Leu Glu Thr Leu Asp
Thr625 630 6351011PRTHomo Sapiens 10Met Gly Ser Gln Trp Ser Leu Trp
Phe Gly Ala1 5 101111PRTHomo Sapiens 11Met Gly Ser Leu Tyr Ser Leu
Trp Phe Gly Ala1 5 101211PRTHomo Sapiens 12Met Gly Ser Leu Cys Ser
Leu Trp Phe Gly Ala1 5 1013649PRTHomo Sapiens 13Met Ala Pro Gly Glu
Lys Ile Lys Ala Lys Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro
Gln Ala Pro Thr Ile Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn
Thr Asn Thr His Gly Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg
Leu Arg Arg Leu Leu Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala
Leu Ile Leu Trp Gln Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75
80Val Ser Val Ser Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala
85 90 95Val Thr Ile Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg
His 100 105 110Leu Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu
Lys Ser Leu 115 120 125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu
Ala Glu Ser Trp Asn 130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg
Phe Ser His Arg Ile Pro Pro145 150 155 160Leu Ile Phe Asp Gln Asp
Glu Lys Gly Lys Ala Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg
Lys Val Gly Gly Ser Ile Ile His Lys Ala Ser Asn 180 185 190Val Met
His Ile Glu Ser Lys Gln Val Val Gly Phe Gln Leu Cys Ser 195 200
205Asn Asp Thr Ser Asp Cys Ala Thr Tyr Thr Phe Ser Leu Gly Ile Asn
210 215 220Ala Ile Gln Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met
Ala Gln225 230 235 240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr
Ser Ala Glu Glu Leu 245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val
Ser Cys Asp Ala Arg Asn Phe 260 265 270Thr Leu Phe His His Pro Met
His Gly Asn Cys Tyr Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr
Ile Leu Ser Thr Ser Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln
Val Ile Leu Tyr Ile Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315
320Leu Val Ser Ser Thr Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu
325 330 335Tyr Pro Phe Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala
Met Val 340
345 350Thr Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu
Pro 355 360 365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val Pro Ile
Arg Asn Ile 370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu
His Ser Cys Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly
Cys Ala Gln Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr
Cys Asn Tyr Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr
Gln Leu His Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln
Ser Val Cys Lys Glu Ala Cys Ser Leu Lys Glu Trp Thr Leu 450 455
460Thr Thr Ser Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp
Leu465 470 475 480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val
Asn Lys Lys Leu 485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile
Phe Tyr Lys Asp Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro
Ala Asn Ser Ile Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln
Leu Gly Leu Trp Met Ser Cys Ser Val Val 530 535 540Cys Val Val Glu
Ile Ile Glu Val Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Ile
Ala Arg Arg Gln Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570
575Gln Ala Pro Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp
580 585 590Asn Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr Phe Asn
Ser Ala 595 600 605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro
Gly Thr Pro Pro 610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg
Ala Phe Ser Asn Gln Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp
Glu Leu 64514649PRTHomo Sapiens 14Met Ala Pro Gly Glu Lys Ile Lys
Ala Lys Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro
Thr Ile Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr
His Gly Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg
Leu Leu Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu
Trp Gln Cys Ala Leu Leu Val Phe Ser Phe Tyr Ala65 70 75 80Val Ser
Val Ser Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val
Thr Ile Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105
110Leu Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu
115 120 125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser
Trp Asn 130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His
Arg Ile Pro Leu145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly
Lys Ala Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly
Gly Ser Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu
Ser Lys Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr
Ser Asp Cys Ala Thr Tyr Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala
Ile Gln Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230
235 240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu
Leu 245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala
Arg Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys
Tyr Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr
Ser Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr
Ile Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser
Thr Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro
Phe Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala Met Val 340 345
350Thr Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro
355 360 365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val Pro Ile Arg
Asn Ile 370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His
Ser Cys Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly Cys
Ala Gln Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys
Asn Tyr Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln
Leu His Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser
Val Cys Lys Glu Ala Cys Ser Phe Lys Glu Trp Thr Leu 450 455 460Thr
Thr Ser Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp Leu465 470
475 480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val Asn Lys Lys
Leu 485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys
Asp Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro Ala Asn Ser
Ile Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln Leu Gly Leu
Trp Met Ser Cys Ser Val Val 530 535 540Cys Val Ile Glu Ile Ile Glu
Val Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Ile Ala Arg Arg
Gln Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570 575Gln Ala
Pro Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp 580 585
590Asn Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr Phe Asn Ser Ala
595 600 605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro Gly Thr
Pro Pro 610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala Phe
Ser Asn Ser Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp Glu Leu
64515649PRTHomo Sapiens 15Met Ala Pro Gly Glu Lys Ile Lys Ala Lys
Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro Thr Ile
Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr His Gly
Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu Leu
Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu Trp Gln
Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75 80Val Ser Val Ser
Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val Thr Ile
Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105 110Leu
Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu 115 120
125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser Trp Asn
130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His Arg Ile
Pro Leu145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly Lys Ala
Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly Gly Ser
Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu Ser Lys
Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr Ser Asp
Cys Ala Thr Tyr Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala Ile Gln
Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230 235
240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu Leu
245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala Arg
Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys Tyr
Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr Ser
Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr Ile
Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser Thr
Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro Phe
Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala Met Val 340 345 350Thr
Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro 355 360
365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val Pro Ile Arg Asn Ile
370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His Ser Cys
Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly Cys Ala Gln
Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr
Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln Leu His
Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser Val Cys
Lys Glu Ala Cys Ser Phe Lys Glu Trp Thr Leu 450 455 460Thr Thr Ser
Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp Leu465 470 475
480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val Asn Lys Lys Leu
485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys Asp
Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro Ala Asn Ser Ile
Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln Leu Gly Leu Trp
Met Ser Cys Ser Val Val 530 535 540Cys Val Ile Glu Ile Ile Glu Val
Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Ile Ala Arg Arg Gln
Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570 575Gln Ala Pro
Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp 580 585 590Asn
Pro Ala Leu Asp Ile Asp Asp Gly Leu Pro Thr Phe Asn Ser Ala 595 600
605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro Gly Thr Pro Pro
610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala Phe Ser Asn
Gln Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp Glu Leu
64516649PRTHomo Sapiens 16Met Ala Pro Gly Glu Lys Ile Lys Ala Lys
Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro Thr Ile
Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr His Gly
Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu Leu
Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu Trp Gln
Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75 80Val Ser Val Ser
Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val Thr Ile
Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105 110Leu
Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu 115 120
125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser Trp Asn
130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His Arg Ile
Pro Leu145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly Lys Ala
Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly Gly Ser
Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu Ser Lys
Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr Ser Asp
Cys Ala Thr Tyr Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala Ile Gln
Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230 235
240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu Leu
245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala Arg
Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys Tyr
Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr Ser
Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr Ile
Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser Thr
Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro Ser
Val Glu Asp Val Gly Thr Glu Ile Glu Thr Thr Met Val 340 345 350Thr
Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro 355 360
365Ser Ser Gln Cys Thr Glu Gly Gly Ser Asp Val Pro Ile Arg Asn Ile
370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His Ser Cys
Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly Cys Ala Gln
Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr
Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln Leu His
Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser Val Cys
Lys Glu Ala Cys Arg Phe Lys Glu Trp Thr Leu 450 455 460Thr Thr Ser
Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp Leu465 470 475
480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val Asn Lys Lys Leu
485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys Asp
Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro Ala Asn Ser Ile
Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln Leu Gly Leu Trp
Met Ser Cys Ser Val Val 530 535 540Cys Val Ile Glu Ile Ile Glu Val
Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Ile Ala Arg Arg Gln
Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570 575Gln Ala Pro
Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp 580 585 590Asn
Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr Phe Asn Ser Ala 595 600
605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro Gly Thr Pro Pro
610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala Phe Ser Asn
Gln Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp Glu Leu
64517638PRTHomo Sapiens 17Met Ala Glu His Arg Ser Met Asp Gly Arg
Met Glu Ala Ala Thr Arg1 5 10 15Gly Gly Ser His Leu Gln Ala Ala Ala
Gln Thr Pro Pro Arg Pro Gly 20 25 30Pro Pro Ser Ala Pro Pro Pro Pro
Pro Lys Glu Gly His Gln Glu Gly 35 40 45Leu Val Glu Leu Pro Ala Ser
Phe Arg Glu Leu Leu Thr Phe Phe Cys 50 55 60Thr Asn Ala Thr Ile His
Gly Ala Ile Arg Leu Val Cys Ser Arg Gly65 70 75 80Asn Arg Leu Lys
Thr Thr Ser Trp Gly Leu Leu Ser Leu Gly Ala Leu 85 90 95Val Ala Leu
Cys Trp Gln Leu Gly Leu Leu Phe Glu Arg His Trp His 100 105 110Arg
Pro Val Leu Met Ala Val Ser Val His Ser Glu Arg Lys Leu Leu 115 120
125Pro Leu Val Thr Leu Cys Asp Gly Asn Pro Arg Arg Pro Ser Pro Val
130 135 140Leu Arg His Leu Glu Leu Leu Asp Glu Phe Ala Arg Glu Asn
Ile Asp145 150 155 160Ser Leu Tyr Asn Val Asn Leu Ser Lys Gly Arg
Ala Ala Leu Ser Ala 165 170 175Thr Val Pro Arg His Glu Pro Pro Phe
His Leu Asp Arg Glu Ile Arg 180 185 190Leu Gln Arg Leu Ser His Ser
Gly Ser Arg Val Arg Val Gly Phe Arg 195 200 205Leu Cys Asn Ser Thr
Gly Gly Asp Cys Phe Tyr Arg Gly Tyr Thr Ser 210
215 220Gly Val Ala Ala Val Gln Asp Trp Tyr His Phe His Tyr Val Asp
Ile225 230 235 240Leu Ala Leu Leu Pro Ala Ala Trp Glu Asp Ser His
Gly Ser Gln Asp 245 250 255Gly His Phe Val Leu Ser Cys Ser Tyr Asp
Gly Leu Asp Cys Gln Ala 260 265 270Arg Gln Phe Arg Thr Ile His His
Pro Thr Tyr Gly Ser Cys Tyr Thr 275 280 285Val Asp Gly Val Trp Thr
Ala Gln Arg Pro Gly Ile Thr His Gly Val 290 295 300Gly Leu Val Leu
Arg Val Glu Gln Gln Pro His Leu Pro Leu Leu Ser305 310 315 320Thr
Leu Ala Gly Ile Arg Val Met Val His Gly Arg Asn His Thr Pro 325 330
335Phe Leu Gly His His Ser Phe Ser Val Arg Pro Gly Thr Glu Ala Thr
340 345 350Ile Arg Ile Arg Glu Asp Glu Val His Arg Leu Gly Ser Pro
Tyr Gly 355 360 365His Cys Thr Ala Gly Gly Glu Gly Val Glu Val Glu
Leu Leu His Asn 370 375 380Thr Ser Tyr Thr Arg Gln Ala Cys Leu Val
Ser Cys Phe Gln Gln Leu385 390 395 400Met Val Glu Thr Cys Ser Cys
Gly Tyr Tyr Leu His Pro Leu Pro Ala 405 410 415Gly Ala Glu Tyr Cys
Ser Ser Ala Arg His Pro Ala Trp Gly His Cys 420 425 430Phe Tyr Arg
Leu Tyr Gln Asp Leu Glu Thr His Arg Leu Pro Cys Thr 435 440 445Ser
Arg Cys Pro Arg Pro Cys Arg Glu Ser Ala Phe Lys Leu Ser Thr 450 455
460Gly Thr Ser Arg Trp Pro Ser Ala Lys Ser Ala Gly Trp Thr Leu
Ala465 470 475 480Thr Leu Gly Glu Gln Gly Leu Pro His Gln Ser His
Arg Gln Arg Ser 485 490 495Ser Leu Ala Lys Ile Asn Ile Val Tyr Gln
Glu Leu Asn Tyr Arg Ser 500 505 510Val Glu Glu Ala Pro Val Tyr Ser
Val Pro Gln Leu Leu Ser Ala Met 515 520 525Gly Ser Leu Cys Ser Leu
Trp Phe Gly Ala Ser Val Leu Ser Leu Leu 530 535 540Glu Leu Leu Glu
Leu Leu Leu Asp Ala Ser Ala Leu Thr Leu Val Leu545 550 555 560Gly
Gly Arg Arg Leu His Arg Ala Trp Phe Ser Trp Pro Arg Ala Ser 565 570
575Pro Ala Ser Gly Ala Ser Ser Ile Lys Pro Glu Ala Ser Gln Met Pro
580 585 590Pro Pro Ala Gly Gly Thr Ser Asp Asp Pro Glu Pro Ser Gly
Pro His 595 600 605Leu Pro Arg Val Met Leu Pro Gly Val Leu Ala Gly
Val Ser Ala Glu 610 615 620Glu Ser Trp Ala Gly Pro Gln Pro Leu Glu
Thr Leu Asp Thr625 630 63518638PRTHomo Sapiens 18Met Ala Glu His
Arg Ser Met Asp Gly Arg Met Glu Ala Ala Thr Arg1 5 10 15Gly Gly Ser
His Leu Gln Ala Ala Ala Gln Thr Pro Pro Arg Pro Gly 20 25 30Pro Pro
Ser Ala Pro Pro Pro Pro Pro Lys Glu Gly His Gln Glu Gly 35 40 45Leu
Val Glu Leu Pro Ala Ser Phe Arg Glu Leu Leu Thr Phe Phe Cys 50 55
60Thr Asn Ala Thr Ile His Gly Ala Ile Arg Leu Val Cys Ser Arg Gly65
70 75 80Asn Arg Leu Lys Thr Thr Ser Trp Gly Leu Leu Ser Leu Gly Ala
Leu 85 90 95Val Ala Leu Cys Trp Gln Leu Gly Leu Leu Phe Glu Arg His
Trp His 100 105 110Arg Pro Val Leu Met Ala Val Ser Val His Ser Glu
Arg Lys Leu Leu 115 120 125Pro Leu Val Thr Leu Cys Asp Gly Asn Pro
Arg Arg Pro Ser Pro Val 130 135 140Leu Arg His Leu Glu Leu Leu Asp
Glu Phe Ala Arg Glu Asn Ile Asp145 150 155 160Ser Leu Tyr Asn Val
Asn Leu Ser Lys Gly Arg Ala Ala Leu Ser Ala 165 170 175Thr Val Pro
Arg His Glu Pro Pro Phe His Leu Asp Arg Glu Ile Arg 180 185 190Leu
Gln Arg Leu Ser His Ser Gly Ser Arg Val Arg Val Gly Phe Arg 195 200
205Leu Cys Asn Ser Thr Gly Gly Asp Cys Phe Tyr Arg Gly Tyr Thr Ser
210 215 220Gly Val Ala Ala Val Gln Asp Trp Tyr His Phe His Tyr Val
Asp Ile225 230 235 240Leu Ala Leu Leu Pro Ala Ala Trp Glu Asp Ser
His Gly Ser Gln Asp 245 250 255Gly His Phe Val Leu Ser Cys Ser Tyr
Asp Gly Leu Asp Cys Gln Ala 260 265 270Arg Gln Phe Arg Thr Phe His
His Pro Thr Tyr Gly Ser Cys Tyr Thr 275 280 285Val Asp Gly Val Trp
Thr Ala Gln Arg Pro Gly Ile Thr His Gly Val 290 295 300Gly Leu Val
Leu Arg Val Glu Gln Gln Pro His Leu Pro Leu Leu Ser305 310 315
320Thr Leu Ala Gly Ile Arg Val Met Val His Gly Arg Asn His Thr Pro
325 330 335Phe Leu Gly His His Ser Phe Ser Val Arg Pro Gly Thr Glu
Ala Thr 340 345 350Ile Ser Ile Arg Glu Asp Glu Val His Arg Leu Gly
Ser Pro Tyr Gly 355 360 365His Cys Thr Ala Gly Gly Glu Gly Val Glu
Val Glu Leu Leu His Asn 370 375 380Thr Ser Tyr Thr Arg Gln Ala Cys
Leu Val Ser Cys Phe Gln Gln Leu385 390 395 400Met Val Glu Thr Cys
Ser Cys Gly Tyr Tyr Leu His Pro Leu Pro Ala 405 410 415Gly Ala Glu
Tyr Cys Ser Ser Ala Arg His Pro Ala Trp Gly His Cys 420 425 430Phe
Tyr Arg Leu Tyr Gln Asp Leu Glu Thr His Arg Leu Pro Cys Thr 435 440
445Ser Arg Cys Pro Arg Pro Cys Arg Glu Ser Ala Phe Lys Leu Ser Thr
450 455 460Gly Thr Ser Arg Trp Pro Ser Ala Lys Ser Ala Gly Trp Thr
Leu Ala465 470 475 480Thr Leu Gly Glu Gln Gly Leu Pro His Gln Ser
His Arg Gln Arg Ser 485 490 495Ser Leu Ala Lys Ile Asn Ile Val Tyr
Gln Glu Leu Asn Tyr Arg Ser 500 505 510Val Glu Glu Ala Pro Val Tyr
Ser Val Pro Gln Leu Leu Ser Ala Met 515 520 525Gly Ser Leu Cys Ser
Leu Trp Phe Gly Ala Ser Val Leu Ser Leu Leu 530 535 540Glu Leu Leu
Glu Leu Leu Leu Asp Ala Ser Ala Leu Thr Leu Val Leu545 550 555
560Gly Gly Arg Arg Leu Arg Arg Ala Trp Phe Ser Trp Pro Arg Ala Ser
565 570 575Pro Ala Ser Gly Ala Ser Ser Ile Lys Pro Glu Ala Ser Gln
Met Pro 580 585 590Pro Pro Ala Gly Gly Thr Ser Asp Asp Pro Glu Pro
Ser Gly Pro His 595 600 605Leu Pro Arg Val Met Leu Pro Gly Val Leu
Ala Gly Val Ser Ala Glu 610 615 620Glu Ser Trp Ala Gly Pro Gln Pro
Leu Glu Thr Leu Asp Thr625 630 63519638PRTHomo Sapiens 19Met Ala
Glu His Arg Ser Met Asp Gly Arg Met Glu Ala Ala Thr Arg1 5 10 15Gly
Gly Pro His Leu Gln Ala Ala Ala Gln Thr Pro Pro Arg Pro Gly 20 25
30Pro Pro Ser Ala Pro Pro Pro Pro Pro Lys Glu Gly His Gln Glu Gly
35 40 45Leu Val Glu Leu Pro Ala Ser Phe Arg Glu Leu Leu Thr Phe Phe
Cys 50 55 60Thr Asn Ala Thr Ile His Gly Ala Ile Arg Leu Val Cys Ser
Arg Gly65 70 75 80Asn Arg Leu Lys Thr Thr Ser Trp Gly Leu Leu Ser
Leu Gly Ala Leu 85 90 95Val Ala Leu Cys Trp Gln Leu Gly Leu Leu Phe
Glu Arg His Trp His 100 105 110Arg Pro Val Leu Met Ala Val Ser Val
His Ser Glu Arg Lys Leu Leu 115 120 125Pro Leu Val Thr Leu Cys Asp
Gly Asn Pro Arg Arg Pro Ser Pro Val 130 135 140Leu Arg His Leu Glu
Leu Leu Asp Glu Phe Ala Arg Glu Asn Ile Asp145 150 155 160Ser Leu
Tyr Asn Val Asn Leu Ser Lys Gly Arg Ala Ala Leu Ser Ala 165 170
175Thr Val Pro Pro His Glu Pro Pro Phe His Leu Asp Arg Glu Ile Arg
180 185 190Leu Gln Ser Leu Ser His Ser Gly Ser Arg Val Arg Val Gly
Phe Arg 195 200 205Leu Cys Asn Ser Thr Gly Gly Asp Cys Phe Tyr Arg
Gly Tyr Thr Ser 210 215 220Gly Val Ala Ala Val Gln Asp Trp Tyr His
Phe His Tyr Val Asp Ile225 230 235 240Leu Ala Leu Leu Pro Ala Ala
Trp Glu Asp Ser His Gly Ser Gln Asp 245 250 255Gly His Phe Val Leu
Ser Cys Ser Tyr Asp Gly Leu Asp Cys Gln Ala 260 265 270Arg Gln Phe
Arg Thr Phe His His Pro Thr Tyr Gly Ser Cys Tyr Thr 275 280 285Val
Asp Gly Val Trp Thr Ala Gln Arg Pro Gly Ile Thr His Gly Val 290 295
300Gly Leu Val Leu Arg Val Glu Gln Gln Pro His Leu Pro Leu Leu
Ser305 310 315 320Thr Leu Ala Gly Ile Arg Val Met Val His Gly Arg
Asn His Thr Pro 325 330 335Phe Leu Gly His His Ser Phe Ser Val Arg
Pro Gly Thr Glu Ala Thr 340 345 350Ile Ser Ile Arg Glu Asp Glu Val
His Arg Leu Gly Ser Pro Tyr Gly 355 360 365His Cys Thr Ala Gly Gly
Glu Gly Val Glu Val Glu Leu Leu His Asn 370 375 380Thr Ser Tyr Thr
Arg Gln Ala Cys Leu Val Ser Cys Phe Gln Gln Leu385 390 395 400Met
Val Glu Thr Cys Ser Cys Gly Tyr Tyr Leu His Pro Leu Pro Ala 405 410
415Gly Ala Glu Tyr Cys Ser Ser Ala Arg His Pro Ala Trp Gly His Cys
420 425 430Phe Tyr Arg Leu Tyr Gln Asp Leu Glu Thr His Arg Leu Pro
Cys Thr 435 440 445Ser Arg Cys Pro Arg Pro Cys Arg Glu Ser Ala Phe
Lys Leu Ser Thr 450 455 460Gly Thr Ser Arg Trp Pro Ser Ala Lys Ser
Ala Gly Trp Thr Leu Ala465 470 475 480Thr Leu Gly Glu Gln Gly Leu
Pro His Gln Ser His Arg Gln Arg Ser 485 490 495Ser Leu Ala Lys Ile
Asn Ile Val Tyr Gln Glu Leu Asn Tyr Arg Ser 500 505 510Val Glu Glu
Ala Pro Val Tyr Ser Val Pro Gln Leu Leu Ser Ala Met 515 520 525Gly
Ser Leu Cys Ser Leu Trp Phe Gly Ala Ser Val Leu Ser Leu Leu 530 535
540Glu Leu Leu Glu Leu Leu Leu Asp Ala Ser Ala Leu Thr Leu Val
Leu545 550 555 560Gly Gly Arg Arg Leu Arg Arg Ala Trp Phe Ser Trp
Pro Arg Ala Ser 565 570 575Pro Ala Ser Gly Ala Ser Ser Ile Lys Pro
Glu Ala Ser Gln Met Pro 580 585 590Pro Pro Ala Gly Gly Thr Ser Asp
Asp Pro Glu Pro Ser Gly Pro His 595 600 605Leu Pro Arg Val Met Leu
Pro Gly Val Leu Ala Gly Val Ser Ala Glu 610 615 620Glu Ser Trp Ala
Gly Pro Gln Pro Leu Glu Thr Leu Asp Lys625 630 63520638PRTHomo
Sapiens 20Met Ala Glu His Arg Ser Met Asp Gly Arg Met Glu Ala Ala
Thr Arg1 5 10 15Gly Gly Pro His Leu Gln Ala Ala Ala Gln Thr Pro Pro
Arg Pro Gly 20 25 30Pro Pro Ser Ala Pro Pro Pro Pro Pro Lys Glu Gly
His Gln Glu Gly 35 40 45Leu Val Glu Leu Pro Ala Ser Phe Arg Glu Leu
Leu Thr Phe Phe Cys 50 55 60Thr Asn Ala Thr Ile His Gly Ala Ile Arg
Leu Val Cys Ser Arg Gly65 70 75 80Asn Arg Leu Lys Thr Thr Ser Trp
Gly Leu Leu Ser Leu Gly Ala Leu 85 90 95Val Ala Leu Cys Trp Gln Leu
Gly Leu Leu Phe Glu Arg His Trp His 100 105 110Arg Pro Val Leu Met
Ala Val Ser Val His Ser Glu Arg Lys Leu Leu 115 120 125Pro Leu Val
Thr Leu Cys Asp Gly Asn Pro Arg Arg Pro Ser Pro Val 130 135 140Leu
Arg His Leu Glu Leu Leu Asp Glu Phe Ala Arg Glu Asn Ile Asp145 150
155 160Ser Leu Tyr Asn Val Asn Leu Ser Lys Gly Arg Ala Ala Leu Ser
Ala 165 170 175Thr Val Pro Arg His Glu Pro Pro Phe His Leu Asp Arg
Glu Ile Arg 180 185 190Leu Gln Arg Leu Ser His Ser Gly Ser Arg Val
Arg Val Gly Phe Arg 195 200 205Leu Cys Asn Ser Thr Gly Gly Asp Cys
Phe Tyr Arg Gly Tyr Thr Ser 210 215 220Gly Val Ala Ala Val Gln Asp
Trp Tyr His Phe His Tyr Val Asp Ile225 230 235 240Leu Ala Leu Leu
Pro Ala Ala Trp Glu Asp Ser His Gly Ser Gln Asp 245 250 255Gly His
Phe Val Leu Ser Cys Ser Tyr Asp Gly Leu Asp Cys Gln Ala 260 265
270Arg Gln Phe Arg Thr Phe His His Pro Thr Tyr Gly Ser Cys Tyr Thr
275 280 285Val Asp Gly Val Trp Thr Ala Gln Arg Pro Gly Ile Thr His
Gly Val 290 295 300Gly Leu Val Leu Arg Val Glu Gln Gln Pro His Leu
Pro Leu Leu Ser305 310 315 320Thr Leu Ala Gly Ile Arg Val Met Val
His Gly Arg Asn His Thr Pro 325 330 335Phe Leu Gly His His Ser Phe
Ser Val Arg Pro Gly Thr Glu Ala Thr 340 345 350Ile Ser Ile Arg Glu
Asp Glu Val His Arg Leu Gly Ser Pro Tyr Gly 355 360 365His Cys Thr
Ala Gly Gly Glu Gly Val Glu Val Glu Leu Leu His Asn 370 375 380Thr
Ser Tyr Thr Arg Gln Ala Cys Leu Val Ser Cys Phe Gln Gln Leu385 390
395 400Met Val Glu Thr Cys Ser Cys Gly Tyr Tyr Leu His Pro Leu Pro
Ala 405 410 415Gly Ala Glu Tyr Cys Ser Ser Ala Arg His Pro Ala Trp
Gly His Cys 420 425 430Phe Tyr Arg Leu Tyr Gln Asp Leu Glu Thr His
Arg Leu Pro Cys Thr 435 440 445Ser Arg Cys Pro Arg Pro Cys Arg Glu
Ser Ala Phe Lys Leu Ser Thr 450 455 460Gly Thr Ser Arg Trp Pro Ser
Ala Lys Ser Ala Gly Trp Thr Leu Ala465 470 475 480Thr Leu Gly Glu
Gln Gly Leu Pro His Gln Ser His Arg Gln Arg Ser 485 490 495Ser Leu
Ala Lys Ile Asn Ile Val Tyr Gln Gly Leu Asn Tyr Arg Ser 500 505
510Val Glu Glu Ala Pro Val Tyr Ser Val Pro Gln Leu Leu Ser Ala Met
515 520 525Gly Ser Leu Cys Ser Leu Trp Phe Gly Ala Ser Val Leu Ser
Leu Leu 530 535 540Glu Leu Leu Glu Leu Leu Leu Asp Ala Ser Ala Leu
Thr Leu Val Leu545 550 555 560Gly Gly Arg Arg Leu Arg Arg Ala Trp
Phe Ser Trp Pro Arg Ala Ser 565 570 575Pro Ala Ser Gly Ala Ser Ser
Ile Lys Pro Glu Ala Ser Gln Met Pro 580 585 590Pro Pro Ala Gly Gly
Thr Ser Asp Asp Pro Glu Pro Ser Gly Pro His 595 600 605Leu Pro Arg
Val Met Leu Pro Gly Val Leu Ala Gly Val Ser Ala Glu 610 615 620Glu
Ser Trp Ala Gly Pro Gln Pro Leu Glu Thr Leu Asp Thr625 630
63521638PRTHomo Sapiens 21Met Ala Glu His Arg Ser Met Asp Gly Arg
Met Glu Ala Ala Thr Arg1 5 10 15Gly Gly Ser His Leu Gln Ala Ala Ala
Gln Thr Pro Pro Arg Pro Gly 20 25 30Pro Pro Ser Ala Pro Pro Pro Pro
Pro Lys Glu Gly His Gln Glu Gly 35 40 45Leu Val Glu Leu Pro Ala Ser
Phe Arg Glu Leu Leu Thr Phe Phe Cys 50 55 60Thr Asn Ala Thr Ile His
Gly Thr Ile Arg Leu Val Cys Ser Arg Gly65 70 75 80Asn Arg Leu Lys
Thr Thr Ser Trp Gly Leu Leu Ser Leu Gly Ala Leu 85 90 95Val Ala Leu
Cys Trp Gln Leu Gly Leu Leu Phe Glu Arg His Trp His 100 105 110Arg
Pro Val Leu Met Ala Val Ser Val His Ser Glu Arg Lys Leu Leu 115 120
125Pro Leu Val Thr Leu Cys Asp Gly Asn Pro Arg Arg Pro Ser Pro
Val
130 135 140Leu Arg His Leu Glu Leu Leu Asp Glu Phe Ala Arg Glu Asn
Ile Asp145 150 155 160Ser Leu Tyr Asn Val Asn Leu Ser Lys Gly Arg
Ala Ala Leu Ser Ala 165 170 175Thr Val Pro Pro His Glu Pro Pro Phe
His Leu Asp Arg Glu Ile Arg 180 185 190Leu Gln Arg Leu Ser His Ser
Gly Ser Arg Val Arg Val Gly Phe Arg 195 200 205Leu Cys Asn Ser Thr
Gly Gly Asp Cys Phe Tyr Arg Gly Tyr Thr Ser 210 215 220Gly Val Ala
Ala Val Gln Asp Trp Tyr His Phe His Tyr Val Asp Ile225 230 235
240Leu Ala Leu Leu Pro Ala Ala Trp Glu Asp Ser His Gly Ser Gln Asp
245 250 255Gly His Phe Val Leu Ser Cys Ser Tyr Asp Gly Leu Asp Cys
Gln Ala 260 265 270Arg Gln Phe Arg Thr Phe His His Pro Thr Tyr Gly
Ser Cys Tyr Thr 275 280 285Val Asp Gly Val Trp Thr Ala Gln Arg Pro
Gly Ile Thr His Gly Val 290 295 300Gly Leu Val Leu Arg Val Glu Gln
Gln Pro His Leu Pro Leu Leu Ser305 310 315 320Thr Leu Ala Gly Ile
Arg Val Met Val His Gly Arg Asn His Thr Pro 325 330 335Phe Leu Gly
His His Ser Phe Ser Val Arg Pro Gly Thr Glu Ala Thr 340 345 350Ile
Ser Ile Arg Glu Asp Glu Val His Arg Leu Gly Ser Pro Tyr Gly 355 360
365His Cys Thr Ala Gly Gly Glu Gly Val Glu Val Gln Leu Leu His Asn
370 375 380Thr Ser Tyr Thr Arg Gln Ala Cys Leu Val Ser Cys Phe Gln
Gln Leu385 390 395 400Met Val Glu Thr Cys Ser Cys Gly Tyr Tyr Leu
His Pro Leu Pro Ala 405 410 415Gly Ala Glu Tyr Cys Ser Ser Ala Arg
His Pro Ala Trp Gly His Cys 420 425 430Phe Tyr Arg Leu Tyr Arg Asp
Leu Glu Thr His Arg Leu Pro Cys Thr 435 440 445Ser Arg Cys Pro Arg
Pro Cys Arg Glu Ser Ala Phe Lys Leu Ser Thr 450 455 460Gly Thr Ser
Arg Trp Pro Ser Ala Lys Ser Ala Gly Trp Thr Leu Ala465 470 475
480Thr Leu Gly Glu Gln Gly Leu Pro His Gln Ser His Arg Gln Arg Ser
485 490 495Ser Leu Ala Lys Ile Asn Ile Val Tyr Gln Glu Leu Asn Tyr
Arg Ser 500 505 510Val Glu Glu Ala Pro Val Tyr Ser Val Pro Gln Leu
Leu Ser Ala Met 515 520 525Gly Ser Leu Cys Ser Leu Trp Phe Gly Ala
Ser Val Leu Ser Leu Leu 530 535 540Glu Leu Leu Glu Leu Leu Leu Asp
Ala Ser Ala Leu Thr Leu Val Leu545 550 555 560Gly Gly Arg Arg Leu
Arg Arg Ala Trp Phe Ser Trp Pro Arg Ala Ser 565 570 575Pro Ala Ser
Gly Ala Ser Ser Ile Lys Pro Glu Ala Gly Gln Met Pro 580 585 590Pro
Pro Ala Gly Gly Thr Ser Asp Asp Pro Glu Pro Ser Gly Pro His 595 600
605Leu Pro Arg Val Met Leu Pro Gly Val Leu Ala Gly Val Ser Ala Glu
610 615 620Glu Ser Trp Ala Gly Pro Gln Pro Leu Glu Thr Leu Asp
Thr625 630 63522638PRTHomo Sapiens 22Met Ala Glu His Arg Ser Met
Asp Gly Arg Met Glu Ala Ala Thr Arg1 5 10 15Gly Gly Ser His Leu Gln
Ala Ala Ala Gln Thr Pro Pro Arg Pro Gly 20 25 30Pro Pro Ser Ala Pro
Pro Pro Pro Pro Lys Glu Gly His Gln Glu Gly 35 40 45Leu Val Glu Leu
Pro Ala Ser Phe Arg Glu Leu Leu Thr Phe Phe Cys 50 55 60Thr Asn Ala
Thr Ile His Gly Ala Ile Arg Leu Val Cys Ser Arg Gly65 70 75 80Asn
Arg Leu Lys Thr Thr Ser Trp Gly Leu Leu Ser Leu Gly Ala Leu 85 90
95Val Ala Leu Cys Trp Gln Leu Gly Leu Leu Phe Glu Arg His Trp His
100 105 110Arg Pro Val Leu Met Ala Val Ser Val His Ser Glu Arg Lys
Leu Leu 115 120 125Pro Leu Val Thr Leu Cys Asp Gly Asn Pro Arg Arg
Pro Ser Pro Val 130 135 140Leu Arg His Leu Glu Leu Leu Asp Glu Phe
Ala Arg Glu Asn Ile Asp145 150 155 160Ser Leu Tyr Asn Val Asn Leu
Ser Lys Gly Arg Ala Ala Leu Ser Ala 165 170 175Thr Val Pro Arg His
Glu Pro Pro Phe His Leu Asp Arg Glu Ile Arg 180 185 190Leu Gln Arg
Leu Ser His Ser Gly Ser Arg Val Arg Val Gly Phe Arg 195 200 205Leu
Cys Asn Ser Thr Gly Gly Asp Cys Phe Tyr Arg Gly Tyr Thr Ser 210 215
220Gly Val Ala Ala Val Gln Asp Trp Tyr His Phe His Tyr Val Asp
Ile225 230 235 240Leu Ala Leu Leu Pro Ala Ala Trp Glu Asp Ser His
Gly Ser Gln Asp 245 250 255Gly His Phe Val Leu Ser Cys Ser Tyr Asp
Gly Leu Asp Cys Gln Ala 260 265 270Arg Gln Phe Arg Thr Phe His His
Pro Thr Tyr Gly Ser Cys Tyr Thr 275 280 285Val Asp Gly Val Trp Thr
Ala Gln Arg Pro Gly Ile Thr His Gly Val 290 295 300Gly Leu Val Leu
Arg Val Glu Gln Gln Pro His Leu Pro Leu Leu Ser305 310 315 320Thr
Leu Ala Gly Ile Arg Val Met Val His Gly Arg Asn His Thr Pro 325 330
335Phe Leu Gly His His Ser Phe Ser Val Arg Pro Gly Thr Glu Ala Thr
340 345 350Ile Ser Ile Arg Glu Asp Glu Val His Arg Leu Gly Ser Pro
Tyr Gly 355 360 365His Cys Thr Ala Gly Gly Glu Gly Val Glu Val Glu
Leu Leu His Asn 370 375 380Thr Ser Tyr Thr Arg Gln Ala Cys Leu Val
Ser Cys Phe Gln Gln Leu385 390 395 400Met Val Glu Thr Cys Ser Cys
Gly Tyr Tyr Leu His Pro Leu Pro Ala 405 410 415Gly Ala Glu Tyr Cys
Ser Ser Ala Arg His Pro Ala Trp Gly His Cys 420 425 430Phe Tyr Arg
Leu Tyr Gln Asp Leu Glu Thr His Arg Leu Pro Cys Thr 435 440 445Ser
Arg Cys Pro Arg Pro Cys Arg Glu Ser Ala Phe Lys Leu Ser Thr 450 455
460Gly Thr Ser Arg Trp Pro Ser Ala Lys Ser Ala Gly Trp Thr Leu
Ala465 470 475 480Thr Leu Gly Glu Gln Gly Leu Pro His Gln Gly His
Arg Gln Arg Ser 485 490 495Ser Leu Ala Lys Ile Asn Ile Val Tyr Gln
Glu Leu Asn Tyr Arg Ser 500 505 510Val Glu Glu Ala Pro Val Tyr Ser
Val Pro Gln Leu Leu Ser Ala Met 515 520 525Gly Ser Leu Cys Ser Leu
Trp Phe Gly Ala Ser Val Leu Ser Leu Leu 530 535 540Glu Leu Leu Glu
Leu Leu Leu Asp Ala Ser Ala Leu Thr Leu Val Leu545 550 555 560Gly
Gly Arg Arg Leu Arg Arg Ala Trp Phe Ser Trp Pro Arg Ala Ser 565 570
575Pro Ala Ser Gly Ala Ser Ser Ile Lys Pro Glu Ala Ser Gln Met Pro
580 585 590Pro Pro Ala Gly Gly Thr Ser Asp Asp Pro Glu Pro Ser Gly
Pro His 595 600 605Leu Pro Arg Val Met Leu Pro Gly Val Leu Ala Gly
Val Ser Ala Glu 610 615 620Glu Ser Trp Ala Gly Pro Gln Pro Leu Glu
Thr Leu Asp Thr625 630 63523638PRTHomo Sapiens 23Met Ala Glu His
Arg Ser Met Asp Gly Arg Met Glu Ala Ala Thr Arg1 5 10 15Gly Gly Ser
His Leu Gln Ala Ala Ala Gln Thr Pro Pro Arg Pro Gly 20 25 30Pro Pro
Ser Ala Pro Pro Pro Pro Pro Lys Glu Gly His Gln Glu Gly 35 40 45Leu
Val Glu Leu Pro Ala Ser Phe Arg Glu Leu Leu Thr Phe Phe Cys 50 55
60Thr Asn Ala Thr Ile His Gly Ala Ile Arg Leu Val Cys Ser Arg Gly65
70 75 80Asn Arg Leu Lys Thr Thr Ser Trp Gly Leu Leu Ser Leu Gly Ala
Leu 85 90 95Val Ala Leu Cys Trp Gln Leu Gly Leu Leu Phe Glu Arg His
Trp His 100 105 110Arg Pro Val Leu Met Ala Val Ser Val His Ser Glu
Arg Lys Leu Leu 115 120 125Pro Leu Val Thr Leu Cys Asp Gly Asn Pro
Arg Arg Pro Ser Pro Val 130 135 140Leu Arg His Leu Glu Leu Leu Asp
Glu Phe Ala Arg Glu Asn Ile Asp145 150 155 160Ser Leu Tyr Asn Val
Asn Leu Ser Lys Gly Arg Ala Ala Leu Ser Ala 165 170 175Thr Val Pro
Pro His Glu Pro Pro Phe His Leu Asp Arg Glu Ile Arg 180 185 190Leu
Gln Arg Leu Ser His Ser Gly Ser Arg Val Arg Val Gly Phe Arg 195 200
205Leu Cys Asn Ser Thr Gly Gly Asp Cys Phe Tyr Arg Gly Tyr Thr Ser
210 215 220Gly Val Ala Ala Val Gln Asp Trp Tyr His Phe His Tyr Val
Asp Ile225 230 235 240Leu Ala Leu Leu Pro Ala Ala Trp Glu Asp Ser
His Gly Ser Gln Asp 245 250 255Gly His Phe Val Leu Ser Cys Ser Tyr
Asp Gly Leu Asp Cys Gln Ala 260 265 270Arg Gln Phe Arg Thr Phe His
His Pro Thr Tyr Gly Ser Cys Tyr Thr 275 280 285Val Asp Gly Val Trp
Thr Ala Gln Arg Pro Gly Ile Thr His Gly Val 290 295 300Gly Leu Val
Leu Arg Val Glu Gln Gln Pro His Leu Pro Leu Leu Ser305 310 315
320Thr Leu Ala Gly Ile Arg Val Met Val His Gly Arg Asn His Thr Pro
325 330 335Phe Leu Gly His His Ser Phe Ser Val Arg Pro Gly Thr Glu
Ala Thr 340 345 350Ile Ser Ile Arg Glu Asp Glu Val His Arg Leu Gly
Ser Pro Tyr Gly 355 360 365His Cys Thr Ala Gly Gly Glu Gly Val Glu
Val Gln Pro Leu His Asn 370 375 380Thr Ser Tyr Thr Arg Gln Ala Cys
Leu Val Ser Cys Phe Gln Gln Leu385 390 395 400Met Val Glu Thr Cys
Ser Cys Gly Tyr Tyr Leu His Pro Leu Pro Ala 405 410 415Gly Ala Glu
Tyr Cys Ser Ser Ala Arg His Pro Ala Trp Gly His Cys 420 425 430Phe
Tyr Arg Leu Tyr Gln Asp Leu Glu Thr His Arg Leu Pro Cys Thr 435 440
445Ser Arg Cys Pro Arg Pro Cys Arg Glu Ser Ala Phe Lys Leu Ser Thr
450 455 460Gly Thr Ser Arg Trp Pro Ser Ala Lys Ser Ala Gly Trp Thr
Leu Ala465 470 475 480Thr Leu Gly Glu Gln Gly Leu Pro His Gln Ser
His Arg Gln Arg Ser 485 490 495Ser Leu Ala Lys Ile Asn Ile Val Tyr
Gln Glu Leu Asn Tyr Arg Ser 500 505 510Val Glu Glu Ala Pro Val Tyr
Ser Val Pro Gln Leu Leu Ser Ala Met 515 520 525Gly Ser Leu Cys Ser
Leu Trp Phe Gly Ala Ser Val Leu Ser Leu Leu 530 535 540Glu Leu Leu
Glu Leu Leu Leu Asp Ala Ser Ala Leu Thr Leu Val Leu545 550 555
560Gly Gly Arg Arg Leu Arg Arg Ala Trp Phe Ser Trp Pro Arg Ala Ser
565 570 575Pro Ala Ser Gly Ala Ser Ser Ile Lys Pro Glu Ala Ser Gln
Met Pro 580 585 590Pro Pro Ala Gly Gly Thr Ser Asp Asp Pro Glu Pro
Ser Gly Pro His 595 600 605Leu Pro Arg Val Met Leu Pro Gly Val Leu
Ala Gly Val Ser Ala Glu 610 615 620Glu Ser Trp Ala Gly Pro Gln Pro
Leu Glu Thr Leu Asp Thr625 630 63524638PRTHomo Sapiens 24Met Ala
Glu His Arg Ser Met Asp Gly Arg Met Glu Ala Ala Thr Arg1 5 10 15Gly
Gly Ser His Leu Gln Ala Ala Ala Gln Thr Pro Pro Arg Pro Gly 20 25
30Pro Pro Ser Ala Pro Pro Pro Pro Pro Lys Glu Gly His Gln Glu Gly
35 40 45Leu Val Glu Leu Pro Ala Ser Phe Arg Glu Leu Leu Thr Phe Phe
Cys 50 55 60Thr Asn Ala Thr Ile His Gly Ala Ile Arg Leu Val Cys Ser
Arg Gly65 70 75 80Asn Arg Leu Lys Thr Thr Ser Trp Gly Leu Leu Ser
Leu Gly Ala Leu 85 90 95Val Ala Leu Cys Trp Gln Leu Gly Leu Leu Phe
Glu Arg His Trp His 100 105 110Arg Pro Val Leu Met Ala Val Ser Val
His Ser Glu Arg Lys Leu Leu 115 120 125Pro Leu Val Thr Leu Cys Asp
Gly Asn Pro Arg Arg Pro Ser Pro Val 130 135 140Leu Arg His Leu Glu
Leu Leu Asp Glu Phe Ala Arg Glu Asn Ile Asp145 150 155 160Ser Leu
Tyr Ser Val Asn Leu Ser Lys Gly Arg Ala Ala Leu Ser Ala 165 170
175Thr Val Pro Arg His Glu Pro Pro Phe His Leu Asp Arg Glu Ile Arg
180 185 190Leu Gln Arg Leu Ser His Ser Gly Ser Arg Val Arg Val Gly
Phe Arg 195 200 205Leu Cys Asn Ser Thr Gly Gly Asp Cys Phe Tyr Arg
Gly Tyr Thr Ser 210 215 220Gly Val Ala Ala Val Gln Asp Trp Tyr His
Phe His Tyr Val Asp Ile225 230 235 240Leu Ala Leu Leu Pro Ala Ala
Trp Glu Asp Ser His Gly Ser Gln Asp 245 250 255Gly His Phe Val Leu
Ser Cys Ser Tyr Asp Gly Leu Asp Cys Gln Ala 260 265 270Arg Gln Phe
Arg Thr Phe His His Pro Thr Tyr Gly Ser Cys Tyr Thr 275 280 285Val
Asp Gly Val Trp Thr Ala Gln Arg Pro Gly Ile Thr His Gly Val 290 295
300Gly Leu Val Leu Arg Val Glu Gln Gln Pro His Leu Pro Leu Leu
Ser305 310 315 320Thr Leu Ala Gly Ile Arg Val Met Val His Gly Arg
Asn His Thr Pro 325 330 335Phe Leu Gly His His Ser Phe Ser Val Arg
Pro Gly Thr Glu Ala Thr 340 345 350Ile Ser Ile Arg Glu Asp Glu Val
His Arg Leu Gly Ser Pro Tyr Gly 355 360 365His Cys Thr Ala Gly Gly
Glu Gly Val Glu Val Glu Leu Leu His Asn 370 375 380Thr Ser Tyr Thr
Arg Gln Pro Cys Leu Val Ser Cys Phe Gln Gln Leu385 390 395 400Met
Val Glu Thr Cys Ser Cys Gly Tyr Tyr Leu His Pro Leu Pro Ala 405 410
415Gly Ala Glu Tyr Cys Ser Ser Ala Arg His Pro Ala Trp Gly His Cys
420 425 430Phe Tyr Arg Leu Tyr Gln Asp Leu Glu Thr His Arg Leu Pro
Cys Thr 435 440 445Ser Arg Cys Pro Arg Pro Cys Arg Glu Ser Ala Phe
Lys Leu Ser Thr 450 455 460Gly Thr Ser Arg Trp Pro Ser Ala Lys Ser
Ala Gly Trp Thr Leu Ala465 470 475 480Thr Leu Gly Glu Gln Gly Leu
Pro His Gln Ser His Arg Gln Arg Ser 485 490 495Ser Leu Ala Lys Ile
Asn Ile Val Tyr Gln Glu Leu Asn Tyr Arg Ser 500 505 510Val Glu Glu
Ala Pro Val Tyr Ser Val Pro Gln Leu Leu Ser Ala Met 515 520 525Gly
Ser Leu Cys Ser Leu Trp Phe Gly Ala Ser Val Leu Ser Leu Leu 530 535
540Glu Leu Leu Glu Leu Leu Leu Asp Ala Ser Ala Leu Thr Leu Val
Leu545 550 555 560Gly Gly Arg Arg Leu Arg Arg Ala Trp Phe Ser Trp
Pro Arg Ala Ser 565 570 575Pro Ala Ser Gly Ala Ser Ser Ile Lys Pro
Glu Ala Ser Gln Met Pro 580 585 590Pro Pro Ala Gly Gly Thr Ser Asp
Asp Pro Glu Pro Ser Gly Pro His 595 600 605Leu Pro Arg Val Met Leu
Pro Gly Val Leu Ala Gly Val Ser Ala Glu 610 615 620Glu Ser Trp Ala
Gly Pro Gln Pro Leu Glu Thr Leu Asp Thr625 630 63525638PRTHomo
Sapiens 25Met Ala Glu His Arg Ser Met Asp Gly Arg Met Glu Ala Ala
Thr Arg1 5 10 15Gly Gly Ser His Leu Gln Ala Ala Ala Gln Thr Pro Pro
Arg Pro Gly 20 25 30Pro Pro Ser Ala Pro Pro Pro Pro Pro Lys Glu Gly
His Gln Glu Gly 35 40
45Leu Val Glu Leu Pro Ala Ser Phe Arg Glu Leu Leu Thr Phe Phe Cys
50 55 60Thr Asn Ala Thr Ile His Gly Ala Ile Arg Leu Val Cys Ser Arg
Gly65 70 75 80Asn Arg Leu Lys Thr Thr Ser Trp Gly Leu Leu Ser Leu
Gly Ala Leu 85 90 95Val Ala Leu Cys Trp Gln Leu Gly Leu Leu Phe Glu
Arg His Trp His 100 105 110Arg Pro Val Leu Met Ala Val Ser Val His
Ser Glu Arg Lys Leu Leu 115 120 125Pro Leu Val Thr Leu Cys Asp Gly
Asn Pro Arg Arg Pro Ser Pro Val 130 135 140Leu Arg His Leu Glu Leu
Leu Asp Glu Phe Ala Arg Glu Asn Ile Asp145 150 155 160Ser Leu Tyr
Asn Val Asn Leu Ser Lys Gly Arg Ala Ala Leu Ser Ala 165 170 175Thr
Val Pro Arg His Glu Pro Pro Phe His Leu Asp Arg Glu Ile Arg 180 185
190Leu Gln Arg Leu Ser His Ser Gly Ser Arg Val Arg Val Gly Phe Arg
195 200 205Leu Cys Asn Ser Thr Gly Gly Asp Cys Phe Tyr Arg Gly Tyr
Thr Ser 210 215 220Gly Val Ala Ala Val Gln Asp Trp Tyr His Phe His
Tyr Val Asp Ile225 230 235 240Leu Ala Leu Leu Pro Ala Ala Trp Glu
Asp Ser His Gly Ser Gln Asp 245 250 255Gly His Phe Val Leu Ser Cys
Ser Tyr Asp Gly Leu Asp Cys Gln Ala 260 265 270Arg Gln Phe Arg Thr
Phe His His Pro Thr Tyr Gly Ser Cys Tyr Thr 275 280 285Val Asp Gly
Val Trp Thr Ala Gln Arg Pro Gly Ile Thr His Gly Val 290 295 300Gly
Leu Val Leu Arg Val Glu Gln Gln Pro His Leu Pro Leu Leu Ser305 310
315 320Thr Leu Ala Gly Ile Arg Val Met Val His Gly Arg Asn His Thr
Pro 325 330 335Phe Leu Gly His His Ser Phe Ser Val Arg Pro Gly Thr
Glu Ala Thr 340 345 350Ile Ser Ile Arg Glu Asp Glu Val His Arg Leu
Gly Ser Pro Tyr Gly 355 360 365His Cys Thr Ala Gly Gly Glu Gly Val
Glu Val Glu Leu Leu His Asn 370 375 380Thr Ser Tyr Thr Arg Gln Ala
Cys Leu Val Ser Cys Phe Gln Gln Leu385 390 395 400Met Val Glu Thr
Cys Ser Cys Gly Tyr Tyr Leu His Pro Leu Pro Ala 405 410 415Gly Ala
Glu Tyr Cys Gly Ser Ala Arg His Pro Ala Trp Gly His Cys 420 425
430Phe Tyr Arg Leu Tyr Gln Asp Leu Glu Thr His Arg Leu Pro Cys Thr
435 440 445Ser Arg Cys Pro Arg Pro Cys Arg Glu Ser Ala Phe Lys Leu
Ser Thr 450 455 460Gly Thr Ser Arg Trp Pro Ser Ala Lys Ser Ala Gly
Trp Thr Leu Ala465 470 475 480Thr Leu Gly Glu Gln Gly Leu Pro His
Gln Ser His Arg Gln Arg Ser 485 490 495Ser Leu Ala Lys Ile Asn Ile
Val Tyr Gln Glu Leu Asn Tyr Arg Ser 500 505 510Val Glu Glu Ala Pro
Val Tyr Ser Val Pro Gln Leu Leu Ser Ala Met 515 520 525Gly Ser Leu
Cys Ser Leu Trp Phe Gly Ala Ser Val Leu Ser Leu Leu 530 535 540Glu
Leu Leu Glu Leu Leu Leu Asp Ala Ser Ala Leu Thr Leu Val Leu545 550
555 560Gly Gly Arg Arg Leu Arg Arg Ala Trp Phe Ser Trp Pro Arg Ala
Ser 565 570 575Pro Ala Ser Gly Ala Ser Ser Ile Lys Pro Glu Ala Ser
Gln Met Pro 580 585 590Pro Pro Ala Gly Gly Thr Ser Asp Asp Pro Glu
Pro Ser Gly Pro His 595 600 605Leu Pro Arg Val Met Leu Pro Gly Val
Leu Ala Gly Val Ser Ala Glu 610 615 620Glu Ser Trp Ala Gly Pro Gln
Pro Leu Glu Thr Leu Asp Thr625 630 63526638PRTHomo Sapiens 26Met
Ala Glu His Arg Ser Met Asp Gly Arg Met Glu Ala Ala Thr Arg1 5 10
15Gly Gly Ser His Leu Gln Ala Ala Ala Gln Thr Pro Pro Arg Pro Gly
20 25 30Pro Pro Ser Ala Pro Pro Pro Pro Pro Lys Glu Gly His Gln Glu
Gly 35 40 45Leu Val Glu Leu Pro Ala Ser Phe Arg Glu Leu Leu Thr Phe
Phe Cys 50 55 60Thr Asn Ala Thr Ile His Gly Ala Ile Arg Leu Val Cys
Ser Arg Gly65 70 75 80Asn Arg Leu Lys Thr Thr Ser Trp Gly Leu Leu
Ser Leu Gly Ala Leu 85 90 95Val Ala Leu Cys Trp Gln Leu Gly Leu Leu
Phe Glu Arg His Trp His 100 105 110Arg Pro Val Leu Met Ala Val Ser
Val His Ser Glu Arg Lys Leu Leu 115 120 125Pro Leu Val Thr Leu Cys
Asp Gly Asn Pro Arg Arg Pro Ser Pro Val 130 135 140Leu Arg His Leu
Glu Leu Leu Asp Glu Phe Ala Arg Glu Asn Ile Asp145 150 155 160Ser
Leu Tyr Asn Val Asn Leu Ser Lys Gly Arg Ala Ala Leu Ser Ala 165 170
175Thr Val Pro Arg His Glu Pro Pro Phe His Leu Asp Arg Glu Ile Arg
180 185 190Leu Gln Arg Leu Ser His Ser Gly Ser Arg Val Arg Val Gly
Phe Arg 195 200 205Leu Cys Asn Ser Thr Gly Gly Asp Cys Phe Tyr Arg
Gly Tyr Thr Ser 210 215 220Gly Val Ala Ala Val Gln Asp Trp Tyr His
Phe His Tyr Val Asp Ile225 230 235 240Leu Ala Leu Leu Pro Ala Ala
Trp Glu Asp Ser His Gly Ser Gln Asp 245 250 255Gly His Phe Val Leu
Ser Cys Ser Tyr Asp Gly Leu Asp Cys Gln Ala 260 265 270Arg Gln Phe
Arg Thr Ile His His Pro Thr Tyr Gly Ser Cys Tyr Thr 275 280 285Val
Asp Gly Val Trp Thr Ala Gln Arg Pro Gly Ile Thr His Gly Val 290 295
300Gly Leu Val Leu Arg Val Glu Gln Gln Pro His Leu Pro Leu Leu
Ser305 310 315 320Thr Leu Ala Gly Ile Arg Val Met Val His Gly Arg
Asn His Thr Pro 325 330 335Phe Leu Gly His His Ser Phe Ser Val Arg
Pro Gly Thr Glu Ala Thr 340 345 350Ile Arg Ile Arg Glu Asp Glu Val
His Arg Leu Gly Ser Pro Tyr Gly 355 360 365His Cys Thr Ala Gly Gly
Glu Gly Val Glu Val Glu Leu Leu His Asn 370 375 380Thr Ser Tyr Thr
Arg Gln Ala Cys Leu Val Ser Cys Phe Gln Gln Leu385 390 395 400Met
Val Glu Thr Cys Ser Cys Gly Tyr Tyr Leu His Pro Leu Pro Ala 405 410
415Gly Ala Glu Tyr Cys Ser Ser Ala Arg His Pro Ala Trp Gly His Cys
420 425 430Phe Tyr Arg Leu Tyr Gln Asp Leu Glu Thr His Arg Leu Pro
Cys Thr 435 440 445Ser Arg Cys Pro Arg Pro Cys Arg Glu Ser Ala Phe
Lys Leu Ser Thr 450 455 460Gly Thr Ser Arg Trp Pro Ser Ala Lys Ser
Ala Gly Trp Thr Leu Ala465 470 475 480Thr Leu Gly Glu Gln Gly Leu
Pro His Gln Ser His Arg Gln Arg Ser 485 490 495Ser Leu Ala Lys Ile
Asn Ile Val Tyr Gln Glu Leu Asn Tyr Arg Ser 500 505 510Val Glu Glu
Ala Pro Val Tyr Ser Val Pro Gln Leu Leu Ser Ala Met 515 520 525Gly
Ser Leu Cys Ser Leu Trp Phe Gly Ala Ser Val Leu Ser Leu Leu 530 535
540Glu Leu Leu Glu Leu Leu Leu Asp Ala Ser Ala Leu Thr Leu Val
Leu545 550 555 560Gly Gly Arg Arg Leu His Arg Ala Trp Phe Ser Trp
Pro Arg Ala Ser 565 570 575Pro Ala Ser Gly Ala Ser Ser Ile Lys Pro
Glu Ala Ser Gln Met Pro 580 585 590Pro Pro Ala Gly Gly Thr Ser Asp
Asp Pro Glu Pro Ser Gly Pro His 595 600 605Leu Pro Arg Val Met Leu
Pro Gly Val Leu Ala Gly Val Ser Ala Glu 610 615 620Glu Ser Trp Ala
Gly Pro Gln Pro Leu Glu Thr Leu Asp Thr625 630 63527649PRTHomo
Sapiens 27Met Ala Pro Gly Glu Lys Ile Lys Ala Lys Ile Lys Lys Asn
Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro Thr Ile Lys Glu Leu Met
Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr His Gly Cys Arg Arg Ile
Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu Leu Trp Ile Gly Phe
Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu Trp Gln Cys Ala Leu Leu
Val Phe Ser Phe Tyr Thr65 70 75 80Val Ser Val Ser Ile Lys Val His
Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val Thr Ile Cys Asn Ile Asn
Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105 110Leu Leu Ala Asp Leu
Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu 115 120 125Tyr Gly Phe
Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser Trp Asn 130 135 140Ser
Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His Arg Ile Pro Leu145 150
155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly Lys Ala Arg Asp Phe Phe
Thr 165 170 175Gly Arg Lys Arg Lys Val Gly Gly Ser Ile Ile His Lys
Ala Ser Asn 180 185 190Val Met His Ile Glu Ser Lys Gln Val Val Gly
Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr Ser Asp Cys Ala Thr Tyr
Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala Ile Gln Glu Trp Tyr Lys
Leu His Tyr Met Asn Ile Met Ala Gln225 230 235 240Val Pro Leu Glu
Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu Leu 245 250 255Leu Val
Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala Arg Asn Phe 260 265
270Thr Leu Phe His His Pro Met His Gly Asn Cys Tyr Thr Phe Asn Asn
275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr Ser Met Gly Gly Ser
Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr Ile Asn Glu Glu Glu
Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser Thr Gly Ala Lys Val
Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro Phe Val Glu Asp Val
Gly Thr Glu Ile Glu Thr Ala Met Val 340 345 350Thr Ser Ile Gly Met
His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro 355 360 365Tyr Ser Gln
Cys Thr Glu Asp Gly Ser Asp Val Pro Ile Arg Asn Ile 370 375 380Tyr
Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His Ser Cys Phe Gln385 390
395 400Thr Lys Met Val Glu Lys Cys Gly Cys Ala Gln Tyr Ser Gln Pro
Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr Gln Gln His Pro
Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln Leu His Arg Ala Phe Val
Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser Val Cys Lys Glu Ala Cys
Ser Phe Lys Glu Trp Thr Leu 450 455 460Thr Thr Ser Leu Ala Gln Trp
Pro Ser Val Val Ser Glu Lys Trp Leu465 470 475 480Leu Pro Val Leu
Thr Trp Asp Gln Gly Arg Gln Val Asn Lys Lys Leu 485 490 495Asn Lys
Thr Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys Asp Leu Asn 500 505
510Gln Arg Ser Ile Met Glu Ser Pro Ala Asn Ser Ile Glu Met Leu Leu
515 520 525Ser Asn Phe Gly Gly Gln Leu Gly Leu Trp Met Ser Cys Ser
Val Val 530 535 540Cys Val Ile Glu Ile Ile Glu Val Phe Phe Ile Asp
Phe Phe Ser Ile545 550 555 560Ile Ala Arg Arg Gln Trp Gln Lys Ala
Lys Glu Trp Trp Ala Trp Lys 565 570 575Gln Ala Pro Pro Cys Pro Glu
Ala Pro Arg Ser Pro Gln Gly Gln Asp 580 585 590Asn Pro Ala Leu Asp
Ile Asp Asp Asp Leu Pro Thr Phe Asn Ser Ala 595 600 605Leu His Leu
Pro Pro Ala Leu Gly Thr Gln Val Pro Gly Thr Pro Pro 610 615 620Pro
Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala Phe Ser Asn Gln Leu625 630
635 640Thr Asp Thr Gln Met Leu Asp Glu Leu 64528649PRTHomo Sapiens
28Met Ala Pro Gly Glu Lys Ile Lys Ala Lys Ile Lys Lys Asn Leu Pro1
5 10 15Val Thr Gly Pro Gln Ala Pro Thr Ile Lys Glu Leu Met Arg Trp
Tyr 20 25 30Cys Leu Asn Thr Asn Thr His Gly Cys Arg Arg Ile Val Val
Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu Leu Trp Ile Gly Phe Thr Leu
Thr Ala Val 50 55 60Ala Leu Ile Leu Trp Gln Cys Ala Leu Leu Val Phe
Ser Phe Tyr Thr65 70 75 80Val Ser Val Ser Ile Lys Val Asn Phe Arg
Lys Leu Asp Phe Pro Ala 85 90 95Val Thr Ile Cys Asn Ile Asn Pro Tyr
Lys Tyr Ser Thr Val Arg His 100 105 110Leu Leu Ala Asp Leu Glu Gln
Glu Thr Arg Glu Ala Leu Lys Ser Leu 115 120 125Tyr Gly Phe Pro Glu
Ser Arg Lys Arg Arg Glu Ala Glu Ser Trp Asn 130 135 140Ser Val Ser
Glu Gly Lys Gln Pro Arg Phe Ser His Arg Ile Pro Leu145 150 155
160Leu Ile Phe Asp Gln Asp Glu Lys Gly Lys Ala Arg Asp Phe Phe Thr
165 170 175Gly Arg Lys Arg Lys Val Gly Gly Ser Ile Ile His Lys Ala
Ser Asn 180 185 190Val Met His Ile Glu Ser Lys Gln Val Val Gly Phe
Gln Leu Cys Ser 195 200 205Asn Asp Thr Ser Asp Cys Ala Thr Tyr Thr
Phe Ser Ser Gly Ile Asn 210 215 220Ala Ile Gln Glu Trp Tyr Lys Leu
His Tyr Met Asn Ile Met Ala Gln225 230 235 240Val Pro Leu Glu Lys
Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu Leu 245 250 255Leu Val Thr
Cys Phe Phe Asp Gly Val Ser Cys Asp Ala Arg Asn Phe 260 265 270Thr
Leu Phe His His Pro Met His Gly Asn Cys Tyr Thr Phe Asn Asn 275 280
285Arg Glu Asn Glu Thr Ile Leu Ser Thr Ser Met Gly Gly Ser Glu Tyr
290 295 300Gly Leu Gln Val Ile Leu Tyr Ile Asn Glu Glu Glu Tyr Asn
Pro Phe305 310 315 320Leu Val Ser Ser Thr Gly Ala Lys Val Ile Ile
His Arg Gln Asp Glu 325 330 335Tyr Pro Phe Val Glu Asp Val Gly Thr
Glu Ile Glu Thr Ala Met Val 340 345 350Thr Ser Ile Gly Met His Leu
Thr Glu Ser Phe Lys Leu Ser Glu Pro 355 360 365Tyr Ser Gln Cys Thr
Glu Asp Gly Ser Asp Val Pro Ile Arg Asn Ile 370 375 380Tyr Asn Ala
Ala Tyr Ser Leu Gln Ile Cys Leu His Ser Cys Phe Gln385 390 395
400Thr Lys Met Val Glu Lys Cys Gly Cys Ala Gln Tyr Ser Gln Pro Leu
405 410 415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr Gln Gln His Pro Asn
Trp Met 420 425 430Tyr Cys Tyr Tyr Gln Leu His Arg Ala Phe Val Gln
Glu Glu Leu Gly 435 440 445Cys Gln Ser Val Cys Lys Glu Ala Cys Ser
Phe Lys Glu Trp Thr Leu 450 455 460Thr Thr Ser Leu Ala Gln Trp Pro
Ser Val Val Ser Glu Lys Trp Leu465 470 475 480Leu Pro Val Leu Thr
Trp Asp Gln Gly Arg Gln Val Asn Lys Lys Leu 485 490 495Asn Lys Thr
Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys Asp Leu Asn 500 505 510Gln
Arg Ser Ile Met Glu Ser Pro Ala Asn Ser Ile Glu Met Leu Leu 515 520
525Ser Asn Phe Gly Gly Gln Leu Gly Leu Trp Met Ser Cys Ser Val Val
530 535 540Cys Val Ile Glu Ile Ile Glu Val Phe Phe Ile Asp Phe Phe
Ser Ile545 550 555 560Ile Ala Arg Arg Gln Trp Gln Lys Ala Lys Glu
Trp Trp Ala Trp Lys 565 570 575Gln Ala Pro Pro Cys Pro Glu Ala Pro
Arg Ser Pro Gln Gly Gln Asp 580
585 590Asn Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr Phe Asn Ser
Ala 595 600 605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro Gly
Thr Pro Pro 610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala
Phe Ser Asn Gln Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp Glu
Leu 64529649PRTHomo Sapiens 29Met Ala Pro Gly Glu Lys Ile Lys Ala
Lys Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro Thr
Ile Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr His
Gly Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu
Leu Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu Trp
Gln Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75 80Val Ser Val
Ser Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val Thr
Ile Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105
110Leu Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu
115 120 125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser
Trp Asn 130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His
Arg Ile Pro Leu145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly
Lys Ala Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly
Gly Ser Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu
Ser Lys Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr
Ser Asp Cys Ala Thr Tyr Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala
Ile Gln Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230
235 240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu
Leu 245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala
Arg Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys
Tyr Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr
Ser Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr
Ile Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser
Thr Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro
Phe Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala Met Val 340 345
350Thr Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro
355 360 365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val Pro Ile Arg
Asn Ile 370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His
Ser Cys Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly Cys
Ala Gln Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys
Asn Tyr Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln
Leu His Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser
Val Cys Lys Glu Ala Cys Ser Phe Lys Glu Trp Thr Leu 450 455 460Thr
Thr Ser Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp Leu465 470
475 480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val Asn Lys Lys
Leu 485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys
Asp Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro Ala Asn Ser
Ile Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln Leu Gly Leu
Trp Met Ser Cys Ser Val Val 530 535 540Cys Val Ile Glu Ile Ile Glu
Val Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Ile Ala Arg Arg
Gln Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570 575Gln Ala
Pro Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp 580 585
590Asn Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr Phe Asn Ser Ala
595 600 605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro Gly Thr
Pro Pro 610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala Phe
Ser Asn Gln Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp Glu Leu
64530649PRTHomo Sapiens 30Met Ala Pro Gly Glu Lys Ile Lys Ala Lys
Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro Thr Ile
Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr His Gly
Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu Leu
Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu Trp Gln
Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75 80Val Ser Val Ser
Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val Thr Ile
Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105 110Leu
Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu 115 120
125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser Trp Asn
130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His Arg Ile
Pro Leu145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly Glu Ala
Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly Gly Ser
Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu Ser Lys
Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr Ser Asp
Cys Ala Thr Tyr Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala Ile Gln
Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230 235
240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu Leu
245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala Arg
Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys Tyr
Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr Ser
Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr Ile
Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser Thr
Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro Phe
Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala Met Val 340 345 350Thr
Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro 355 360
365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val Pro Ile Arg Asn Ile
370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His Ser Cys
Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly Cys Ala Gln
Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr
Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln Leu His
Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser Val Cys
Lys Glu Ala Cys Ser Phe Lys Glu Trp Thr Leu 450 455 460Thr Thr Ser
Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp Leu465 470 475
480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val Asn Lys Lys Leu
485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys Asp
Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro Ala Asn Ser Ile
Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln Leu Gly Leu Trp
Met Ser Cys Ser Val Val 530 535 540Cys Val Ile Glu Ile Ile Glu Val
Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Ile Ala Arg Arg Gln
Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570 575Gln Ala Pro
Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp 580 585 590Asn
Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr Phe Asn Ser Ala 595 600
605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro Ser Thr Pro Pro
610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala Phe Ser Asn
Gln Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp Glu Leu
64531649PRTHomo Sapiens 31Met Ala Pro Gly Glu Lys Ile Lys Ala Lys
Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro Thr Ile
Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr His Gly
Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu Leu
Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu Trp Gln
Cys Ala Leu Leu Val Phe Ser Phe Tyr Ala65 70 75 80Val Ser Val Ser
Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val Thr Ile
Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105 110Leu
Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu 115 120
125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser Trp Asn
130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His Arg Ile
Pro Leu145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly Lys Ala
Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly Gly Ser
Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu Ser Lys
Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr Ser Asp
Cys Ala Thr Tyr Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala Ile Gln
Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230 235
240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu Leu
245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala Arg
Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys Tyr
Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr Ser
Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr Ile
Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser Thr
Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro Phe
Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala Met Val 340 345 350Thr
Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro 355 360
365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val Pro Ile Arg Asn Ile
370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His Ser Cys
Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly Cys Ala Gln
Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr
Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln Leu His
Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser Val Cys
Lys Glu Ala Cys Ser Phe Lys Glu Trp Thr Leu 450 455 460Thr Thr Ser
Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp Leu465 470 475
480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val Asn Lys Lys Leu
485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys Asp
Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro Ala Asn Ser Ile
Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln Leu Gly Leu Trp
Met Ser Cys Ser Val Val 530 535 540Cys Val Ile Glu Ile Ile Glu Val
Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Thr Ala Arg Arg Gln
Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570 575Gln Ala Pro
Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp 580 585 590Asn
Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr Phe Asn Ser Ala 595 600
605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro Gly Thr Pro Pro
610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala Phe Ser Asn
Ser Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp Glu Leu
64532649PRTHomo Sapiens 32Met Ala Pro Gly Glu Lys Ile Lys Ala Lys
Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro Thr Ile
Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr His Gly
Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu Leu
Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu Trp Gln
Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75 80Val Ser Val Ser
Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val Thr Ile
Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105 110Leu
Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu 115 120
125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser Trp Asn
130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His Arg Ile
Pro Leu145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly Lys Ala
Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly Gly Ser
Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu Ser Lys
Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr Ser Asp
Cys Ala Thr Tyr Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala Ile Gln
Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230 235
240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu Leu
245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala Arg
Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys Tyr
Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr Ser
Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr Ile
Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser Thr
Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro Phe
Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala Met Val 340 345 350Thr
Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro 355 360
365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val Pro Ile Arg Asn Ile
370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His Ser Cys
Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly Cys Ala Gln
Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr
Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln Leu His
Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser Val Cys
Lys Glu Ala Cys Ser Phe Lys Glu Trp Thr Leu 450
455 460Thr Thr Ser Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp
Leu465 470 475 480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val
Asn Lys Lys Leu 485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile
Phe Tyr Lys Asp Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro
Ala Asn Ser Ile Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln
Leu Gly Leu Trp Met Ser Cys Ser Val Val 530 535 540Cys Val Ile Glu
Ile Ile Glu Val Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Ile
Ala Arg Arg Gln Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570
575Gln Ala Pro Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp
580 585 590Asn Pro Ala Leu Asp Ile Asp Asp Gly Leu Pro Thr Phe Asn
Ser Ala 595 600 605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro
Gly Thr Pro Pro 610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg
Ala Phe Ser Asn Gln Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp
Glu Leu 64533649PRTHomo Sapiens 33Met Ala Pro Gly Glu Lys Ile Lys
Ala Lys Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro
Thr Ile Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr
His Gly Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg
Leu Leu Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu
Trp Gln Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75 80Val Ser
Val Ser Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val
Thr Ile Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105
110Leu Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu
115 120 125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser
Trp Asn 130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His
Arg Ile Pro Pro145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly
Lys Ala Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly
Gly Ser Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu
Ser Lys Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr
Ser Asp Cys Ala Thr Tyr Thr Phe Ser Leu Gly Ile Asn 210 215 220Ala
Ile Gln Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230
235 240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu
Leu 245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala
Arg Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys
Tyr Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr
Ser Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr
Ile Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser
Thr Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro
Phe Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala Met Val 340 345
350Thr Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro
355 360 365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val Pro Ile Arg
Asn Ile 370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His
Ser Cys Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly Cys
Ala Gln Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys
Asn Tyr Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln
Leu His Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser
Val Cys Lys Glu Ala Cys Ser Leu Lys Glu Trp Thr Leu 450 455 460Thr
Thr Ser Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp Leu465 470
475 480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val Asn Lys Lys
Leu 485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys
Asp Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro Ala Asn Ser
Ile Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln Leu Gly Leu
Trp Met Ser Cys Ser Val Val 530 535 540Cys Val Ile Glu Ile Ile Glu
Val Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Thr Ala Arg Arg
Gln Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570 575Gln Ala
Pro Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp 580 585
590Asn Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr Phe Asn Ser Ala
595 600 605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro Gly Thr
Pro Pro 610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala Phe
Ser Asn Gln Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp Glu Leu
64534649PRTHomo Sapiens 34Met Ala Pro Gly Glu Lys Ile Lys Ala Lys
Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro Thr Ile
Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr His Gly
Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu Leu
Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu Trp Gln
Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75 80Val Ser Val Ser
Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val Thr Ile
Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105 110Leu
Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu 115 120
125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser Trp Asn
130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His Arg Ile
Pro Leu145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly Lys Ala
Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly Gly Ser
Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu Ser Lys
Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr Ser Asp
Cys Ala Thr Tyr Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala Ile Gln
Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230 235
240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu Leu
245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala Arg
Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys Tyr
Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr Ser
Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr Ile
Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser Thr
Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro Phe
Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala Met Val 340 345 350Thr
Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro 355 360
365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val Pro Ile Arg Asn Ile
370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His Ser Cys
Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly Cys Ala Gln
Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr
Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln Leu His
Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser Val Cys
Lys Glu Ala Cys Ser Phe Lys Glu Trp Thr Leu 450 455 460Thr Thr Ser
Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp Leu465 470 475
480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val Asn Lys Lys Leu
485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys Asp
Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro Ala Asn Ser Ile
Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln Leu Gly Leu Trp
Met Ser Cys Ser Val Val 530 535 540Cys Val Ile Glu Ile Ile Glu Val
Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Ile Ala Arg Arg Gln
Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570 575Gln Ala Pro
Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp 580 585 590Asn
Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr Phe Asn Ser Ala 595 600
605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro Gly Thr Pro Pro
610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala Phe Ser Asn
Gln Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp Glu Leu
64535649PRTHomo Sapiens 35Met Ala Pro Gly Glu Lys Ile Lys Ala Lys
Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro Thr Ile
Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr His Gly
Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu Leu
Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu Trp Gln
Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75 80Val Ser Val Ser
Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val Thr Ile
Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105 110Leu
Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu 115 120
125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser Trp Asn
130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His Arg Ile
Pro Leu145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly Lys Ala
Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly Gly Ser
Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu Ser Lys
Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr Ser Asp
Cys Ala Thr Tyr Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala Ile Gln
Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230 235
240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu Leu
245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala Arg
Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys Tyr
Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr Ser
Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr Ile
Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser Thr
Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325 330 335Tyr Pro Phe
Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala Met Val 340 345 350Thr
Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu Ser Glu Pro 355 360
365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val Pro Ile Arg Asn Ile
370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile Cys Leu His Ser Cys
Phe Gln385 390 395 400Thr Lys Met Val Glu Lys Cys Gly Cys Ala Gln
Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr
Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys Tyr Tyr Gln Leu His
Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440 445Cys Gln Ser Val Cys
Lys Glu Ala Cys Ser Phe Lys Glu Trp Thr Leu 450 455 460Thr Thr Ser
Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys Trp Leu465 470 475
480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln Val Asn Lys Lys Leu
485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu Ile Phe Tyr Lys Asp
Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser Pro Ala Asn Ser Ile
Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly Gln Leu Gly Leu Trp
Met Ser Cys Ser Val Val 530 535 540Cys Val Ile Glu Ile Ile Glu Val
Phe Phe Ile Asp Phe Phe Ser Ile545 550 555 560Ile Ala Arg Arg Gln
Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys 565 570 575Gln Ala Pro
Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly Gln Asp 580 585 590Asn
Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr Phe Asn Ser Ala 595 600
605Leu His Leu Pro Pro Ala Leu Gly Thr Gln Val Pro Gly Thr Pro Pro
610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu Glu Arg Ala Phe Ser Asn
Gln Leu625 630 635 640Thr Asp Thr Gln Met Leu Asp Glu Leu
64536649PRTHomo Sapiens 36Met Ala Pro Gly Glu Lys Ile Lys Ala Lys
Ile Lys Lys Asn Leu Pro1 5 10 15Val Thr Gly Pro Gln Ala Pro Thr Ile
Lys Glu Leu Met Arg Trp Tyr 20 25 30Cys Leu Asn Thr Asn Thr His Gly
Cys Arg Arg Ile Val Val Ser Arg 35 40 45Gly Arg Leu Arg Arg Leu Leu
Trp Ile Gly Phe Thr Leu Thr Ala Val 50 55 60Ala Leu Ile Leu Arg Gln
Cys Ala Leu Leu Val Phe Ser Phe Tyr Thr65 70 75 80Val Ser Val Ser
Ile Lys Val His Phe Arg Lys Leu Asp Phe Pro Ala 85 90 95Val Thr Ile
Cys Asn Ile Asn Pro Tyr Lys Tyr Ser Thr Val Arg His 100 105 110Leu
Leu Ala Asp Leu Glu Gln Glu Thr Arg Glu Ala Leu Lys Ser Leu 115 120
125Tyr Gly Phe Pro Glu Ser Arg Lys Arg Arg Glu Ala Glu Ser Trp Asn
130 135 140Ser Val Ser Glu Gly Lys Gln Pro Arg Phe Ser His Arg Ile
Pro Leu145 150 155 160Leu Ile Phe Asp Gln Asp Glu Lys Gly Lys Ala
Arg Asp Phe Phe Thr 165 170 175Gly Arg Lys Arg Lys Val Gly Gly Ser
Ile Ile His Lys Ala Ser Asn 180 185 190Val Met His Ile Glu Ser Lys
Gln Val Val Gly Phe Gln Leu Cys Ser 195 200 205Asn Asp Thr Ser Asp
Cys Ala Thr Tyr Thr Phe Ser Ser Gly Ile Asn 210 215 220Ala Ile Gln
Glu Trp Tyr Lys Leu His Tyr Met Asn Ile Met Ala Gln225 230 235
240Val Pro Leu Glu Lys Lys Ile Asn Met Ser Tyr Ser Ala Glu Glu Leu
245 250 255Leu Val Thr Cys Phe Phe Asp Gly Val Ser Cys Asp Ala Arg
Asn Phe 260 265 270Thr Leu Phe His His Pro Met His Gly Asn Cys Tyr
Thr Phe Asn Asn 275 280 285Arg Glu Asn Glu Thr Ile Leu Ser Thr Ser
Met Gly Gly Ser Glu Tyr 290 295 300Gly Leu Gln Val Ile Leu Tyr Ile
Asn Glu Glu Glu Tyr Asn Pro Phe305 310 315 320Leu Val Ser Ser Thr
Gly Ala Lys Val Ile Ile His Arg Gln Asp Glu 325
330 335Tyr Pro Phe Val Glu Asp Val Gly Thr Glu Ile Glu Thr Ala Met
Val 340 345 350Thr Ser Ile Gly Met His Leu Thr Glu Ser Phe Lys Leu
Ser Glu Pro 355 360 365Tyr Ser Gln Cys Thr Glu Asp Gly Ser Asp Val
Pro Ile Arg Asn Ile 370 375 380Tyr Asn Ala Ala Tyr Ser Leu Gln Ile
Cys Leu His Ser Cys Phe Gln385 390 395 400Thr Lys Met Val Glu Lys
Cys Gly Cys Ala Gln Tyr Ser Gln Pro Leu 405 410 415Pro Pro Ala Ala
Asn Tyr Cys Asn Tyr Gln Gln His Pro Asn Trp Met 420 425 430Tyr Cys
Tyr Tyr Gln Leu His Arg Ala Phe Val Gln Glu Glu Leu Gly 435 440
445Cys Gln Ser Val Cys Lys Glu Ala Cys Ser Phe Lys Glu Trp Thr Leu
450 455 460Thr Thr Ser Leu Ala Gln Trp Pro Ser Val Val Ser Glu Lys
Trp Leu465 470 475 480Leu Pro Val Leu Thr Trp Asp Gln Gly Arg Gln
Val Asn Lys Lys Leu 485 490 495Asn Lys Thr Asp Leu Ala Lys Leu Leu
Ile Phe Tyr Lys Asp Leu Asn 500 505 510Gln Arg Ser Ile Met Glu Ser
Pro Ala Asn Ser Ile Glu Met Leu Leu 515 520 525Ser Asn Phe Gly Gly
Gln Leu Gly Leu Trp Met Ser Cys Ser Val Val 530 535 540Cys Val Ile
Glu Ile Ile Glu Val Phe Phe Ile Asp Phe Phe Ser Ile545 550 555
560Ile Ala Arg Arg Gln Trp Gln Lys Ala Lys Glu Trp Trp Ala Trp Lys
565 570 575Gln Ala Pro Pro Cys Pro Glu Ala Pro Arg Ser Pro Gln Gly
Gln Asp 580 585 590Asn Pro Ala Leu Asp Ile Asp Asp Asp Leu Pro Thr
Phe Asn Ser Ala 595 600 605Leu His Leu Pro Pro Ala Leu Gly Thr Gln
Val Pro Gly Thr Pro Pro 610 615 620Pro Lys Tyr Asn Thr Leu Arg Leu
Glu Arg Ala Phe Ser Asn Gln Leu625 630 635 640Thr Asp Thr Gln Met
Leu Asp Glu Leu 64537638PRTHomo sapiens 37Met Ala Glu His Arg Ser
Met Asp Gly Arg Met Glu Ala Ala Thr Arg1 5 10 15Gly Gly Ser His Leu
Gln Ala Ala Ala Gln Thr Pro Pro Arg Pro Gly 20 25 30Pro Pro Ser Ala
Pro Pro Pro Pro Pro Lys Glu Gly His Gln Glu Gly 35 40 45Leu Val Glu
Leu Pro Ala Ser Phe Arg Glu Leu Leu Thr Phe Phe Cys 50 55 60Thr Asn
Ala Thr Ile His Gly Ala Ile Arg Leu Val Cys Ser Arg Gly65 70 75
80Asn Arg Leu Lys Thr Thr Ser Trp Gly Leu Leu Ser Leu Gly Ala Leu
85 90 95Val Ala Leu Cys Trp Gln Leu Gly Leu Leu Phe Glu Arg His Trp
His 100 105 110Arg Pro Val Leu Met Ala Val Ser Val His Ser Glu Arg
Lys Leu Leu 115 120 125Pro Leu Val Thr Leu Cys Asp Gly Asn Pro Arg
Arg Pro Ser Pro Val 130 135 140Leu Arg His Leu Glu Leu Leu Asp Glu
Phe Ala Arg Glu Asn Ile Asp145 150 155 160Ser Leu Tyr Asn Val Asn
Leu Ser Lys Gly Arg Ala Ala Leu Ser Ala 165 170 175Thr Val Pro Arg
His Glu Pro Pro Phe His Leu Asp Arg Glu Ile Arg 180 185 190Leu Gln
Arg Leu Ser His Ser Gly Ser Arg Val Arg Val Gly Phe Arg 195 200
205Leu Cys Asn Ser Thr Gly Gly Asp Cys Phe Tyr Arg Gly Tyr Thr Ser
210 215 220Gly Val Ala Ala Val Gln Asp Trp Tyr His Phe His Tyr Val
Asp Ile225 230 235 240Leu Ala Leu Leu Pro Ala Ala Trp Glu Asp Ser
His Gly Ser Gln Asp 245 250 255Gly His Phe Val Leu Ser Cys Ser Tyr
Asp Gly Leu Asp Cys Gln Ala 260 265 270Arg Gln Phe Arg Thr Phe His
His Pro Thr Tyr Gly Ser Cys Tyr Thr 275 280 285Val Asp Gly Val Trp
Thr Ala Gln Arg Pro Gly Ile Thr His Gly Val 290 295 300Gly Leu Val
Leu Arg Val Glu Gln Gln Pro His Leu Pro Leu Leu Ser305 310 315
320Thr Leu Ala Gly Ile Arg Val Met Val His Gly Arg Asn His Thr Pro
325 330 335Phe Leu Gly His His Ser Phe Ser Val Arg Pro Gly Thr Glu
Ala Thr 340 345 350Ile Ser Ile Arg Glu Asp Glu Val His Arg Leu Gly
Ser Pro Tyr Gly 355 360 365His Cys Thr Ala Gly Gly Glu Gly Val Glu
Val Glu Leu Leu His Asn 370 375 380Thr Ser Tyr Thr Arg Gln Ala Cys
Leu Val Ser Cys Phe Gln Gln Leu385 390 395 400Met Val Glu Thr Cys
Ser Cys Gly Tyr Tyr Leu His Pro Leu Pro Ala 405 410 415Gly Ala Glu
Tyr Cys Ser Ser Ala Arg His Pro Ala Trp Gly His Cys 420 425 430Phe
Tyr Arg Leu Tyr Gln Asp Leu Glu Thr His Arg Leu Pro Cys Thr 435 440
445Ser Arg Cys Pro Arg Pro Cys Arg Glu Ser Ala Phe Lys Leu Ser Thr
450 455 460Gly Thr Ser Arg Trp Pro Ser Ala Lys Ser Ala Gly Trp Thr
Leu Ala465 470 475 480Thr Leu Gly Glu Gln Gly Leu Pro His Gln Ser
His Arg Gln Arg Ser 485 490 495Ser Leu Ala Lys Ile Asn Ile Val Tyr
Gln Glu Leu Asn Tyr Arg Ser 500 505 510Val Glu Glu Ala Pro Val Tyr
Ser Val Pro Gln Leu Leu Ser Ala Met 515 520 525Gly Ser Leu Tyr Ser
Leu Trp Phe Gly Ala Ser Val Leu Ser Leu Leu 530 535 540Glu Leu Leu
Glu Leu Leu Leu Asp Ala Ser Ala Leu Thr Leu Val Leu545 550 555
560Gly Gly Arg Arg Leu Arg Arg Ala Trp Phe Ser Trp Pro Arg Ala Ser
565 570 575Pro Ala Ser Gly Ala Ser Ser Ile Lys Pro Glu Ala Ser Gln
Met Pro 580 585 590Pro Pro Ala Gly Gly Thr Ser Asp Asp Pro Glu Pro
Ser Gly Pro His 595 600 605Leu Pro Arg Val Met Leu Pro Gly Val Leu
Ala Gly Val Ser Ala Glu 610 615 620Glu Ser Trp Ala Gly Pro Gln Pro
Leu Glu Thr Leu Asp Thr625 630 63538649PRTHomo sapiens 38Met Ala
Pro Gly Glu Lys Ile Lys Ala Lys Ile Lys Lys Asn Leu Pro1 5 10 15Val
Thr Gly Pro Gln Ala Pro Thr Ile Lys Glu Leu Met Arg Trp Tyr 20 25
30Cys Leu Asn Thr Asn Thr His Gly Cys Arg Arg Ile Val Val Ser Arg
35 40 45Gly Arg Leu Arg Arg Leu Leu Trp Ile Gly Phe Thr Leu Thr Ala
Val 50 55 60Ala Leu Ile Leu Trp Gln Cys Ala Leu Leu Val Phe Ser Phe
Tyr Thr65 70 75 80Val Ser Val Ser Ile Lys Val His Phe Arg Lys Leu
Asp Phe Pro Ala 85 90 95Val Thr Ile Cys Asn Ile Asn Pro Tyr Lys Tyr
Ser Thr Val Arg His 100 105 110Leu Leu Ala Asp Leu Glu Gln Glu Thr
Arg Glu Ala Leu Lys Ser Leu 115 120 125Tyr Gly Phe Pro Glu Ser Arg
Lys Arg Arg Glu Ala Glu Ser Trp Asn 130 135 140Ser Val Ser Glu Gly
Lys Gln Pro Arg Phe Ser His Arg Ile Pro Leu145 150 155 160Leu Ile
Phe Asp Gln Asp Glu Lys Gly Lys Ala Arg Asp Phe Phe Thr 165 170
175Gly Arg Lys Arg Lys Val Gly Gly Ser Ile Ile His Lys Ala Ser Asn
180 185 190Val Met His Ile Glu Ser Lys Gln Val Val Gly Phe Gln Leu
Cys Ser 195 200 205Asn Asp Thr Ser Asp Cys Ala Thr Tyr Thr Phe Ser
Ser Gly Ile Asn 210 215 220Ala Ile Gln Glu Trp Tyr Lys Leu His Tyr
Met Asn Ile Met Ala Gln225 230 235 240Val Pro Leu Glu Lys Lys Ile
Asn Met Ser Tyr Ser Ala Glu Glu Leu 245 250 255Leu Val Thr Cys Phe
Phe Asp Gly Val Ser Cys Asp Ala Arg Asn Phe 260 265 270Thr Leu Phe
His His Pro Met His Gly Asn Cys Tyr Thr Phe Asn Asn 275 280 285Arg
Glu Asn Glu Thr Ile Leu Ser Thr Ser Met Gly Gly Ser Glu Tyr 290 295
300Gly Leu Gln Val Ile Leu Tyr Ile Asn Glu Glu Glu Tyr Asn Pro
Phe305 310 315 320Leu Val Ser Ser Thr Gly Ala Lys Val Ile Ile His
Arg Gln Asp Glu 325 330 335Tyr Pro Ser Val Glu Asp Val Gly Thr Glu
Ile Glu Thr Thr Met Val 340 345 350Thr Ser Ile Gly Met His Leu Thr
Glu Ser Phe Lys Leu Ser Glu Pro 355 360 365Ser Ser Gln Cys Thr Glu
Gly Gly Ser Asp Val Pro Ile Arg Asn Ile 370 375 380Tyr Asn Ala Ala
Tyr Ser Leu Gln Ile Cys Leu His Ser Cys Phe Gln385 390 395 400Thr
Lys Met Val Glu Lys Cys Gly Cys Ala Gln Tyr Ser Gln Pro Leu 405 410
415Pro Pro Ala Ala Asn Tyr Cys Asn Tyr Gln Gln His Pro Asn Trp Met
420 425 430Tyr Cys Tyr Tyr Gln Leu His Arg Ala Phe Val Gln Glu Glu
Leu Gly 435 440 445Cys Gln Ser Val Cys Lys Glu Ala Cys Arg Phe Lys
Glu Trp Thr Leu 450 455 460Thr Thr Ser Leu Ala Gln Trp Pro Ser Val
Val Ser Glu Lys Trp Leu465 470 475 480Leu Pro Val Leu Thr Trp Asp
Gln Gly Arg Gln Val Asn Lys Lys Leu 485 490 495Asn Lys Thr Asp Leu
Ala Lys Leu Leu Ile Phe Tyr Lys Asp Leu Asn 500 505 510Gln Arg Ser
Ile Met Glu Ser Pro Ala Asn Ser Ile Glu Met Leu Leu 515 520 525Ser
Asn Phe Gly Gly Gln Leu Gly Leu Trp Met Ser Cys Ser Val Val 530 535
540Cys Val Ile Glu Ile Ile Glu Val Phe Phe Ile Asp Phe Phe Ser
Ile545 550 555 560Ile Ala Arg Arg Gln Trp Gln Lys Ala Lys Glu Trp
Trp Ala Trp Lys 565 570 575Gln Ala Pro Pro Cys Pro Glu Ala Pro Arg
Ser Pro Gln Gly Gln Asp 580 585 590Asn Pro Ala Leu Asp Ile Asp Asp
Asp Leu Pro Thr Phe Asn Ser Ala 595 600 605Leu His Leu Pro Pro Ala
Leu Gly Thr Gln Val Pro Gly Thr Pro Pro 610 615 620Pro Lys Tyr Asn
Thr Leu Arg Leu Glu Arg Ala Phe Ser Asn Gln Leu625 630 635 640Thr
Asp Thr Gln Met Leu Asp Glu Leu 645
* * * * *