U.S. patent application number 11/595651 was filed with the patent office on 2007-05-31 for sodium-independent small neutral amino acid transporter transporting l- and d- amino acids.
This patent application is currently assigned to Japan Science and Technology Corporation. Invention is credited to Hitoshi Endou, Yoshikatsu Kanai.
Application Number | 20070122422 11/595651 |
Document ID | / |
Family ID | 18554163 |
Filed Date | 2007-05-31 |
United States Patent
Application |
20070122422 |
Kind Code |
A1 |
Endou; Hitoshi ; et
al. |
May 31, 2007 |
Sodium-independent small neutral amino acid transporter
transporting L- and D- amino acids
Abstract
Novel sodium-independent small neutral amino acid transporters
which transport L- and D-amino acids. A protein comprising the
amino acid sequence represented by SEQ ID NO:1 or 4 or an amino
acid derived therefrom by deletion, substitution or addition of one
or more amino acids and being capable of sodium-independently
transporting L- and D-small neutral amino acids and analogs
thereof; a gene encoding the above protein; a method of screening
substances inhibiting or promoting the function of the above
protein; an antibody against the above protein; and a method of
regulating cell function by using the above antibody, function
inhibitors, function promoters, etc.
Inventors: |
Endou; Hitoshi; (Kanagawa,
JP) ; Kanai; Yoshikatsu; (Tokyo, JP) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Japan Science and Technology
Corporation
Kawaguchi-shi
JP
|
Family ID: |
18554163 |
Appl. No.: |
11/595651 |
Filed: |
November 9, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10214867 |
Aug 7, 2002 |
7138494 |
|
|
11595651 |
Nov 9, 2006 |
|
|
|
PCT/JP01/00031 |
Jan 9, 2001 |
|
|
|
10214867 |
Aug 7, 2002 |
|
|
|
Current U.S.
Class: |
424/185.1 ;
424/144.1; 435/320.1; 435/325; 435/6.16; 435/69.1; 530/350;
530/388.22; 536/23.5 |
Current CPC
Class: |
C07K 14/47 20130101 |
Class at
Publication: |
424/185.1 ;
435/006; 435/069.1; 435/320.1; 435/325; 530/350; 536/023.5;
424/144.1; 530/388.22 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; A61K 39/00 20060101 A61K039/00; C07K 16/28 20060101
C07K016/28; C07K 14/705 20060101 C07K014/705 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 7, 2000 |
JP |
2000-028822 |
Claims
1.-14. (canceled)
15. A method for detection, identification or quantification of
action as a substrate of a substance to be tested to the ability of
transporting a small neutral amino acid and analogs thereof in a
sodium-independent manner using a protein which comprises an amino
acid sequence represented by SEQ ID NO: 1 or 4 or an amino acid
sequence where one or more amino acid(s) is/are deleted,
substituted or added in the said amino acid sequence and which is
capable of transporting a small neutral amino acid or analog
thereof in a sodium-independent manner.
16.-22. (canceled)
23. The method of claim 15, wherein the protein is derived from
human being or from mouse.
24. The method of claim 15, wherein the protein is derived from
orphan, tissue or cultured cell.
25. A method for determining whether a test substance is a
substrate for a protein which comprises an amino acid sequence
represented by SEQ ID NO: 1 or 4 or an amino acid sequence where
one or more amino acid(s) is/are deleted, substituted or added in
the said amino acid sequence and which is capable of transporting a
small neutral amino acid or analog thereof in a sodium-independent
manner, the method comprising the step of contacting the protein
with the test substance, and determining whether the test substance
is a substrate for the protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of co-pending U.S.
application Ser. No. 10/214,867, filed Aug. 7, 2002, now allowed,
which is a continuation of PCT Application No. PCT/JP01/00031,
filed Jan. 9, 2001, which claims the benefit of Japanese
Application No. 2000-028822 filed Feb. 7, 2000. The contents of all
of these applications are hereby incorporated by reference in their
entirety.
TECHNICAL FIELD
[0002] The present invention relates to a gene concerning a
sodium-independent transport of a small neutral amino acid and
analogs thereof, to protein encoding the genes and to an antibody
against the protein. The present invention further relates to a
method for screening the substance to be tested using the said
protein.
BACKGROUND ART
[0003] Cells are required that amino acids are to be always
incorporated thereinto as nutrition and such a function is carried
by an amino acid transporter which is a membrane protein existing
in cell membrane. The amino acid transporter is aligned in a
specific site in each tissue in multicellular living things and
plays an important role in expression of specific functions in each
tissue.
[0004] A transport system asc is an amino acid transport system
which transports small neutral amino acids mostly alanine, serine
and cysteine and, originally, it was found in erythrocyte membrane
and mentioned in many literatures. After that, its existence was
confirmed in cultured cells as well (Christensen, Physiol. Rev.,
volume 70, page 43, 1990). The transport system asc is a
transporter which is sodium-independent or, in other words, it does
not need sodium ion for its function. Its transport substrate
selectivity and transport characteristic have been known to have
some differences depending upon cells and animal species.
[0005] The transport system asc shows a high affinity to a
transport substrate such as alanine, serine or cysteine and, as a
transport system similar thereto, there is a transport system C
where small neutral amino acid such as alanine, serine or cysteine
is a transport substrate as well but affinity to a transport
substrate is low (Young et al., Biochem. J., volume 154, page 43,
1976; Young et al., Biochem. J., volume 162, page 33, 1977). The
transport system C is believed to be a subsystem for the transport
system asc. Sheep where the transport system C is genetically
deficient was found, reduction in glutathione content in its
erythrocytes was shown and the importance of incorporation of
cysteine mediated by cell membrane in the production of glutathione
was proved (Young, et al., Nature, volume 254, page 156, 1975).
[0006] However, in the conventional methods, it is difficult to
analyze the details of transport of amino acid and analogs thereof
mediated by the amino acid transport system asc and the functional
role in vivo and there has been a demand to isolate gene of neutral
amino acid transporter carrying a function of the amino acid
transport system asc so as to make the detailed function analysis
possible.
[0007] As to a small neutral amino acid transporter, there were
cloned ASCT1 and ASCT2 (Kanai, Curr. Opin. Cell Biol., volume 9,
page 565, 1997). However, they are sodium-dependent transporters
and are entirely different from a sodium-independent amino acid
transport system asc. In the meanwhile, glycine transporter and
proline transporter were cloned (Amara and Kuhar, Annu. Rev.
Neurosci., volume 16, page 73, 1993) but both of them transport
only glycine and proline in a sodium-dependent manner and are
different from the transport system asc.
[0008] cDNA of rBAT and 4F2hc which are type II membrane
glycoproteins having only one transmembrane structure which is
considered to be an activating factor of an amino acid transporter
were cloned although they are not transporters per se and it was
known that, when they were expressed in oocytes of Xenopus,
incorporation of basic amino acid together with neutral amino acid
was activated (Palacin, J. Exp. Biol., volume 196, page 123,
1994).
[0009] With regard to a transporter which selectively transports
neutral amino acid, there were cloned neutral amino acid
transporter LAT1 (Kanai et al., J. Biol. Chem., volume 273, pages
23629-23632, 1998) and LAT 2 (Segawa et al., J. Biol. Chem., volume
274, pages 19745-19751, 1999) corresponding to a transport system
L. It was further shown that LAT1 and LAT2 functioned only when
they coexist together with a cofactor 4F2hc. Both are not dependent
on Na.sup.+ but LAT1 shows an exchange transport activity for
transporting the large neutral amino acids such as leucine,
isoleucine, valine, phenylalanine, tyrosine, tryptophane,
methionine and histidine and LAT2 has a wide substrate selectivity
transporting the small neutral amino acids such as glycine,
alanine, serine, cysteine and threonine in addition to large
neutral amino acids. However, even they are different from the
amino acid transport system asc in terms of substrate
selectivity.
[0010] With regard to proteins analogous to the neutral amino acid
transporters LAT1 and LAT2, there were cloned the above-mentioned
y.sup.+LAT1 and y.sup.+LAT2 having a function of a transport system
y.sup.+L transporting the neutral amino acids and basic amino acids
(Torrents et al., J. Biol. Chem., volume 273, pages 32437-32445,
1998). It was also shown that both y.sup.+LAT1 and y.sup.+LAT2
functioned only when coexisted together with the cofactor 4F2hc.
y.sup.+LAT1 and y.sup.+LAT2 mainly transport glutamine, leucine and
isoleucine as neutral amino acids and are different from the amino
acid transport system asc in terms of the substrate
selectivity.
[0011] With regard to a transporter demanding the cofactor 4F2hc
for the expression of the function, there was cloned xCT which is a
protein analogous to the neutral amino acid transporters LAT1 and
LAT2 (Sato et al., J. Biol. Chem., 274: 11455-11458, 1999). The xCT
transports cystine and glutamic acid and is different from the
amino acid transport system asc in terms of the substrate
selectivity.
[0012] With regard to a transporter demanding other cofactor rBAT
having a structure analogous to 4F2hc for expressing the function,
there was cloned BAT1 which is a protein analogous to the neutral
amino acid transporters LAT1 and LAT2 (Chairoungdua et al., J.
Biol. Chem., 274: 28845-28848, 1999). BAT1 transports cystine,
neutral amino acids and basic amino acids and is different from the
amino acid transport system asc in terms of substrate
selectivity.
[0013] As such, molecular substances of the transporters which
function upon linking to 4F2hc and rBAT were made clear and it was
clarified that there was a group of transporters which achieve a
transporting function when a molecular complex is formed with the
type II glycoprotein.
DISCLOSURE OF THE INVENTION
[0014] An object of the present invention is to provide a gene of a
transporter which transports small neutral amino acid in a
sodium-independent manner and shows a function of transport system
asc and to provide a sodium-independent small neutral amino acid
transporter which is a polypeptide encoded by the gene. Other
objects will be apparent from the following description.
[0015] The present inventors have checked the EST (expressed
sequence tag) database using the base sequence of translated region
of cDNA of LAT1 and identified a base sequence analogous to LAT1.
They have prepared a probe corresponding to that, screened a cDNA
library and cloned a gene coding for a novel protein. They have
further expressed this genetic product in oocytes of Xenopus and
made clear that 4F2hc is essential when the genetic product
achieves the function and that, although the expressed function
corresponds to the neutral amino acid transport system asc, it
makes not only L-amino acids but also D-amino acids into
high-affinity transport substrates unlike the conventionally
mentioned property of the transport system asc whereupon the
present invention has been achieved.
[0016] Thus, the present invention relates to a protein comprising
an amino acid sequence represented by SEQ ID NO: 1 or 4 or an amino
acid sequence where one or more amino acid(s) therein is/are
deleted, substituted or added and being capable of transporting the
small neutral amino acid and analogs thereof in a
Na.sup.+-independent manner. The protein of the present invention
is a protein having an ability of transporting the small neutral
amino acids and analogs thereof in a sodium-independent manner when
co-existed together with a protein having an amino acid sequence
represented by SEQ ID NO: [[3]] 7 or [[6]] 8 or a protein
comprising an amino acid sequence where one or more amino acid(s)
is/are deleted, substituted or added.
[0017] The present invention further relates to a gene coding for
the above-mentioned protein of the present invention. To be more
specific, the present invention relates to a gene coding for a
protein having an ability of transporting the small neutral amino
acid and analogs thereof in a sodium-independent manner comprising
a base sequence represented by SEQ ID NO: 2 or 5 or a base sequence
being able to hybridize with DNA comprising the said base sequence
under a stringent condition.
[0018] The novel protein of the present invention being capable of
transporting small neutral amino acids and analogs thereof in a
sodium-independent manner or an asc-type amino acid transporter 1
has an ability of transporting (incorporating) the small neutral
amino acid such as glycine, L-alanine, L-serine, L-cysteine and
L-threonine in a highly affinitive manner when co-existed together
with an amino acid transport activating factor 4F2hc. It further
transports L-valine, L-methionine, L-isoleucine, L-leucine,
L-histidine and L-phenylalanine in a lowly affinitive manner. The
asc-1 further transports D-alanine, D-serine, D-cysteine and
D-threonine and particularly D-serine in a highly affinitive
manner. In addition, the asc-1 transports the substances analogous
to amino acid such as a-aminoisobutyric acid, .beta.-alanine and
alanine methyl ester.
[0019] Further, the sodium-independent small neutral amino acid
transporter asc-1 of the present invention which transports L- and
D-amino acids is mostly expressed in brain, lung, small intestine
and placenta in vivo. Particularly, asc-1 transports D-serine which
is believed to be an endogenous function-modifying substance for an
NMDA-type glutamic acid receptor and, therefore, there is a
possibility that it participates in the kinetics of D-serine in
brain and affects the functioning state of the NMDA receptor.
Furthermore, since asc-1 transports cysteine, it is believed to be
a factor regulating the production amount of glutathione produced
from cysteine as a material.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a drawing which shows a comparison of amino acid
sequence of mouse asc-1 (SEQ ID NO: 1) with those of rat LAT2 (SEQ
ID NO: 9), rat LAT1 (SEQ ID NO: 10), human y.sup.+LAT1(SEQ ID NO:
11), human y.sup.+LAT2 (SEQ ID NO: 12) and mouse xCT (SEQ ID NO:
13). The presumed transmembrane sites are shown by the lines
added.
[0021] FIG. 2 is a drawing which shows a comparison of amino acid
sequences of mouse asc-1 (SEQ ID NO: 1) and human asc-1 (SEQ ID NO:
4).
[0022] FIG. 3 is a picture which is a substitute for a drawing
which shows the result of analysis of expression of asc-1 gene mRNA
in various organ tissues of mouse by a northern blotting.
[0023] FIG. 4 is a picture which is a substitute for a drawing
which shows the result of by a western blotting analysis using
mouse brain membrane sample conducted under a non-reductive
condition (-) and a reductive condition (+) using anti-asc-1
antibody (left) and anti-4F2hc antibody (right).
[0024] FIG. 5 is a drawing which shows the result of an experiment
for incorporation of alanine by oocytes into which cRNA of mouse
asc-1 gene and/or mouse 4F2hc gene are/is injected.
[0025] FIG. 6 is a drawing which shows the result of checking the
influence of salt added in an experiment of incorporation of
alanine by oocytes into which cRNA of asc-1 gene of mouse and cRNA
of 4F2hc gene of mouse are injected.
[0026] FIG. 7 is a drawing which shows the result of checking the
influence of concentration of alanine substrate in an experiment of
incorporation of alanine by oocytes into which cRNA of asc-1 gene
of mouse and cRNA of 4F2hc gene of mouse are injected.
[0027] FIG. 8 is a drawing which shows the result of checking the
influence of addition of various L-amino acids or analogous
compounds thereof to the system in an experiment of incorporation
of alanine by oocytes into which cRNA of asc-1 gene of mouse and
cRNA of 4F2hc gene of mouse are injected.
[0028] FIG. 9 is a drawing which shows the result of checking the
influence of addition of various D-amino acids to the system in an
experiment of incorporation of alanine by oocytes into which cRNA
of asc-1 gene of mouse and cRNA of 4F2hc gene of mouse are
injected.
[0029] FIG. 10 is a drawing which shows the result of checking the
influence of addition of alanine or analogous compounds thereof to
the system in an experiment of incorporation of alanine by oocytes
into which cRNA of asc-1 gene of mouse and cRNA of 4F2hc gene of
mouse are injected.
[0030] FIG. 11 is a drawing which shows the result of checking the
incorporation of radiolabeled L-amino acid by oocytes into which
cRNA of asc-1 gene of mouse and cRNA of 4F2hc gene of mouse are
injected.
[0031] FIG. 12 is a drawing which shows the result of checking the
incorporation of radiolabeled D-amino acid by oocytes into which
cRNA of asc-1 gene of mouse and cRNA of 4F2hc gene of mouse are
injected.
[0032] FIG. 13 is a drawing which shows the result of checking the
incorporation of radiolabeled L-alanine or analogous compounds
thereof by oocytes into which cRNA of asc-1 gene of mouse and cRNA
of 4F2hc gene of mouse are injected.
[0033] FIG. 14 is a drawing which shows the result of checking the
influence of pH in an experiment of incorporation of alanine by
oocytes into which cRNA of asc-1 gene of mouse and cRNA of 4F2hc
gene of mouse are injected.
[0034] FIG. 15 is a drawing which shows the result of checking the
release of .sup.14C-alanine by oocytes into which cRNA of asc-1
gene of mouse and cRNA of 4F2hc gene of mouse are injected. The
ordinate shows the rate (%) of the released radioactivity to the
radioactivity injected into the oocytes.
[0035] FIG. 16 is a drawing which shows the result of checking the
progress of release of .sup.14C-alanine with a lapse of time by
oocytes into which cRNA of asc-1 gene of mouse and cRNA of 4F2hc
gene of mouse are injected. In the drawing, .largecircle. is the
case where, in the release of .sup.14C-alanine in the oocytes into
which water is injected as a control instead of cRNA of asc-1 gene
of mouse and cRNA of 4F2hc gene of mouse, a Na.sup.+-free uptake
solution to which no alanine is added is used; .circle-solid. is
the case where, in the release of .sup.14C-alanine in the oocytes
into which water is injected as a control instead of cRNA of asc-1
gene of mouse and cRNA of 4F2hc gene of mouse, a Na.sup.+-free
uptake solution to which alanine is added is used; .quadrature. is
the case where, in the release of .sup.14C-alanine in the oocytes
into which cRNA of asc-1 gene of mouse and cRNA of 4F2hc gene of
mouse are injected, a Na.sup.+-free uptake solution to which no
alanine is added is used; and .quadrature. is the case where, in
the release of .sup.14C-alanine in the oocytes into which cRNA of
asc-1 gene of mouse and cRNA of 4F2hc gene of mouse are injected, a
Na.sup.+-free uptake solution to which alanine is added is used.
The ordinate shows the rate (%) of the released radioactivity to
the radioactivity injected into the oocytes.
[0036] FIG. 17 is a drawing which shows the result of checking the
progress of release of .sup.14C-leucine with a lapse of time by
oocytes into which cRNA of LAT1 gene of rat and cRNA of 4F2hc gene
of rat are injected. In the drawing, 0 is the case where, in the
release of .sup.14C-leucine in the oocytes into which water is
injected as a control instead of cRNA of LAT1 gene of rat and cRNA
of 4F2hc gene of rat, a Na.sup.+-free uptake solution to which no
leucine is added is used; .circle-solid. is the case where, in the
release of .sup.14C-leucine in the oocytes into which water is
injected as a control instead of cRNA of LAT1 gene of rat and cRNA
of 4F2hc gene of rat, a Na.sup.+-free uptake solution to which
leucine is added is used; .quadrature. is the case where, in the
release of .sup.14C-leucine in the oocytes into which cRNA of LAT1
gene of rat and cRNA of 4F2hc gene of rat are injected, a
Na.sup.+-free uptake solution to which no leucine is added is used;
and .box-solid. is the case where, in the release of
.sup.14C-leucine in the oocytes into which cRNA of LAT1 gene of rat
and cRNA of 4F2hc gene of rat are injected, a Na.sup.+-free uptake
solution to which leucine is added is used. The ordinate shows the
rate (%) of the released radioactivity to the radioactivity
injected into the oocytes.
[0037] FIG. 18 is a drawing which shows the result of checking the
release of .sup.14C-alanine when various L-amino acids are added to
a Na.sup.+-free uptake solution containing no sodium ion by oocytes
into which cRNA of asc-1 gene of mouse and cRNA of 4F2hc gene of
mouse are injected (black bars) or by oocytes into which water is
injected instead of cRNA as a control (white bars). (-) shows the
release of .sup.14C-alanine mediated by asc-1 of mouse when no
amino acid is added to a Na.sup.+-free uptake solution. The
ordinate shows the rate (%) of the released radioactivity to the
radioactivity injected into the oocytes.
[0038] FIG. 19 is a drawing which shows the result of checking the
release of .sup.14C-alanine mediated by mouse asc-1 when various
kinds of D-amino acids are added to a Na.sup.+-free uptake
solution. (-) shows the release of .sup.14C-alanine mediated by
asc-1 of mouse when no amino acid is added to a Na.sup.+-free
uptake solution. The ordinate shows the rate (%) of the released
radioactivity to the radioactivity injected into the oocytes.
[0039] FIG. 20 is a drawing which shows the result of checking the
release of .sup.14C-alanine mediated by asc-1 of mouse when various
kinds of alanine-analogous compounds are added to a Na.sup.+-free
uptake solution. (-) shows the release of .sup.14C-alanine mediated
by asc-1 of mouse when no amino acid is added to a Na.sup.+-free
uptake solution. The ordinate shows the rate (%) of the released
radioactivity to the radioactivity injected into the oocytes.
[0040] FIG. 21 is a drawing which shows the result of checking the
release of the injected .sup.14C-amino acid from oocytes into which
cRNA of asc-1 gene of mouse and cRNA of 4F2hc gene of mouse are
injected. Black bars show the case where alanine is added to a
Na.sup.+-free uptake solution while bars with oblique lines show
the case where alanine is not added to a Na.sup.+-free uptake
solution.
BEST MODE FOR CARRYING OUT THE INVENTION
[0041] SEQ ID NO: 2 and NO: 1 in the Sequence Listing which will be
mentioned later stand for a full-length cDNA base sequence (about
1.6 kbp) of gene of sodium-independent small neutral amino acid
transporter (asc-1 of mouse) transporting L- and D-amino acid
derived from brain of mouse and an amino acid sequence (530 amino
acids) of protein encoded in its translated region.
[0042] SEQ ID NO: 5 and NO: 4 in the Sequence Listing which will be
mentioned later stand for a full-length cDNA base sequence (about
1.9 kbp) of gene of sodium-independent small neutral amino acid
transporter (human asc-1) transporting L- and D-amino acid derived
from human brain and an amino acid sequence (523 amino acids) of
protein encoded in its translated region.
[0043] When the base sequence or the amino acid sequence
represented by the above SEQ ID NO: 1 or 2 or SEQ ID NO: 4 or 5 was
subjected to a homology check for all sequences contained in the
known DNA databases (GenBank.TM. and EMBL) and protein databases
(NBRF and SWISS-PROT), there was nothing which was identical
whereby all those sequences are believed to be novel.
[0044] With regard to the protein of the present invention, that
which has an amino acid sequence represented by SEQ ID NO: 1 or 4
and, in addition, protein having an amino acid sequence where one
or more amino acid(s) is/are deficient, substituted or added in the
amino acid sequence represented by SEQ ID NO: 1 or 4 for example
may be listed. Defect, substitution or addition of amino acid may
be within such an extent that the neutral amino acid transport
activity is not lost and that is usually from 1 to about 106 or,
preferably, from 1 to about 53. Like the amino acid sequence
represented by SEQ ID NO: 1 or 4, such a protein has usually 80%
or, preferably, 90% homology of amino acid.
[0045] With regard to the gene of the present invention, that which
contains DNA being hybridizable under a stringent condition with
DNA comprising a base sequence represented by SEQ ID NO: 2 or 5 may
be exemplified in addition to that having a base sequence
represented by SEQ ID NO: 2 or 5. The DNA which is hybridizable as
such may be in such an extent that the protein encoded by the DNA
is capable of transporting the neutral amino acid. Such a DNA has
usually not less than 70% or, preferably, not less than 80% of
homology of a base sequence to the base sequence represented by SEQ
ID NO: 2 or 5. Such a DNA includes mutant gene found in nature,
artificially modified mutant and homologous gene derived from
living things of difference species.
[0046] Hybridization under a stringent condition according to the
present invention is usually carried out in such a manner that
hybridization is carried out in a hybridization solution of
5.times.SSC or the equivalent salt concentration at the temperature
condition of 37-42.degree. C. for about 12 hours, a preliminary
washing is carried out upon necessity by a solution of 5.times.SSC
or the equivalent salt concentration and a washing is carried out
in a solution of 1.times.SSC or the equivalent salt
concentration.
[0047] The sodium-independent small neutral amino acid transporter
gene of the present invention which transports L- and D-amino acids
can be isolated and obtained by carrying out a screening using
appropriate mammalian tissues or cells as a gene source. With
regard to mammals, human being may be listed in addition to
non-human animals such as dog, cow, horse, goat, sheep, monkey,
pig, rabbit, rat and mouse.
[0048] Screening and isolation of gene may be appropriately carried
out by a homology cloning, etc.
[0049] For example, brain of mouse or human being is used as a gene
source and mRNA (poly(A).sup.+ RNA) is prepared therefrom. A cDNA
library is constructed therefrom and a screening is carried out for
the cDNA library using a probe corresponding to LAT1-homologous
sequence (such as GenBank.TM./EBI/DDBJ accession No. N32639)
obtained by checking an EST (expressed sequence tag) database
whereupon clone containing cDNA of asc-1 gene is obtained.
[0050] With regard to the obtained cDNA, its base sequence is
determined by a conventional method, the translated region is
analyzed and an amino acid sequence of the protein encoded thereby,
i.e. asc-1, can be determined.
[0051] The fact that the resulting cDNA is a cDNA of a
sodium-independent small neutral amino acid transporter gene which
transports L- and D-amino acids or, in other words, it is a genetic
product encoded by cDNA is a sodium-independent small neutral amino
acid transporter which transports L- and D-amino acids is, for
example, able to be tested as follows. Thus, RNA (cRNA) (a capped
one) complementary thereto prepared from the resulting cDNA of
asc-1 gene is expressed by introducing into oocytes together with
cRNA having a base sequence of 4F2hc represented by SEQ ID NO: 3 or
6 and an ability of transporting (incorporating) a neutral amino
acid into cells can be confirmed by measuring the incorporation of
a substrate into the cells by means of a conventional incorporation
test (Kanai and Hediger, Nature, volume 360, pages 467-471, 1992)
where an appropriate neutral amino acid is a substrate.
[0052] An asc-1 protein is synthesized by means of an in vitro
translation method (Hediger, et al., Biochim. Biophys. Acta, volume
1064, page 360, 1991) using RNA (cRNA) which is complementary
thereto prepared from the resulting cDNA of asc-1 whereupon it is
possible to check the size of protein, addition or non-addition of
sugar, etc. by means of electrophoresis.
[0053] Since the cDNA of gene of 4F2hc was reported already (Broer,
et al., Biochem. J., volume 312, page 863, 1995), it is possible to
prepare a gene of 4F2hc easily by means of PCR or the like. cRNA (a
capped one) can be synthesized from the resulting cDNA of
4F2hc.
[0054] The same incorporation experiment is applied to expression
cells whereupon it is possible to check the characteristics of the
asc-1 such as a characteristic that asc-1 conducts transport of an
exchange type of amino acid as well as substrate selectivity of
asc-1 and pH-dependency.
[0055] When an appropriate genomic DNA library or cDNA library
prepared from different gene source is screened using the resulting
cDNA of asc-1 gene, it is possible to isolate homologous gene or
chromosomal gene derived from different living things.
[0056] When a synthetic primer designed on the basis of information
of the disclosed base sequence of gene of the present invention (a
base sequence represented by SEQ ID NO: 2 or 5 or a part thereof)
is used and a conventional PCR (polymerase chain reaction) is
carried out, it is possible to isolate a gene from a cDNA library
or a genomic DNA library.
[0057] DNA libraries such as a cDNA library and a genomic DNA
library can be prepared by a method mentioned in, for example,
"Molecular Cloning" (by Sambrook, J., Fritsh, E. F and Manitis, T.;
published by Cold Spring Harbor Press). Alternatively, when a
commercially available library is available, that may be used.
[0058] The sodium-independent small neutral amino acid transporter
(asc-1) transporting D- and L-amino acids according to the present
invention may be produced by, for example, a gene recombination
technique using the cDNA coding therefor. For example, DNA (such as
cDNA) coding for asc-1 is incorporated into an appropriate
expression vector and the resulting recombinant DNA can be
introduced into an appropriate host cell. Examples of the
expression system (host-vector system) for the production of
polypeptide are expression systems of bacteria, yeasts, insect
cells and mammalian cells. Among them, the use of insect cells and
mammalian cells is preferred for the preparation of functional
protein.
[0059] For example, in the case of expression of polypeptide in
mammalian cells, DNA coding for a sodium-independent small neutral
amino acid transporter asc-1 which transports the L- and D-amino
acids is inserted into the downstream of an appropriate promoter
(such as cytomegalovirus promoter, SV 40 promoter, LTR promoter or
elongation 1a promoter) in an appropriate expression vector (such
as vector of adenovirus type, vector of retrovirus type, papilloma
virus vector, vaccinia virus vector or vector of SV 40 type)
whereupon an expression vector is constructed. After that, an
appropriate animal cell is transformed by the resulting expression
vector and the transformant is incubated in an appropriate medium
whereupon an aimed polypeptide is produced. Examples of the
mammalian cell used as a host are cell strains such as simian COS-7
cell, CHO cell of Chinese hamster and human HeLa cell.
[0060] With regard to the DNA coding for a sodium-independent small
amino acid transporter asc-1 which transports the L- and D-amino
acids, the cDNA having a base sequence represented by SEQ ID NO: 2
or 5 may be used for example and, in addition, it is not limited to
the above-mentioned DNA sequence but DNA corresponding to amino
acid may be designed and used as a DNA coding for the polypeptide.
In that case, as to the codon for coding for one amino acid, from 1
to 6 kinds of codon(s) is/are known for each and, although the
codon used may be selected freely, it is possible to design a
sequence having higher expression efficiency by taking the
frequency of use of codon of the host utilized for the expression
into consideration. DNA having a designed base sequence can be
prepared by chemical synthesis of DNA, by fragmentation of the
above-mentioned cDNA and combination thereof, by a partial
modification of a base sequence, etc. Artificial modification of a
base sequence and introduction of variation can be carried out by
means of a site-specific mutagenesis (Mars, D. F., et al.,
Proceedings of National Academy of Sciences, volume 81, page 5662,
1984), etc. utilizing a primer comprising a synthetic
oligonucleotide coding for the desired modification.
[0061] The present invention further relates to nucleotide
containing a partial sequence of continuous 14 or more bases,
preferably 20 or more bases or, more preferably, 30 or more bases
in the base sequence represented by SEQ ID NO: 2 or 5 of the
Sequence Listing or a complementary sequence thereof. The
nucleotide of the present invention may be used as a probe for the
detection of gene coding for a protein being capable of
transporting a sodium-independent small neutral amino acid and
analogs thereof.
[0062] When the sodium-independent small neutral amino acid
transporter of the present invention transporting the L- and
D-amino acids or a polypeptide having the immunological homology
thereto is used, an antibody against that can be prepared. The
antibody can be utilized for the detection or the purification of
the sodium-independent small neutral amino acid transporter which
transports the L- and D-amino acids. The antibody can be
manufactured using the sodium-independent small neutral amino acid
transporter of the present invention transporting the L- and
D-amino acids, a fragment thereof, a synthetic peptide having a
partial sequence thereof or the like as an antigen. A polyclonal
antibody can be manufactured by a conventional method where antigen
is inoculated to a host animal (such as rat or rabbit) and the
immunized serum is recovered therefrom while a monoclonal antibody
can be manufactured by a conventional technique such as a hybridoma
method.
[0063] The sodium-independent small neutral amino acid transporter
asc-1 of the present invention transporting the L- and D-amino
acids, gene thereof and expression cell thereof can be used in an
in vitro test for permeation efficiency at the cell membrane where
asc-1 is present or at the site where asc-1 is presumed to be
present.
[0064] Further, the sodium-independent small neutral amino acid
transporter asc-1 of the present invention transporting the L- and
D-amino acids, gene thereof and expression cell thereof can be used
in the development of compounds which efficiently permeate the cell
membrane where asc-1 is present or at the site where asc-1 is
presumed to be present. Furthermore, the sodium-independent small
neutral amino acid transporter asc-1 of the present invention
transporting the L- and D-amino acids, gene thereof and expression
cell thereof can be used in an in vitro test for a drug interaction
at the cell membrane where asc-1 is present or at the site where
asc-1 is presumed to be present.
[0065] When the sodium-independent small neutral amino acid
transporter asc-1 of the present invention which transports the L-
and D-amino acids is inhibited, it is possible to limit the
permeation of specific compounds at the cell membrane where asc-1
is expressed or at the site where asc-1 is presumed to be present.
Further, the sodium-independent small neutral amino acid
transporter asc-1 of the present invention which transports the L-
and D-amino acids, gene thereof and expression cell thereof can be
used in the development of drugs (such as specific inhibitor for
asc-1) which limit the passing of the cell membrane or the
permeation at the site where asc-1 is presumed to be present of a
compound transported by asc-1.
[0066] Accordingly, the present invention provides a method for
detection, identification or quantification of action as a
substrate of a substance to be tested to the ability of the present
invention for transporting a small neutral amino acid and analogs
thereof in a sodium-independent manner using the protein of the
present protein. In accordance with the method of the present
invention, it is possible to screen a substance which promotes the
function of the protein of the present invention or a substance
which inhibits that. When an uptake solution containing an amino
acid which is labeled by radioactivity or by fluorescence such as
.sup.14C-alanine is used and the amount of the said incorporated or
released amino acid is measured in the presence of the substance to
be tested, it is now possible to test the action of the said
substance to be tested to the protein of the present invention.
[0067] The present invention further provides a method for
controlling the resistance of cells to oxidative stress where the
protein of the present invention, a specific antibody thereof, a
substance for promoting the function thereof or a substance for
inhibiting the function thereof is used to modulate an ability of
transporting a small neutral amino acid of the said protein or
analogs thereof.
[0068] The present invention furthermore provides a method for
controlling the activity of a glutamic acid receptor of an NMDA
type in a nervous system where the protein of the present
invention, a specific antibody thereof, a substance for promoting
the function thereof or a substance for inhibiting the function
thereof is used to modulate an ability of transporting a small
neutral amino acid of the said protein or analogs thereof; a method
for controlling the plasticity of synaptic transmission in which a
glutamic acid receptor of an NMDA type is participated by means of
the above-mentioned method; and a method for controlling the
neuronal death in which a glutamic acid receptor of an NMDA type is
participated by means of the above-mentioned method.
[0069] The present invention provides a method for controlling such
as inhibition or promotion of growth of cells where the protein of
the present invention, a specific antibody thereof, a substance for
promoting the function thereof or a substance for inhibiting the
function thereof is used to modulate an ability of transporting a
small neutral amino acid of the said protein or analogs
thereof.
[0070] The present invention provides a method for changing the
fate of a drug transported by the protein in vivo where the protein
of the present invention, a specific antibody thereof, a substance
for promoting the function thereof or a substance for inhibiting
the function thereof is used to modulate an ability of transporting
a neutral amino acid of the said protein or analogs thereof.
[0071] The present invention provides a method for changing the
fate of a toxin or an exogenous substance transported by the
protein in vivo where the protein of the present invention, a
specific antibody thereof, a substance for promoting the function
thereof or a substance for inhibiting the function thereof is used
to modulate an ability of transporting a neutral amino acid of the
said protein or analogs thereof.
[0072] The present invention will now be further illustrated by way
of the Examples although the present invention is not limited
thereto.
[0073] In the following Examples, each of the operation was carried
out, unless otherwise clearly mentioned, by a method descried in
"Molecular Cloning" (by Sambrook, J., Fritsh, E. F. and Manitis,
T.; published by Cold Spring Harbor Press in 1989) or, when a
commercially available reagent or kit is used, it was carried out
according to the direction for use of the said commercially
available product.
EXAMPLES
Example 1
Mouse and Human cDNA Cloning of Sodium-Independent Small Neutral
Amino Acid Transporter which Transports L- and D-Amino Acids
[0074] (1) Isolation of cDNA of 4F2hc of Mouse and Human Being and
Preparation of cRNA
[0075] A cDNA library was prepared from poly(A).sup.+ RNA purified
from brain of mouse or from poly(A).sup.+ RNA derived from human
placenta (purchased from Clontec) using a kit for the synthesis of
cDNA (trade name: Superscript Choice System; manufactured by Gibco)
and was integrated into a site cleaved by a restriction enzyme
EcoRI of phage vector .lamda.ZipLox (manufactured by Gibco). A
segment which corresponds to the bases of from 135th to 580th bases
of 4F2hc gene of rat (Broer, et al., Biochem. J., volume 312, page
863, 1995) was amplified and labeled with .sup.32P-dCTP and the
resulting one was used as a probe whereby a cDNA library of brain
of mouse and a cDNA library of human placenta were screened.
Hybridization was carried out for one night in a solution for
hybridization and a filter membrane was washed at 37.degree. C.
with 0.1.times.SSC/0.1% SDS. As to the solution for hybridization,
there was used a buffer of pH 6.5 containing 5.times.SSC, 3.times.
Denhard's solution, 0.2% SDS, 10% dextran sulfate, 50% formamide,
0.01% Abtiform B (trade name; Sigma) (antifoaming agent), 0.2 mg/ml
salmon sperm modified DNA, 2.5 mM sodium pyrophosphate and 25
mM-MES. A cDNA moiety of .lamda.ZipLox phage into which cDNA was
integrated was integrated into a plasmid pZL1. In cDNA of human
4F2hc, a cDNA moiety of .lamda.ZipLox phage into which cDNA was
integrated was recombined to a plasmid pZL1.
[0076] The resulting clone or the clone containing cDNA of 4F2hc of
mouse and human being was subjected to a base sequence
determination of cDNA by a diterminator cycle sequencing method
(Applied Biosystems) using a synthetic primer for determination of
base sequence. As a result, the cloned cDNA was confirmed to be
that of gene of 4F2hc of mouse or human being. The base sequences
of the resulting 4F2hc are represented by SEQ ID NO: 3 and 6 of the
Sequence Listing which will be mentioned later.
[0077] From the plasmid containing cDNA of 4F2hc of mouse and human
being prepared as above, cRNA (RNA complementary to cDNA) was
prepared using a T7RNA polymerase.
[0078] (2) Isolation of cDNA of Mouse of Sodium-Independent Small
Neutral Amino Acid Transporter Asc-1 which Transports L- and
D-Amino Acids and Preparation of cRNA
[0079] A sense primer (5'-CTCTTCACATGCATCTCCAC-3') (SEQ ID NO: 14)
corresponding to 35-54 bp of LAT1-analogous sequence
GenBank.TM./EBI/DDBI accession No. N32639 obtained by retrieval of
EST (expressed sequence tag) database using a base sequence of
translated region of LAT1, an antisense primer
(5'-GGTACACGACCACACACATC-3') (SEQ ID NO: 15) corresponding to
397-416 bp thereof and an IMAGE (Integrated and Molecular Analysis
of Genomes and their Expression) cDNA clone No. 267666 were used as
templates whereby DNA fragment was amplified by PCR. The resulting
DNA fragment was labeled with .sup.32P-dCTP and was used as a probe
to screen a cDNA library of brain of mouse.
[0080] The cDNA library was prepared from poly(A).sup.+ RNA derived
from brain of mouse using a kit for the synthesis of cDNA (trade
name: Superscript Choice System; manufactured by Gibco) and was
integrated into a site of phage vector .lamda.ZipLox (Gibco)
cleaved by a restriction enzyme EcoRI. Hybridization by a probe
labeled with .sup.32P_dCTP was carried out for one night in a
solution of hybridization at 37.degree. C. and a filter membrane
was washed at 37.degree. C. with 0.1.times.SSC/0.1% SDS. As to the
solution for hybridization, there was used a buffer of pH 6.5
containing 5.times.SSC, 3.times. Denhard's solution, 0.2% SDS, 10%
dextran sulfate, 50% formamide, 0.01% Abtiform B (trade name;
Sigma) (antifoaming agent), 0.2 mg/ml salmon sperm modified DNA,
2.5 mM sodium pyrophosphate and 25 mM-MES. A cDNA moiety of %
ZipLox phage into which cDNA was integrated was integrated into a
plasmid pZL1 and was further subcloned to a plasmid pBluescript II
SK.sup.- (manufactured by Stratagene).
[0081] The resulting clone or the clone containing cDNA of asc-1 of
mouse was subjected to a base sequence determination of cDNA by a
diterminator cycle sequencing method (Applied Biosystems) using a
synthetic primer for determination of base sequence.
[0082] As a result, a base sequence of asc-1 gene of mouse was
obtained. Further, a base sequence of cDNA was analyzed by a
conventional method to determine a translated region of cDNA and an
amino acid sequence of asc-1 encoded there.
[0083] Those sequences are represented by SEQ ID NO: 1 (amino acid
sequence) and 2 (base sequence) in the Sequence Listing which will
be mentioned later.
[0084] The asc-1 had a homology of 45% in terms of amino acid
sequence to a rat transporter LAT1 corresponding to a neutral amino
acid transport system .sup.-L and the homology of 65% to LAT2.
Further, the asc-1 had a homology of 45% to a human transporter
y.sup.+LAT1 corresponding to a neutral and basic amino acid
transport system y.sup.-L and the homology of 45% to y+LAT2.
Furthermore, the asc-1 had a homology of 45% to a transporter xCT
of mouse corresponding to cystine and an acidic amino acid
transport system x.sup.-c and a homology of 44% in terms of amino
acid sequence to a transporter BAT1 of rat corresponding to cystine
and a neutral and basic amino acid transport system b.sup.0.+.
[0085] Comparison of asc-1 with LAT2 of rat, LAT1 of rat, human
y.sup.+LAT1, human y.sup.+LAT2 and xCT of mouse in terms of amino
acid sequence is shown in FIG. 1.
[0086] As a result of analysis of an amino acid sequence of asc-1
by an SOSUI algorithm (Hirokawa, T. et al., Bioinformatics, volume
14, page 378 (1998)), 12 membrane-spanning domains were expected as
shown by the lines in FIG. 1. There were also the sites which were
believed to be tyrosine phosphorylated site in the second
hydrophilic loop, protein kinase C-dependent phosphorylated site in
N-terminal intracellular region, the eighth hydrophilic loop and
C-terminal intracellular region and cAMP-dependent phosphorylated
site in N-terminal intracellular region.
[0087] (3) Isolation of Human cDNA of Sodium-Independent Small
Neutral Amino Acid Transporter Asc-1 which Transports L- and
D-Amino Acids and Preparation of cRNA.
[0088] Fragment cleaved by NcoI of asc-1 cDNA of mouse
(corresponding to 523-1366 bp of asc-1 cDNA of mouse) was labeled
with .sup.32P-dCTP and this was used as a probe for screening a
human brain cDNA library.
[0089] The cDNA library was prepared from poly(A).sup.+ RNA
(purchased from Clontech) derived from human brain using a kit for
the synthesis of cDNA (trade name: Superscript Choice System;
manufactured by Gibco) and integrated into a site of phage vector
.lamda.ZipLox (manufactured by Gibco) cleaved by a restriction
enzyme EcoRI. Hybridization by a probe labeled with .sup.32P-dCTP
was carried out at 37.degree. C. in a solution for hybridization
for one night and a filter membrane was washed at 37.degree. C.
with 0.1.times.SSC/0.1% SDS. As to the solution for hybridization,
there was used a buffer of pH 6.5 containing 5.times.SSC, 3.times.
Denhard's solution, 0.2% SDS, 10% dextran sulfate, 50% formamide,
0.01% Abtiform B (trade name; Sigma) (antifoaming agent), 0.2 mg/ml
salmon sperm modified DNA, 2.5 mM sodium pyrophosphate and 25
mM-MES. A cDNA moiety of .lamda.ZipLox phage into which cDNA was
integrated was integrated into a plasmid pZL1.
[0090] The resulting clone or the clone containing cDNA of human
asc-1 was subjected to a base sequence determination of cDNA by a
diterminator cycle sequencing method (Applied Biosystems) using a
synthetic primer for determination of base sequence.
[0091] As a result, a base sequence of human asc-1 gene was
obtained. Further, a base sequence of cDNA was analyzed by a
conventional method to determine a translated region of cDNA and an
amino acid sequence of asc-1 encoded there.
[0092] Those sequences are represented by SEQ ID NO: 4 (amino acid
sequence) and 5 (base sequence) in the Sequence Listing which will
be mentioned later.
[0093] Comparison of the expected amino acid sequence of human
asc-1 and asc-1 of rat is shown in FIG. 2.
[0094] (4) Expression of Asc-1 Gene in Various Tissues of Mouse
(Analysis by a Northern Blotting)
[0095] cDNA fragment corresponding to 1-512 bases of asc-1 gene was
excised by restriction enzymes EcoRI and XhoI and labeled with
.sup.32P-dCTP and the resulting one is used as a probe for
conducting a northern blotting as follows to RNA extracted from
various tissues of mouse. Thus, 3 .mu.g of poly(A).sup.+ RNA were
subjected to electrophoresis with 1% agarose/formaldehyde gel and
transferred to a nitrocellulose filter. This filter was subjected
to hybridization for one night using a hybridization solution
containing asc-1 cDNA fragment labeled with .sup.32P_dCTP. The
filter was washed at 65.degree. C. with 0.1.times.SSC containing
0.1% SDS.
[0096] Result of the northern blotting is shown in FIG. 3 by a
picture which is a substitute for a drawing. As a result, a band
was detected near 1.9 kb in brain, lung and placenta. In addition,
a band was detected at about 4.4 kb in small intestine.
[0097] (5) Expression of asc-1 and 4F2hc Protein in Brain of
Mouse
[0098] Specific antibody to a synthetic oligopeptide
[PSPLPITDKPLKTQC] (SEQ ID NO: 16) corresponding to 517-530 amino
acid residues of asc-1 of mouse and to a synthetic oligopeptide
[CEGLLLQFPFVA] (SEQ ID NO: 17) (cysteine residue of C-terminal or
N-terminal was introduced for a conjugation with KLH (keyhole
limpet hemocyanine)) corresponding to 516-526 amino acid residue of
4F2hc of mouse was prepared according to a method of Altman, et al.
(Altman, et al., Proc. Natl. Acad. Sci. USA, volume 81, pages
2176-2180, 1984).
[0099] Fraction of cerebral membrane of mouse was prepared
according to a method of Thorens, et al. (Thorens, et al., Cell,
volume 55, pages 281-290, 1988). A protein sample was treated at
100.degree. C. for 5 minutes in the presence (under reducing
condition) or absence (under non-reducing condition) of 5%
2-mercaptoethanol, subjected to electrophoresis by
SDS-polyacrylamide gel, subjected to blotting to a Hybond-P PVDV
transfer membrane and treated with an anti-asc-1 antiserum
(1:10,000) or an anti-4F2hc antiserum (1:10,000).
[0100] The result is shown in a picture of FIG. 4 as a substitute
for a drawing. Left side of FIG. 4 is for the anti-asc-1 antibody
while right side thereof is for the anti-4F2hc antibody. They were
carried out under non-reducing condition (-) and reducing condition
(+), respectively.
[0101] As shown in FIG. 4, in the anti-asc-1 antiserum, a band of
118 kDa observed under a non-reducing condition disappeared under a
reducing condition and transferred to a band of 33 kDa. In the
anti-4F2hc antiserum, a band of 118 kDa observed under a
non-reducing condition disappeared under a reducing condition and a
band of 85 kDa appeared. Those results suggest that asc-1 and 4F2hc
were connected by a disulfide bond to form a heterodimer.
Example 2
Characterization of Sodium-Independent Small Neutral Amino Acid
Transporter Asc-1 which Transports L- and D-Amino Acids
[0102] (1) Role of 4F2hc in Transport Activity of asc-1
[0103] Incorporation of alanine when asc-1 gene cRNA of mouse was
solely expressed in oocytes of Xenopus and when asc-1 gene cRNA of
mouse and 4F2hc gene cRNA of mouse were expressed in oocytes of
Xenopus together was compared.
[0104] Into oocytes were injected 12 ng of asc-1 gene cRNA of
mouse, 13 ng of 4F2hc gene cRNA of mouse or 12 ng asc-1 gene cRNA
of mouse/13 ng of 4F2hc gene cRNA of mouse to express followed by
incubating for 3 days. With regard to the oocytes in which asc-1
gene cRNA, 4F2hc gene cRNA or asc-1 gene cRNA/4F2hc gene cRNA was
injected, there was carried out an experiment for incorporation of
the substrate according to a method by Kanai, et al. (Kanai and
Hediger, Nature, volume 360, pages 467-471, 1992) using alanine as
a substrate as follows. Thus, the oocytes were allowed to stand for
30 minutes in a Na.sup.+-free uptake solution [100 mM choline
chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM
magnesium chloride, 5 mM HEPES; pH 7.4] containing .sup.14C-alanine
(100 .mu.M) as a substrate and the rate of the substrate
incorporated into the cells was measured by way of counting the
radioactivity incorporated thereinto.
[0105] The result is shown in FIG. 5. In the oocytes where only
asc-1 was expressed, incorporation of alanine was in the same level
as in the case of the oocytes into which water was injected as a
control while, in the oocytes where both asc-1 and 4F2hc were
expressed together, a big incorporation of alanine was shown
whereby it is believed that 4F2hc is necessary for asc-1 to achieve
its function.
[0106] (2) Dependency of Transport Activity Asc-1 on Salt
[0107] In an experiment of incorporation of alanine by oocytes into
which both asc-1 gene cRNA and 4F2hc gene cRNA of mouse were
injected together, influence of the salt added to the medium was
investigated.
[0108] The experiment of incorporation of alanine was carried out
in accordance with the method mentioned in the above Example 2(1)
using oocytes into which both asc-1 gene cRNA and 4F2hc gene cRNA
of mouse were injected together. When the influence of sodium ion
was checked however, a standard uptake solution (where 100 mM
choline chloride was substituted with 100 mM sodium chloride) was
used in place of the Na.sup.+-free uptake solution as the uptake
solution. When the influence of chlorine ion was checked, a
gluconic acid uptake solution (where 100 mM sodium chloride was
substituted with 100 mM sodium gluconate) was used in place of the
standard uptake solution.
[0109] The result is shown in FIG. 6. Even when choline outside the
cells was changed to sodium or even when chlorine ion outside the
cells was changed to gluconic acid ion, that does no affect the
incorporation of alanine at all. From those, it is shown that asc-1
is a transporter which acts independently of sodium ion and
chlorine ion.
[0110] (3) A Michaelis-Menten Kinetic Test for Asc-1
[0111] A Michaelis-Menten kinetic test was carried out for a
sodium-independent small neutral amino acid transporter asc-1 which
transports L- and D-amino acids. By checking the changes in the
rate of incorporation of alanine due to the difference in
concentration of the alanine substrate, the Michaelis-Menten
kinetic test of asc-1 was carried out.
[0112] An experiment for the incorporation of alanine was carried
out according to the method mentioned in the above Example 2(1)
using the oocytes into which both asc-1 gene cRNA of mouse and
4F2hc gene cRNA of mouse were injected. The result is shown in FIG.
7. The result was that the Km value was 23.0.+-.5.1 .mu.M (mean
value.+-.standard error; n=4).
[0113] With regard to amino acids other than alanine which were
used as substrates, a Michaelis-Menten kinetic was carried out as
well in the same manner whereupon Km values and Vmax values were
calculated. The result is shown in the following Table 1. Each Vmax
value in Table 1 was shown in terms of the ratio when the Vmax
value of alanine was defined as 1.00. TABLE-US-00001 TABLE 1 Km
Values and Vmax values of Amino Acids Used as Substrates Km Amino
Acid .mu.M Vmax.sup.a L-Alanine 23.0 (1.00) Glycine 7.8 0.89
L-Serine 11.3 1.02 L-Threonine 19.3 0.86 L-Cysteine 23.7 0.82
L-Valine 112 1.17 L-Methionine 139 1.15 L-Isoleucine 160 1.33
L-Leucine 245 0.58 L-Histidine 368 0.79 L-Phenylalanine 464 1.09
AIB 22.7 0.81 D-Alanine 100 0.86 D-Serine 52.0 1.22 .beta.-Alanine
281 0.92 .sup.aThe Vmax value for each amino acid was shown in
terms of the ratio to the Vmax value for alanine
[0114] (4) Substrate Selectivity of Asc-1 (Experiment for
Inhibition by Addition of Amino Acid and Analogs Thereof)
[0115] In an experiment of incorporation of alanine by oocytes into
which both asc-1 gene cRNA of mouse and 4F2hc gene cRNA of mouse
were injected, influence of addition of various amino acid and
analogs thereof was investigated.
[0116] In an experiment of incorporation of alanine, a method
according to that mentioned in the above Example 2(1) was carried
out using oocytes into which both asc-1 gene cRNA of mouse and
4F2hc gene cRNA of mouse were injected. Here, a Na.sup.+-free
uptake solution was used and incorporation of .sup.14C-alanine (50
.mu.M) was measured in the presence and absence of 5 mM of various
compounds (non-labeled).
[0117] The result in the presence and absence (-) of various
L-amino acids or analogous compounds thereof is shown in FIG. 8.
The result in the presence and absence (-) of various D-amino acids
is shown in FIG. 9. The result in the presence and absence (-) of
alanine or analogous compounds thereof is shown in FIG. 10.
[0118] In various neutral L-amino acids, a cis-inhibiting effect
was observed. Glycine, alanine, serine, threonine and cysteine
particularly strongly inhibited the incorporation of
.sup.14C-alanine mediated by asc-1 (refer to FIG. 8).
[0119] Among the D-amino acids, D-alanine and D-serine strongly
inhibited the incorporation of .sup.14C-alanine mediated by asc-1.
D-Threonine and D-cysteine mediumly inhibited the incorporation of
.sup.14C-alanine mediated by asc-1 (refer to FIG. 9).
[0120] Even in the case of the substances other than standard amino
acids, .beta.-alanine, alanine methyl ester and
.alpha.-aminoisobutyric acid (.alpha.-methylalanine) also inhibited
the incorporation of .sup.14C-alanine mediated by asc-1 (refer to
FIG. 10). Acidic amino acids, basic amino acids, transport system
L-specific inhibitor 2-amino-2-norbornane-carboxylic acid (BCH),
.gamma.-aminoisobutyric acid and N-methylamino acids
(N-methylalanine, .alpha.-aminomethylisobutyric acid and sarcosine)
did not affect the incorporation of .sup.14C-alanine mediated by
asc-1 (refer to FIG. 8 and FIG. 10).
[0121] (5) Substrate Selectivity of Asc-1 (Test for Incorporation
Using Various Amino Acids and Analogs as Substrate)
[0122] Incorporation by asc-1 was investigated using various amino
acids and analogs thereof as substrates. Experiment for
incorporation of various amino acids and analogs thereof was
carried out according to the method mentioned in the above Example
2(1) using oocytes into which asc-1 gene cRNA of mouse and 4F2hc
gene cRNA of mouse were injected together. With regard to the
substrates however, various compounds labeled with radioactivity
were used in place of .sup.14C-alanine.
[0123] Result of incorporation of the radiolabeled L-amino acids is
shown in FIG. 11. Result of incorporation of the radiolabeled
D-amino acids is shown in FIG. 12. Result of incorporation of the
radiolabeled L-alanine or analogous compound thereof is shown in
FIG. 13.
[0124] As a result, a big incorporation into the oocytes was noted
when glycine (a .sup.14C compound), L-alanine (a .sup.14C
compound), L-serine (a .sup.14C compound), L-threonine (a .sup.14C
compound), L-cysteine (a .sup.14C compound) (for those, refer to
FIG. 11), D-alanine (a .sup.14C compound), D-serine (a .sup.14C
compound) (for those, refer to FIG. 12), .beta.-alanine (a .sup.14C
compound) and a-aminoisobutyric acid (a .sup.14C compound) (for
those, refer to FIG. 13) were used as substrates.
[0125] (6) pH-Dependency of Transport Activity of Asc-1
[0126] Influence of pH was checked in an experiment for
incorporation of alanine by oocytes into which both asc-1 gene cRNA
and 4F2hc gene cRNA of mouse were injected together. With regard to
an experiment for incorporation of alanine, the method according to
that mentioned in the above Example 2 (1) was carried out using
oocytes into which both asc-1 gene cRNA of mouse and 4F2hc gene
cRNA of mouse were injected together.
[0127] Result where the influence of pH in the experiment for
incorporation of alanine was checked is shown in FIG. 14. As a
result thereof, there was no significant dependency on pH in the
incorporation of alanine (refer to FIG. 14).
[0128] (7) Test of Release of Amino Acid Mediated by Asc-1
[0129] Release of a preloaded .sup.14C-alanine mediated by asc-1
was checked in the oocytes into which both asc-1 gene cRNA and
4F2hc gene cRNA of mouse were injected together. .sup.14C-Alanine
(-3nCi) of 100 .mu.M in an amount of 100 nl was injected into the
oocytes into which both asc-1 gene cRNA and 4F2hc gene cRNA of
mouse were injected together, washed with an ice-cooled
Na.sup.+-free uptake solution containing no alanine and transferred
to a Na.sup.+-free uptake solution to which alanine (100 .mu.M) was
added or not added at room temperature (18.degree. C.-22.degree.
C.) and the amount of .sup.14C-alanine released outside the cells
was measured.
[0130] Further, .sup.14C-leucine was similarly injected into
oocytes into which LAT1 gene cRNA and 4F2hc gene cRNA of rat were
injected together (Kanai et al., J. Biol. Chem., volume 273, page
23629, 1988), washed with an ice-cooled Na.sup.+-free uptake
solution containing no leucine and transferred to a Na.sup.+-free
uptake solution to which leucine (100 .mu.M) was added or not added
at room temperature (18.degree. C.-22.degree. C.) whereupon the
amount of .sup.14C-leucine released outside the cells was
measured.
[0131] Those results are shown in FIG. 15, FIG. 16 and FIG. 17.
[0132] FIG. 15 shows the result of checking the release of
.sup.14C-alanine from the oocytes into which cRNA of asc-1 gene of
mouse and cRNA of 4F2hc gene of mouse were injected and the
ordinate in the drawing shows a rate (%) of the released
radioactivity to the radioactivity injected into the oocytes. Left
side of FIG. 15 shows the case in the absence of Na (-) while right
side thereof shows that in the presence of Na (+) and, in each
graph, L-Ala(-) shows the case where L-alanine was not added while
L-Ala(+) shows the case where L-alanine was added.
[0133] FIG. 16 shows the result of checking the progress of release
of .sup.14C-alanine with a lapse of time by oocytes into which cRNA
of mouse asc-1 gene and cRNA of mouse 4F2hc gene are injected. In
the drawing, .largecircle. is the case where, in the release of
.sup.14C-alanine in the oocytes into which water is injected as a
control instead of cRNA of mouse asc-1 gene and cRNA of mouse 4F2hc
gene, a Na.sup.+-free uptake solution to which no alanine is added
is used; .circle-solid. is the case where, in the release of
.sup.14C-alanine in the oocytes into which water is injected as a
control instead of cRNA of mouse asc-1 gene and cRNA of mouse 4F2hc
gene, a Na.sup.+-free uptake solution to which alanine is added is
used; .quadrature. is the case where, in the release of
.sup.14C-alanine in the oocytes into which cRNA of mouse asc-1 gene
and cRNA of mouse 4F2hc gene are injected, a Na.sup.+-free uptake
solution to which no alanine is added is used; and .box-solid. is
the case where, in the release of .sup.14C-alanine in the oocytes
into which cRNA of mouse asc-1 gene and cRNA of mouse 4F2hc gene
are injected, a Na.sup.+-free uptake solution to which alanine is
added is used. The ordinate in the drawing shows the rate (%) of
the released radioactivity to the radioactivity injected into the
oocytes.
[0134] FIG. 17 shows the result of checking the progress of release
of .sup.14C-leucine with a lapse of time by oocytes into which cRNA
of rat LAT1 gene and cRNA of rat 4F2hc gene are injected. In the
drawing, 0 is the case where, in the release of .sup.14C-leucine in
the oocytes into which water is injected as a control instead of
cRNA of rat LAT1 gene and cRNA of rat 4F2hc gene, a Na.sup.+-free
uptake solution to which no leucine is added is used;
.circle-solid. is the case where, in the release of
.sup.14C-leucine in the oocytes into which water is injected as a
control instead of cRNA of rat LAT1 gene and cRNA of rat 4F2hc
gene, a Na.sup.+-free uptake solution to which leucine is added is
used; .circle-solid. is the case where, in the release of
.sup.14C-leucine in the oocytes into which cRNA of rat LAT1 gene
and cRNA of rat 4F2hc gene are injected, a Na.sup.+-free uptake
solution to which no leucine is added is used; and .box-solid. is
the case where, in the release of .sup.14C-leucine in the oocytes
into which cRNA of rat LAT1 gene and cRNA of rat 4F2hc gene are
injected, a Na.sup.+-free uptake solution to which leucine is added
is used. The ordinate in the drawing shows the rate (%) of the
released radioactivity to the radioactivity injected into the
oocytes.
[0135] As a result, even when alanine was not added to the outside
of the cells, a significant release of .sup.14C-alanine was
observed in the case of asc-1 and such a release significantly
increased by addition of alanine to the outside of the cells (refer
to FIG. 15 and FIG. 16). On the other hand, in the case of LAT1
which is a complete exchange transport mediating the forced
exchange, release of leucine was observed only when leucine was
added to the outside of the cells (refer to FIG. 17). Accordingly,
although asc-1 is mostly in an exchange transport mode, it was
found to be a transporter where a transport mode of a promotion
diffusion type is mixed as well.
[0136] (8) Investigation of Substrate Selectivity of Asc-1
Utilizing the Release Test of Amino Acids
[0137] In oocytes into which cRNA of asc-1 gene and cRNA of 4F2hc
gene of mouse were injected together, release of preloaded
.sup.14C-alanine mediated by asc-1 was investigated whereupon it
was checked whether the compound which inhibited the incorporation
of .sup.14C-alanine mediated by asc-1 was a substrate of asc-1.
[0138] .sup.14C-Alanine (-3nCi) of 100 .mu.M in an amount of 100 nl
was injected into the oocytes into which both asc-1 gene cRNA and
4F2hc gene cRNA of mouse were injected together, washed with an
ice-cooled Na.sup.+-free uptake solution containing no alanine and
transferred to a Na.sup.+-free uptake solution to which amino acid
or amino acid analog (100 .mu.M) was added or not added at room
temperature (18.degree. C.-22.degree. C.) and the amount of
.sup.14C-alanine released outside the cells was measured.
[0139] The result is shown in FIG. 18. Black bars in FIG. 18 are
the case where there were used oocytes into which cRNA of mouse
asc-1 gene and cRNA of mouse 4F2hc gene were injected while white
bars are the case where there were used oocytes into which water
was injected instead of cRNA as a control. (-) shows the case where
no amino acid was added to a Na.sup.+-free uptake solution. The
ordinate in FIG. 18 shows the rate (%) of the released
radioactivity to the radioactivity injected into the oocytes.
[0140] As a result, a high increase in the release of
.sup.14C-alanine was observed in glycine, alanine, serine and
threonine and a medium increase therein was observed in methionine
and valine (refer to FIG. 18). Such a result coincides with the
result of the test for incorporation of amino acids (refer to FIG.
11) and the test for release of amino acids was shown to be able to
be used for determination of substrate selectivity of asc-1.
[0141] The result where further investigation was conducted for
D-amino acids and amino acid analogs using the said method is shown
in FIG. 19 and FIG. 20. With regard to D-amino acids, D-alanine,
D-serine, D-threonine and D-cysteine resulted in a significant
increase in the release of .sup.14C-alanine (refer to FIG. 19).
With regard to amino acid analogs, .beta.-alanine, alanine methyl
ester and .alpha.-aminoisobutyric acid (AIB) resulted in a
significant increase in the release of .sup.14C-alanine (refer to
FIG. 20). It was therefore found that D-threonine, D-cysteine and
alanine methyl ester which have been unable to be subjected to an
incorporation experiment using radiolabeled ones because of
unavailability of radiolabeled compounds are now able to be
substrates for asc-1. As such, when an amino acid releasing test is
used, it is now possible to screen whether a compound can be a
substrate for asc-1 or, in other words, whether it can be
transported by asc-1 even in the case of the compound where no
radiolabeled one is available for the investigation.
[0142] (9) Investigation of Substrate Selectivity of Intracellular
Substrate-Binding Site of Asc-1 Utilizing the Release Test of Amino
Acids
[0143] In oocytes into which cRNA of asc-1 gene and cRNA of 4F2hc
gene of mouse were injected together, release of preloaded
.sup.14C-amino acid mediated by asc-1 was checked whereby the
substrate selectivity of intracellular substrate-binding site of
asc-1 was checked.
[0144] .sup.14C-Amino acid (.about.3nCi) of 100 .mu.M in an amount
of 100 nl was injected into the oocytes into which both asc-1 gene
cRNA and 4F2hc gene cRNA of mouse were injected together, washed
with an ice-cooled Na.sup.+-free uptake solution containing no
alanine and transferred to a Na.sup.+-free uptake solution to which
alanine (100 .mu.M) was added or not added at room temperature
(18.degree. C.-22.degree. C.) and the amount of .sup.14C-amino acid
released outside the cells was measured.
[0145] The result is shown in FIG. 21. Black bars in FIG. 21 show
the case where alanine was added to a Na.sup.+-free uptake solution
while bars with oblique lines show the case where alanine was not
added to a Na.sup.+-free uptake solution. The ordinate of FIG. 21
shows the rate (%) of the release radioactivity to the
radioactivity injected into oocytes.
[0146] As a result, an increase in release of .sup.14C-labeled
glycine, alanine, serine, threonine and cysteine injected into the
cells by extracellular alanine was observed. Therefore, it was
shown that the intracellular substrate-binding site shows a
substrate selectivity for receiving small neutral amino acid such
as glycine, alanine, serine, threonine and cysteine as same as in
the extracellular case.
[0147] (10) Confirmation of Human Asc-1
[0148] From a plasmid containing cDNA of human asc-1 obtained in
Example 1(3), cRNA (RNA complementary to cDNA) was prepared using a
T7 RNA polymerase. Comparison was made between incorporation of
.sup.14C-alanine in the case where human asc-1 gene cRNA was solely
expressed in oocytes with that in the case where both human asc-1
gene cRNA and human 4F2hc gene cRNA were expressed in oocytes.
[0149] Expression was carried out by injection of 12.5 ng of human
asc-1 gene cRNA, 12.5 ng of human 4F2hc gene cRNA or 12.5 ng of
human asc-1 gene cRNA/12.5 ng of human 4F2hc gene cRNA into oocytes
and incubation was conducted for three days. With regard to the
oocytes into which human asc-1 gene cRNA, 4F2hc gene cRNA or human
asc-1 gene cRNA/4F2hc gene cRNA was injected, an experiment for
incorporation of substrate was carried out according to Example
2(1) using alanine as a substrate.
[0150] The result is as follows. Like in the case of asc-1 of
mouse, the oocytes where only asc-1 was expressed showed
incorporation of alanine in the same level as in the case of
oocytes into which water was injected as a control while, in the
oocytes where both asc-1 and 4F2hc were expressed together, a big
incorporation of alanine was observed. Accordingly, like asc-1 of
mouse, human asc-1 was also shown to achieve the function only when
it was present together with 4F2hc. It is also noted that the human
asc-1 shows the same property as the above-mentioned asc-1 of
mouse.
INDUSTRIAL APPLICABILITY
[0151] The sodium-independent small neutral amino acid transporter
according to the present invention which transports L- and D-amino
acids and gene thereof makes it possible to conduct an in vitro
investigation of transport of small neutral amino acids of L- and
D-forms and amino acid analogous compounds including exogenous
matters at the expressed part of the said transporter and also to
conduct an in vitro presumption of fate of those compounds in vivo
on the basis of the above. Further, that is useful for the
development of medicaments which efficiently permeate the expressed
part of the said transporter and the present invention provides
novel amino acid transporters. Furthermore, as a result of
modulation of an ability of the said transporter for transporting
the small neutral L- and D-amino acids and analogs thereof, it is
useful as a method for controlling the resistance of cells to
oxidative stress, a method for controlling the activity of glutamic
acid receptor of an NMDA type in nervous system, a method for
controlling the cell growth and a method for screening the
medicament having such activities.
Sequence CWU 1
1
29 1 530 PRT Mus sp. 1 Met Arg Arg Asp Ser Asp Met Ala Ser His Ile
Gln Gln Pro Gly Gly 1 5 10 15 His Gly Asn Pro Gly Pro Ala Pro Ser
Pro Ser Pro Gly Pro Gly Pro 20 25 30 Gly Pro Gly Ala Ser Glu Arg
Val Ala Leu Lys Lys Glu Ile Gly Leu 35 40 45 Val Ser Ala Cys Thr
Ile Ile Ile Gly Asn Ile Ile Gly Ser Gly Ile 50 55 60 Phe Ile Ser
Pro Lys Gly Val Leu Glu His Ser Gly Ser Val Gly Leu 65 70 75 80 Ala
Leu Phe Val Trp Val Leu Gly Gly Gly Val Thr Ala Leu Gly Ser 85 90
95 Leu Cys Tyr Ala Glu Leu Gly Val Ala Ile Pro Lys Ser Gly Gly Asp
100 105 110 Tyr Ala Tyr Val Thr Glu Ile Phe Gly Gly Leu Ala Gly Phe
Leu Leu 115 120 125 Leu Trp Ser Ala Val Leu Ile Met Tyr Pro Thr Ser
Leu Ala Val Ile 130 135 140 Ser Met Thr Phe Ser Asn Tyr Val Leu Gln
Pro Val Phe Pro Asn Cys 145 150 155 160 Ile Pro Pro Ala Thr Ala Ser
Arg Val Leu Ser Met Ala Cys Leu Met 165 170 175 Leu Leu Thr Trp Val
Asn Ser Ser Ser Val Arg Trp Ala Thr Arg Ile 180 185 190 Gln Val Ile
Phe Thr Gly Gly Lys Leu Leu Ala Leu Ser Leu Ile Ile 195 200 205 Thr
Val Gly Phe Val Gln Ile Phe Gln Gly His Phe Glu Glu Leu Arg 210 215
220 Pro Thr Asn Ala Phe Ala Phe Trp Met Thr Pro Ser Val Gly His Leu
225 230 235 240 Ala Leu Ala Phe Leu Gln Gly Ser Phe Ala Phe Ser Gly
Trp Asn Phe 245 250 255 Leu Asn Tyr Val Thr Glu Glu Leu Val Asp Pro
Arg Lys Asn Leu Pro 260 265 270 Arg Ala Ile Phe Ile Ser Ile Pro Leu
Val Thr Phe Val Tyr Thr Phe 275 280 285 Thr Asn Val Ala Tyr Phe Thr
Ala Met Ser Pro Gln Glu Leu Leu Ser 290 295 300 Ser Asn Ala Val Ala
Val Thr Phe Gly Glu Lys Leu Leu Gly Tyr Phe 305 310 315 320 Ser Trp
Val Met Pro Val Ser Val Ala Leu Ser Thr Phe Gly Gly Ile 325 330 335
Asn Gly Tyr Leu Phe Thr Ser Ser Arg Leu Cys Phe Ser Gly Ala Arg 340
345 350 Glu Gly His Leu Pro Ser Phe Leu Ala Met Ile His Val Arg Arg
Cys 355 360 365 Thr Pro Ile Pro Ala Leu Leu Val Cys Cys Gly Ala Thr
Ala Val Ile 370 375 380 Met Leu Val Gly Asp Thr Tyr Thr Leu Ile Asn
Tyr Val Ser Phe Ile 385 390 395 400 Asn Tyr Leu Cys Tyr Gly Val Thr
Ile Leu Gly Leu Leu Val Leu Arg 405 410 415 Trp Arg Arg Pro Ala Leu
His Arg Pro Ile Lys Val Asn Leu Leu Val 420 425 430 Pro Val Val Tyr
Leu Val Phe Trp Ala Phe Leu Leu Val Phe Ser Phe 435 440 445 Ile Ser
Glu Pro Met Val Cys Gly Val Gly Ile Ile Ile Ile Leu Thr 450 455 460
Gly Val Pro Ile Phe Phe Leu Gly Val Phe Trp Arg Ser Lys Pro Lys 465
470 475 480 Cys Val His Arg Phe Thr Glu Ser Met Thr Arg Trp Gly Gln
Glu Leu 485 490 495 Cys Phe Val Val Tyr Pro Gln Gly Ser Leu Glu Glu
Glu Glu Asn Gly 500 505 510 Pro Met Gly Gln Pro Ser Pro Leu Pro Ile
Thr Asp Lys Pro Leu Lys 515 520 525 Thr Gln 530 2 1716 DNA Mus sp.
2 agggaactgg gatgaggcgg gacagcgaca tggcaagcca catacaacag ccaggcgggc
60 acgggaaccc cggccctgcg ccctcgcctt ccccgggccc tggtcccggc
ccgggcgcct 120 cggagcgggt ggcactcaag aaagagatcg ggctggtgag
cgcttgcacc atcatcatcg 180 ggaacatcat tggctcaggc atcttcatct
cacccaaggg tgtcctggaa cactcgggct 240 ccgtgggttt ggccctcttc
gtctgggtcc tgggtggggg cgtgacagct ctgggctctc 300 tctgctatgc
agagctgggt gtcgccatcc ccaagtctgg tggggactac gcctatgtca 360
ctgagatctt cgggggcctg gctggattcc tactgctctg gagtgctgtc ctcatcatgt
420 accccaccag cctggctgtc atctccatga ccttctccaa ctatgtgctt
cagcctgtct 480 ttcccaactg tatcccccca gccacagcct ctcgagtact
ctccatggcc tgcctgatgc 540 tcctgacgtg ggtgaacagc tccagcgtac
gctgggccac gcgcatccag gttatcttca 600 ctggtgggaa gctgctggcg
ctgtctctca tcatcactgt tggctttgtc cagatcttcc 660 aaggacactt
tgaagagctg agacccacca atgccttcgc cttctggatg acaccgtctg 720
tgggtcacct ggccctggct ttcctccaag gttcttttgc cttcagtggc tggaacttcc
780 tcaactatgt cacggaggag ctggttgacc cacgcaagaa cctacctcgt
gccatcttca 840 tttccatccc actggtcacc tttgtgtaca cattcaccaa
tgtcgcctac ttcactgcca 900 tgtcccccca ggagttgctg tcctccaacg
ccgtggcggt gaccttcggc gagaagctgc 960 tgggctactt ttcgtgggtc
atgcccgtct ctgtggccct ctctactttt ggagggatca 1020 atggctacct
gttcacctca tccaggctat gcttctctgg agcccgagag ggacacttac 1080
ccagcttcct ggccatgatt catgtcagac gctgcacccc aatccctgcc ctccttgtct
1140 gttgcggggc cacagcggtc atcatgctcg tgggtgacac atacacactc
atcaactatg 1200 tgtccttcat caactacctc tgctacggag tcactatcct
gggcctgctt gtgctgcgct 1260 ggagacggcc ggcactccac aggcccatta
aggtgaacct cctcgttcct gttgtgtact 1320 tggtgttctg ggcattccta
ctggtcttca gcttcatctc ggagcccatg gtctgtgggg 1380 tcggcatcat
cattatcctc actggggttc ccatcttctt cctgggagtg ttctggagaa 1440
gcaaaccaaa gtgtgtacac agattcacag agtccatgac acgctggggc caggagctgt
1500 gtttcgtggt ttacccccag ggctccctag aggaggagga aaatggcccc
atgggccagc 1560 cctccccatt gcccatcacg gacaagccct tgaagacaca
atgagacctt gtagagactg 1620 gaacagccga ttctgtttac atgttgttta
ttgagaaggg ggttgtgttt tgttttgttt 1680 tcaaaaattt tttttctgca
aaaaaaaaaa aaaaaa 1716 3 1852 DNA Mus sp. CDS (106)..(1683) 3
gctagcctca cggccacggg acgcctctct gaacggggat ccaggcagga ttagagctgc
60 ctcactgact acaggccgtg tcgtgtcacc gtttctgcag gcacc atg agc cag
gac 117 Met Ser Gln Asp 1 acc gaa gtg gac atg aaa gat gtg gag ctg
aac gag cta gaa ccg gag 165 Thr Glu Val Asp Met Lys Asp Val Glu Leu
Asn Glu Leu Glu Pro Glu 5 10 15 20 aag cag ccc atg aat gca gcg gac
ggg gcg gcg gcc ggg gag aag aac 213 Lys Gln Pro Met Asn Ala Ala Asp
Gly Ala Ala Ala Gly Glu Lys Asn 25 30 35 ggt ctg gtg aag atc aag
gtg gcg gag gac gag acg gag gcc ggg gtc 261 Gly Leu Val Lys Ile Lys
Val Ala Glu Asp Glu Thr Glu Ala Gly Val 40 45 50 aag ttc acc ggc
tta tcc aag gag gag cta ctg aag gta gcg ggc agc 309 Lys Phe Thr Gly
Leu Ser Lys Glu Glu Leu Leu Lys Val Ala Gly Ser 55 60 65 cct ggc
tgg gtg cgc acc cgc tgg gcg ctg ctg ctg ctc ttc tgg ctc 357 Pro Gly
Trp Val Arg Thr Arg Trp Ala Leu Leu Leu Leu Phe Trp Leu 70 75 80
ggt tgg ctg ggc atg ctg gcg ggc gcc gtg gtt atc atc gtt cgg gcg 405
Gly Trp Leu Gly Met Leu Ala Gly Ala Val Val Ile Ile Val Arg Ala 85
90 95 100 ccg cgc tgc cgt gag ctg cct gta cag agg tgg tgg cac aag
ggc gcc 453 Pro Arg Cys Arg Glu Leu Pro Val Gln Arg Trp Trp His Lys
Gly Ala 105 110 115 ctc tac cgc atc ggc gac ctt cag gcc ttt gta ggc
cgg gat gcg gga 501 Leu Tyr Arg Ile Gly Asp Leu Gln Ala Phe Val Gly
Arg Asp Ala Gly 120 125 130 ggc ata gct ggt ctg aag agc cat ctg gag
tac ttg agc acc ctg aag 549 Gly Ile Ala Gly Leu Lys Ser His Leu Glu
Tyr Leu Ser Thr Leu Lys 135 140 145 gtg aag ggc ctg gtg tta ggc cca
att cac aag aac cag aag gat gaa 597 Val Lys Gly Leu Val Leu Gly Pro
Ile His Lys Asn Gln Lys Asp Glu 150 155 160 atc aat gaa acc gac ctg
aaa cag att aat ccc act ttg ggc tcc cag 645 Ile Asn Glu Thr Asp Leu
Lys Gln Ile Asn Pro Thr Leu Gly Ser Gln 165 170 175 180 gaa gat ttt
aaa gac ctt cta caa agt gcc aag aaa aag agc att cac 693 Glu Asp Phe
Lys Asp Leu Leu Gln Ser Ala Lys Lys Lys Ser Ile His 185 190 195 atc
att ttg gac ctc act ccc aac tac cag ggc cag aat gcg tgg ttc 741 Ile
Ile Leu Asp Leu Thr Pro Asn Tyr Gln Gly Gln Asn Ala Trp Phe 200 205
210 ctc cct gct cag gct gac att gta gcc acc aaa atg aag gaa gct ctg
789 Leu Pro Ala Gln Ala Asp Ile Val Ala Thr Lys Met Lys Glu Ala Leu
215 220 225 agt tct tgg ttg cag gac ggt gtg gat ggt ttc caa ttc cgg
gat gtg 837 Ser Ser Trp Leu Gln Asp Gly Val Asp Gly Phe Gln Phe Arg
Asp Val 230 235 240 gga aag ctg atg aat gca ccc ttg tac ttg gct gag
tgg cag aat atc 885 Gly Lys Leu Met Asn Ala Pro Leu Tyr Leu Ala Glu
Trp Gln Asn Ile 245 250 255 260 acc aag aac tta agt gag gac agg ctt
ttg att gca ggg act gag tcc 933 Thr Lys Asn Leu Ser Glu Asp Arg Leu
Leu Ile Ala Gly Thr Glu Ser 265 270 275 tct gac ctg cag caa att gtc
aac ata ctt gaa tcc acc agc gac ctg 981 Ser Asp Leu Gln Gln Ile Val
Asn Ile Leu Glu Ser Thr Ser Asp Leu 280 285 290 ctg ttg acc agc tcc
tac ctg tca aat tcc act ttc act ggg gag cgt 1029 Leu Leu Thr Ser
Ser Tyr Leu Ser Asn Ser Thr Phe Thr Gly Glu Arg 295 300 305 act gaa
tcc cta gtc act agg ttt ttg aat gcc act ggc agc caa tgg 1077 Thr
Glu Ser Leu Val Thr Arg Phe Leu Asn Ala Thr Gly Ser Gln Trp 310 315
320 tgc agc tgg agt gtg tcg caa gca gga ctc ctc gca gac ttt ata ccg
1125 Cys Ser Trp Ser Val Ser Gln Ala Gly Leu Leu Ala Asp Phe Ile
Pro 325 330 335 340 gac cat ctt ctc cga ctc tac cag ctg ctg ctc ttc
act ctg cca ggg 1173 Asp His Leu Leu Arg Leu Tyr Gln Leu Leu Leu
Phe Thr Leu Pro Gly 345 350 355 act cct gtt ttt agc tac ggg gat gag
ctt ggc ctt cag ggt gcc ctt 1221 Thr Pro Val Phe Ser Tyr Gly Asp
Glu Leu Gly Leu Gln Gly Ala Leu 360 365 370 cct gga cag cct gcg aag
gcc cca ctc atg ccg tgg aat gag tcc agc 1269 Pro Gly Gln Pro Ala
Lys Ala Pro Leu Met Pro Trp Asn Glu Ser Ser 375 380 385 atc ttt cac
atc cca aga cct gta agc ctc aac atg aca gtg aag ggc 1317 Ile Phe
His Ile Pro Arg Pro Val Ser Leu Asn Met Thr Val Lys Gly 390 395 400
cag aat gaa gac cct ggc tcc ctt ctt acc cag ttc cgg cgg ctg agt
1365 Gln Asn Glu Asp Pro Gly Ser Leu Leu Thr Gln Phe Arg Arg Leu
Ser 405 410 415 420 gac ctt cgg ggt aag gag cgc tct ctg ttg cac ggt
gac ttc cat gca 1413 Asp Leu Arg Gly Lys Glu Arg Ser Leu Leu His
Gly Asp Phe His Ala 425 430 435 ctg tct tcc tca cct gac ctc ttc tcc
tac ata cga cac tgg gac cag 1461 Leu Ser Ser Ser Pro Asp Leu Phe
Ser Tyr Ile Arg His Trp Asp Gln 440 445 450 aat gag cgt tac ctg gtg
gtg ctc aac ttc cga gat tcg ggc cgg tca 1509 Asn Glu Arg Tyr Leu
Val Val Leu Asn Phe Arg Asp Ser Gly Arg Ser 455 460 465 gcc agg cta
ggg gcc tcc aac ctc cct gct ggc ata agc ctg cca gcc 1557 Ala Arg
Leu Gly Ala Ser Asn Leu Pro Ala Gly Ile Ser Leu Pro Ala 470 475 480
agc gct aaa ctt ttg ctt agt acc gac agt gcc cgg caa agc cgt gag
1605 Ser Ala Lys Leu Leu Leu Ser Thr Asp Ser Ala Arg Gln Ser Arg
Glu 485 490 495 500 gag gac acc tcc ctg aag ctg gaa aac ctg agc ctg
aat cct tat gag 1653 Glu Asp Thr Ser Leu Lys Leu Glu Asn Leu Ser
Leu Asn Pro Tyr Glu 505 510 515 ggc ttg ctg tta cag ttc ccc ttt gtg
gcc tgatccttcc tatgcagaac 1703 Gly Leu Leu Leu Gln Phe Pro Phe Val
Ala 520 525 ctaccaccct cctttgttct ccccaggcct tttggattct agtcttcctc
tccttgtttt 1763 taaacttttg cagattacat acgaattctt atactgggtg
tttttgtctt caaataaaaa 1823 catcacccct gcctcaaaaa aaaaaaaaa 1852 4
523 PRT Homo sapiens 4 Met Ala Gly His Thr Gln Gln Pro Ser Gly Arg
Gly Asn Pro Arg Pro 1 5 10 15 Ala Pro Ser Pro Ser Pro Val Pro Gly
Thr Val Pro Gly Ala Ser Glu 20 25 30 Arg Val Ala Leu Lys Lys Glu
Ile Gly Leu Leu Ser Ala Cys Thr Ile 35 40 45 Ile Ile Gly Asn Ile
Ile Gly Ser Gly Ile Phe Ile Ser Pro Lys Gly 50 55 60 Val Leu Glu
His Ser Gly Ser Val Gly Leu Ala Leu Phe Val Trp Val 65 70 75 80 Leu
Gly Gly Gly Val Thr Ala Leu Gly Ser Leu Cys Tyr Ala Glu Leu 85 90
95 Gly Val Ala Ile Pro Lys Ser Gly Gly Asp Tyr Ala Tyr Val Thr Glu
100 105 110 Ile Phe Gly Gly Leu Ala Gly Phe Leu Leu Leu Trp Ser Ala
Val Leu 115 120 125 Ile Met Tyr Pro Thr Ser Leu Ala Val Ile Ser Met
Thr Phe Ser Asn 130 135 140 Tyr Val Leu Gln Pro Val Phe Pro Asn Cys
Ile Pro Pro Thr Thr Ala 145 150 155 160 Ser Arg Val Leu Ser Met Ala
Cys Leu Met Leu Leu Thr Trp Val Asn 165 170 175 Ser Ser Ser Val Arg
Trp Ala Thr Arg Ile Gln Asp Met Phe Thr Gly 180 185 190 Gly Lys Leu
Leu Ala Leu Ser Leu Ile Ile Gly Val Gly Leu Leu Gln 195 200 205 Ile
Phe Gln Gly His Phe Glu Glu Leu Arg Pro Ser Asn Ala Phe Ala 210 215
220 Phe Trp Met Thr Pro Ser Val Gly His Leu Ala Leu Ala Phe Leu Gln
225 230 235 240 Gly Ser Phe Ala Phe Ser Gly Trp Asn Phe Leu Asn Tyr
Val Thr Glu 245 250 255 Glu Met Val Asp Ala Arg Lys Asn Leu Pro Arg
Ala Ile Phe Ile Ser 260 265 270 Ile Pro Leu Val Thr Phe Val Tyr Thr
Phe Thr Asn Ile Ala Tyr Phe 275 280 285 Thr Ala Met Ser Pro Gln Glu
Leu Leu Ser Ser Asn Ala Val Ala Val 290 295 300 Thr Phe Gly Glu Lys
Leu Leu Gly Tyr Phe Ser Trp Val Met Pro Val 305 310 315 320 Ser Val
Ala Leu Ser Thr Phe Gly Gly Ile Asn Gly Tyr Leu Phe Thr 325 330 335
Tyr Ser Arg Leu Cys Phe Ser Gly Ala Arg Glu Gly His Leu Pro Ser 340
345 350 Leu Leu Ala Met Ile His Val Arg His Cys Thr Pro Ile Pro Ala
Leu 355 360 365 Leu Val Cys Cys Gly Ala Thr Ala Val Ile Met Leu Val
Gly Asp Thr 370 375 380 Tyr Thr Leu Ile Asn Tyr Val Ser Phe Ile Asn
Tyr Leu Cys Tyr Gly 385 390 395 400 Val Thr Ile Leu Gly Leu Leu Leu
Leu Arg Trp Arg Arg Pro Ala Leu 405 410 415 His Arg Pro Ile Lys Val
Asn Leu Leu Ile Pro Val Ala Tyr Leu Val 420 425 430 Phe Trp Ala Phe
Leu Leu Val Phe Ser Phe Ile Ser Glu Pro Met Val 435 440 445 Cys Gly
Val Gly Val Ile Ile Ile Leu Thr Gly Val Pro Ile Phe Phe 450 455 460
Leu Gly Val Phe Trp Arg Ser Lys Pro Lys Cys Val His Arg Leu Thr 465
470 475 480 Glu Ser Met Thr His Trp Gly Gln Glu Leu Cys Phe Val Val
Tyr Pro 485 490 495 Gln Asp Ala Pro Glu Glu Glu Glu Asn Gly Pro Cys
Pro Pro Ser Leu 500 505 510 Leu Pro Ala Thr Asp Lys Pro Ser Lys Pro
Gln 515 520 5 1918 DNA Homo sapiens 5 cggctgcgag ggccgtgagc
tcacggaccg acggaccgac gggcggccgg ccggacagac 60 ggggcagcgc
agggagcggg gacgcggcgg gacagcgaca tggccggcca cacgcagcag 120
ccgagcgggc gcgggaaccc caggcctgcg ccctcgccct ccccagtccc agggaccgtc
180 cccggcgcct cggagcgggt ggcgctcaag aaggagatcg ggctgctgag
cgcctgcacc 240 atcatcatcg ggaacatcat cggctcgggc atcttcatct
cgcccaaggg ggtcctggag 300 cactcaggct ccgtgggtct ggccctgttc
gtctgggtcc tgggtggggg cgtgacggct 360 ctgggctccc tctgctatgc
agagctggga gtcgccatcc ccaagtctgg cggggactac 420 gcctacgtca
cagagatctt cgggggcctg gctggctttc tgctgctctg gagcgccgtc 480
ctcatcatgt accccaccag ccttgctgtc atctccatga ccttctccaa ctacgtgctg
540 cagcccgtgt tccccaactg catccccccc accacagcct cccgggtgct
gtccatggcc 600 tgcctgatgc tcctgacatg ggtgaacagc tccagtgtgc
gctgggccac gcgcatccag 660 gacatgttca caggcgggaa gctgctggcc
ttgtccctca tcatcggcgt gggccttctc 720 cagatcttcc aaggacactt
cgaggagctg aggcccagca atgcctttgc tttctggatg 780 acgccctccg
tgggacacct ggccctggcc ttcctccagg gctccttcgc cttcagtggc 840
tggaacttcc tcaactatgt caccgaggag atggttgacg cccgaaagaa cctacctcgc
900 gccatcttca tctccatccc actggtgacc ttcgtgtaca cgttcaccaa
cattgcctac 960 ttcacggcca tgtcccccca ggagctgctc tcctccaatg
cggtggctgt gaccttcggg 1020 gagaagctgc tgggctactt ttcttgggtc
atgcctgtct ccgtggctct gtcaaccttc 1080 ggagggatca atggttacct
gttcacctac tccaggctgt gcttctctgg
agcccgcgag 1140 gggcacctgc ccagcctgct ggccatgatc cacgtcagac
actgcacccc catccccgcc 1200 ctcctcgtct gttgcggggc cacagccgtc
atcatgctcg tgggcgacac gtacacgctc 1260 atcaactatg tgtccttcat
caactacctc tgctacggcg tcaccatcct gggcctgctg 1320 ctgctgcgct
ggaggcggcc tgcactccac aggcccatca aggtgaacct tctcatcccc 1380
gtggcgtact tggtcttctg ggccttcctg ctggtcttca gcttcatctc agagcctatg
1440 gtctgtgggg tcggcgtcat catcatcctt acgggggtgc ccattttctt
tctgggagtg 1500 ttctggagaa gcaaaccaaa gtgtgtgcac agactcacag
agtccatgac acactggggc 1560 caggagctgt gtttcgtggt ctacccccag
gacgcccccg aagaggagga gaatggcccc 1620 tgcccaccct ccctgctgcc
tgccacagac aagccctcga agccacaatg agatttttgt 1680 agagactgaa
gcagttgttt ctgtttacat gttgtttatt gaggaggtgt tttggcaaaa 1740
aagttttgtt ttgttttttt ctggaaaaaa aagaaaaaag atacgactct cagaagcctg
1800 ttttaaggaa gccctaaaat gtggactggg tttcctgtct tagcactgcc
ctgctagctc 1860 ttcctgaaaa ggcctataaa taaacagggc tggctgttaa
aaaaaaaaaa aaaaaaaa 1918 6 1897 DNA Homo sapiens CDS (112)..(1698)
6 ctgcgcggag gcacagaggc cggggagagc gttctgggtc cgagggtcca ggtaggggtt
60 gagccaccat ctgaccgcaa gctgcgtcgt gtcgccggtt ctgcaggcac c atg agc
117 Met Ser 1 cag gac acc gag gtg gat atg aag gag gtg gag ctg aat
gag tta gag 165 Gln Asp Thr Glu Val Asp Met Lys Glu Val Glu Leu Asn
Glu Leu Glu 5 10 15 ccc gag aag cag ccg atg aac gcg gcg tct ggg gcg
gcc atg tcc ctg 213 Pro Glu Lys Gln Pro Met Asn Ala Ala Ser Gly Ala
Ala Met Ser Leu 20 25 30 gcg gga gcc gag aag aat ggt ctg gtg aag
atc aag gtg gcg gaa gac 261 Ala Gly Ala Glu Lys Asn Gly Leu Val Lys
Ile Lys Val Ala Glu Asp 35 40 45 50 gag gcg gag gcg gca gcc gcg gct
aag ttc acg ggc ctg tcc aag gag 309 Glu Ala Glu Ala Ala Ala Ala Ala
Lys Phe Thr Gly Leu Ser Lys Glu 55 60 65 gag ctg ctg aag gtg gca
ggc agc ccc ggc tgg gta cgc acc cgc tgg 357 Glu Leu Leu Lys Val Ala
Gly Ser Pro Gly Trp Val Arg Thr Arg Trp 70 75 80 gca ctg ctg ctg
ctc ttc tgg ctc ggc tgg ctc ggc atg ctt gct ggt 405 Ala Leu Leu Leu
Leu Phe Trp Leu Gly Trp Leu Gly Met Leu Ala Gly 85 90 95 gcc gtg
gtc ata atc gtg cga gcg ccg cgt tgt cgc gag cta ccg gcg 453 Ala Val
Val Ile Ile Val Arg Ala Pro Arg Cys Arg Glu Leu Pro Ala 100 105 110
cag aag tgg tgg cac acg ggc gcc ctc tac cgc atc ggc gac ctt cag 501
Gln Lys Trp Trp His Thr Gly Ala Leu Tyr Arg Ile Gly Asp Leu Gln 115
120 125 130 gcc ttc cag ggc cac ggc gcg ggc aac ctg gcg ggt ctg aag
ggg cgt 549 Ala Phe Gln Gly His Gly Ala Gly Asn Leu Ala Gly Leu Lys
Gly Arg 135 140 145 ctc gat tac ctg agc tct ctg aag gtg aag ggc ctt
gtg ctg ggt cca 597 Leu Asp Tyr Leu Ser Ser Leu Lys Val Lys Gly Leu
Val Leu Gly Pro 150 155 160 att cac aag aac cag aag gat gat gtc gct
cag act gac ttg ctg cag 645 Ile His Lys Asn Gln Lys Asp Asp Val Ala
Gln Thr Asp Leu Leu Gln 165 170 175 atc gac ccc aat ttt ggc tcc aag
gaa gat ttt gac agt ctc ttg caa 693 Ile Asp Pro Asn Phe Gly Ser Lys
Glu Asp Phe Asp Ser Leu Leu Gln 180 185 190 tcg gct aaa aaa aag agc
atc cgt gtc att ctg gac ctt act ccc aac 741 Ser Ala Lys Lys Lys Ser
Ile Arg Val Ile Leu Asp Leu Thr Pro Asn 195 200 205 210 tac cgg ggt
gag aac tcg tgg ttc tcc act cag gtt gac act gtg gcc 789 Tyr Arg Gly
Glu Asn Ser Trp Phe Ser Thr Gln Val Asp Thr Val Ala 215 220 225 acc
aag gtg aag gat gct ctg gag ttt tgg ctg caa gct ggc gtg gat 837 Thr
Lys Val Lys Asp Ala Leu Glu Phe Trp Leu Gln Ala Gly Val Asp 230 235
240 ggg ttc cag gtt cgg gac ata gag aat ctg aag gat gca tcc tca ttc
885 Gly Phe Gln Val Arg Asp Ile Glu Asn Leu Lys Asp Ala Ser Ser Phe
245 250 255 ttg gct gag tgg caa aat atc acc aag ggc ttc agt gaa gac
agg ctc 933 Leu Ala Glu Trp Gln Asn Ile Thr Lys Gly Phe Ser Glu Asp
Arg Leu 260 265 270 ttg att gcg ggg act aac tcc tcc gac ctt cag cag
atc ctg agc cta 981 Leu Ile Ala Gly Thr Asn Ser Ser Asp Leu Gln Gln
Ile Leu Ser Leu 275 280 285 290 ctc gaa tcc aac aaa gac ttg ctg ttg
act agc tca tac ctg tct gat 1029 Leu Glu Ser Asn Lys Asp Leu Leu
Leu Thr Ser Ser Tyr Leu Ser Asp 295 300 305 tct ggt tct act ggg gag
cat aca aaa tcc cta gtc aca cag tat ttg 1077 Ser Gly Ser Thr Gly
Glu His Thr Lys Ser Leu Val Thr Gln Tyr Leu 310 315 320 aat gcc act
ggc aat cgc tgg tgc agc tgg agt ttg tct cag gca agg 1125 Asn Ala
Thr Gly Asn Arg Trp Cys Ser Trp Ser Leu Ser Gln Ala Arg 325 330 335
ctc ctg act tcc ttc ttg ccg gct caa ctt ctc cga ctc tac cag ctg
1173 Leu Leu Thr Ser Phe Leu Pro Ala Gln Leu Leu Arg Leu Tyr Gln
Leu 340 345 350 atg ctc ttc acc ctg cca ggg acc cct gtt ttc agc tac
ggg gat gag 1221 Met Leu Phe Thr Leu Pro Gly Thr Pro Val Phe Ser
Tyr Gly Asp Glu 355 360 365 370 att ggc ctg gat gca gct gcc ctt cct
gga cag cct atg gag gct cca 1269 Ile Gly Leu Asp Ala Ala Ala Leu
Pro Gly Gln Pro Met Glu Ala Pro 375 380 385 gtc atg ctg tgg gat gag
tcc agc ttc cct gac atc cca ggg gct gta 1317 Val Met Leu Trp Asp
Glu Ser Ser Phe Pro Asp Ile Pro Gly Ala Val 390 395 400 agt gcc aac
atg act gtg aag ggc cag agt gaa gac cct ggc tcc ctc 1365 Ser Ala
Asn Met Thr Val Lys Gly Gln Ser Glu Asp Pro Gly Ser Leu 405 410 415
ctt tcc ttg ttc cgg cgg ctg agt gac cag cgg agt aag gag cgc tcc
1413 Leu Ser Leu Phe Arg Arg Leu Ser Asp Gln Arg Ser Lys Glu Arg
Ser 420 425 430 cta ctg cat ggg gac ttc cac gcg ttc tcc gct ggg cct
gga ctc ttc 1461 Leu Leu His Gly Asp Phe His Ala Phe Ser Ala Gly
Pro Gly Leu Phe 435 440 445 450 tcc tat atc cgc cac tgg gac cag aat
gag cgt ttt ctg gta gtg ctt 1509 Ser Tyr Ile Arg His Trp Asp Gln
Asn Glu Arg Phe Leu Val Val Leu 455 460 465 aac ttt ggg gat gtg ggc
ctc tcg gct gga ctg cag gcc tcc gac ctg 1557 Asn Phe Gly Asp Val
Gly Leu Ser Ala Gly Leu Gln Ala Ser Asp Leu 470 475 480 cct gcc agc
gcc agc ctg cca gcc aag gct gac ctc ctg ctc agc acc 1605 Pro Ala
Ser Ala Ser Leu Pro Ala Lys Ala Asp Leu Leu Leu Ser Thr 485 490 495
cag cca ggc cgt gag gag ggc tcc cct ctt gag ctg gaa cgc ctg aaa
1653 Gln Pro Gly Arg Glu Glu Gly Ser Pro Leu Glu Leu Glu Arg Leu
Lys 500 505 510 ctg gag cct cac gaa ggg ctg ctg ctc cgc ttc ccc tac
gcg gcc 1698 Leu Glu Pro His Glu Gly Leu Leu Leu Arg Phe Pro Tyr
Ala Ala 515 520 525 tgacttcagc ctgacatgga cccactaccc ttctcctttc
cttcccaggc cctttggctt 1758 ctgatttttc tcttttttaa aaacaaacaa
acaaactgtt gcagattatg agtgaacccc 1818 caaatagggt gttttctgcc
ttcaaataaa agtcacccct gcatggtgaa gtcttccctc 1878 taaaaaaaaa
aaaaaaaaa 1897 7 526 PRT Mus sp. 7 Met Ser Gln Asp Thr Glu Val Asp
Met Lys Asp Val Glu Leu Asn Glu 1 5 10 15 Leu Glu Pro Glu Lys Gln
Pro Met Asn Ala Ala Asp Gly Ala Ala Ala 20 25 30 Gly Glu Lys Asn
Gly Leu Val Lys Ile Lys Val Ala Glu Asp Glu Thr 35 40 45 Glu Ala
Gly Val Lys Phe Thr Gly Leu Ser Lys Glu Glu Leu Leu Lys 50 55 60
Val Ala Gly Ser Pro Gly Trp Val Arg Thr Arg Trp Ala Leu Leu Leu 65
70 75 80 Leu Phe Trp Leu Gly Trp Leu Gly Met Leu Ala Gly Ala Val
Val Ile 85 90 95 Ile Val Arg Ala Pro Arg Cys Arg Glu Leu Pro Val
Gln Arg Trp Trp 100 105 110 His Lys Gly Ala Leu Tyr Arg Ile Gly Asp
Leu Gln Ala Phe Val Gly 115 120 125 Arg Asp Ala Gly Gly Ile Ala Gly
Leu Lys Ser His Leu Glu Tyr Leu 130 135 140 Ser Thr Leu Lys Val Lys
Gly Leu Val Leu Gly Pro Ile His Lys Asn 145 150 155 160 Gln Lys Asp
Glu Ile Asn Glu Thr Asp Leu Lys Gln Ile Asn Pro Thr 165 170 175 Leu
Gly Ser Gln Glu Asp Phe Lys Asp Leu Leu Gln Ser Ala Lys Lys 180 185
190 Lys Ser Ile His Ile Ile Leu Asp Leu Thr Pro Asn Tyr Gln Gly Gln
195 200 205 Asn Ala Trp Phe Leu Pro Ala Gln Ala Asp Ile Val Ala Thr
Lys Met 210 215 220 Lys Glu Ala Leu Ser Ser Trp Leu Gln Asp Gly Val
Asp Gly Phe Gln 225 230 235 240 Phe Arg Asp Val Gly Lys Leu Met Asn
Ala Pro Leu Tyr Leu Ala Glu 245 250 255 Trp Gln Asn Ile Thr Lys Asn
Leu Ser Glu Asp Arg Leu Leu Ile Ala 260 265 270 Gly Thr Glu Ser Ser
Asp Leu Gln Gln Ile Val Asn Ile Leu Glu Ser 275 280 285 Thr Ser Asp
Leu Leu Leu Thr Ser Ser Tyr Leu Ser Asn Ser Thr Phe 290 295 300 Thr
Gly Glu Arg Thr Glu Ser Leu Val Thr Arg Phe Leu Asn Ala Thr 305 310
315 320 Gly Ser Gln Trp Cys Ser Trp Ser Val Ser Gln Ala Gly Leu Leu
Ala 325 330 335 Asp Phe Ile Pro Asp His Leu Leu Arg Leu Tyr Gln Leu
Leu Leu Phe 340 345 350 Thr Leu Pro Gly Thr Pro Val Phe Ser Tyr Gly
Asp Glu Leu Gly Leu 355 360 365 Gln Gly Ala Leu Pro Gly Gln Pro Ala
Lys Ala Pro Leu Met Pro Trp 370 375 380 Asn Glu Ser Ser Ile Phe His
Ile Pro Arg Pro Val Ser Leu Asn Met 385 390 395 400 Thr Val Lys Gly
Gln Asn Glu Asp Pro Gly Ser Leu Leu Thr Gln Phe 405 410 415 Arg Arg
Leu Ser Asp Leu Arg Gly Lys Glu Arg Ser Leu Leu His Gly 420 425 430
Asp Phe His Ala Leu Ser Ser Ser Pro Asp Leu Phe Ser Tyr Ile Arg 435
440 445 His Trp Asp Gln Asn Glu Arg Tyr Leu Val Val Leu Asn Phe Arg
Asp 450 455 460 Ser Gly Arg Ser Ala Arg Leu Gly Ala Ser Asn Leu Pro
Ala Gly Ile 465 470 475 480 Ser Leu Pro Ala Ser Ala Lys Leu Leu Leu
Ser Thr Asp Ser Ala Arg 485 490 495 Gln Ser Arg Glu Glu Asp Thr Ser
Leu Lys Leu Glu Asn Leu Ser Leu 500 505 510 Asn Pro Tyr Glu Gly Leu
Leu Leu Gln Phe Pro Phe Val Ala 515 520 525 8 529 PRT Homo sapiens
8 Met Ser Gln Asp Thr Glu Val Asp Met Lys Glu Val Glu Leu Asn Glu 1
5 10 15 Leu Glu Pro Glu Lys Gln Pro Met Asn Ala Ala Ser Gly Ala Ala
Met 20 25 30 Ser Leu Ala Gly Ala Glu Lys Asn Gly Leu Val Lys Ile
Lys Val Ala 35 40 45 Glu Asp Glu Ala Glu Ala Ala Ala Ala Ala Lys
Phe Thr Gly Leu Ser 50 55 60 Lys Glu Glu Leu Leu Lys Val Ala Gly
Ser Pro Gly Trp Val Arg Thr 65 70 75 80 Arg Trp Ala Leu Leu Leu Leu
Phe Trp Leu Gly Trp Leu Gly Met Leu 85 90 95 Ala Gly Ala Val Val
Ile Ile Val Arg Ala Pro Arg Cys Arg Glu Leu 100 105 110 Pro Ala Gln
Lys Trp Trp His Thr Gly Ala Leu Tyr Arg Ile Gly Asp 115 120 125 Leu
Gln Ala Phe Gln Gly His Gly Ala Gly Asn Leu Ala Gly Leu Lys 130 135
140 Gly Arg Leu Asp Tyr Leu Ser Ser Leu Lys Val Lys Gly Leu Val Leu
145 150 155 160 Gly Pro Ile His Lys Asn Gln Lys Asp Asp Val Ala Gln
Thr Asp Leu 165 170 175 Leu Gln Ile Asp Pro Asn Phe Gly Ser Lys Glu
Asp Phe Asp Ser Leu 180 185 190 Leu Gln Ser Ala Lys Lys Lys Ser Ile
Arg Val Ile Leu Asp Leu Thr 195 200 205 Pro Asn Tyr Arg Gly Glu Asn
Ser Trp Phe Ser Thr Gln Val Asp Thr 210 215 220 Val Ala Thr Lys Val
Lys Asp Ala Leu Glu Phe Trp Leu Gln Ala Gly 225 230 235 240 Val Asp
Gly Phe Gln Val Arg Asp Ile Glu Asn Leu Lys Asp Ala Ser 245 250 255
Ser Phe Leu Ala Glu Trp Gln Asn Ile Thr Lys Gly Phe Ser Glu Asp 260
265 270 Arg Leu Leu Ile Ala Gly Thr Asn Ser Ser Asp Leu Gln Gln Ile
Leu 275 280 285 Ser Leu Leu Glu Ser Asn Lys Asp Leu Leu Leu Thr Ser
Ser Tyr Leu 290 295 300 Ser Asp Ser Gly Ser Thr Gly Glu His Thr Lys
Ser Leu Val Thr Gln 305 310 315 320 Tyr Leu Asn Ala Thr Gly Asn Arg
Trp Cys Ser Trp Ser Leu Ser Gln 325 330 335 Ala Arg Leu Leu Thr Ser
Phe Leu Pro Ala Gln Leu Leu Arg Leu Tyr 340 345 350 Gln Leu Met Leu
Phe Thr Leu Pro Gly Thr Pro Val Phe Ser Tyr Gly 355 360 365 Asp Glu
Ile Gly Leu Asp Ala Ala Ala Leu Pro Gly Gln Pro Met Glu 370 375 380
Ala Pro Val Met Leu Trp Asp Glu Ser Ser Phe Pro Asp Ile Pro Gly 385
390 395 400 Ala Val Ser Ala Asn Met Thr Val Lys Gly Gln Ser Glu Asp
Pro Gly 405 410 415 Ser Leu Leu Ser Leu Phe Arg Arg Leu Ser Asp Gln
Arg Ser Lys Glu 420 425 430 Arg Ser Leu Leu His Gly Asp Phe His Ala
Phe Ser Ala Gly Pro Gly 435 440 445 Leu Phe Ser Tyr Ile Arg His Trp
Asp Gln Asn Glu Arg Phe Leu Val 450 455 460 Val Leu Asn Phe Gly Asp
Val Gly Leu Ser Ala Gly Leu Gln Ala Ser 465 470 475 480 Asp Leu Pro
Ala Ser Ala Ser Leu Pro Ala Lys Ala Asp Leu Leu Leu 485 490 495 Ser
Thr Gln Pro Gly Arg Glu Glu Gly Ser Pro Leu Glu Leu Glu Arg 500 505
510 Leu Lys Leu Glu Pro His Glu Gly Leu Leu Leu Arg Phe Pro Tyr Ala
515 520 525 Ala 9 533 PRT Rattus sp. 9 Met Glu Lys Gly Thr Arg Gln
Arg Asn Asn Thr Ala Lys Asn His Pro 1 5 10 15 Asp Arg Gly Ser Asp
Thr Ser Pro Glu Ala Glu Ala Ser Ser Gly Gly 20 25 30 Gly Gly Val
Ala Leu Lys Lys Glu Ile Gly Leu Val Ser Ala Cys Gly 35 40 45 Ile
Ile Val Gly Asn Ile Ile Gly Ser Gly Ile Phe Val Ser Pro Lys 50 55
60 Gly Val Leu Glu Asn Ala Gly Ser Val Gly Leu Ala Leu Ile Val Trp
65 70 75 80 Ile Val Thr Gly Val Ile Thr Ala Val Gly Ala Leu Cys Tyr
Ala Glu 85 90 95 Leu Gly Val Thr Ile Pro Lys Ser Gly Gly Asp Tyr
Ser Tyr Val Lys 100 105 110 Asp Ile Phe Gly Gly Leu Ala Gly Phe Leu
Arg Leu Trp Ile Ala Val 115 120 125 Leu Val Ile Tyr Pro Thr Asn Gln
Ala Val Ile Ala Leu Thr Phe Ser 130 135 140 Asn Tyr Val Leu Gln Phe
Leu Phe Pro Thr Cys Phe Pro Pro Glu Ser 145 150 155 160 Gly Leu Arg
Leu Leu Ala Ala Ile Cys Leu Leu Leu Leu Thr Trp Val 165 170 175 Asn
Cys Ser Ser Val Arg Trp Ala Thr Arg Val Gln Asp Ile Phe Thr 180 185
190 Ala Gly Lys Leu Leu Ala Leu Ala Leu Ile Ile Ile Met Gly Val Val
195 200 205 Gln Ile Cys Lys Gly Glu Phe Phe Trp Leu Glu Pro Lys Asn
Ala Phe 210 215 220 Glu Asn Phe Gln Glu Pro Asp Ile Gly Leu Val Ala
Leu Ala Phe Leu 225 230 235 240 Gln Gly Ser Phe Ala Tyr Gly Gly Trp
Asn Phe Leu Asn Tyr Val Thr 245 250 255 Glu Glu Leu Val Asp Pro Tyr
Lys Asn Leu Pro Arg Ala Ile Phe Ile 260 265 270 Ser Ile Pro Leu Val
Thr Phe Val Tyr Val Phe Ala Asn Ile Ala Tyr 275 280 285 Val Thr Ala
Met Ser Pro Gln Glu Leu Leu Ala Ser Asn Ala Val Ala 290 295 300 Val
Thr Phe Gly Glu Lys Leu Leu Gly Val Met Ala Trp Ile Met Pro 305 310
315 320 Ile Ser Val Ala Leu Ser Thr Phe Gly Gly Val Asn Gly Ser
Leu Phe 325 330 335 Thr Ser Ser Arg Leu Phe Phe Ala Gly Ala Arg Glu
Gly His Leu Pro 340 345 350 Ser Val Leu Ala Met Ile His Val Lys Arg
Cys Thr Pro Ile Pro Ala 355 360 365 Leu Leu Phe Thr Cys Leu Ser Thr
Leu Leu Met Leu Val Thr Ser Asp 370 375 380 Met Tyr Thr Leu Ile Asn
Tyr Val Gly Phe Ile Asn Tyr Leu Phe Tyr 385 390 395 400 Gly Val Thr
Val Ala Gly Gln Ile Val Leu Arg Trp Lys Lys Pro Asp 405 410 415 Ile
Pro Arg Pro Ile Lys Ile Ser Leu Leu Phe Pro Ile Ile Tyr Leu 420 425
430 Leu Phe Trp Ala Phe Leu Leu Ile Phe Ser Leu Trp Ser Glu Pro Val
435 440 445 Val Cys Gly Ile Gly Leu Ala Ile Met Leu Thr Gly Val Pro
Val Tyr 450 455 460 Phe Leu Gly Val Tyr Trp Gln His Lys Pro Lys Cys
Phe Asn Asp Phe 465 470 475 480 Ile Glu Ser Leu Thr Leu Val Ser Gln
Lys Met Cys Val Val Val Tyr 485 490 495 Pro Gln Glu Gly Asp Ser Gly
Thr Glu Glu Thr Ile Asp Asp Val Glu 500 505 510 Glu Gln His Lys Pro
Ile Phe Gln Pro Thr Pro Val Lys Asp Pro Asp 515 520 525 Ser Glu Glu
Gln Pro 530 10 512 PRT Rattus sp. 10 Met Ala Val Ala Gly Ala Lys
Arg Arg Ala Val Ala Ala Pro Ala Thr 1 5 10 15 Thr Ala Ala Glu Glu
Glu Arg Gln Ala Arg Glu Lys Met Leu Glu Ala 20 25 30 Arg Arg Gly
Asp Gly Ala Asp Pro Glu Gly Glu Gly Val Thr Leu Gln 35 40 45 Arg
Asn Ile Thr Leu Ile Asn Gly Val Ala Ile Ile Val Gly Thr Ile 50 55
60 Ile Gly Ser Gly Ile Phe Val Thr Pro Thr Gly Val Leu Lys Glu Ala
65 70 75 80 Gly Ser Pro Gly Leu Ser Leu Val Val Trp Ala Val Cys Gly
Val Phe 85 90 95 Ser Ile Val Gly Ala Leu Cys Tyr Ala Glu Leu Gly
Thr Thr Ile Ser 100 105 110 Lys Ser Gly Gly Asp Tyr Ala Tyr Met Leu
Glu Val Tyr Gly Ser Leu 115 120 125 Pro Ala Phe Leu Lys Leu Trp Ile
Glu Leu Leu Ile Ile Arg Pro Ser 130 135 140 Ser Gln Tyr Ile Val Ala
Leu Val Phe Ala Thr Tyr Leu Leu Lys Pro 145 150 155 160 Val Phe Pro
Thr Cys Pro Val Pro Glu Glu Ala Ala Lys Leu Val Ala 165 170 175 Cys
Leu Cys Val Leu Leu Leu Thr Ala Val Asn Cys Tyr Ser Val Lys 180 185
190 Ala Ala Thr Arg Val Gln Asp Ala Phe Ala Ala Ala Lys Leu Leu Ala
195 200 205 Leu Ala Leu Ile Ile Leu Leu Gly Phe Ile Gln Met Gly Lys
Asp Ile 210 215 220 Gly Gln Gly Asp Ala Ser Asn Leu His Gln Lys Leu
Ser Phe Glu Gly 225 230 235 240 Thr Asn Leu Asp Val Gly Asn Ile Val
Leu Ala Leu Tyr Ser Gly Leu 245 250 255 Phe Ala Tyr Gly Gly Trp Asn
Tyr Leu Asn Phe Val Thr Glu Glu Met 260 265 270 Ile Asn Pro Tyr Arg
Asn Leu Pro Leu Ala Ile Ile Ile Ser Leu Pro 275 280 285 Ile Val Thr
Leu Val Tyr Val Leu Thr Asn Leu Ala Tyr Phe Thr Thr 290 295 300 Leu
Ser Thr Asn Gln Met Leu Thr Ser Glu Ala Val Ala Val Asp Phe 305 310
315 320 Gly Asn Tyr His Leu Gly Val Met Ser Trp Ile Ile Pro Val Phe
Val 325 330 335 Gly Leu Ser Cys Phe Gly Ser Val Asn Gly Ser Leu Phe
Thr Ser Ser 340 345 350 Arg Leu Phe Phe Val Gly Ser Arg Glu Gly His
Leu Pro Ser Ile Leu 355 360 365 Ser Met Ile His Pro Gln Leu Leu Thr
Pro Val Pro Ser Leu Val Phe 370 375 380 Thr Cys Val Met Thr Leu Met
Tyr Ala Phe Ser Arg Asp Ile Phe Ser 385 390 395 400 Ile Ile Asn Phe
Phe Ser Phe Phe Asn Trp Leu Cys Val Ala Leu Ala 405 410 415 Ile Ile
Gly Met Met Trp Leu Arg Phe Lys Lys Pro Glu Leu Glu Arg 420 425 430
Pro Ile Lys Val Asn Leu Ala Leu Pro Val Phe Phe Ile Leu Ala Cys 435
440 445 Leu Phe Leu Ile Ala Val Ser Phe Trp Lys Thr Pro Leu Glu Cys
Gly 450 455 460 Ile Gly Phe Ala Ile Ile Leu Ser Gly Leu Pro Val Tyr
Phe Phe Gly 465 470 475 480 Val Trp Trp Lys Asn Lys Pro Lys Trp Ile
Leu Gln Val Ile Phe Ser 485 490 495 Val Thr Val Leu Cys Gln Lys Leu
Met Cys Val Val Pro Gln Glu Thr 500 505 510 11 511 PRT Homo sapiens
11 Met Val Asp Ser Thr Glu Tyr Glu Val Ala Ser Gln Pro Glu Val Glu
1 5 10 15 Thr Ser Pro Leu Gly Asp Gly Ala Ser Pro Gly Pro Glu Gln
Val Lys 20 25 30 Leu Lys Lys Glu Ile Ser Leu Leu Asn Gly Val Cys
Leu Ile Val Gly 35 40 45 Asn Met Ile Gly Ser Gly Ile Phe Val Ser
Pro Lys Gly Val Leu Ile 50 55 60 Tyr Ser Ala Ser Phe Gly Leu Ser
Leu Val Ile Trp Ala Val Gly Gly 65 70 75 80 Leu Phe Ser Val Phe Gly
Ala Leu Cys Tyr Ala Glu Leu Gly Thr Thr 85 90 95 Ile Lys Lys Ser
Gly Ala Ser Tyr Ala Tyr Ile Leu Glu Ala Phe Gly 100 105 110 Gly Phe
Leu Ala Phe Ile Arg Leu Trp Thr Ser Leu Leu Ile Ile Glu 115 120 125
Pro Thr Ser Gln Ala Ile Ile Ala Ile Thr Phe Ala Asn Tyr Met Val 130
135 140 Gln Phe Leu Phe Pro Ser Cys Phe Ala Pro Tyr Ala Ala Ser Arg
Leu 145 150 155 160 Leu Ala Ala Ala Cys Ile Cys Leu Leu Thr Phe Ile
Asn Cys Ala Tyr 165 170 175 Val Lys Trp Gly Thr Leu Val Gln Asp Ile
Phe Thr Tyr Ala Lys Val 180 185 190 Leu Ala Leu Ile Ala Val Ile Val
Ala Gly Ile Val Arg Leu Gly Gln 195 200 205 Gly Ala Ser Thr His Phe
Glu Asn Ser Phe Glu Gly Ser Ser Phe Ala 210 215 220 Val Gly Asp Ile
Ala Leu Ala Leu Tyr Ser Ala Leu Phe Ser Tyr Ser 225 230 235 240 Gly
Trp Asp Thr Leu Asn Tyr Val Thr Glu Glu Ile Lys Asn Pro Glu 245 250
255 Arg Asn Leu Pro Leu Ser Ile Gly Ile Ser Met Pro Ile Val Thr Ile
260 265 270 Ile Tyr Ile Leu Thr Asn Val Ala Tyr Tyr Thr Val Leu Asp
Met Arg 275 280 285 Asp Ile Leu Ala Ser Asp Ala Val Ala Val Thr Phe
Ala Asp Gln Ile 290 295 300 Phe Gly Ile Phe Asn Trp Ile Ile Pro Leu
Ser Val Ala Leu Ser Cys 305 310 315 320 Phe Gly Gly Leu Asn Ala Ser
Ile Val Ala Ala Ser Arg Leu Phe Phe 325 330 335 Val Gly Ser Arg Glu
Gly His Leu Pro Asp Ala Ile Cys Met Ile His 340 345 350 Val Glu Arg
Phe Thr Pro Val Pro Ser Leu Leu Phe Asn Gly Ile Met 355 360 365 Ala
Leu Ile Tyr Leu Cys Val Glu Asp Ile Phe Gln Leu Ile Asn Tyr 370 375
380 Tyr Ser Phe Ser Tyr Trp Phe Phe Val Gly Leu Ser Ile Val Gly Gln
385 390 395 400 Leu Tyr Leu Arg Trp Lys Glu Pro Cys Arg Pro Arg Pro
Leu Lys Leu 405 410 415 Ser Val Phe Phe Pro Ile Val Phe Cys Leu Cys
Thr Ile Phe Leu Val 420 425 430 Ala Val Pro Leu Tyr Ser Asp Thr Ile
Asn Ser Leu Ile Gly Ile Ala 435 440 445 Ile Ala Leu Ser Gly Leu Pro
Phe Tyr Phe Leu Ile Ile Arg Val Pro 450 455 460 Glu His Lys Arg Pro
Leu Tyr Leu Arg Arg Ile Val Gly Ser Ala Thr 465 470 475 480 Arg Tyr
Leu Gln Val Leu Cys Met Ser Val Ala Ala Glu Met Asp Leu 485 490 495
Glu Asp Gly Gly Glu Met Pro Lys Gln Arg Asp Pro Lys Ser Asn 500 505
510 12 515 PRT Homo sapiens 12 Met Glu Ala Arg Glu Pro Gly Arg Pro
Thr Pro Thr Tyr His Leu Val 1 5 10 15 Pro Asn Thr Ser Gln Ser Gln
Val Glu Glu Asp Val Ser Ser Pro Pro 20 25 30 Gln Arg Ser Ser Glu
Thr Met Gln Leu Lys Lys Glu Ile Ser Leu Leu 35 40 45 Asn Gly Val
Ser Leu Val Val Gly Asn Met Ile Gly Ser Gly Ile Phe 50 55 60 Val
Ser Pro Lys Gly Val Leu Val His Thr Ala Ser Tyr Gly Met Ser 65 70
75 80 Leu Ile Val Trp Ala Ile Gly Gly Leu Phe Ser Val Val Gly Ala
Leu 85 90 95 Cys Tyr Ala Glu Leu Gly Thr Thr Ile Thr Lys Ser Gly
Ala Ser Tyr 100 105 110 Ala Tyr Ile Leu Glu Ala Phe Gly Gly Phe Ile
Ala Phe Ile Arg Leu 115 120 125 Trp Val Ser Leu Leu Val Val Glu Pro
Thr Gly Gln Ala Ile Ile Ala 130 135 140 Ile Thr Phe Ala Asn Tyr Ile
Ile Gln Pro Ser Phe Pro Ser Cys Asp 145 150 155 160 Pro Pro Tyr Leu
Ala Cys Arg Leu Leu Ala Ala Ala Cys Ile Cys Leu 165 170 175 Leu Thr
Phe Val Asn Cys Ala Tyr Val Lys Trp Gly Thr Arg Val Gln 180 185 190
Asp Thr Phe Thr Tyr Ala Lys Val Val Ala Leu Ile Ala Ile Ile Val 195
200 205 Met Gly Leu Val Lys Leu Cys Gln Gly His Ser Glu His Phe Gln
Asp 210 215 220 Ala Phe Glu Gly Ser Ser Trp Asp Met Gly Asn Leu Ser
Leu Ala Leu 225 230 235 240 Tyr Ser Ala Leu Phe Ser Tyr Ser Gly Trp
Asp Thr Leu Asn Phe Val 245 250 255 Thr Glu Glu Ile Lys Asn Pro Glu
Arg Asn Leu Pro Leu Ala Ile Gly 260 265 270 Ile Ser Met Pro Ile Val
Thr Leu Ile Tyr Ile Leu Thr Asn Val Ala 275 280 285 Tyr Tyr Thr Val
Leu Asn Ile Ser Asp Val Leu Ser Ser Asp Ala Val 290 295 300 Ala Val
Thr Phe Ala Asp Gln Thr Phe Gly Met Phe Ser Trp Thr Ile 305 310 315
320 Pro Ile Ala Val Ala Leu Ser Cys Phe Gly Gly Leu Asn Ala Ser Ile
325 330 335 Phe Ala Ser Ser Arg Leu Phe Phe Val Gly Ser Arg Glu Gly
His Leu 340 345 350 Pro Asp Leu Leu Ser Met Ile His Ile Glu Arg Phe
Thr Pro Ile Pro 355 360 365 Ala Leu Leu Phe Asn Cys Thr Met Ala Leu
Ile Tyr Leu Ile Val Glu 370 375 380 Asp Val Phe Gln Leu Ile Asn Tyr
Phe Ser Phe Ser Tyr Trp Phe Phe 385 390 395 400 Val Gly Leu Ser Val
Val Gly Gln Leu Tyr Leu Arg Trp Lys Glu Pro 405 410 415 Lys Arg Pro
Arg Pro Leu Lys Leu Ser Val Phe Phe Pro Ile Val Phe 420 425 430 Cys
Ile Cys Ser Val Phe Leu Val Ile Val Pro Leu Phe Thr Asp Thr 435 440
445 Ile Asn Ser Leu Ile Gly Ile Gly Ile Ala Leu Ser Gly Val Pro Phe
450 455 460 Tyr Phe Met Gly Val Tyr Leu Pro Glu Ser Arg Arg Pro Leu
Phe Ile 465 470 475 480 Arg Asn Val Leu Ala Ala Ile Thr Arg Gly Thr
Gln Gln Leu Cys Phe 485 490 495 Cys Val Leu Thr Glu Leu Asp Val Ala
Glu Glu Lys Lys Asp Glu Arg 500 505 510 Lys Thr Asp 515 13 502 PRT
Mus sp. 13 Met Val Arg Lys Pro Val Val Ala Thr Ile Ser Lys Gly Gly
Tyr Leu 1 5 10 15 Gln Gly Asn Met Ser Gly Arg Leu Pro Ser Met Gly
Asp Gln Glu Pro 20 25 30 Pro Gly Gln Glu Lys Val Val Leu Lys Lys
Lys Ile Thr Leu Leu Arg 35 40 45 Gly Val Ser Ile Ile Ile Gly Thr
Val Ile Gly Ser Gly Ile Phe Ile 50 55 60 Ser Pro Lys Gly Ile Leu
Gln Asn Thr Gly Ser Val Gly Met Ser Leu 65 70 75 80 Val Phe Trp Ser
Ala Cys Gly Val Leu Ser Leu Phe Gly Ala Leu Ser 85 90 95 Tyr Ala
Glu Leu Gly Thr Ser Ile Lys Lys Ser Gly Gly His Tyr Thr 100 105 110
Tyr Ile Leu Glu Val Phe Gly Pro Leu Leu Ala Phe Val Arg Val Trp 115
120 125 Val Glu Leu Leu Val Ile Arg Pro Gly Ala Thr Ala Val Ile Ser
Leu 130 135 140 Ala Phe Gly Arg Tyr Ile Leu Glu Pro Phe Phe Ile Gln
Cys Glu Ile 145 150 155 160 Pro Glu Leu Ala Ile Lys Leu Val Thr Ala
Val Gly Ile Thr Val Val 165 170 175 Met Val Leu Asn Ser Thr Ser Val
Ser Trp Ser Ala Arg Ile Gln Ile 180 185 190 Phe Leu Thr Phe Cys Lys
Leu Thr Ala Ile Leu Ile Ile Ile Val Pro 195 200 205 Gly Val Ile Gln
Leu Ile Lys Gly Gln Thr His His Phe Lys Asp Ala 210 215 220 Phe Ser
Gly Arg Asp Thr Ser Leu Met Gly Leu Pro Leu Ala Phe Tyr 225 230 235
240 Tyr Gly Met Tyr Ala Tyr Ala Gly Trp Phe Tyr Leu Asn Phe Ile Thr
245 250 255 Glu Glu Val Asp Asn Pro Glu Lys Thr Ile Pro Leu Ala Ile
Cys Ile 260 265 270 Ser Met Ala Ile Ile Thr Val Gly Tyr Val Leu Thr
Asn Val Ala Tyr 275 280 285 Phe Thr Thr Ile Ser Ala Glu Glu Leu Leu
Gln Ser Ser Ala Val Ala 290 295 300 Val Thr Phe Ser Glu Arg Leu Leu
Gly Lys Phe Ser Leu Ala Val Pro 305 310 315 320 Ile Phe Val Ala Leu
Ser Cys Phe Gly Ser Met Asn Gly Gly Val Phe 325 330 335 Ala Val Ser
Arg Leu Phe Tyr Val Ala Ser Arg Glu Gly His Leu Pro 340 345 350 Glu
Ile Leu Ser Met Ile His Val His Lys His Thr Pro Leu Pro Ala 355 360
365 Val Ile Val Leu His Pro Leu Thr Met Val Met Leu Phe Ser Gly Asp
370 375 380 Leu Tyr Ser Leu Leu Asn Phe Leu Ser Phe Ala Arg Trp Leu
Phe Met 385 390 395 400 Gly Leu Ala Val Ala Gly Leu Ile Tyr Leu Arg
Tyr Lys Arg Pro Asp 405 410 415 Met His Arg Pro Phe Lys Val Pro Leu
Phe Ile Pro Ala Leu Phe Ser 420 425 430 Phe Thr Cys Leu Phe Met Val
Val Leu Ser Leu Tyr Ser Cys Pro Phe 435 440 445 Ser Thr Gly Val Gly
Phe Leu Ile Thr Leu Thr Gly Val Pro Ala Tyr 450 455 460 Tyr Leu Phe
Ile Val Trp Asp Lys Lys Pro Lys Trp Phe Arg Arg Leu 465 470 475 480
Ser Asp Arg Ile Thr Arg Thr Leu Gln Ile Ile Leu Glu Val Val Pro 485
490 495 Glu Asp Ser Lys Glu Leu 500 14 20 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 14 ctcttcacat
gcatctccac 20 15 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 15 ggtacacgac cacacacatc 20 16
15 PRT Artificial Sequence Description of Artificial Sequence
Synthetic oligopeptide 16 Pro Ser Pro Leu Pro Ile Thr Asp Lys Pro
Leu Lys Thr Gln Cys 1 5 10 15 17 12 PRT Artificial Sequence
Description of Artificial Sequence Synthetic oligopeptide 17 Cys
Glu Gly Leu Leu Leu Gln Phe Pro Phe Val Ala 1 5 10 18 23 PRT Homo
sapiens 18 Glu Ile Gly Leu Leu Ser Ala Cys Thr Ile Ile Ile Gly Asn
Ile Ile 1 5 10 15 Gly Ser Gly Ile Phe Ile Ser 20 19 23 PRT Homo
sapiens 19 Leu Phe Val Trp Val Leu Gly Gly Gly Val Thr Ala Leu Gly
Ser Leu 1 5 10 15 Cys Tyr Ala Glu Leu Gly Val 20 20 23 PRT Homo
sapiens 20 Phe Gly Gly Leu Ala Gly Phe Leu Leu Leu Trp Ser Ala Val
Leu Ile 1 5 10 15 Met Tyr Pro Thr Ser Leu Ala 20 21 23 PRT Homo
sapiens 21 Thr Ala Ser Arg Val Leu Ser Met Ala Cys Leu Met Leu Leu
Thr Trp 1
5 10 15 Val Asn Ser Ser Ser Val Arg 20 22 23 PRT Homo sapiens 22
Thr Gly Gly Lys Leu Leu Ala Leu Ser Leu Ile Ile Gly Val Gly Leu 1 5
10 15 Leu Gln Ile Phe Gln Gly His 20 23 23 PRT Homo sapiens 23 Leu
Ala Leu Ala Phe Leu Gln Gly Ser Phe Ala Phe Ser Gly Trp Asn 1 5 10
15 Phe Leu Asn Tyr Val Thr Glu 20 24 23 PRT Homo sapiens 24 Asn Leu
Pro Arg Ala Ile Phe Ile Ser Ile Pro Leu Val Thr Phe Val 1 5 10 15
Tyr Thr Phe Thr Asn Ile Ala 20 25 23 PRT Homo sapiens 25 Lys Leu
Leu Gly Tyr Phe Ser Trp Val Met Pro Val Ser Val Ala Leu 1 5 10 15
Ser Thr Phe Gly Gly Ile Asn 20 26 23 PRT Homo sapiens 26 Cys Thr
Pro Ile Pro Ala Leu Leu Val Cys Cys Gly Ala Thr Ala Val 1 5 10 15
Ile Met Leu Val Gly Asp Thr 20 27 22 PRT Homo sapiens 27 Asn Tyr
Val Ser Phe Ile Asn Tyr Leu Cys Tyr Gly Val Thr Ile Leu 1 5 10 15
Gly Leu Leu Leu Leu Arg 20 28 23 PRT Homo sapiens 28 Lys Val Asn
Leu Leu Ile Pro Val Ala Tyr Leu Val Phe Trp Ala Phe 1 5 10 15 Leu
Leu Val Phe Ser Phe Ile 20 29 22 PRT Homo sapiens 29 Cys Gly Val
Gly Val Ile Ile Ile Leu Thr Gly Val Pro Ile Phe Phe 1 5 10 15 Leu
Gly Val Phe Trp Arg 20
* * * * *