U.S. patent application number 12/228769 was filed with the patent office on 2010-08-26 for sodium-independent transporter carrying acidic amino acid and its gene.
This patent application is currently assigned to J-Pharma Co., Ltd.. Invention is credited to Hitoshi Endou, Yoshikatsu Kanai.
Application Number | 20100216976 12/228769 |
Document ID | / |
Family ID | 27678310 |
Filed Date | 2010-08-26 |
United States Patent
Application |
20100216976 |
Kind Code |
A1 |
Endou; Hitoshi ; et
al. |
August 26, 2010 |
Sodium-independent transporter carrying acidic amino acid and its
gene
Abstract
It is intended to provide a sodium-independent transporter
carrying an acidic amino acid and its gene. A protein having the
amino acid sequence represented by SEQ ID NO: 1 and being capable
of sodium-independently transporting an acidic amino acid and its
analogs; a gene encoding this protein; a fused protein of the above
protein with an auxiliary factor enabling the expression of its
function; a gene encoding the same; a method of analyzing the
function of a transporter using the same: and utilization
thereof.
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: |
J-Pharma Co., Ltd.
Tokyo
JP
|
Family ID: |
27678310 |
Appl. No.: |
12/228769 |
Filed: |
August 15, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10921772 |
Aug 18, 2004 |
7419790 |
|
|
12228769 |
|
|
|
|
PCT/JP2003/001614 |
Feb 17, 2003 |
|
|
|
10921772 |
|
|
|
|
Current U.S.
Class: |
530/387.9 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 39/02 20180101; C07K 14/47 20130101; A61P 35/00 20180101; A61P
25/30 20180101 |
Class at
Publication: |
530/387.9 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 18, 2002 |
JP |
2002-040608 |
Claims
1-17. (canceled)
18. An antibody directed to a protein selected from the group
consisting of the following (A) and (B): (A) a protein consisting
of the amino acid sequence represented by SEQ ID NO: 1; and (B) a
protein having the ability to transport acidic amino acids and
their analogues in a sodium-independent manner which consists of an
amino acid sequence where one or several amino acids are deleted,
substituted or added in the amino acid sequence represented by SEQ
ID NO: 1.
19-28. (canceled)
29. The antibody of claim 18, wherein the protein is derived from
mouse.
30. The antibody of claim 18, wherein the protein is derived from
organ, tissue or cultured cell.
31. An antibody directed to a protein selected from the group
consisting of the following (C) and (D): (C) a fusion protein of a
protein consisting of the amino acid sequence represented by SEQ ID
NO: 1 and a protein consisting of the amino acid sequence
represented by SEQ ID NO: 3; and, (D) a protein having the ability
to transport acidic amino acids and their analogues in a
sodium-independent manner which consists of an amino acid sequence
where one or several amino acids are deleted, substituted or added
in the amino acid sequence of a fusion protein of a protein
consisting of the amino acid sequence represented by SEQ ID NO: 1
and a protein consisting of the amino acid sequence represented by
SEQ ID NO: 3.
32. The antibody of claim 31, wherein the protein is derived from
mouse.
33. The antibody of claim 31, wherein the protein is derived from
organ, tissue or cultured cell.
34. An antibody directed to a protein selected from the group
consisting of the following (E) and (F): (E) a fusion protein of a
protein consisting of the amino acid sequence represented by SEQ ID
NO: 1 and a protein consisting of the amino acid sequence
represented by SEQ ID NO: 5; and, (F) a protein having the ability
to transport acidic amino acids and their analogues in a
sodium-independent manner which consists of an amino acid sequence
where one or several amino acids are deleted, substituted or added
and in the amino acid sequence of a fusion protein of a protein
consisting of the amino acid sequence represented by SEQ ID NO: 1
and a protein consisting of the amino acid sequence represented by
SEQ ID NO: 5.
35. The antibody of claim 34, wherein the protein is derived from
mouse.
36. The antibody of claim 34, wherein the protein is derived from
organ, tissue or cultured cell.
Description
TECHNICAL FIELD
[0001] The present invention relates to a protein associated with
the sodium-independent transport of acidic amino acids and its
analogue, fusion protein thereof, as well as a gene encoding said
protein. The present invention also relates to a method for
controlling the cell proliferation or for altering the in vivo
pharmacokinetics of a pharmaceutical, toxic substance or
xenobiotics by modulating an ability to transport acidic amino
acids and its analogue possessed by a protein associated with the
sodium-independent transport of acidic amino acids and its
analogue, by means of employing said protein, its fusion protein,
its specific antibody, or its function-promoting substance or
function-suppressing substance, as well as an agent for controlling
an ability to transport acidic amino acids and its analogue
comprising said substances.
BACKGROUND ART
[0002] A cell always requires the uptake of an amino acid as a
nutrition, and such a function is exerted by an amino acid
transporter which is a membrane protein existing in a cell
membrane. The amino acid transporter is distributed in a specific
site in each tissue in a multicellular organism and plays an
important role in expressing the specific function of each tissue.
For example, in kidney cells and small intestine, it plays a role
for epithelial absorption of amino acid in lumen and, in nerve
tissues, it is in charge of recovery of amino acid as a
neurotransmitter released as a result of neurotransmission and also
of supply of amino acid as a precursor for neurotransmitter to
nerve cells. Further, it exists in blood-brain barrier and
placental barrier and makes permeation of the amino acid
possible.
[0003] With regard to an amino acid transport mechanism, its
identification and classification have been conducted using
cultured cells and membrane specimens since 1960's and, reflecting
the multiplicity of amino acid molecules, many transport systems
have been described. However, there has been no independent
transport system for each amino acid but most of the amino acid
transports have been conducted by a few kinds of transport systems
which transport several amino acids having similar side chains
(Christensen, Physiol. Rev., volume 70, page 43, 1990).
[0004] Transport of acidic amino acids such as glutamic acid and
aspartic acid having carboxyl group on a side chain has been
believed to be carried out by both of a sodium-dependent
transporter which requires sodium ion for its function and a
sodium-independent transporter which does not require sodium ion
for its function.
[0005] However, in a conventional method, it is difficult to
analyze the details of the transport of an amino acid or its
analogue via the acidic amino acid transport system and the in vivo
functional roles, and it has been desired to enable a detailed
functional analysis by isolating a gene of acidic amino acid
transporter responsible for the function of the acidic amino acid
transport system.
[0006] With regard to sodium-dependent acidic amino acid
transporters, five kinds of glutamate transporters--EAAC1, GLT-1,
GLAST, EAAT4 and EAAT5--have been cloned (Kanai, Curr. Opin. Cell
Biol., volume 9, page 565, 1997; Kanai and Endou, Curr. Drug
Metab., volume 2, page 339, 2001).
[0007] With regard to sodium-independent transporters, 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) have been cloned as neutral amino acid
transporters corresponding to a transport system L. It was also
shown that LAT1 and LAT2 function only when they coexist with a
cofactor 4F2hc which is a single membrane-spanning type protein.
LAT1 shows an exchange transport activity which transports
large-sized neutral amino acids such as leucine, isoleucine,
valine, phenylalanine, tyrosine, tryptophan, methionine and
histidine while LAT2 shows a broad substrate selectivity
transporting small-sized neutral amino acids such as glycine,
alanine, serine, cysteine and threonine in addition to large-sized
neutral amino acids and they are not acidic amino transporters.
[0008] With regard to proteins analogous to LAT1 and LAT2, the
above-mentioned y.sup.+LAT1 and y.sup.+LAT2 having the functions of
a transport system y.sup.+L which transports neutral amino acids
and basic amino acids have been cloned (Torrents, et al., J. Biol.
Chem., volume 273, pages 32437-32445, 1998). It was also revealed
that both of y.sup.+LAT1 and y.sup.+LAT2 function only when being
coexisting with a cofactor 4F2hc. y.sup.+LAT1 and y.sup.+LAT2
mainly transport glutamine, leucine and isoleucine as neutral amino
acids and do not transport acidic amino acids.
[0009] With regard to a transporter which requires a cofactor 4F2hc
for expressing its function, Asc-1 which is a protein analogous to
LAT1 to LAT2 was cloned (Fukasawa, et al., J. Biol. Chem., 275:
9690-9698, 2000). Asc-1 selectively transports alanine, serine,
cysteine, threonine, glycine, etc., shows a substrate selectivity
of amino acid transport system asc and does not transport acidic
amino acids.
[0010] With regard to a transporter which requires another cofactor
rBAT having an analogous structure to 4F2hc for expressing its
function, BAT1 which is a protein analogous to LAT1 and LAT2 was
cloned (Chairoungdua, et al., J. Biol. Chem., 274: 28845-28848,
1999). BAT1 transports cystine, neutral amino acids and basic amino
acids and does not transport acidic amino acids.
[0011] As described above, molecular entity of a transporter which
functions by binding to 4F2hc and rBAT was characterized and, the
presence of a group of transporters which achieves a transport
ability by forming heterodimer with a single membrane-spanning type
protein and a heterodimeric amino acid transporter family was
established.
[0012] Further, with regard to a transporter requiring a cofactor
4F2hc for expressing its function, xCT which is a protein analogous
to LAT1 and LAT2 was cloned (Sato, et al., J. Biol. Chem., 274;
11455-11458, 1999). xCT transports cystine, glutamic acid and
sodium aminoadipate in a sodium-independent manner and corresponds
to an amino acid transport system Xc. xCT needs a negative charge
of side chain of amino acid for recognition of substrate and is
classified under sodium-independent acidic amino acid transporters
(Kanai and Endou, Curr. Drug Metab., volume 2, page 339, 2001).
[0013] xCT transports glutamic acid but does not transport aspartic
acid and its transport is suppressed by cystine. In addition, xCT
is a transporter where expression is induced by oxidative stress
and, except a few cases, its expression in common normal tissues is
not detected. However, it has been reported that there is a
sodium-independent glutamic acid and aspartic acid transporter
which is not suppressed by cystine (Christensen, Physiol. Rev.,
volume 70, page 43, 1990) and it has been suggested that there is a
sodium-independent acidic amino acid transporter other than xCT
which has not been identified.
[0014] Further, Asc-2 which is a protein having an analogous
structure to LAT1 and LAT2 and binds to unidentified protein other
than rBAT or 4F2hc was cloned (Chairoungdua, et al., J. Biol.
Chem., 276: 49390-49399, 2001). Asc-2 is not expressed in a cell
membrane by itself, however, by preparing a fusion protein with
4F2hc or rBAT, it transfers to a cell membrane as a fusion protein
and a transport activity can be detected. When Asc-2 is expressed
in a cell membrane as a fusion protein with 4F2hc or rBAT, it shows
a characteristic of a sodium-independent neutral amino acid
transport system asc.
DISCLOSURE OF THE INVENTION
[0015] An object of the present invention is to provide a gene of a
transporter which transports acidic amino acids such as glutamic
acid and aspartic acid in a sodium-independent manner and also to
provide a sodium-independent acidic amino acid transporter which is
a polypeptide encoded by the gene.
[0016] Other objects will be apparent from the following
description.
[0017] The present inventors have searched the EST (expressed
sequence tag) database using a base sequence of translation region
of cDNA of BAT1 and identified a base sequence analogous to BAT1. A
base sequence of cDNA clone corresponding to the sequence has been
decided and clarified that it encodes a novel protein. Further, a
fusion protein of the translated product of the gene with 4F2hc or
rBAT has been prepared and expressed in a cell membrane of oocyte
of Xenopus. As a result, it has been clarified that the function of
the translated product of the gene is a sodium-independent
transporter which transports acidic amino acids such as glutamic
acid and aspartic acid whereby the present invention has been
achieved.
[0018] Thus, the present invention relates to a protein selected
from the following (A) or (B).
[0019] (A) protein comprising an amino acid sequence represented by
SEQ ID NO: 1.
[0020] (B) protein comprising an amino acid sequence where one or
several amino acid(s) is/are deleted, substituted or added in the
amino acid sequence represented by SEQ ID NO: 1 and having an
ability of transport of acidic amino acids or its analogue in a
sodium-independent manner.
[0021] The present invention also relates to a gene comprising DNA
selected from the following (a) and (b).
[0022] (a) DNA comprising a base sequence represented by SEQ ID NO:
2.
[0023] (b) DNA hybridizing with DNA comprising the base sequence
represented by SEQ ID NO: 2 under a stringent condition and encodes
a protein having an ability of transport of acidic amino acids or
its analogue in a sodium-independent manner.
[0024] The novel protein of the present invention having an ability
of transport of acidic amino acids and its analogue in a
sodium-independent manner or, in other words, an amino acid
transporter AGT1 (aspartate/glutamate transporter 1) is expressed
in a cell membrane and has an ability of transport (uptake) of
acidic amino acids such as glutamic acid and aspartic acid in a
highly affinitive manner by preparing a fusion protein with 4F2hc
or rBAT.
[0025] Incidentally, the sodium-independent transporter AGT1 of the
present invention which transports acidic amino acids is mainly
expressed in the kidney in vivo.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows the amino acid sequences of mouse AGT1, mouse
Asc-2, rat LAT1, rat y.sup.+LAT1, mouse xCT and rat BAT1 for
comparison with each other. The assumed membrane-spanning sites are
shown by lines. A conserved cystine residue is shown by *, assumed
cAMP-dependent phosphorylation site is shown by #, an assumed
C-kinase-dependent phosphorylation site is shown by + and an
assumed tyrosine phosphorylation site is shown by &.
[0027] FIG. 2 is a photographic picture as a substitute for drawing
which shows the result of analysis of expression of AGT1 gene mRNA
in various organ tissues of mouse by means of a northern
blotting.
[0028] FIG. 3 is a photograph as a substitute for drawing which
shows the result of the western blotting analysis by an anti-AGT1
antibody. It was carried out under a non-reducing condition (-) and
a reducing condition (+) in a mouse kidney membrane specimen.
[0029] FIG. 4 is a photograph as a substitute for drawing which
shows the result of immunohistological analysis of AGT1 by an
anti-AGT1 antibody in a mouse kidney. a: slightly magnified image.
Stainings are observed in proximal tubule of outer layer of medulla
and in distal tubule of cortex. b: absorption experiment by antigen
peptide. Stainings observed in "a" disappeared and specificity of
staining was shown. c and d: highly magnified images of proximal
tubule (c) and distal tubule (d). Stainings are observed in the
side of basolateral membrane.
[0030] FIG. 5 is a schematic drawing of a fusion protein prepared
by connecting AGT1 with 4F2hc or rBAT. Amino acid sequences and
gene base sequences of the connection parts of AGT1-4F2hc fusion
protein and AGT1-rBAT fusion protein are shown below in FIG. 5.
[0031] FIG. 6 shows the result of experiment of the uptake of
aspartic acid by an oocyte into which mouse 4F2hc gene cRNA, AGT1
gene cRNA, AGT1 gene cRNA/mouse 4F2hc gene cRNA, AGT1-4F2hc fusion
protein gene cRNA or AGT1-rBAT fusion protein gene cRNA is
injected.
[0032] FIG. 7 shows the result of experiment of the uptake of
aspartic acid by COS-7 cells into which mouse 4F2hc gene, mouse
rBAT gene, AGT1 gene, AGT1 gene/mouse 4F2hc gene or AGT1 gene/mouse
rBAT gene is injected.
[0033] FIG. 8 is a drawing which shows the result of investigating
the expression of a fusion protein of AGT1 with 4F2hc (AGT1-4F2hc)
in an oocyte cell membrane by a immunofluorescence analysis. As
controls, investigations by a immunofluorescence analysis were
carried out using an anti-4F2hc antibody (a, c and e) or an
anti-AGT1 antibody in the oocyte into which water was injected (a
and b), the oocyte into which AGT1 gene cRNA was injected and
expressed (c and d) and the oocyte into which a fusion protein
(AGT1-4F2hc) gene cRNA of AGT1 with 4F2hc was injected and
expressed (e and f).
[0034] FIG. 9 shows the result of investigating the influence of
added salt in an experiment of the uptake of aspartic acid by an
oocyte into which a fusion protein (AGT1-4F2hc) gene cRNA of AGT1
with 4F2hc was injected.
[0035] FIG. 10 shows the result of investigating the influence of
the concentration of substrate aspartic acid in an experiment of
the uptake of aspartic acid by an oocyte into which a fusion
protein (AGT1-4F2hc) gene cRNA of AGT1 with 4F2hc was injected.
[0036] FIG. 11 shows the result of investigating the influence of
addition of various amino acids and analogous compounds on a system
in an experiment of the uptake of aspartic acid by an oocyte into
which a fusion protein (AGT1-4F2hc) gene cRNA of AGT1 with 4F2hc
was injected.
[0037] FIG. 12 shows the result of investigating the influence of
addition of various acidic amino acids and analogous compounds on a
system in an experiment of the uptake of aspartic acid by an oocyte
into which a fusion protein (AGT1-4F2hc) gene cRNA of AGT1 with
4F2hc was injected.
[0038] PDC: L-trans-pyrrolidine-2,4-dicarboxylate; DHK:
dihydrokainate; **: the case where p<0.01 in Student's t-test to
the data which does not constitute a pair.
[0039] FIG. 13 shows the result of investigating the uptake of
radio-labeled amino acid by an oocyte into which a fusion protein
(AGT1-4F2hc) gene cRNA of AGT1 with 4F2hc was injected.
BEST MODE FOR CARRYING OUT THE INVENTION
[0040] SEQ ID NO: 1 in the Sequence Listing which will be shown
later represents an amino acid sequence (478 amino acids) of a
sodium-independent transporter (mouse AGT1) derived from mouse
transporting acidic amino acids and SEQ ID NO: 2 represents an
amino acid sequence (478 amino acids) of protein encoded in a
full-length cDNA base sequence (about 2.1 kbp) of the gene and a
translation region thereof.
[0041] When the amino acid sequence represented by SEQ ID NO: 1 or
the base sequence represented by SEQ ID NO: 2 which will be
mentioned later was subjected to a homology search for all
sequences included in the known protein databases (NBRF and
SWISS-PROT) and DNA databases (GenBank and EMBL), no sequence was
identical, whereby the sequence is believed to be novel.
[0042] With regard to the protein of the present invention, there
may be exemplified, in addition to the one having an amino acid
sequence represented by SEQ ID NO: 1, a protein having the amino
acid sequence where one or several amino acid(s) is/are deleted,
substituted or added in the amino acid sequence represented by SEQ
ID NO: 1. Deletion, substitution or addition of amino acid(s) may
be within such an extent that transport activity of a neutral amino
acid is not lost and, usually, it is from 1 to about 96 or,
preferably, from 1 to about 48. Such a protein usually has a
homology to the amino acid sequence represented by SEQ ID NO: 1 to
an extent of 1 to 80%, preferably, 1 to 90%.
[0043] With regard to the gene of the present invention, in
addition to the one having a base sequence represented by SEQ ID
NO: 2, it includes a gene containing DNA which can be hybridized
with DNA having a base sequence represented by SEQ ID NO: 2 under a
stringent condition. With regard to the DNA which can be
hybridized, any substance will do so far as the protein encoded by
the DNA has an ability of transporting a neutral amino acid. Such a
DNA usually has a homology of base sequence of more than 70%,
preferably more than 80% with the base sequence represented by SEQ
ID NO: 2. Such a DNA includes a variant gene found in nature, an
artificially modified variant gene, a homologous gene derived from
other organism, and the like.
[0044] In the present invention, a hybridization under a stringent
condition is usually carried out in such a manner that a
hybridization is carried out for about 12 hours under the
temperature of 37 to 42.degree. C. in a hybridization solution of
5.times.SSC or having the identical salt concentration therewith, a
preliminary washing is carried out upon necessity using a solution
of 5.times.SSC or having the identical salt concentration therewith
and then washing is carried out in a solution of 1.times.SSC or
having the identical salt concentration therewith.
[0045] The sodium-independent transporter gene which transports
acidic amino acids according to the present invention can be
isolated and obtained by carrying out a screening using a tissue or
a cell of appropriate mammals as a gene source. Examples of the
mammals are non-human animals such as dog, cattle, horse, goat,
sheep, monkey, pig, rabbit, rat and mouse, and in addition to
those, human beings.
[0046] Screening and isolation of gene is able to be advantageously
carried out by, for example, a homology cloning method.
[0047] For example, mouse or human kidney is used as a gene source
and mRNA (poly(A).sup.+RNA) is prepared therefrom. Then a cDNA
library is constructed therefrom and cDNA is screened using a probe
corresponding to a sequence analogous to BAT1 (such as
GenBank.TM./EBI/DDBJ accession No AI314100) obtained by searching
the EST (expressed sequence tag) database whereby a clone
containing cDNA of Asc-2 gene can be obtained.
[0048] With regard to the obtained cDNA, the base sequence is
determined by a conventional method and translation region is
analyzed, whereby an amino acid sequence of the protein encoded
thereby, i.e. AGT1, can be determined.
[0049] The fact that the resulting cDNA is a sodium-independent
transporter which transports acidic amino acids, in other words,
the gene product encoded with cDNA is a sodium-independent
transporter which transports acidic amino acids can be tested, for
example, by the following method. Thus, cDNA encoding a fusion
protein of AGT1 with 4F2hc or rBAT is prepared using the resulting
cDNA of AGT1 gene, then RNA (cRNA) which is prepared from the cDNA
and complementary thereto is introduced into the oocyte to be
expressed and an ability of transport (uptake) of acidic amino
acids into the cells can be confirmed by measuring the uptake of
the substrate into cells by a common uptake test using an
appropriate acidic amino acid as a substrate (Kanai and Hediger,
Nature, volume 360, pages 467-471, 1992).
[0050] AGT1 protein is synthesized by an in vitro translation
method (Hediger, et al., Biochim. Biophys. Acta, volume 1064, page
360, 1991) using RNA (cRNA) prepared from the resulting cDNA of
AGT1 gene and being complementary thereto and the size of the
protein or the presence of sugar, etc. can be investigated by means
of electrophoresis.
[0051] Since cDNA of 4F2hc gene has been reported already
(Fukasawa, et al., J. Biol. Chem., 275: 9690-9698, 2000), it is
possible to easily prepare a gene of 4F2hc from the sequence
information by a PCR or the like.
[0052] Since cDNA of rBAT gene has been also reported already
(Segawa, H., et al., Biochem. J, 328: 657-664, 2000), it is
possible to easily prepare a gene of rBAT from the sequence
information by a PCR or the like.
[0053] cDNA encoding a fusion protein of AGT1 with 4F2hc or rBAT is
easily prepared by a PCR or the like from cDNA of AGT1 gene, cDNA
of 4F2hc gene or cDNA of rBAT gene.
[0054] The characteristic of AGT1 such as substrate selectivity of
AGT1 can be investigated by applying the similar uptake experiment
to the expressed cells.
[0055] Homologous gene, chromosome gene, etc. derived from
different tissues and different organisms can be isolated by
screening an appropriate cDNA library or genomic DNA library
prepared from different gene sources using the resulting cDNA of
AGT1 gene.
[0056] It is also possible to isolate a gene from a cDNA library or
a genomic DNA library by a conventional PCR (polymerase chain
reaction) method using a synthetic primer designed on the basis of
information of the disclosed base sequence of the gene of the
present invention (the base sequence represented by SEQ ID NO: 2 or
a part thereof).
[0057] DNA library such as a cDNA library or a genomic DNA library
may be prepared by a method mentioned, for example, in "Molecular
Cloning" by Sambrook, J., Fritsh, E. F. and Manitis, T. (Cold
Spring Harbor Press, 1989). When there is a commercially available
library, it can be used as well.
[0058] The sodium-independent transporter which transports acidic
amino acids according to the present invention and the gene (AGT1)
thereof may be produced by, for example, a gene recombination
technique using cDNA encoding therefor. For example, DNA (such as
cDNA) encoding AGT1 is incorporated into an appropriate expression
vector and the resulting recombinant DNA can be introduced into an
appropriate host cell. With regard to an expression system
(host-vector system) for the production of polypeptide, there may
be exemplified expression systems of bacteria, yeasts, insect cells
and mammalian cells. Among those, it is preferred to use insect
cells and mammalian cells for the preparation of functional
proteins.
[0059] A fusion protein of the sodium-independent transporter which
transports acidic amino acids according to the present invention
with 4F2hc or rBAT or a gene thereof (AGT1-4F2hc or AGT1-rBAT) may
be produced, for example, by a gene recombination technique using
cDNA encoding it. For example, DNA (such as cDNA) encoding
AGT1-4F2hc or AGT1-rBAT is incorporated into an appropriate
expression vector and the resulting recombinant DNA can be
introduced into an appropriate host cell. With regard to an
expression system (host-vector system) for the production of
polypeptide, there may be exemplified expression systems of
bacteria, yeasts, insect cells and mammalian cells. Among those, it
is preferred to use insect cells and mammalian cells for the
preparation of functional proteins.
[0060] For example, when polypeptide is expressed in mammalian
cells, DNA encoding the sodium-independent transporter AGT1 which
transports acidic amino acids according to the present invention or
DNA encoding a fusion protein of AGT1 with 4F2hc or rBAT is
inserted into a downstream side of an appropriate promoter (such as
cytomegalovirus promoter, SV40 promoter, LTR promoter, elongation 1
a promoter, etc.) in an appropriate expression vector (such as
adenovirus vector, retrovirus vector, papilloma virus vector,
vaccinia virus vector, SV40 vector, etc.) so that expression vector
is constructed. Then, an appropriate animal cell is transformed
using the resulting expression vector and the transformant is
incubated in an appropriate medium whereby a desired polypeptide is
produced. Examples of the mammalian cell used as a host are cell
strains such as simian COS-7 cell, Chinese hamster CHO cell and
human HeLa cell.
[0061] Accordingly, the present invention provides a vector,
preferably an expression vector, which contains a gene encoding the
above-mentioned gene of the present invention or for a protein in
the gene and also provides a host cell (transformant) which is
transformed using the vector.
[0062] With regard to the DNA encoding the sodium-independent
transporter AGT1 which transports acidic amino acids, cDNA having
the base sequence represented by SEQ ID NO: 2 may be used, for
example, in addition to that, DNA corresponding to the amino acid
sequence is designed and may be used as DNA encoding polypeptide
without limiting to the above-mentioned cDNA sequence. In that
case, with regard to a codon encoding one amino acid, 1 to 6 kinds
are known for each, and although the used codon may be optionally
selected, frequency of use of a codon of a host utilized for the
expression may be taken into consideration to design a sequence
having higher expression efficiency. DNA having the designed base
sequence can be prepared by chemical synthesis of DNA, binding to
fragmentation of the above-mentioned cDNA, partial modification of
the base sequence, and the like. The artificial partial
modification of and introduction of variation into base sequence
may be carried out utilizing a primer comprising synthetic
oligonucleotide encoding the desired modification by a
site-specific mutagenesis (Mark, D. F., et al., Proceedings of
National Academy of Sciences, volume 81, page 5662 (1984),
etc.).
[0063] DNA encoding a fusion protein (AGT1-4F2hc or AGT1-rBAT) of
the sodium-independent transporter AGT1 which transports acidic
amino acids with 4F2hc or rBAT may be prepared, for example, using
a base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 or
using cDNA having a base sequence represented by SEQ ID NO: 6 and,
moreover, DNA corresponding to the amino acid sequence is designed
and can be used as DNA encoding polypeptide without limiting to the
above-mentioned cDNA sequences. In that case, with regard to a
codon encoding one amino acid, 1 to 6 kinds are known for each, and
although the used codon may be optionally selected, frequency of
use of a codon of a host utilized for the expression may be taken
into consideration to design a sequence having higher expression
efficiency may be designed. DNA having the designed base sequence
can be prepared by chemical synthesis of DNA, binding to
fragmentation of the above-mentioned cDNA, partial modification of
the base sequence, and the like. The artificial partial
modification and introduction of variation into base sequence may
be carried out utilizing a primer comprising synthetic
oligonucleotide encoding the desired modification by a
site-specific mutagenesis (Mark, D. F., et al., Proceedings of
National Academy of Sciences, volume 81, page 5662 (1984),
etc.).
[0064] The present invention also provides a nucleotide containing
a partial sequence of continuous 14 or more bases, preferably 20 or
more bases, in the base sequence represented by SEQ ID NO: 2 or the
complementary sequence thereof.
[0065] The nucleotide of the present invention can be used as a
probe for detection of a gene encoding a protein having an ability
of transport of acidic amino acids or its analogue in a
sodium-independent manner. It can be also used as a primer for
obtaining a gene encoding the protein and the gene encoding a
protein having high homology thereto. Further, it can be used for
modulation of expression of a gene encoding a protein having an
ability to transport acidic amino acids and its analogue in a
sodium-independent manner by its anti-sense chain, etc.
[0066] It is possible to prepare the corresponding antibody using
the sodium-independent transporter which transports acidic amino
acids of the present invention or using a polypeptide having an
immunological homology thereto. The antibody can be utilized for
detection, purification, and the like. of the sodium-independent
transporter which transports acidic amino acids. The antibody can
be manufactured using the sodium-independent transporter which
transports acidic amino acids according to the present invention, a
fragment thereof, synthetic peptide having a partial sequence
thereof, etc. as an antigen. Polyclonal antibody can be
manufactured by a conventional method where antigen is inoculated
to a host animal (such as rat and rabbit) and immune serum is
recovered therefrom while monoclonal antibody can be manufactured
by a conventional technique such as a hybridoma method.
[0067] The protein of the present invention has an ability of
transporting acidic amino acids and its analogue in a
sodium-independently manner and the ability is strongly affected in
the presence of various substances. By screening a substance which
inhibits or accelerates the ability, the ability of the present
protein for transporting the substance can be controlled.
[0068] Accordingly, the present invention provides a method for
detecting an effect of a test substance as a substrate on an
ability of the protein of the present invention for transporting
acidic amino acids or its analogue in a sodium-independent manner
using the above-mentioned protein of the present invention.
[0069] The amino acid which is transported by the protein of the
present invention is the substance essential for proliferation and
growth of cells and for maintenance of life, and by controlling the
uptake of such a substance into cells, proliferation, growth, etc.
of cells can be controlled. Accordingly, the present invention
provides a method for controlling the cell proliferation by
modulating an ability of the protein for transporting the acidic
amino acids and analogous substance thereto using the
above-mentioned protein of the present invention, a specific
antibody thereof or a function-promoting or function-suppressing
substance thereof.
[0070] Gene of a fusion protein of the sodium-independent
transporter AGT1 transporting the acidic amino acids with 4F2hc or
rBAT according to the present invention and the expressed cell
thereof can be used for an in vitro test for the efficiency of
permeation of a substance at a cell membrane where AGT1 is present
or at the site where the presence of AGT1 is assumed. In addition,
a gene of a fusion protein of the sodium-independent transporter
AGT1 transporting the acidic amino acids with 4F2hc or rBAT and the
expressed cell thereof can be used for the development of a
compound which efficiently permeates through a cell membrane where
AGT1 is present or at the site where the presence of AGT1 is
assumed. Further, a gene of a fusion protein of the
sodium-independent transporter AGT1 transporting the acidic amino
acids with 4F2hc or rBAT and the expressed cell thereof can be used
for an in vitro test of pharmaceutical interaction at a cell
membrane where AGT1 is present or at the site where the presence of
AGT1 is assumed.
[0071] Accordingly, the present invention provides a method for
changing the pharmacokinetics of pharmaceuticals or xenobiotics
transported by the above-mentioned protein of the present invention
by using the protein, a specific antibody thereof or a
function-promoting or function-suppressing substance thereof, by
modulating an ability of the protein for transport acidic amino
acids or its analogue.
[0072] As described above, since the protein of the present
invention has an ability to transport acidic amino acids or its
analogue in a sodium-independent manner and this ability can be
suppressed or promoted not only by the number of a protein existing
in a cell, but also by the presence of various substances (in the
presence of a function-suppressing substance, etc. or in the
presence of a function-promoting substance, etc., respectively),
the present invention provides a controlling agent for transport
ability of a protein for acidic amino acids or its analogue
possessed by the above-mentioned protein of the present invention
which comprises the protein, a specific antibody thereof or a
function-promoting substance or function-suppressing substance
thereof.
[0073] Since the controlling agent for transport ability of the
present invention can control the proliferation, growth, and the
like. of cells, it can be used as a controlling agent for cell
proliferation, and since the agent can modulate and control the
pharmacokinetics of pharmaceutical, toxic substance or xenobiotics,
it can be used as a controlling agent for pharmacokinetics of
pharmaceutical, toxin or xenobiotics.
[0074] By suppressing the sodium-independent transporter AGT1 of
the present invention which transports acidic amino acids, the
permeation of a specific compound through the cell membrane where
AGT1 is expressed or through the site where AGT1 is assumed to be
present can be limited. In addition, a gene of a fusion protein of
the sodium-independent transporter AGT1 of the present invention
transporting the acidic amino acids with 4F2hc or rBAT and its
expression cell can be used for the development of a pharmaceutical
(such as a specific inhibitor for AGT1) which limits the permeation
of a compound transported by AGT1 through a cell membrane or the
site where AGT1 is assumed to be present.
[0075] Further, in accordance with the present invention, it has
been found that the protein having an amino acid sequence
represented by SEQ ID NO: 3 or NO: 5 comprises an ability to
promote the transfer AGT1 into a cell membrane. Accordingly, the
present invention provides a promoting agent for the transfer of
AGT1 into a cell membrane containing a protein having an amino acid
sequence represented by SEQ ID NO: 3 or NO: 5 or a protein having
an amino acid sequence where one or several amino acid(s) of the
above protein is/are deleted, substituted or added.
[0076] All of the contents mentioned in the specification of the
Japanese patent application No. 2002-040,608 shall be incorporated
into the present specification.
EXAMPLES
[0077] The present invention will now be illustrated in more detail
by way of the following Examples although those Examples do not
limit the present invention.
[0078] In the following Examples, each operation was carried out,
unless otherwise clearly mentioned, according to a method mentioned
in "Molecular Cloning" by Sambrook, J., Fritsh, E. F. and Manitis,
T. (Cold Spring Harbor Press, 1989) or according to the Directions
for Use of the commercially available products when the
commercially available reagents or kits are used.
Example 1
[0079] Cloning and expression analysis of a sodium-independent
transporter which transports acidic amino acids
[0080] (1) Identification of Mouse cDNA of a Sodium-Independent
Transporter which Transports Acidic Amino Acids
[0081] cDNA clone corresponding to the base sequence
GenBank.TM./EBI/DDBJ accession No. AI314100 derived from mouse
analogous to rat BAT1 obtained by searching the EST (expressed
sequence tag) database using a base sequence of a translation
region of rat BAT1 (Chairoungdua, et al., J. Biol. Chem., 274:
28845-28848, 1999) was purchased from IMAGE (Integrated and
Molecular Analysis of Genomes and their Expression) (IMAGE clone I.
D.: 1907807) and its fragment (1.8-kb) cleaved by a restriction
enzyme XhoI was labeled with .sup.32P-dCTP and used as a probe
whereby a mouse kidney cDNA library was screened.
[0082] The cDNA library was prepared from poly(A).sup.+RNA derived
from mouse kidney using a kit for the synthesis of cDNA (trade
name: Superscript Choice System, manufactured by Gibco) and
incorporated into a site of phage vector AZipLox (manufactured by
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 for hybridization of 37.degree. C. and the filter membrane
was washed with 0.1.times.SSC/0.1% SDS at 37.degree. C. With regard
to the solution for hybridization, 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 was used. The cDNA portion of
.lamda.ZipLox phage into which cDNA was incorporated was
incorporated into a plasmid pZL1. The resulting cDNA-inserted
fragment of clone was further incorporated into an NotI-cleaved
site of a plasmid pcDNA 3.1 (Invitrogen).
[0083] A base sequence for the full-length cDNA was determined by a
dye terminator cycle sequencing method (Applied Biosystems) using a
synthetic primer for the determination of base sequence. Further, a
base sequence of cDNA was analyzed by a conventional method and
translation region of cDNA and amino acid sequence of the protein
encoded thereby were determined.
[0084] Those sequences are represented in SEQ ID NO: 1 of the
Sequence Listing which will be shown later.
[0085] AGT1 comprised a 48% homology to a mouse transporter Asc-2
corresponding to a neutral amino acid transport system asc.
Further, AGT1 comprised a 35% homology to a rat transporter LAT1
and 37% to LAT2 corresponding to a neutral amino acid transport
system L, 37% homology to a rat transporter y.sup.+LAT1 and 36% to
a human transporter y.sup.+LAT2 corresponding to neutral and basic
amino acid transport system y.sup.+L. Furthermore, AGT1 comprised a
37% homology to a mouse transporter Asc-1 corresponding to a
neutral amino acid transport system asc, 37% to a mouse transporter
xCT corresponding to a cystine and acidic amino acid transport
system x, and 36% to a rat transporter BAT1 corresponding to a
cystine, neutral and basic amino acid transport system b.sup.0,+.
Still further, Asc-2 comprised a 30% homology to a mouse and human
transporter CAT1 to 4 corresponding to a basic amino acid transport
system y.sup.+.
[0086] Comparison of AGT1 with mouse Asc-2, rat LAT1, rat
y.sup.+LAT1, mouse xCT and rat BAT1 in terms of amino acid
sequences is shown in FIG. 1.
[0087] When an amino acid sequence of AGT1 was analyzed by an SOSUI
algorithm (Hirokawa, T., et al., Bioinformatics, volume 14, page
378 (1998)), 12 membrane-spanning domains were assumed as shown in
FIG. 1. In the third hydrophilic loop, conserved cysteine residues
were present among Asc-2, LAT1, Asc-2, y.sup.+LAT1, xCT and BAT1.
It is assumed that, via the cysteine residue, Asc-2 is binded to
unknown cofactor via a disulfide bond. In addition, there were
sites believed to be a cAMP-dependent phosphorylation site in the
eighth hydrophilic loop, C-kinase-dependent phosphorylation sites
in an N-terminal intracellular region and the sixth hydrophilic
loop and a tyrosine phosphorylation site in an N-terminal
intracellular region respectively.
[0088] (2) Expression of AGT1 Gene in Various Tissues of Mouse
(Analysis by Northern Blotting)
[0089] cDNA fragments corresponding to 43rd to 1836th base pair of
AGT1 gene were amplified by a PCR, labeled with .sup.32P-dCTP and
using as a probe, a northern blotting was carried out in the
following manner to RNA extracted from various tissues of mouse. 3
.mu.g of poly(A).sup.+RNA was subjected to an electrophoresis using
1% agarose/formaldehyde gel and then transferred to a
nitrocellulose filter. The filter was subjected to a hybridization
for one night using a hybridization solution containing Asc-2 cDNA
fragments labeled with .sup.32P-dCTP. The filter was washed at
65.degree. C. with 0.1.times.SSC containing 0.1% SDS.
[0090] As a result of the northern blotting (FIG. 2), a band was
detected at about 2.2 kb in the kidney.
[0091] (3) Expression of AGT1 Protein in the Mouse Kidney
[0092] A specific antibody to synthetic oligopeptide
(CIPDVSDDHIHEES) (mentioned in SEQ ID NO: 7 of Sequence Listing)
corresponding to 465-478 of mouse AGT1 was prepared according to a
method of Altman, et al. (Altman, et al., Proc. Natl. Acad. Sci.
USA, volume 81, pages 2176-2180, 1984).
[0093] Membrane fraction of mouse kidney was prepared according to
a method of Thorens, et al. (Thorens, et al., Cell, volume 55,
pages 281-290, 1988). The protein sample was treated at 100.degree.
C. for 5 minutes in the presence (under a reducing condition) or
absence (under a non-reducing condition) of 5% 2-mercaptoethanol,
subjected to electrophoresis by an SDS-polyacrylamide gel, blotted
to Hybond-P PVDV transfer membrane and treated with an anti-AGT1
antiserum (1:10,000).
[0094] As a result, in the mouse kidney, a band was detected near
250 kDa under a non-reducing condition by an anti-AGT1 antibody as
shown in FIG. 3. Under a reducing condition, a band was detected
near 40 kDa. From those results, it is suggested that AGT1 is
binded to some protein by a disulfide bond.
[0095] (4) Immunohistological Analysis of AGT1 Protein in the Mouse
Kidney
[0096] According to a conventional method, a mouse kidney paraffin
slice was treated with an anti-AGT1 antiserum (1:1,000) and colored
with diaminobenzidine. Further, with an object of investigating the
specificity of color development, an experiment of treating with an
anti-AGT1 antiserum (1:1,000) in the presence of 50 mg/ml of an
antigen peptide was also carried out.
[0097] As a result, in the mouse kidney, stainings were noted in
proximal tubule of outer layer of medulla and in distal tubule of
cortex as shown in FIG. 4a. As the stainings were not detected when
an anti-AGT1 antiserum was made to act in the presence of an
antigen peptide, the specificity in staining was shown (FIG. 4b).
Further, when an observation was conducted with highly magnified,
it was clarified that AGT1 protein was present in basolateral
membrane of proximal tubule (FIG. 4c) and distal tubule (FIG.
4d).
Example 2
Preparation of a Fusion Protein of Sodium-Independent Transporter
AGT1 Transporting Acidic Amino Acids with 4F2hc or with rBAT and
Analysis of its Function
[0098] (1) Preparation of a Fusion Protein of Sodium-Independent
Transporter AGT1 Transporting Acidic Amino Acids with 4F2hc or
rBAT
[0099] In order to prepare a fusion protein of AGT1 with
rBAT(AGT1-rBAT), a PCR was carried out using synthetic oligo-DNA
primers 5'-GCGCGAAGCTTACCTATAGGCAGAAACATTC-3' (in which, to a
sequence corresponding to 4th to 23rd base pair of AGT1 cDNA were
added a sequence corresponding to cleaved site with HindIII and
GCGC at 5'-side; mentioned in SEQ ID NO: 8 of the Sequence Listing)
and 5'-ATATGCGGCCGCACTTTCTTCATGTATGTGGT-3' (in which, to a sequence
corresponding to 1473rd to 1492nd base pair of AGT1 cDNA were added
a sequence corresponding to the cleaved site with NotI and ATAT at
5'-side; mentioned in SEQ ID NO: 9 of the Sequence Listing) where
AGT1 cDNA was used as a template. The resulting PCR product was
cleaved with HindIII and NotI and ligated to HindIII and NotI sites
of mammalian cell expression vector pcDNA3.1(+) (Invitrogen).
Further, a PCR was carried out using a synthetic oligo-DNA primers
5'-ATATGCGGCCGCAGATGAGGACAAAGGCAAGAG-3' (in which, to a sequence
corresponding to the base pair immediately after translation
initiation codon ATG of mouse rBAT to 21st as shown in SEQ ID NO: 6
were added a sequence corresponding to a site cleaved by NotI and
ATAT at 5'-side; mentioned in SEQ ID NO: 10 in the Sequence
Listing) and 5'-GCGCGCTCTAGAAATGCTTTAGTATTTGGCATAATC-3' (in which,
to a sequence of 2228th to 2251st base pair of mouse rBAT as shown
by SEQ ID NO: 6 were added a sequence corresponding to a site
cleaved with XbaI and GCGC at 5'-side; mentioned in SEQ ID NO: 11
in the Sequence Listing) where rBAT cDNA was used as a template.
The resulting PCR product was cleaved with NotI and XbaI and
ligated to NotI and XbaI sites of the mammalian cell expression
vector pcDNA3.1(+) into which the above-mentioned AGT1 PCR product
was incorporated to prepare cDNA encoding a fusion protein of AGT1
with rBAT (FIG. 5).
[0100] In order to prepare a fusion protein of AGT1 with
4F2hc(AGT1-4F2hc), a PCR was carried out using synthetic oligo-DNA
primers 5'-GCGCGAAGCTTACCTATAGGCAGAAACATTC-3' (in which, to a
sequence corresponding to 4th to 23rd base pair of AGT1 cDNA were
added a sequence corresponding to a site cleaved by HindIII and
GCGC at 5'-side; mentioned in SEQ ID NO: 8 of the Sequence Listing)
and 5'-ATATGCGGCCGCACTTTCTTCATGTATGTGGT-3' (in which, to a sequence
corresponding to 1473rd to 1492nd base pair of AGT1 cDNA were added
a sequence corresponding to a site cleaved by NotI and ATAT at
5'-side; mentioned in SEQ ID NO: 9 of the Sequence Listing) where
AGT1 cDNA was used as a template. The resulting PCR product was
cleaved with HindIII and NotI and ligated to HindIII and NotI sites
of mammalian cell expression vector pcDNA3.1(+) (Invitrogen).
Further, a PCR was carried out using synthetic oligo-DNA primers
5'-ATATGCGGCCGCAAGCCAGGACACCGAAGTGGA-3' (in which, to a sequence
corresponding to the base pair immediately after translation
initiation codon ATG of mouse 4F2hc to 21st as shown in SEQ ID NO:
4 were added a sequence corresponding to a site cleaved by NotI and
ATAT at 5'-side; mentioned in SEQ ID NO: 12 in the Sequence
Listing) and 55'-GCGCTCTAGACATGAGGCAGGGGTGATGTTIT-3' (in which, to
a sequence corresponding to 1820th to 1838th base pair of mouse
4F2hc shown in SEQ ID NO: 4 were added a sequence corresponding to
a site cleaved by XbaI and GCGC at 5'-side; mentioned in SEQ ID NO:
13 of the Sequence Listing) where 4F2hc cDNA was used as a
template. The resulting PCR product was cleaved with NotI and XbaI
and ligated to NotI and XbaI sites of mammalian cell expression
vector pcDNA3.1(+) into which the above-mentioned AGT1 PCR product
was incorporated to give cDNA encoding a fusion protein of AGT1
with 4F2hc (FIG. 5).
[0101] (2) Expression of a Function of a Fusion Protein of a
Sodium-Independent Transporter AGT1 Transporting Acidic Amino Acids
with 4F2hc or rBAT
[0102] Comparisons were conducted for the uptake of aspartic acid
when mouse AGT1 gene cRNA was expressed in the oocyte, when mouse
AGT1 gene cRNA and mouse 4F2hc gene cRNA were expressed in the
oocyte and when a fusion protein of ACT1 with 4F2hc or rBAT was
expressed in the oocyte.
[0103] 25 ng of mouse 4F2hc gene cRNA, 25 ng of AGT1 gene cRNA,
12.5 ng of AGT1 gene cRNA/12.5 ng of mouse 4F2hc gene cRNA, 25 ng
of AGT1-4F2hc fusion protein gene cRNA or 25 ng of AGT1-rBAT fusion
protein gene cRNA was injected into the oocyte to express and
incubation was conducted for 3 days.
[0104] With regard to the oocyte into which mouse 4F2hc gene cRNA,
AGT1 gene cRNA, AGT1 gene cRNA/mouse 4F2hc gene cRNA, AGT1-4F2hc
fusion protein gene cRNA or AGT1-rBAT fusion protein gene cRNA was
injected, experiments for the uptake of a substrate was carried out
using aspartic acid as a substrate according to a method of Kanai,
et al. (Kanai and Hediger, Nature, volume 360, pages 467-471, 1992)
as follows. The oocyte were allowed to stand for 30 minutes in a
sodium-free uptake solution (100 mM choline chloride, 2 mM
potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium
chloride and 5 mM HEPES; pH 7.4) containing .sup.14C-aspartic acid
(20 mM) as a substrate and an uptake rate of the substrate was
measured by the count of radioactivity incorporated into the
cells.
[0105] As a result (FIG. 6), the levels of the uptake of aspartic
acid in the oocyte where only 4F2hc was expressed, the oocyte where
only AGT1 was expressed and the oocyte where both AGT1 and 4F2hc
were co-expressed were similar to that in the control oocyte into
which water was injected, while a higher uptake of aspartic acid
was noted in the oocyte where AGT1-rBAT or AGT1-4F2hc was
expressed.
[0106] It was investigated that rBAT or 4F2hc cannot be a direct
cofactor of AGT1 using COS-7 cells. According to a method mentioned
in Mizoguchi, et al., Kidney Int, 59: 1821-1833, 2001, plasmid DNA
(each 1 mg) containing AGT1 cDNA, rBAT cDNA or 4F2hc cDNA was
introduced into COS-7 cells using LIPOFECTAMINE 2000 Reagent (Life
Technologies). After the introduction, the cells were incubated for
two days in a 24-well plate and the uptake of .sup.14C-aspartic
acid (20 mM) was measured. Measurement of the uptake was conducted
according to a method of Mizoguchi, et al., Kidney Int, 59:
1821-1833, 2001, in which it was started by removing the culture
liquid and adding Dulbecco's PBS (manufactured by Gibco) containing
.sup.14C-aspartic acid, and completed by removing it and washing
with ice-cooled Dulbecco's PBS. After the washing, it was dissolved
with 0.1N NaOH and radioactivity was measured by a liquid
scintillation counter.
[0107] As a result (FIG. 7), the levels of the uptake of aspartic
acid in the oocyte where only 4F2hc was expressed, the oocyte where
only rBAT was expressed, the oocyte where only AGT1 was expressed,
the oocyte where both AGT1 and 4F2hc were co-expressed and the
oocyte where both AGT1 and rBAT were co-expressed were similar to
that the control oocyte into which a pcDNA 3.1 plasmid containing
no inserted cDNA whereby it was confirmed that rBAT or 4F2hc was
not a direct cofactor for AGT1.
[0108] (3) Identification of Expression of a Fusion Protein of
Sodium-Independent Transporter AGT1 Transporting Acidic Amino Acids
with 4F2hc (AGT1-4F2hc) in Oocyte Cell Membrane by a
Immunofluorescence Analysis
[0109] Whether the fact that when AGT1 was expressed in the oocyte,
no function was observed while a fusion protein of AGT1 with 4F2hc
(AGT1-4F2hc) showed a functional activity is due to the fact that
AGT1 is not transported to a cell membrane while AGT1-4F2hc is
transported to a cell membrane or not was investigated by a
immunofluorescence analysis.
[0110] 25 ng of AGT1 gene cRNA or 25 ng of the cRNA of a gene of a
fusion protein of AGT1 with 4F2hc(AGT1-4F2hc) was injected into the
oocyte to express, incubated for 3 days and, the oocyte was fixed
in a 4% paraformaldehyde-phosphate buffer and prepared a paraffin
section (3 mm) according to a conventional method. After removing
the paraffin, the section was subjected to a blocking with 5% goat
serum in 0.05M Tris buffer in a physiological saline containing
0.1% Tween 20 and treated with an affinity-purified anti-Asc-2
antibody or an affinity-purified anti-4F2hc antibody (Fukasawa, et
al., J. Biol. Chem., 275: 9690-9698, 2000). Then, the section was
treated with Alexa Fluor 488-labeled goat anti-rabbit IgG
(Molecular Probe, Inc.), washed with 0.05M Tris buffer in
physiological saline containing 0.1% Tween 20 and observed with
Olympus Fluoview (FV500) confocal laser microscope (Olympus).
Excitation was effected with argon laser. at 488 nm and
fluorescence from Alexa Fluor 488 was detected using a BA505IF
filter.
[0111] As a result (FIG. 8), in the oocyte in which AGT1 was
expressed, an AGT1 protein detected in an anti-AGT1 antibody was
not present in a cell membrane but remained inside the cell
membrane (FIG. 8d), while in an oocyte in which a fusion protein of
AGT1 with 4F2hc(AGT1-4F2hc) was expressed, an AGT1-4F2hc fusion
protein expressed in a cell membrane was detected in both
anti-4F2hc antibody (FIG. 8 e) and anti-AGT1 antibody (FIG. 80. In
the control oocyte into which water was injected, no specific color
development by anti-4F2hc antibody (FIG. 8a) or anti-AGT1 antibody
(FIG. 8b) was observed. Accordingly, it was proven that the fact
that no function was observed when AGT1 was expressed in the oocyte
while a fusion protein of AGT1 with 4F2hc(AGT1-4F2hc) showed a
functional activity is due to the fact that AGT1 is not transported
to a cell membrane by itself, while its fusion protein with 4F2hc
(AGT1-4F2hc) is transported to a cell membrane.
[0112] (4) Salt-Dependency of Transport Activity of AGT1
[0113] In an uptake experiment of aspartic acid by the oocyte into
which a cRNA of a gene of a fusion protein of AGT1 with 4F2hc or
with rBAT (AGT1-4F2hc or AGT1-rBAT) was injected, influence of salt
added to the medium was investigated.
[0114] An uptake experiment of aspartic acid was carried out
according to the method mentioned in the above Example 2(2) using
an oocyte into which a cRNA of a gene of a fusion protein of AGT1
with 4F2hc or with rBAT (AGT1-4F2hc or AGT1-rBAT) was injected.
With regard to the uptake solution, a standard uptake solution (100
mM of choline chloride was exchanged with 100 mM sodium chloride)
was used instead of a sodium-free uptake solution when the effect
of sodium ion was investigated. A gluconic acid uptake solution
(100 mM sodium chloride was exchanged with 100 mM sodium gluconate)
was used instead of a standard uptake solution when the effect of
chlorine ion was investigated.
[0115] As a result (FIG. 9), even when extracellular choline was
exchanged with sodium or even when extracellular chlorine was
exchanged with gluconate ion, it did not affect the uptake of
aspartic acid at all. From the above, it was noted that Asc-2 is a
transporter which acts independently on sodium ion and chlorine
ion.
[0116] (5) Michaelis-Menten Kinetic Analysis of AGT1
[0117] A Michaelis-Menten kinetic analysis of sodium-independent
transporter AGT1 which transports acidic amino acids was carried
out. The Michaelis-Menten kinetic analysis was conducted by
investigating the change in the ratio of uptake of aspartic acid by
the difference in the substrate aspartic acid concentration.
[0118] The aspartic acid uptake experiment was carried according to
the method mentioned in the above Example 2(2) using the oocyte
into which a cRNA of a gene of a fusion protein of AGT1 with 4F2hc
or with rBAT (AGT1-4F2hc or AGT1-rBAT) was injected. As a result
(FIG. 10), the Km value of aspartic acid transport by AGT1-4F2hc
was 25.5+5.9 mM (mean value+standard error). The Km value of
aspartic acid transport by AGT1-rBAT was 20.1+6.1.
[0119] The Michaelis-Menten kinetic analysis was similarly carried
out in a fusion protein of AGT1 with 4F2hc (AGT1-4F2hc) in glutamic
acid and Km value and Vmax value were calculated. Result of the
above is shown in the following Table 1.
Table 1
[0120] Km values and Vmax values of substrate amino acid
TABLE-US-00001 Amino Acid Km.sup.a (.mu.M) Vmax.sup.b L-Aspartic
acid 25.5 .+-. 5.9 (1.00) L-Glutamic acid 21.8 .+-. 6.5 0.63 .+-.
0.10 .sup.a, bV max value of L-glutamic acid is shown by the ratio
to Vmax value of L-aspartic acid. Both Km and Vmax values are
represented by mean value .+-. standard error.
[0121] (6) Substrate Selectivity of AGT1 (Inhibition Experiment
Using Added Amino Acid and Its Analogue)
[0122] In an uptake experiment of aspartic acid by an oocyte into
which a cRNA of a gene of a fusion protein of AGT1 with 4F2hc
(AGT1-4F2hc) was injected, the effect of addition of various amino
acids and their analogues on the system was investigated.
[0123] An aspartic acid uptake experiment was carried out according
to the method mentioned in the above Example 2(2) using an oocyte
into which a cRNA of a gene of a fusion protein of AGT1 with 4F2hc
(AGT1-4F2hc) was injected. However, a sodium-free uptake solution
was used and the uptake of .sup.14C-aspartic acid (20 mM) was
measured in the presence and absence of 2 mM of various compounds
(non-labeled).
[0124] As a result (FIG. 11), in aspartic acid, glutamic acid and
cysteine, a significant cis-inhibiting effect was observed.
[0125] Basic amino acids, neutral amino acids except cysteine,
cystine, 2-amino-2-norbornane-carboxylic acid (BCH) which is a
transport system L-specific inhibitor, .gamma.-aminoisobutyric
acid, .alpha.-aminomethylisobutyric acid, D-aspartic acid and
D-glutamic acid did no affect the uptake of .sup.14C-aspartic acid
mediated by AGT1-4F2hc (FIG. 11).
[0126] In an oocyte into which cRNA of a gene of a fusion protein
of AGT1 with rBAT (AGT1-rBAT) were injected together, the effect of
adding various amino acids and their analogues on the system was
also investigated in an aspartic acid uptake experiment by an
oocyte in a similar manner as in the case of AGT1-4F2hc.
[0127] As a result, in the case of AGT1-rBAT, the same result as in
the case of AGT1-4F2hc was obtained, and in the case of a fusion
protein of AGT1 with 4F2hc or with rBAT (AGT1-4F2hc or AGT1-rBAT),
the 4F2hc or rBAT moiety did not affect the characteristic of
substrate-binding site and, with regard to the information
concerning the substrate selectivity obtained in a fusion protein,
the AGT1 itself also reflects the transport characteristic.
[0128] In the substances analogous to acidic amino acids,
threo-.beta.-hydroxyaspartate (THA), L-serine-O-sulfate (SOS),
L-cysteine sulfate and L-cysteate strongly inhibited the uptake of
.sup.14C-aspartic acid mediated by AGT1.
[0129] On the contrary, in the case of L-.alpha.-aminoadipate,
L-homocysteate, L-trans-pyrrolidine-2,4-dicarboxylate (PDC) and
dihydrokainate (DHK), no inhibition effect on the uptake of
.sup.14C-aspartic acid mediated by AGT1 was observed (FIG. 12).
[0130] (7) Substrate Selectivity of AGT1 (Uptake Experiment Using
Various Amino Acids and their Analogues as Substrates)
[0131] Various kinds of amino acids and their analogues were used
as substrates and uptake by an oocyte into which a cRNA of a gene
of a fusion protein of AGT1 with 4F2hc (AGT1-4F2hc) was injected
was investigated.
[0132] The uptake experiment of various amino acids and their
analogues was carried out according to the method mentioned in the
above-mentioned Example 2(2) using an oocyte into which a cRNA of a
gene of a fusion protein of AGT1 with 4F2hc (AGT1-4F2hc) was
injected. With regard to a substrate, various compounds which were
labeled with radioactivity were used instead of .sup.14C-aspartic
acid.
[0133] As a result, when L-glutamic acid (14C compound) was used as
a substrate in addition to L-aspartic acid (14C compound) (FIG.
13), a substantial uptake into the oocyte was observed.
INDUSTRIAL APPLICABILITY
[0134] The sodium-independent transporter of the present invention
which transports acidic amino acids and a gene thereof enables an
in vitro investigation of transport of acidic amino acids and amino
acid analogues including xenobiotics at the site where the
transporter is expressed, and based on which, also enables an in
vitro assumption of pharmacokinetic of these compounds. Further,
the present invention is useful for the development of
pharmaceutical which permeates efficiently through a site where the
transporter is expressed. Furthermore, by modulating an ability to
transport acidic amino acids and its analogue possessed by the
transporter, the invention can be utilized for the development of a
method for controlling a cell proliferation.
Sequence CWU 1
1
221478PRTMus sp.AGT1 1Met Ala Met Asp Ser Lys Lys Glu Ile Arg Leu
Lys Arg Glu Leu Gly1 5 10 15Tyr Phe Trp Gly Thr Asn Phe Leu Ile Ile
Asn Ile Ile Gly Ala Gly 20 25 30Ile Phe Val Ser Pro Lys Gly Val Leu
Gln His Ser Ser Met Asn Val 35 40 45Gly Val Ser Leu Cys Val Trp Ala
Val Cys Ala Val Leu Thr Leu Thr 50 55 60Ser Ala Leu Cys Ser Ala Glu
Ile Gly Ile Thr Phe Pro Tyr Ser Gly65 70 75 80Ala His Tyr Tyr Phe
Leu Lys Arg Cys Phe Gly Pro Leu Val Ala Phe 85 90 95Leu Arg Leu Trp
Thr Ser Leu Phe Leu Gly Pro Gly Leu Ile Ala Ser 100 105 110Gln Ala
Leu Leu Leu Ala Glu Tyr Gly Val Gln Pro Phe Tyr Pro Ser 115 120
125Cys Ser Ala Pro Ile Leu Pro Arg Lys Cys Leu Ala Leu Ala Met Leu
130 135 140Trp Ile Val Gly Ile Leu Asn Ser Arg Gly Val Lys Glu Leu
Ser Trp145 150 155 160Leu Gln Thr Val Ser Ser Val Leu Lys Val Gly
Ile Leu Gly Val Ile 165 170 175Ser Leu Ser Gly Leu Phe Leu Leu Val
Arg Gly Lys Lys Glu Asn Val 180 185 190Gln Arg Leu Gln Asn Ala Phe
Asp Ala Glu Phe Pro Glu Val Ser Gln 195 200 205Leu Ile Glu Ala Ile
Phe Gln Gly Tyr Phe Ala Phe Ser Gly Gly Gly 210 215 220Cys Phe Thr
Cys Ile Ala Gly Glu Leu Lys Lys Pro Ser Lys Thr Ile225 230 235
240Pro Arg Cys Ile Phe Thr Gly Leu Pro Leu Val Thr Val Val Tyr Leu
245 250 255Leu Ala Asn Ile Ser Tyr Leu Thr Val Leu Thr Pro Gln Glu
Met Leu 260 265 270Ser Ser Asp Ala Val Ala Leu Thr Trp Thr Asp Arg
Val Ile Pro Gln 275 280 285Phe Thr Trp Thr Val Pro Phe Ala Ile Ser
Ala Ser Leu Phe Ile Asn 290 295 300Leu Val Ile Asn Val Leu Glu Thr
Ser Arg Val Leu Tyr Ile Ala Ser305 310 315 320Glu Asn Gly Gln Leu
Pro Leu Leu Phe Cys Ala Leu Asn Val His Ser 325 330 335Ser Pro Phe
Ile Ala Val Leu Leu Ile Ile Ser Met Ala Ser Ile Leu 340 345 350Ile
Val Leu Thr Asn Leu Ile Asp Leu Ile Asn Tyr Leu Tyr Phe Val 355 360
365Val Ser Ile Trp Thr Ala Leu Ser Ile Ile Gly Ile Leu Lys Leu Arg
370 375 380Tyr Gln Glu Pro Asn Leu His Arg Pro Tyr Lys Val Phe Leu
Pro Phe385 390 395 400Thr Phe Ile Ala Leu Gly Ile Thr Leu Ser Leu
Val Leu Ile Pro Leu 405 410 415Val Lys Ser Pro Lys Leu His Tyr Ile
Tyr Val Phe Leu Phe Leu Leu 420 425 430Ser Gly Leu Val Phe Tyr Val
Pro Leu Ile His Phe Lys Val Lys Phe 435 440 445Val Trp Phe Gln Lys
Leu Thr Cys Tyr Leu Gln Leu Leu Phe Asn Ile 450 455 460Cys Ile Pro
Asp Val Ser Asp Asp His Ile His Glu Glu Ser465 470 47522141DNAMus
sp.CDS(59)..(1492)AGT1 2caaacctata ggcagaaaca ttcacagagt acaattttgt
gaatgtaaac ttctctca 58atg gca atg gat tca aag aag gaa atc cgt ctt
aag aga gaa ctt gga 106Met Ala Met Asp Ser Lys Lys Glu Ile Arg Leu
Lys Arg Glu Leu Gly1 5 10 15tat ttc tgg ggg aca aac ttt tta att att
aat ata att ggt gca gga 154Tyr Phe Trp Gly Thr Asn Phe Leu Ile Ile
Asn Ile Ile Gly Ala Gly 20 25 30ata ttt gtg tcc ccc aag gga gtg ctc
cag cac tct tcc atg aat gtt 202Ile Phe Val Ser Pro Lys Gly Val Leu
Gln His Ser Ser Met Asn Val 35 40 45gga gtc tcc ttg tgt gtt tgg gct
gtc tgt gca gtg ctg acc ttg acc 250Gly Val Ser Leu Cys Val Trp Ala
Val Cys Ala Val Leu Thr Leu Thr 50 55 60agt gct ctc tgc tct gca gag
atc ggg ata acc ttc cca tac agt ggg 298Ser Ala Leu Cys Ser Ala Glu
Ile Gly Ile Thr Phe Pro Tyr Ser Gly65 70 75 80gct cac tat tat ttt
tta aag cga tgc ttc ggc cct ctc gtg gca ttc 346Ala His Tyr Tyr Phe
Leu Lys Arg Cys Phe Gly Pro Leu Val Ala Phe 85 90 95ctg agg ctt tgg
act agc ttg ttt ctc ggc cca ggc tta att gct agc 394Leu Arg Leu Trp
Thr Ser Leu Phe Leu Gly Pro Gly Leu Ile Ala Ser 100 105 110caa gct
ctg cta ctg gct gag tat ggc gtt cag cct ttt tat ccc agc 442Gln Ala
Leu Leu Leu Ala Glu Tyr Gly Val Gln Pro Phe Tyr Pro Ser 115 120
125tgc tct gcc ccg att cta cca aga aaa tgt ctg gcc ctg gcg atg ctg
490Cys Ser Ala Pro Ile Leu Pro Arg Lys Cys Leu Ala Leu Ala Met Leu
130 135 140tgg att gtg gga att ctg aat tct cgt ggt gta aaa gag ctg
tca tgg 538Trp Ile Val Gly Ile Leu Asn Ser Arg Gly Val Lys Glu Leu
Ser Trp145 150 155 160ctt cag aca gtg agc tca gtg ctg aag gtg ggc
ata ctc ggt gtc att 586Leu Gln Thr Val Ser Ser Val Leu Lys Val Gly
Ile Leu Gly Val Ile 165 170 175tcc ctc agc ggc ctg ttc ttg ctg gtg
aga ggg aag aag gag aat gta 634Ser Leu Ser Gly Leu Phe Leu Leu Val
Arg Gly Lys Lys Glu Asn Val 180 185 190caa agg ctt cag aat gcg ttt
gat gcc gag ttc cca gag gtc tct cag 682Gln Arg Leu Gln Asn Ala Phe
Asp Ala Glu Phe Pro Glu Val Ser Gln 195 200 205tta ata gaa gct att
ttc caa gga tac ttt gcg ttt tct ggc ggg gga 730Leu Ile Glu Ala Ile
Phe Gln Gly Tyr Phe Ala Phe Ser Gly Gly Gly 210 215 220tgc ttt aca
tgt ata gca ggg gag ctg aag aaa ccc agt aaa aca att 778Cys Phe Thr
Cys Ile Ala Gly Glu Leu Lys Lys Pro Ser Lys Thr Ile225 230 235
240cct aga tgc atc ttt aca gga ctg cct ctg gta act gtc gtg tac tta
826Pro Arg Cys Ile Phe Thr Gly Leu Pro Leu Val Thr Val Val Tyr Leu
245 250 255ctg gct aat att tcc tac ctg aca gtt ctg aca ccc cag gaa
atg ctc 874Leu Ala Asn Ile Ser Tyr Leu Thr Val Leu Thr Pro Gln Glu
Met Leu 260 265 270tct tca gat gct gtt gct ctt aca tgg aca gac agg
gtc att ccc caa 922Ser Ser Asp Ala Val Ala Leu Thr Trp Thr Asp Arg
Val Ile Pro Gln 275 280 285ttc aca tgg act gtt cct ttt gct att tct
gct tca ctg ttt atc aac 970Phe Thr Trp Thr Val Pro Phe Ala Ile Ser
Ala Ser Leu Phe Ile Asn 290 295 300ctt gtg att aat gta ctt gag aca
tca aga gtg tta tat att gca agt 1018Leu Val Ile Asn Val Leu Glu Thr
Ser Arg Val Leu Tyr Ile Ala Ser305 310 315 320gag aat ggc cag ctg
cct ttg ttg ttt tgt gcc ctg aat gtc cat tcc 1066Glu Asn Gly Gln Leu
Pro Leu Leu Phe Cys Ala Leu Asn Val His Ser 325 330 335tct ccc ttt
ata gct gtg cta cta att atc agt atg gca tcc att tta 1114Ser Pro Phe
Ile Ala Val Leu Leu Ile Ile Ser Met Ala Ser Ile Leu 340 345 350att
gtc tta aca aac cta att gat ctg ata aac tat ctc tat ttt gtt 1162Ile
Val Leu Thr Asn Leu Ile Asp Leu Ile Asn Tyr Leu Tyr Phe Val 355 360
365gtt tcc att tgg act gcc tta tca ata ata gga atc ttg aaa ctg agg
1210Val Ser Ile Trp Thr Ala Leu Ser Ile Ile Gly Ile Leu Lys Leu Arg
370 375 380tac caa gag ccc aat cta cac aga cca tat aag gtg ttt tta
ccg ttc 1258Tyr Gln Glu Pro Asn Leu His Arg Pro Tyr Lys Val Phe Leu
Pro Phe385 390 395 400aca ttc ata gcg ttg ggc atc acc ctg agc ttg
gtt ttg atc ccg ctt 1306Thr Phe Ile Ala Leu Gly Ile Thr Leu Ser Leu
Val Leu Ile Pro Leu 405 410 415gtc aag tct cca aag ttg cat tat atc
tat gtg ttc ctc ttc ctt ctc 1354Val Lys Ser Pro Lys Leu His Tyr Ile
Tyr Val Phe Leu Phe Leu Leu 420 425 430agt ggg ctg gtg ttt tac gtg
cca ttg ata cac ttt aaa gtg aag ttc 1402Ser Gly Leu Val Phe Tyr Val
Pro Leu Ile His Phe Lys Val Lys Phe 435 440 445gtt tgg ttt cag aag
ttg act tgc tat ctg cag tta ctg ttt aat att 1450Val Trp Phe Gln Lys
Leu Thr Cys Tyr Leu Gln Leu Leu Phe Asn Ile 450 455 460tgc atc cct
gat gtg tct gat gac cac ata cat gaa gaa agt 1492Cys Ile Pro Asp Val
Ser Asp Asp His Ile His Glu Glu Ser465 470 475tgaggaagaa tagcctttgt
agccatactg tgttccagat aaaggttaag tgtaagctaa 1552aatagtaatg
atggcaatgc tataaactga aatggtatat agaaaagtac ccaggaaatt
1612cctagttttt aaaatatcaa aggaaatggc tgggtagatg actgtgctgt
taagggcacc 1672agatgtcctt ccaggggact gaagttcaat tcacaggccc
cacttagaag ttcataacta 1732tctgtaattc tagtcacaga ggatccaata
tcctctactg gattctacca gcactgcatg 1792catctggagt agagacatac
atgtaggcac ccatgcacat ttacaagaag aaagaaagag 1852agaaagaaag
aaagaaagaa agaaagaaag aaagaaagaa agaaagaaag aaagggagag
1912ggagagagga aggaaggaag gaaataagga aagaaggaag gaggaaagaa
agacagtaaa 1972gaaagtattt cacataacta aactgttttt attaaaaata
aaatttctag cttgtatagc 2032ttcatgtagt aagaatatct ttctcattct
tctgtttatc tcatcgattt tctactgaat 2092gtattcttat aataaaagtt
actgatggaa attaaaaaaa aaaaaaaaa 21413526PRTMus sp.4F2hc 3Met Ser
Gln Asp Thr Glu Val Asp Met Lys Asp Val Glu Leu Asn Glu1 5 10 15Leu
Glu Pro Glu Lys Gln Pro Met Asn Ala Ala Asp Gly Ala Ala Ala 20 25
30Gly Glu Lys Asn Gly Leu Val Lys Ile Lys Val Ala Glu Asp Glu Thr
35 40 45Glu Ala Gly Val Lys Phe Thr Gly Leu Ser Lys Glu Glu Leu Leu
Lys 50 55 60Val Ala Gly Ser Pro Gly Trp Val Arg Thr Arg Trp Ala Leu
Leu Leu65 70 75 80Leu Phe Trp Leu Gly Trp Leu Gly Met Leu Ala Gly
Ala Val Val Ile 85 90 95Ile Val Arg Ala Pro Arg Cys Arg Glu Leu Pro
Val Gln Arg Trp Trp 100 105 110His Lys Gly Ala Leu Tyr Arg Ile Gly
Asp Leu Gln Ala Phe Val Gly 115 120 125Arg Asp Ala Gly Gly Ile Ala
Gly Leu Lys Ser His Leu Glu Tyr Leu 130 135 140Ser Thr Leu Lys Val
Lys Gly Leu Val Leu Gly Pro Ile His Lys Asn145 150 155 160Gln Lys
Asp Glu Ile Asn Glu Thr Asp Leu Lys Gln Ile Asn Pro Thr 165 170
175Leu Gly Ser Gln Glu Asp Phe Lys Asp Leu Leu Gln Ser Ala Lys Lys
180 185 190Lys Ser Ile His Ile Ile Leu Asp Leu Thr Pro Asn Tyr Gln
Gly Gln 195 200 205Asn Ala Trp Phe Leu Pro Ala Gln Ala Asp Ile Val
Ala Thr Lys Met 210 215 220Lys Glu Ala Leu Ser Ser Trp Leu Gln Asp
Gly Val Asp Gly Phe Gln225 230 235 240Phe Arg Asp Val Gly Lys Leu
Met Asn Ala Pro Leu Tyr Leu Ala Glu 245 250 255Trp Gln Asn Ile Thr
Lys Asn Leu Ser Glu Asp Arg Leu Leu Ile Ala 260 265 270Gly Thr Glu
Ser Ser Asp Leu Gln Gln Ile Val Asn Ile Leu Glu Ser 275 280 285Thr
Ser Asp Leu Leu Leu Thr Ser Ser Tyr Leu Ser Asn Ser Thr Phe 290 295
300Thr Gly Glu Arg Thr Glu Ser Leu Val Thr Arg Phe Leu Asn Ala
Thr305 310 315 320Gly Ser Gln Trp Cys Ser Trp Ser Val Ser Gln Ala
Gly Leu Leu Ala 325 330 335Asp Phe Ile Pro Asp His Leu Leu Arg Leu
Tyr Gln Leu Leu Leu Phe 340 345 350Thr Leu Pro Gly Thr Pro Val Phe
Ser Tyr Gly Asp Glu Leu Gly Leu 355 360 365Gln Gly Ala Leu Pro Gly
Gln Pro Ala Lys Ala Pro Leu Met Pro Trp 370 375 380Asn Glu Ser Ser
Ile Phe His Ile Pro Arg Pro Val Ser Leu Asn Met385 390 395 400Thr
Val Lys Gly Gln Asn Glu Asp Pro Gly Ser Leu Leu Thr Gln Phe 405 410
415Arg Arg Leu Ser Asp Leu Arg Gly Lys Glu Arg Ser Leu Leu His Gly
420 425 430Asp Phe His Ala Leu Ser Ser Ser Pro Asp Leu Phe Ser Tyr
Ile Arg 435 440 445His Trp Asp Gln Asn Glu Arg Tyr Leu Val Val Leu
Asn Phe Arg Asp 450 455 460Ser Gly Arg Ser Ala Arg Leu Gly Ala Ser
Asn Leu Pro Ala Gly Ile465 470 475 480Ser Leu Pro Ala Ser Ala Lys
Leu Leu Leu Ser Thr Asp Ser Ala Arg 485 490 495Gln Ser Arg Glu Glu
Asp Thr Ser Leu Lys Leu Glu Asn Leu Ser Leu 500 505 510Asn Pro Tyr
Glu Gly Leu Leu Leu Gln Phe Pro Phe Val Ala 515 520 52541852DNAMus
sp.CDS(106)..(1683)4F2hc 4gctagcctca cggccacggg acgcctctct
gaacggggat ccaggcagga ttagagctgc 60ctcactgact acaggccgtg tcgtgtcacc
gtttctgcag gcacc atg agc cag gac 117 Met Ser Gln Asp 1acc gaa gtg
gac atg aaa gat gtg gag ctg aac gag cta gaa ccg gag 165Thr Glu Val
Asp Met Lys Asp Val Glu Leu Asn Glu Leu Glu Pro Glu5 10 15 20aag
cag ccc atg aat gca gcg gac ggg gcg gcg gcc ggg gag aag aac 213Lys
Gln Pro Met Asn Ala Ala Asp Gly Ala Ala Ala Gly Glu Lys Asn 25 30
35ggt ctg gtg aag atc aag gtg gcg gag gac gag acg gag gcc ggg gtc
261Gly Leu Val Lys Ile Lys Val Ala Glu Asp Glu Thr Glu Ala Gly Val
40 45 50aag ttc acc ggc tta tcc aag gag gag cta ctg aag gta gcg ggc
agc 309Lys Phe Thr Gly Leu Ser Lys Glu Glu Leu Leu Lys Val Ala Gly
Ser 55 60 65cct ggc tgg gtg cgc acc cgc tgg gcg ctg ctg ctg ctc ttc
tgg ctc 357Pro Gly Trp Val Arg Thr Arg Trp Ala Leu Leu Leu Leu Phe
Trp Leu 70 75 80ggt tgg ctg ggc atg ctg gcg ggc gcc gtg gtt atc atc
gtt cgg gcg 405Gly Trp Leu Gly Met Leu Ala Gly Ala Val Val Ile Ile
Val Arg Ala85 90 95 100ccg cgc tgc cgt gag ctg cct gta cag agg tgg
tgg cac aag ggc gcc 453Pro Arg Cys Arg Glu Leu Pro Val Gln Arg Trp
Trp His Lys Gly Ala 105 110 115ctc tac cgc atc ggc gac ctt cag gcc
ttt gta ggc cgg gat gcg gga 501Leu Tyr Arg Ile Gly Asp Leu Gln Ala
Phe Val Gly Arg Asp Ala Gly 120 125 130ggc ata gct ggt ctg aag agc
cat ctg gag tac ttg agc acc ctg aag 549Gly Ile Ala Gly Leu Lys Ser
His Leu Glu Tyr Leu Ser Thr Leu Lys 135 140 145gtg aag ggc ctg gtg
tta ggc cca att cac aag aac cag aag gat gaa 597Val Lys Gly Leu Val
Leu Gly Pro Ile His Lys Asn Gln Lys Asp Glu 150 155 160atc aat gaa
acc gac ctg aaa cag att aat ccc act ttg ggc tcc cag 645Ile Asn Glu
Thr Asp Leu Lys Gln Ile Asn Pro Thr Leu Gly Ser Gln165 170 175
180gaa gat ttt aaa gac ctt cta caa agt gcc aag aaa aag agc att cac
693Glu Asp Phe Lys Asp Leu Leu Gln Ser Ala Lys Lys Lys Ser Ile His
185 190 195atc att ttg gac ctc act ccc aac tac cag ggc cag aat gcg
tgg ttc 741Ile Ile Leu Asp Leu Thr Pro Asn Tyr Gln Gly Gln Asn Ala
Trp Phe 200 205 210ctc cct gct cag gct gac att gta gcc acc aaa atg
aag gaa gct ctg 789Leu Pro Ala Gln Ala Asp Ile Val Ala Thr Lys Met
Lys Glu Ala Leu 215 220 225agt tct tgg ttg cag gac ggt gtg gat ggt
ttc caa ttc cgg gat gtg 837Ser Ser Trp Leu Gln Asp Gly Val Asp Gly
Phe Gln Phe Arg Asp Val 230 235 240gga aag ctg atg aat gca ccc ttg
tac ttg gct gag tgg cag aat atc 885Gly Lys Leu Met Asn Ala Pro Leu
Tyr Leu Ala Glu Trp Gln Asn Ile245 250 255 260acc aag aac tta agt
gag gac agg ctt ttg att gca ggg act gag tcc 933Thr Lys Asn Leu Ser
Glu Asp Arg Leu Leu Ile Ala Gly Thr Glu Ser 265 270 275tct gac ctg
cag caa att gtc aac ata ctt gaa tcc acc agc gac ctg 981Ser Asp Leu
Gln Gln Ile Val Asn Ile Leu Glu Ser Thr Ser Asp Leu 280 285 290ctg
ttg acc agc tcc tac ctg tca aat tcc act ttc act ggg gag cgt 1029Leu
Leu Thr Ser Ser Tyr Leu Ser Asn Ser Thr Phe Thr Gly Glu Arg 295 300
305act gaa tcc cta gtc act agg ttt ttg aat gcc act ggc agc caa tgg
1077Thr Glu Ser Leu Val Thr Arg Phe Leu Asn Ala Thr Gly Ser Gln Trp
310 315 320tgc agc tgg agt gtg tcg caa gca gga ctc ctc gca gac ttt
ata ccg 1125Cys Ser Trp Ser Val Ser Gln Ala Gly Leu Leu Ala Asp Phe
Ile Pro325
330 335 340gac cat ctt ctc cga ctc tac cag ctg ctg ctc ttc act ctg
cca ggg 1173Asp His Leu Leu Arg Leu Tyr Gln Leu Leu Leu Phe Thr Leu
Pro Gly 345 350 355act cct gtt ttt agc tac ggg gat gag ctt ggc ctt
cag ggt gcc ctt 1221Thr Pro Val Phe Ser Tyr Gly Asp Glu Leu Gly Leu
Gln Gly Ala Leu 360 365 370cct gga cag cct gcg aag gcc cca ctc atg
ccg tgg aat gag tcc agc 1269Pro Gly Gln Pro Ala Lys Ala Pro Leu Met
Pro Trp Asn Glu Ser Ser 375 380 385atc ttt cac atc cca aga cct gta
agc ctc aac atg aca gtg aag ggc 1317Ile Phe His Ile Pro Arg Pro Val
Ser Leu Asn Met Thr Val Lys Gly 390 395 400cag aat gaa gac cct ggc
tcc ctt ctt acc cag ttc cgg cgg ctg agt 1365Gln Asn Glu Asp Pro Gly
Ser Leu Leu Thr Gln Phe Arg Arg Leu Ser405 410 415 420gac ctt cgg
ggt aag gag cgc tct ctg ttg cac ggt gac ttc cat gca 1413Asp Leu Arg
Gly Lys Glu Arg Ser Leu Leu His Gly Asp Phe His Ala 425 430 435ctg
tct tcc tca cct gac ctc ttc tcc tac ata cga cac tgg gac cag 1461Leu
Ser Ser Ser Pro Asp Leu Phe Ser Tyr Ile Arg His Trp Asp Gln 440 445
450aat gag cgt tac ctg gtg gtg ctc aac ttc cga gat tcg ggc cgg tca
1509Asn Glu Arg Tyr Leu Val Val Leu Asn Phe Arg Asp Ser Gly Arg Ser
455 460 465gcc agg cta ggg gcc tcc aac ctc cct gct ggc ata agc ctg
cca gcc 1557Ala Arg Leu Gly Ala Ser Asn Leu Pro Ala Gly Ile Ser Leu
Pro Ala 470 475 480agc gct aaa ctt ttg ctt agt acc gac agt gcc cgg
caa agc cgt gag 1605Ser Ala Lys Leu Leu Leu Ser Thr Asp Ser Ala Arg
Gln Ser Arg Glu485 490 495 500gag gac acc tcc ctg aag ctg gaa aac
ctg agc ctg aat cct tat gag 1653Glu Asp Thr Ser Leu Lys Leu Glu Asn
Leu Ser Leu Asn Pro Tyr Glu 505 510 515ggc ttg ctg tta cag ttc ccc
ttt gtg gcc tgatccttcc tatgcagaac 1703Gly Leu Leu Leu Gln Phe Pro
Phe Val Ala 520 525ctaccaccct cctttgttct ccccaggcct tttggattct
agtcttcctc tccttgtttt 1763taaacttttg cagattacat acgaattctt
atactgggtg tttttgtctt caaataaaaa 1823catcacccct gcctcaaaaa
aaaaaaaaa 18525685PRTMus sp.rBAT 5Met Asp Glu Asp Lys Gly Lys Arg
Asp Pro Ile Gln Met Ser Met Lys1 5 10 15Gly Cys Arg Thr Asn Asn Gly
Phe Val Gln Asn Glu Asp Ile Pro Glu 20 25 30Gln Asp Pro Asp Pro Gly
Ser Arg Asp Thr Pro Gln Pro Asn Ala Val 35 40 45Ser Ile Pro Ala Pro
Glu Glu Pro His Leu Lys Ala Val Arg Pro Tyr 50 55 60Ala Gly Met Pro
Lys Glu Val Leu Phe Gln Phe Ser Gly Gln Ala Arg65 70 75 80Tyr Arg
Val Pro Arg Glu Ile Leu Phe Trp Leu Thr Val Val Ser Val 85 90 95Phe
Leu Leu Ile Gly Ala Thr Ile Ala Ile Ile Val Ile Ser Pro Lys 100 105
110Cys Leu Asp Trp Trp Gln Ala Gly Pro Ile Tyr Gln Ile Tyr Pro Arg
115 120 125Ser Phe Lys Asp Ser Asp Lys Asp Gly Asn Gly Asp Leu Lys
Gly Ile 130 135 140Gln Glu Lys Leu Asp Tyr Ile Thr Ala Leu Asn Ile
Lys Thr Leu Trp145 150 155 160Ile Thr Ser Phe Tyr Lys Ser Ile Phe
Glu Asp Phe Arg Tyr Ala Val 165 170 175Glu Asp Ile Lys Glu Ile Asp
Pro Ile Phe Gly Thr Met Lys Asp Phe 180 185 190Glu Asn Leu Val Ala
Ala Ile His Asp Lys Gly Leu Lys Leu Ile Ile 195 200 205Asp Phe Ile
Pro Asn His Thr Ser Asp Lys His Pro Trp Phe Gln Ser 210 215 220Ser
Arg Thr Arg Ser Gly Lys Tyr Thr Asp Tyr Tyr Ile Trp His Asn225 230
235 240Cys Thr His Cys Gln Arg Val Pro Thr Pro Pro Asn Asn Trp Leu
Ser 245 250 255Val Tyr Gly His Ser Ser Trp His Phe Asp Glu Val Arg
Glu Gln Cys 260 265 270Tyr Phe His Gln Phe Leu Arg Glu Gln Pro Asp
Leu Tyr Phe Arg Asn 275 280 285Pro Ala Val Gln Glu Glu Ile Lys Glu
Ile Ile Thr Phe Trp Leu Ser 290 295 300Lys Gly Val Asp Gly Phe Ser
Phe Asp Ala Val Lys Phe Leu Leu Glu305 310 315 320Ala Lys Asp Leu
Arg Asn Glu Ile Gln Val Asn Thr Ser Gln Ile Pro 325 330 335Asp Thr
Val Thr His Tyr Ser Glu Leu Tyr His Asp Phe Thr Thr Thr 340 345
350Gln Val Gly Met His Asp Ile Val Arg Asp Phe Arg Gln Thr Met Asn
355 360 365Gln Tyr Ser Arg Glu Pro Gly Arg Tyr Arg Phe Met Gly Ala
Glu Ala 370 375 380Ser Ala Glu Ser Ile Glu Arg Thr Met Met Tyr Tyr
Gly Leu Pro Phe385 390 395 400Ile Gln Glu Ala Asp Phe Pro Phe Asn
Lys Tyr Phe Thr Thr Ile Gly 405 410 415Thr Leu Ser Gly His Thr Val
Tyr Glu Val Ile Thr Ser Trp Met Glu 420 425 430Asn Met Pro Glu Gly
Lys Trp Pro Asn Trp Met Thr Gly Gly Pro Glu 435 440 445Thr Pro Arg
Leu Thr Ser Arg Val Gly Ser Glu Tyr Val Asn Ala Met 450 455 460His
Met Leu Leu Phe Thr Leu Pro Gly Thr Pro Ile Thr Tyr Tyr Gly465 470
475 480Glu Glu Ile Gly Met Gly Asp Ile Ser Val Thr Asn Phe Asn Glu
Ser 485 490 495Tyr Asp Ser Thr Thr Leu Val Ser Lys Ser Pro Met Gln
Trp Asp Asn 500 505 510Ser Ser Asn Ala Gly Phe Thr Glu Ala Asn His
Thr Trp Leu Pro Pro 515 520 525Asn Ser Asp Tyr His Thr Val Asn Val
Asp Val Gln Lys Thr Gln Pro 530 535 540Ser Ser Ala Leu Arg Leu Tyr
Gln Asp Leu Ser Leu Leu His Ala Thr545 550 555 560Glu Leu Val Leu
Ser Arg Gly Trp Phe Cys Leu Leu Arg Asp Asp Ser 565 570 575His Ser
Val Val Tyr Thr Arg Glu Leu Asp Gly Ile Asp Asn Val Phe 580 585
590Leu Val Val Leu Asn Phe Gly Glu Ser Ser Thr Val Leu Asn Leu Gln
595 600 605Gly Ile Ile Ser Asp Leu Pro Pro Glu Leu Arg Ile Arg Leu
Ser Thr 610 615 620Asn Ser Ala Ser Lys Gly Ser Ala Val Asp Thr Arg
Ala Ile Ser Leu625 630 635 640Glu Lys Gly Glu Gly Leu Val Leu Glu
His Ser Thr Lys Ala Pro Leu 645 650 655His Gln Gln Ala Ala Phe Arg
Asp Arg Cys Phe Val Ser Ser Arg Ala 660 665 670Cys Tyr Ser Ser Ala
Leu Asp Ile Leu Tyr Ser Ser Cys 675 680 68562287DNAMus
sp.CDS(46)..(2100)rBAT 6gatccccctg ctggaaagca ccaggaagag ctacacaggg
tagac atg gat gag gac 57 Met Asp Glu Asp 1aaa ggc aag aga gac ccc
atc caa atg agt atg aag gga tgc cga acc 105Lys Gly Lys Arg Asp Pro
Ile Gln Met Ser Met Lys Gly Cys Arg Thr5 10 15 20aat aac ggg ttt
gtc caa aat gaa gac att ccg gag cag gac cca gac 153Asn Asn Gly Phe
Val Gln Asn Glu Asp Ile Pro Glu Gln Asp Pro Asp 25 30 35cca ggc tcc
agg gac acc cca cag ccc aac gcc gtg agt atc cct gct 201Pro Gly Ser
Arg Asp Thr Pro Gln Pro Asn Ala Val Ser Ile Pro Ala 40 45 50cca gag
gag cct cac cta aag gcg gtg cgg ccc tat gca ggg atg ccc 249Pro Glu
Glu Pro His Leu Lys Ala Val Arg Pro Tyr Ala Gly Met Pro 55 60 65aag
gaa gta ctc ttc cag ttc tcc ggc cag gct cgc tac cgg gtg ccc 297Lys
Glu Val Leu Phe Gln Phe Ser Gly Gln Ala Arg Tyr Arg Val Pro 70 75
80cga gag atc ctc ttc tgg ctc acc gtg gtt tcc gtg ttc ctg ctc att
345Arg Glu Ile Leu Phe Trp Leu Thr Val Val Ser Val Phe Leu Leu
Ile85 90 95 100gga gcc acc ata gcc atc atc gtc atc tct cca aaa tgc
ctt gac tgg 393Gly Ala Thr Ile Ala Ile Ile Val Ile Ser Pro Lys Cys
Leu Asp Trp 105 110 115tgg caa gca ggt ccc ata tac cag atc tac ccg
agg tct ttt aag gac 441Trp Gln Ala Gly Pro Ile Tyr Gln Ile Tyr Pro
Arg Ser Phe Lys Asp 120 125 130agt gac aag gat ggg aat gga gac ctg
aaa ggt atc cag gag aag ctg 489Ser Asp Lys Asp Gly Asn Gly Asp Leu
Lys Gly Ile Gln Glu Lys Leu 135 140 145gac tat atc act gct tta aac
ata aag act ctt tgg atc act tcc ttt 537Asp Tyr Ile Thr Ala Leu Asn
Ile Lys Thr Leu Trp Ile Thr Ser Phe 150 155 160tat aaa tcg atc ttt
gaa gac ttc aga tac gct gtt gag gat atc aaa 585Tyr Lys Ser Ile Phe
Glu Asp Phe Arg Tyr Ala Val Glu Asp Ile Lys165 170 175 180gaa att
gac cct att ttt gga aca atg aaa gat ttt gag aat ttg gtt 633Glu Ile
Asp Pro Ile Phe Gly Thr Met Lys Asp Phe Glu Asn Leu Val 185 190
195gct gcc atc cat gac aaa ggt tta aaa tta ata att gat ttc ata cca
681Ala Ala Ile His Asp Lys Gly Leu Lys Leu Ile Ile Asp Phe Ile Pro
200 205 210aac cac act agt gac aaa cat cct tgg ttc caa tcg agt agg
aca cgg 729Asn His Thr Ser Asp Lys His Pro Trp Phe Gln Ser Ser Arg
Thr Arg 215 220 225agc gga aaa tac acc gat tac tac atc tgg cac aac
tgt acc cat tgt 777Ser Gly Lys Tyr Thr Asp Tyr Tyr Ile Trp His Asn
Cys Thr His Cys 230 235 240caa cgt gta ccc acc cct ccc aac aac tgg
ctg agt gtg tat gga cac 825Gln Arg Val Pro Thr Pro Pro Asn Asn Trp
Leu Ser Val Tyr Gly His245 250 255 260tcc agc tgg cac ttt gat gaa
gta cga gag caa tgt tat ttt cac cag 873Ser Ser Trp His Phe Asp Glu
Val Arg Glu Gln Cys Tyr Phe His Gln 265 270 275ttt ttg aga gag caa
cca gat tta tat ttc cga aat cct gct gtt caa 921Phe Leu Arg Glu Gln
Pro Asp Leu Tyr Phe Arg Asn Pro Ala Val Gln 280 285 290gag gaa ata
aag gaa ata ata acg ttc tgg ctc tcg aag ggt gtt gat 969Glu Glu Ile
Lys Glu Ile Ile Thr Phe Trp Leu Ser Lys Gly Val Asp 295 300 305ggg
ttt agt ttt gat gca gtt aaa ttt ctt ctg gaa gcg aag gat ctg 1017Gly
Phe Ser Phe Asp Ala Val Lys Phe Leu Leu Glu Ala Lys Asp Leu 310 315
320aga aat gaa atc caa gtg aat aca tcc caa att ccg gac acg gtc acc
1065Arg Asn Glu Ile Gln Val Asn Thr Ser Gln Ile Pro Asp Thr Val
Thr325 330 335 340cac tac tca gag ctg tac cat gac ttc acc aca act
cag gtg gga atg 1113His Tyr Ser Glu Leu Tyr His Asp Phe Thr Thr Thr
Gln Val Gly Met 345 350 355cat gac atc gtc cga gac ttc cgg cag acc
atg aac cag tac agc agg 1161His Asp Ile Val Arg Asp Phe Arg Gln Thr
Met Asn Gln Tyr Ser Arg 360 365 370gag cct ggc aga tac cgg ttc atg
ggg gcc gaa gcc tca gct gag agc 1209Glu Pro Gly Arg Tyr Arg Phe Met
Gly Ala Glu Ala Ser Ala Glu Ser 375 380 385atc gag agg acc atg atg
tac tat ggc ttg cca ttt atc cag gaa gcc 1257Ile Glu Arg Thr Met Met
Tyr Tyr Gly Leu Pro Phe Ile Gln Glu Ala 390 395 400gac ttt cct ttc
aac aag tac ttc acc aca ata ggc act ctc tct ggg 1305Asp Phe Pro Phe
Asn Lys Tyr Phe Thr Thr Ile Gly Thr Leu Ser Gly405 410 415 420cat
act gtc tat gaa gtt atc aca tcc tgg atg gaa aac atg cct gaa 1353His
Thr Val Tyr Glu Val Ile Thr Ser Trp Met Glu Asn Met Pro Glu 425 430
435gga aaa tgg ccc aat tgg atg act ggc gga ccg gag act cct cgg ctg
1401Gly Lys Trp Pro Asn Trp Met Thr Gly Gly Pro Glu Thr Pro Arg Leu
440 445 450act tct cga gta ggg agt gag tat gtc aac gcc atg cac atg
ctc ctg 1449Thr Ser Arg Val Gly Ser Glu Tyr Val Asn Ala Met His Met
Leu Leu 455 460 465ttc aca ctc ccg gga acg ccc atc act tac tat gga
gag gaa atc ggg 1497Phe Thr Leu Pro Gly Thr Pro Ile Thr Tyr Tyr Gly
Glu Glu Ile Gly 470 475 480atg gga gac att tcc gtt aca aat ttc aac
gag agc tat gat agt act 1545Met Gly Asp Ile Ser Val Thr Asn Phe Asn
Glu Ser Tyr Asp Ser Thr485 490 495 500acc ctt gtc tcc aag tca ccg
atg cag tgg gac aat agt tcc aat gct 1593Thr Leu Val Ser Lys Ser Pro
Met Gln Trp Asp Asn Ser Ser Asn Ala 505 510 515ggg ttt act gag gcc
aac cac acc tgg cta cca cca aac tct gac tac 1641Gly Phe Thr Glu Ala
Asn His Thr Trp Leu Pro Pro Asn Ser Asp Tyr 520 525 530cac acc gtc
aat gtg gat gtc caa aag acc cag ccg agc tcc gca ctg 1689His Thr Val
Asn Val Asp Val Gln Lys Thr Gln Pro Ser Ser Ala Leu 535 540 545agg
ctg tat cag gat ctg agt cta ctc cat gcc aca gag ctg gtc ctc 1737Arg
Leu Tyr Gln Asp Leu Ser Leu Leu His Ala Thr Glu Leu Val Leu 550 555
560agc cgg ggc tgg ttt tgc ctc ttg aga gac gac agt cac tct gtg gtg
1785Ser Arg Gly Trp Phe Cys Leu Leu Arg Asp Asp Ser His Ser Val
Val565 570 575 580tac aca aga gag ctg gac ggc ata gat aac gtc ttc
ctc gtg gtt ctg 1833Tyr Thr Arg Glu Leu Asp Gly Ile Asp Asn Val Phe
Leu Val Val Leu 585 590 595aat ttt gga gaa tca tca act gtg cta aat
cta cag ggg atc att tca 1881Asn Phe Gly Glu Ser Ser Thr Val Leu Asn
Leu Gln Gly Ile Ile Ser 600 605 610gat ctt cct cca gag ctg aga ata
agg tta agt acc aac tca gcc tcc 1929Asp Leu Pro Pro Glu Leu Arg Ile
Arg Leu Ser Thr Asn Ser Ala Ser 615 620 625aaa ggc agt gct gtt gac
acc cgt gcc att tct ctg gag aag gga gag 1977Lys Gly Ser Ala Val Asp
Thr Arg Ala Ile Ser Leu Glu Lys Gly Glu 630 635 640ggc ctg gtc ttg
gag cac agc acg aag gct ccc ctc cat cag cag gcc 2025Gly Leu Val Leu
Glu His Ser Thr Lys Ala Pro Leu His Gln Gln Ala645 650 655 660gct
ttc aga gac aga tgc ttt gtt tcc agt cgg gcg tgc tac tcc agt 2073Ala
Phe Arg Asp Arg Cys Phe Val Ser Ser Arg Ala Cys Tyr Ser Ser 665 670
675gca ctg gac atc ctc tat agc tcg tgt tagggaggaa gctccctaag
2120Ala Leu Asp Ile Leu Tyr Ser Ser Cys 680 685agatggccac
ccagaacatc acgtacgcac aggctgagca gactcatgaa tggcatcaat
2180tcttagatat ttctgtagca cgatgcacgt tttttaaagt gtttaaagat
tatgccaaat 2240actaaagcat ttaaatatga aaaaaaaaaa aaaaaagcgg cgcgccg
2287714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic partial peptide 7Cys Ile Pro Asp Val Ser Asp Asp His Ile
His Glu Glu Ser1 5 10831DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 8gcgcgaagct tacctatagg
cagaaacatt c 31932DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 9atatgcggcc gcactttctt catgtatgtg gt
321033DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10atatgcggcc gcagatgagg acaaaggcaa gag
331136DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11gcgcgctcta gaaatgcttt agtatttggc ataatc
361233DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 12atatgcggcc gcaagccagg acaccgaagt gga
331332DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13gcgctctaga catgaggcag gggtgatgtt tt
3214465PRTMus sp. 14Met Gln Leu Leu Arg Ala Leu Gly Val Phe His Val
Ser Met Ile Leu1 5 10 15Phe Ser Ala Thr Leu Gly Thr Gly Ile Phe Val
Thr Pro Lys Ala Val 20 25 30Leu Lys Tyr Ser Ser Leu Asn Ile Pro Val
Ser Leu Ser Ile Trp Ala 35 40 45Gly Cys Gly Leu Leu Ser Ile Met Ser
Ala Leu Cys Asn Ala Glu Ile 50 55 60Ala Thr Thr Tyr Pro Leu Ser Gly
Ala Ser Tyr Tyr Phe Leu Lys Arg65 70 75 80Thr Leu Gly Ser Ser Val
Ala Phe Leu Ser Leu Trp Ile Lys Leu Phe 85 90 95Ala His Phe Leu Gly
Ile Gly Ala Gln Cys Leu Leu Ile Ala Thr Ser 100 105 110Val Ile Gln
Cys Phe Tyr Ser Gly Cys Pro Ala Pro Glu Leu Pro Thr 115 120 125Lys
Cys Leu Ala Leu Ala Ile
Leu Trp Ser Phe Gly Ile Val Ser Ala 130 135 140Arg Gly Ile Lys Thr
Val Ala Trp Phe Asn Thr Val Ser Ser Phe Ile145 150 155 160Lys Leu
Ser Val Leu Cys Leu Ile Ser Leu Thr Val Leu Leu Val Asn 165 170
175Gly Lys Lys Glu Asn Val Ser Arg Phe Glu Asn Ala Leu Asp Ala Glu
180 185 190Leu Pro Asn Ala Ser Gln Ile Ala Asp Ala Ile Leu Gln Val
Ser Tyr 195 200 205Ser Tyr Leu Gly Ser Ser Val Leu Ile Val Ile Ala
Gly Glu Ile Lys 210 215 220Arg Pro Thr Glu Thr Ile Pro Lys Thr Leu
Ile Tyr Gly Ile Ser Ile225 230 235 240Val Thr Val Leu Tyr Leu Leu
Thr Asn Ile Ser Tyr Leu Ala Val Leu 245 250 255Thr Ser Gln Glu Ile
Ile Phe Ser Asp Ser Val Gly Val Thr Trp Met 260 265 270Asn Arg Val
Phe Pro Ser Ile Gln Trp Ile Ser Ser Phe Leu Ile Ser 275 280 285Ala
Phe Leu Leu Gly Ser Val Ser Cys Gly Ile Val Ser Ala Ser Arg 290 295
300Val Phe Tyr Ser Ala Ser Gly Glu Gly Glu Phe Pro Ser Ile Tyr
Ser305 310 315 320Met Leu Asn Asp His His Ser Pro Ala Val Ala Asp
Ile Gln Ile Val 325 330 335Ile Leu Ser Ser Val Ala Ile Ile Ser Ser
Ser Ile Ile Tyr Leu Val 340 345 350Lys Tyr Val Ser Leu Gly Ser Phe
Cys Ile Asn Leu Leu Gln Met Ile 355 360 365Gly Leu Leu Lys Ile Arg
Tyr Gln Asn Pro Asp Ile Pro Arg Pro Tyr 370 375 380Lys Val Trp Leu
Pro Phe Ile Phe Gly Ser Ile Ala Leu Ser Leu Phe385 390 395 400Leu
Ile Phe Thr Pro Val Ile Gln Ser Pro Ser Ile Glu His Val Tyr 405 410
415Gln Val Val Phe Leu Phe Cys Gly Phe Leu Cys Tyr Trp Leu Gln Ala
420 425 430Asn Leu Asn Gly His Ala Thr Cys Phe Asp Thr Ile Thr Cys
Tyr Cys 435 440 445Gln Leu Leu Phe Asn Ile Ser Pro Ser Glu Asp Pro
Glu Glu Gln Lys 450 455 460Asn46515512PRTMus sp. 15Met Ala Val Ala
Gly Ala Lys Arg Arg Ala Val Ala Ala Pro Ala Thr1 5 10 15Thr Ala Ala
Glu Glu Glu Arg Gln Ala Arg Glu Lys Met Leu Glu Ala 20 25 30Arg Arg
Gly Asp Gly Ala Asp Pro Glu Gly Glu Gly Val Thr Leu Gln 35 40 45Arg
Asn Ile Thr Leu Ile Asn Gly Val Ala Ile Ile Val Gly Thr Ile 50 55
60Ile Gly Ser Gly Ile Phe Val Thr Pro Thr Gly Val Leu Lys Glu Ala65
70 75 80Gly Ser Pro Gly Leu Ser Leu Val Val Trp Ala Val Cys Gly Val
Phe 85 90 95Ser Ile Val Gly Ala Leu Cys Tyr Ala Glu Leu Gly Thr Thr
Ile Ser 100 105 110Lys Ser Gly Gly Asp Tyr Ala Tyr Met Leu Glu Val
Tyr Gly Ser Leu 115 120 125Pro Ala Phe Leu Lys Leu Trp Ile Glu Leu
Leu Ile Ile Arg Pro Ser 130 135 140Ser Gln Tyr Ile Val Ala Leu Val
Phe Ala Thr Tyr Leu Leu Lys Pro145 150 155 160Val Phe Pro Thr Cys
Pro Val Pro Glu Glu Ala Ala Lys Leu Val Ala 165 170 175Cys Leu Cys
Val Leu Leu Leu Thr Ala Val Asn Cys Tyr Ser Val Lys 180 185 190Ala
Ala Thr Arg Val Gln Asp Ala Phe Ala Ala Ala Lys Leu Leu Ala 195 200
205Leu Ala Leu Ile Ile Leu Leu Gly Phe Ile Gln Met Gly Lys Asp Ile
210 215 220Gly Gln Gly Asp Ala Ser Asn Leu His Gln Lys Leu Ser Phe
Glu Gly225 230 235 240Thr Asn Leu Asp Val Gly Asn Ile Val Leu Ala
Leu Tyr Ser Gly Leu 245 250 255Phe Ala Tyr Gly Gly Trp Asn Tyr Leu
Asn Phe Val Thr Glu Glu Met 260 265 270Ile Asn Pro Tyr Arg Asn Leu
Pro Leu Ala Ile Ile Ile Ser Leu Pro 275 280 285Ile Val Thr Leu Val
Tyr Val Leu Thr Asn Leu Ala Tyr Phe Thr Thr 290 295 300Leu Ser Thr
Asn Gln Met Leu Thr Ser Glu Ala Val Ala Val Asp Phe305 310 315
320Gly Asn Tyr His Leu Gly Val Met Ser Trp Ile Ile Pro Val Phe Val
325 330 335Gly Leu Ser Cys Phe Gly Ser Val Asn Gly Ser Leu Phe Thr
Ser Ser 340 345 350Arg Leu Phe Phe Val Gly Ser Arg Glu Gly His Leu
Pro Ser Ile Leu 355 360 365Ser Met Ile His Pro Gln Leu Leu Thr Pro
Val Pro Ser Leu Val Phe 370 375 380Thr Cys Val Met Thr Leu Met Tyr
Ala Phe Ser Arg Asp Ile Phe Ser385 390 395 400Ile Ile Asn Phe Phe
Ser Phe Phe Asn Trp Leu Cys Val Ala Leu Ala 405 410 415Ile Ile Gly
Met Met Trp Leu Arg Phe Lys Lys Pro Glu Leu Glu Arg 420 425 430Pro
Ile Lys Val Asn Leu Ala Leu Pro Val Phe Phe Ile Leu Ala Cys 435 440
445Leu Phe Leu Ile Ala Val Ser Phe Trp Lys Thr Pro Leu Glu Cys Gly
450 455 460Ile Gly Phe Ala Ile Ile Leu Ser Gly Leu Pro Val Tyr Phe
Phe Gly465 470 475 480Val Trp Trp Lys Asn Lys Pro Lys Trp Ile Leu
Gln Val Ile Phe Ser 485 490 495Val Thr Val Leu Cys Gln Lys Leu Met
Gln Val Val Pro Gln Glu Thr 500 505 51016512PRTRattus sp. 16Met Val
Ala Ser Thr Lys Tyr Glu Val Ala Ala Gln Asn Glu Ala Asp1 5 10 15Glu
Ala Asp Gly Ser Ala Gln Gly Asp Gly Ala Gly Pro Ala Ala Glu 20 25
30Gln Val Lys Leu Lys Lys Glu Ile Ser Leu Leu Asn Gly Val Cys Leu
35 40 45Ile Val Gly Asn Met Ile Gly Ser Gly Ile Phe Val Ser Pro Lys
Gly 50 55 60Val Leu Met Tyr Ser Ala Ser Phe Gly Leu Ser Leu Val Ile
Trp Ala65 70 75 80Val Gly Gly Ile Phe Ser Val Phe Gly Ala Leu Cys
Tyr Ala Glu Leu 85 90 95Gly Thr Thr Ile Lys Lys Ser Gly Ala Ser Tyr
Ala Tyr Ile Leu Glu 100 105 110Ala Phe Gly Gly Phe Leu Ala Phe Ile
Arg Leu Trp Thr Ser Leu Leu 115 120 125Ile Ile Glu Pro Thr Ser Gln
Ala Val Ile Ala Ile Thr Phe Ala Asn 130 135 140Tyr Met Val Gln Pro
Leu Phe Pro Ser Cys Gly Ala Pro Tyr Ala Ala145 150 155 160Gly Arg
Leu Leu Ala Ala Ala Cys Ile Cys Leu Leu Thr Phe Ile Asn 165 170
175Cys Ala Tyr Val Lys Trp Gly Thr Leu Val Gln Asp Ile Phe Thr Tyr
180 185 190Ala Lys Val Leu Ala Leu Ile Ala Val Ile Ile Ala Gly Ile
Val Arg 195 200 205Leu Gly Gln Gly Ala Thr Thr Asn Phe Glu Asp Ser
Phe Glu Gly Ser 210 215 220Ser Phe Ala Met Gly Asp Ile Ala Leu Ala
Leu Tyr Ser Ala Leu Phe225 230 235 240Ser Tyr Ser Gly Trp Asp Thr
Leu Asn Tyr Val Thr Glu Glu Ile Arg 245 250 255Asn Pro Glu Arg Asn
Leu Pro Leu Ser Ile Gly Ile Ser Met Pro Ile 260 265 270Val Thr Ile
Ile Tyr Leu Leu Thr Asn Val Ala Tyr Tyr Ser Val Leu 275 280 285Asp
Ile Lys Asp Ile Leu Ala Ser Asp Ala Val Ala Val Thr Phe Ala 290 295
300Asp Gln Ile Phe Gly Ile Phe Asn Trp Thr Ile Pro Leu Ala Val
Ala305 310 315 320Leu Ser Cys Phe Gly Gly Leu Asn Ala Ser Ile Val
Ala Ala Ser Arg 325 330 335Leu Leu Phe Val Gly Ser Arg Glu Gly His
Leu Pro Asp Ala Ile Cys 340 345 350Met Ile His Val Glu Arg Phe Thr
Pro Val Pro Ser Leu Leu Phe Asn 355 360 365Gly Ile Leu Ala Leu Val
Tyr Leu Cys Val Glu Asp Ile Phe Gln Leu 370 375 380Ile Asn Tyr Tyr
Ser Phe Ser Tyr Trp Phe Phe Val Gly Leu Ser Ile385 390 395 400Val
Gly Gln Leu Tyr Leu Arg Trp Lys Glu Pro Asp Arg Pro Arg Pro 405 410
415Leu Lys Leu Ser Leu Phe Phe Pro Ile Val Phe Cys Leu Cys Thr Ile
420 425 430Phe Leu Val Ala Val Pro Leu Tyr Ser Asp Thr Ile Asn Ser
Leu Ile 435 440 445Gly Ile Gly Ile Ala Leu Ser Gly Leu Pro Phe Tyr
Phe Leu Ile Ile 450 455 460Arg Val Pro Glu His Lys Arg Pro Leu Cys
Leu Arg Arg Ile Val Ala465 470 475 480Ser Thr Thr Arg Tyr Leu Gln
Ile Ile Cys Met Ser Val Ala Ala Glu 485 490 495Met Asp Leu Glu Asp
Gly Glu Leu Pro Lys Gln Gly Pro Lys Ser Lys 500 505
51017502PRTRattus sp. 17Met Val Arg Lys Pro Val Val Ala Thr Ile Ser
Lys Gly Gly Tyr Leu1 5 10 15Gln Gly Asn Met Ser Gly Arg Leu Pro Ser
Met Gly Asp Gln Glu Pro 20 25 30Pro Gly Gln Glu Lys Val Val Leu Lys
Lys Lys Ile Thr Leu Leu Arg 35 40 45Gly Val Ser Ile Ile Ile Gly Thr
Val Ile Gly Ser Gly Ile Phe Ile 50 55 60Ser Pro Lys Gly Ile Leu Gln
Asn Thr Gly Ser Val Gly Met Ser Leu65 70 75 80Val Phe Trp Ser Ala
Cys Gly Val Leu Ser Leu Phe Gly Ala Leu Ser 85 90 95Tyr Ala Glu Leu
Gly Thr Ser Ile Lys Lys Ser Gly Gly His Tyr Thr 100 105 110Tyr Ile
Leu Glu Val Phe Gly Pro Leu Leu Ala Phe Val Arg Val Trp 115 120
125Val Glu Leu Leu Val Ile Arg Pro Gly Ala Thr Ala Val Ile Ser Leu
130 135 140Ala Phe Gly Arg Tyr Ile Leu Glu Pro Phe Phe Ile Gln Cys
Glu Ile145 150 155 160Pro Glu Leu Ala Ile Lys Leu Val Thr Ala Val
Gly Ile Thr Val Val 165 170 175Met Val Leu Asn Ser Thr Ser Val Ser
Trp Ser Ala Arg Ile Gln Ile 180 185 190Phe Leu Ile Phe Cys Lys Leu
Thr Ala Ile Leu Ile Ile Ile Val Pro 195 200 205Gly Val Ile Gln Leu
Ile Lys Gly Gln Thr His His Phe Lys Asp Ala 210 215 220Phe Ser Gly
Arg Asp Thr Ser Leu Met Gly Leu Pro Leu Ala Phe Tyr225 230 235
240Tyr Gly Met Tyr Ala Tyr Ala Gly Trp Phe Tyr Leu Asn Phe Ile Thr
245 250 255Glu Glu Val Asp Asn Pro Glu Lys Thr Ile Pro Leu Ala Ile
Cys Ile 260 265 270Ser Met Ala Ile Ile Thr Val Gly Tyr Val Leu Thr
Asn Val Ala Tyr 275 280 285Phe Ile Thr Ile Ser Ala Glu Glu Leu Leu
Gln Ser Ser Ala Val Ala 290 295 300Val Ile Phe Ser Glu Arg Leu Leu
Gly Lys Phe Ser Leu Ala Val Pro305 310 315 320Ile Phe Val Ala Leu
Ser Cys Phe Gly Ser Met Asn Gly Gly Val Phe 325 330 335Ala Val Ser
Arg Leu Phe Tyr Val Ala Ser Arg Glu Gly His Leu Pro 340 345 350Glu
Ile Leu Ser Met Ile His Val His Lys His Thr Pro Leu Pro Ala 355 360
365Val Ile Val Leu His Pro Leu Thr Met Val Met Leu Phe Ser Gly Asp
370 375 380Leu Tyr Ser Leu Leu Asn Phe Leu Ser Phe Ala Arg Trp Leu
Phe Met385 390 395 400Gly Leu Ala Val Ala Gly Leu Ile Tyr Leu Arg
Tyr Lys Arg Pro Asp 405 410 415Met His Arg Pro Phe Lys Val Pro Leu
Phe Ile Pro Ala Leu Phe Ser 420 425 430Phe Thr Cys Leu Phe Met Val
Val Leu Ser Leu Tyr Ser Asp Pro Phe 435 440 445Ser Thr Gly Val Gly
Phe Leu Ile Thr Leu Thr Gly Val Pro Ala Tyr 450 455 460Tyr Leu Phe
Ile Val Trp Asp Lys Lys Pro Lys Trp Phe Arg Arg Leu465 470 475
480Ser Asp Arg Ile Thr Arg Thr Leu Gln Ile Ile Leu Glu Val Val Pro
485 490 495Glu Asp Ser Lys Glu Leu 50018487PRTRattus sp. 18Met Glu
Glu Thr Ser Pro Arg Arg Arg Arg Glu Asp Glu Lys Ser Val1 5 10 15His
Ser Thr Glu Pro Lys Thr Thr Ser Leu Gln Lys Glu Val Gly Leu 20 25
30Leu Ser Gly Ile Cys Ile Ile Val Gly Thr Ile Ile Gly Ser Gly Ile
35 40 45Phe Ile Ser Pro Lys Ser Val Leu Ala Asn Thr Glu Ser Val Gly
Pro 50 55 60Cys Leu Ile Ile Trp Ala Ala Cys Gly Val Leu Ala Thr Leu
Gly Ala65 70 75 80Leu Cys Phe Ala Glu Leu Gly Thr Met Ile Thr Lys
Ser Gly Gly Glu 85 90 95Tyr Pro Tyr Leu Met Glu Ala Phe Gly Pro Ile
Pro Ala Tyr Leu Phe 100 105 110Ser Trp Thr Ser Leu Ile Val Met Lys
Pro Ser Ser Phe Ala Ile Ile 115 120 125Cys Leu Ser Phe Ser Glu Tyr
Tyr Cys Ala Ala Phe Tyr Leu Gly Cys 130 135 140Arg Pro Pro Ala Val
Val Val Lys Leu Leu Ala Ala Ala Ala Ile Leu145 150 155 160Leu Ile
Thr Thr Val Asn Ala Leu Ser Val Arg Leu Gly Ser Tyr Val 165 170
175Gln Asn Val Phe Thr Ala Ala Lys Leu Val Ile Val Ala Ile Ile Ile
180 185 190Ile Ser Gly Leu Val Leu Leu Ala Gln Gly Asn Val Lys Asn
Phe Gln 195 200 205Asn Ser Phe Glu Gly Ser Gln Thr Ser Val Gly Ser
Ile Ser Leu Ala 210 215 220Phe Tyr Asn Gly Leu Trp Ala Tyr Asp Gly
Trp Asn Gln Leu Asn Tyr225 230 235 240Ile Thr Glu Glu Leu Arg Asn
Pro Tyr Arg Asn Leu Pro Met Ala Ile 245 250 255Val Ile Gly Ile Pro
Leu Val Thr Val Cys Tyr Ile Leu Met Asn Ile 260 265 270Ala Tyr Phe
Thr Val Met Thr Pro Thr Glu Leu Leu Gln Ser Gln Ala 275 280 285Val
Ala Val Thr Phe Gly Asp Arg Val Leu Tyr Pro Ala Ser Trp Val 290 295
300Val Pro Leu Phe Val Ala Phe Ser Thr Ile Gly Ala Ala Asn Gly
Thr305 310 315 320Cys Phe Thr Ala Gly Arg Leu Ile Tyr Val Ala Gly
Arg Glu Gly His 325 330 335Met Leu Lys Val Leu Ser Tyr Ile Ser Val
Lys Arg Leu Thr Pro Ala 340 345 350Pro Ala Leu Val Phe Tyr Gly Ile
Ile Ala Ile Ile Tyr Ile Ile Pro 355 360 365Gly Asp Ile Asn Ser Leu
Val Asn Tyr Phe Ser Phe Ala Ala Trp Leu 370 375 380Phe Tyr Gly Met
Thr Ile Leu Gly Leu Val Val Met Arg Phe Thr Arg385 390 395 400Lys
Asp Leu Glu Arg Pro Ile Lys Val Pro Ile Phe Ile Pro Ile Ile 405 410
415Val Ile Leu Val Ser Val Phe Leu Ile Leu Ala Pro Ile Ile Ser Ser
420 425 430Pro Ala Trp Glu Tyr Leu Tyr Cys Val Leu Phe Ile Leu Ser
Gly Leu 435 440 445Ile Phe Tyr Phe Leu Phe Val His Tyr Lys Phe Arg
Trp Ala Gln Lys 450 455 460Ile Ser Arg Pro Ile Thr Lys His Leu Gln
Met Leu Met Glu Val Val465 470 475 480Pro Pro Glu Lys Asp Pro Glu
4851927DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 19gaa gaa agt gcg gcc gca agc cag gac
27Glu Glu Ser Ala Ala Ala Ser Gln Asp1 5209PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Glu
Glu Ser Ala Ala Ala Ser Gln Asp1 52127DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 21gaa gaa agt gcg gcc gca gat gag gac 27Glu Glu Ser
Ala Ala Ala Asp Glu Asp1 5229PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 22Glu Glu Ser Ala Ala Ala Asp
Glu Asp1 5
* * * * *