Patent application title: Functional domain and associated molecule of dock2 essentially required in lymphocyte migration
Inventors:
Yoshinori Fukui (Fukuoka-Shi, JP)
Takehiko Sasazuki (Tokyo, JP)
IPC8 Class: AG01N3353FI
USPC Class:
435 71
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving antigen-antibody binding, specific binding protein assay or specific ligand-receptor binding assay
Publication date: 2009-09-17
Patent application number: 20090233314
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Patent application title: Functional domain and associated molecule of dock2 essentially required in lymphocyte migration
Inventors:
Yoshinori Fukui
Takehiko Sasazuki
Agents:
FOLEY AND LARDNER LLP;SUITE 500
Assignees:
Origin: WASHINGTON, DC US
IPC8 Class: AG01N3353FI
USPC Class:
435 71
Abstract:
The present invention is related to provide a method for screening a
substance interfering in the association of DOCK2 and ELMO1, a method for
screening a substance interfering in the association of ELMO1 and Tiam1,
and a method for searching a therapeutic agent for immune related
diseases such as allergy, autoimmune diseases, GvH, graft rejection with
the use of these searching methods, and so on. It was found that in
DOCK2-mutant lacking 504 amino acid residues at the N terminus of DOCK2,
Rac-activating ability was significantly decreased, and that actin
polymerization could not be induced, and ELMO1 was identified as a
molecule binding to this domain. It was found that DOCK2 was associated
to ELMO1 via SH3 domain. Moreover, it was found that ELMO1 is bound with
Tiam1 functioning as Rac-specific GDP/GTP exchange factor (GEF). It was
found that DOCK2 activates Rac by recruiting Tiam1 via ELMO1.Claims:
1-12. (canceled)
13. A method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor, comprising the steps of contacting ELMO, GDP/GTP exchange factor and a test substance, and then estimating the level of formation of association of ELMO and GDP/GTP exchange factor.
14. A method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor, comprising the steps of contacting N terminus domain of ELMO, GDP/GTP exchange factor and a test substance, and then estimating the level of formation of association of N terminus domain of ELMO and GDP/GTP exchange factor.
15. The method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 13, wherein ELMO or its N terminus domain and/or GDP/GTP exchange factor is fused with another peptide.
16. The method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 13, wherein an antibody against ELMO or its N terminus domain is acted to a GDP/GTP exchange factor fractionated by an antibody against GDP/GTP exchange factor or by an antibody against another peptide fused with GDP/GTP exchange factor, and the level of formation of association is estimated.
17. The method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 13, wherein the level of formation of association is estimated by detecting GTP-binding form of activated-Rac.
18. The method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 13, wherein the substance interfering in the association of ELMO and GDP/GTP exchange factor is a substance promoting or suppressing the function of regulating lymphocyte migration.
19. The method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 13, wherein the substance interfering in the association of ELMO and GDP/GTP exchange factor is a substance inhibiting the binding of ELMO and GDP/GTP exchange factor.
20. The method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 13, wherein ELMO is an ELMO bound with DOCK2.
21. The method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 13, wherein ELMO is ELMO1.
22. The method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 13, wherein the GDP/GTP exchange factor is a Rac-specific GDP/GTP exchange factor.
23. The method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 22, wherein the Rac-specific GDP/GTP exchange factor is Tiam1.
24. A method for searching a therapeutic agent for immune related diseases such as allergy, autoimmune diseases, GvH, and graft rejection, wherein the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 13 is used.
25. A method for searching a therapeutic agent for diseases caused by the suppression of lymphocyte migration, which promotes cytoskeletal reorganization by activating Rac, wherein the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to claim 13 is used.
26-39. (canceled)
Description:
TECHNICAL FIELD
[0001]The present invention relates to the identification of DOCK2 domain by using a deletion mutant, and a method for screening a substance interfering in the binding of DOCK 2 and SH3 domain of DOCK 2, particularly to a method for screening a substance interfering in the association of DOCK2 and ELMO, a method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor such as Tiam, or to a method for searching therapeutic agents for immune-related diseases, such as allergy, autoimmune diseases, GvH or graft rejection, with the use of these screening methods.
BACKGROUND ART
[0002]Immune response is a regulatory mechanism indispensable against infection for a living body, and immune cells are patrolling constantly in the living body, to respond rapidly to various sources of infection. Such characteristics that constitutive cells are moving continuously are not recognized in other complex living systems, and have been developed specifically in the immune system. Among the immune cells, cells such as neutrophils, macrophages are known to function during primary defense of infection, while T- and B-lymphocytes trigger antigen-specific immune response by recognizing external foreign substances via the antigen receptor. The above T- and B-lymphocytes differentiate in primary lymphoid organs such as thymus and bone marrow, and transfer to a particular compartment in second lymphoid organs such as spleen, lymph nodes, Payer's patch (lymphoid organs in the small intestine), and by recognizing antigens gathered there from various organs via the antigen receptor, induce specific immune response. At that time, the transfer of lymphocytes to a particular site of second lymphoid organ is very important for the formation of immune response. Heretofore, the transfer of the lymphocytes was known to be induced by protein called generally various chemokines, while the molecule mechanism that controls the mobility of the lymphocytes themselves remained unknown.
[0003]Change of cell polarization and cytoskeletal reorganization were indispensable for the cells movement (Cell 84, 359-369, 1996), and these were known to be controlled by G protein of low molecular weight such as Rho, Rac and Cdc42 (Proc. Natl. Acad. Sci. USA 92, 5027-5031, 1995; Science 279, 509-514, 1998; J. Cell Biol. 141, 1147-1157, 1998; Science 287, 1037-1040, 2000). Among these, Rac particularly provides driving force at the time of cell migration, by forming an actin-rich protrusion, called foliar protrusion (Science 279, 509-514, 1998; Cell 103, 227-238, 2000). On the other hand, molecules showing structural homology called CED5, DOCK 180 and Myoblast city (MBC) were identified in Caenorhabditis elegans, human and Drosophila melanogaster. These molecules are called CDM family molecules by their initials, and all of them are thought to be related to cytoskeletal reorganization by functioning upstream of Rac (Cell 84, 359-369, 1996; J. Cell Biol. 138, 589-603, 1997; Nature 392, 501-504, 1998; Genes Dev. 12, 3331-3336, 1998; Genes Dev. 12, 3337-3342, 1998; Nature Cell Biol. 2, 131-136, 2000). Although genetic analysis with the use of a deletion mutant has shown that the above CED-5 and Myoblast City are crucial for cell migration of particular types of cells, (J. Cell Biol. 138, 589-603, 1997; Nature 392, 501-504, 1998; Nature Cell Biol. 2, 131-136, 2000), physiological relevance of the CDM family proteins in mammals remained unknown.
[0004]It is known that DOCK2 (KIAA0209; DNA Res. 3, 321-329) encodes another member of the CDM family proteins, which is specifically expressed in human haematopoietic cells, and that the DOCK2 binds to activate Rac in 293T kidney cells (Biochem, Biophys. Acta 1452, 179-187, 1999). On the other hand, the present inventors isolated a new gene Hch belonging to the CDM family from mouse thymus cDNA library, and found that the gene product comprises 1828 amino acids, and encodes SH3 domain at the N terminus (Nature, 412, 826-831, 2001). Moreover, the present inventors confirmed by Northern Blot analysis using mouse organs that whereas DOCK180 was expressed in various organs, the expression of Hch was restricted to thymus and spleen. Further, by an analysis using cell lines they confirmed that Hch expression was observed in all T-, B- and macrophage cells, with the exception of two mutant T-cell lines. Furthermore, it has been revealed that a significant change in cell morphology and enhancement of adhesion were observed by introducing Hch into mutant T-cell line lacking Hch expression. Though 1677 of the 1828 amino acids encoded by Hch are identical to human DOCK2, and Hch was thought to be mouse DOCK2 homologue, the physiological function remained unknown.
[0005]The present inventors identified DOCK2 as a molecule belonging to the CDM family, expressing specifically in lymphocytes as mentioned above, and by generating the knockout mice, they revealed that the molecule was indispensable to lymphocyte migration (Nature, 412, 826-831, 2001). In DOCK2-deleted lymphocytes, active Rac is not detected by any of chemokine stimulation. Therefore, it can be thought that DOCK2 regulates lymphocyte migration via Rac activation. However, it remains unknown by which mechanism DOCK2 activates Rac. Rac functions as a molecule switch, and is activated by a GDP/GTP exchange factor (GEF). Though DOCK2 binds with Rac, it is hard from its structure, to think that it functions as GEF. Therefore, it is estimated that DOCK2 activates Rac by recruiting GEF via other molecules.
[0006]Recently, CED-12 being a molecule that associates with CED-5, which is one of the CDM family molecules, and that regulates cytoskeleton has been identified in C. elegans, and ELMO-1, -2 and -3 were reported as their mammalian homologues (Cell, 107, 27-41, 2001). Moreover, several dozens of GDP/GTP exchange factors (GEF) were known heretofore, and among these GEFs, as a molecule functioning as Rac-specific GEF, the following are known: Tiam-1 and -2 that determines the invasion to thymoma cell lines (Cell, 77, 537-549, 1994; Nature, 375, 338-340, 1995); Vav1 that regulates T cell receptor signal (Nature, 385, 169-172, 1997) besides Vav2, Vav3; Trio (J. Cell Science, 113, 729-739, 2000); STEF (J. Biol. Chem., 277, 2860-2868, 2002); and P-Rex1 (Cell, 108, 809-821, 2002). All these five molecules have a common domain, and comprise a function to provide GTP to Rac.
[0007]Autoimmune diseases and graft rejection are caused by the invasion of lymphocytes into the target organ. Therefore, it is thought that DOCK2 might be a suitable target molecule to treat or prevent such diseases or pathology. The object of the present invention is to identify the functional domain of DOCK2 by using a deletion mutant, to screen a substance interfering in the binding of DOCK2 and SH3 domain of DOCK2, particularly to provide a method for screening a substance interfering in the association of DOCK2 and ELMO, a method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor such as Tiam, or a method for searching therapeutic agents for immune-related diseases, such as allergy, autoimmune diseases, GvH or graft rejection with the use of these screening methods, and the like.
[0008]DOCK2 is a molecule expressed specifically in lymphocytes, comprised of 1828 amino acid residues including SH3 domain, that activates Rac and regulates cytoskeleton to determine lymphocyte mobility. The present inventors have made a keen study to solve the above object, found that Rac-activating ability was significantly decreased in DOCK2 mutant lacking 504 amino acid residues in the N terminus including SH3 domain of DOCK 2, and that actin polymerization could not be induced, and they identified ELMO1 as a molecule binding to this domain. Moreover, as the binding of DOCK2 and ELMO1 was completely inhibited by the single amino acid mutation of SH3 domain, they have found that DOCK2 associates with ELMO1 via SH3 domain. Furthermore, they have found that ELMO1 binds with Tiam1 functioning as Rac-specific GDP/GTP exchange factor (GEF). In other words, they have found that DOCK2 activates Rac by recruiting Tiam1 via ELMO1. Therefore, they found that by inhibiting intermolecular interaction of SH3 domain of DOCK2, ELMO1 and Tiam1, the artificial control of lymphocyte migration was possible. The present invention has been thus completed with this knowledge.
DISCLOSURE OF THE INVENTION
[0009]In other words, the present invention relates to a method for screening a substance interfering in the association of DOCK2 and ELMO, comprising the steps of contacting DOCK2, ELMO and a test substance, and then estimating the level of formation of association of DOCK2 and ELMO ("1"); a method for screening a substance interfering in the association of DOCK2 and ELMO, comprising the steps of contacting SH3 domain of DOCK2, ELMO and a test substance, and then estimating the level of formation of association of SH3 domain of DOCK2 and ELMO ("2"); a method for screening a substance interfering in the association of DOCK2 and C terminus domain of ELMO, comprising the steps of contacting DOCK2, C terminus domain of ELMO and a test substance, and then estimating the level of formation of association of DOCK2 and C terminus domain of ELMO ("3"); a method for screening a substance interfering in the association of DOCK2 and ELMO, comprising the steps of contacting SH3 domain of DOCK2, C terminus domain of ELMO and a test substance, and then estimating the level of formation of association of SH3 domain of DOCK2 and C terminus domain of ELMO ("4"); the method for screening a substance interfering in the association of DOCK2 and ELMO according to any one of "1" to "4", wherein DOCK2 or its SH3 domain and/or ELMO or its C-terminus domain is bound with a marker protein and/or peptide tag ("5"); the method for screening a substance interfering in the association of DOCK2 and ELMO according to anyone of "1" to "5", wherein an antibody against ELMO or its C terminus domain is acted to DOCK2 or its SH3 domain fractionated by an antibody against DOCK2 or its SH3 domain, or an antibody against other peptides fused with DOCK2 or its SH3 domain, and the level of formation of association is estimated ("6"); the method for screening a substance interfering in the association of DOCK2 and ELMO according to any one of "1" to "6", wherein the level of formation of association is estimated by detecting GTP-binding form of activated-Rac ("7"); the method for screening a substance interfering in the association of DOCK2 and ELMO according to any one of "1" to "7", wherein the substance interfering in the association of DOCK2 and ELMO is a substance promoting or suppressing the function of regulating lymphocyte migration ("8"); the method for screening a substance interfering in the association of DOCK2 and ELMO according to any one of "1" to "7", wherein the substance interfering in the association of DOCK2 and ELMO is a substance inhibiting the binding of DOCK2 and ELMO ("9"); the method for screening a substance interfering in the association of DOCK2 and ELMO according to any one of "1" to "9", wherein ELMO is ELMO1 ("10"); a method for searching a therapeutic agent for immune related diseases such as allergy, autoimmune diseases, GvH, and graft rejection wherein the method for screening a substance interfering in the association of DOCK2 and ELMO according to any one of "1" to "10" is used ("11"); and a method for searching a therapeutic agent for diseases caused by the suppression of lymphocyte migration, which promotes cytoskeletal reorganization by activating Rac, wherein the method for screening a substance interfering in the association of DOCK2 and ELMO according to any one of "1" to "10" is used ("12").
[0010]Moreover, the present invention is related to a method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor, comprising the steps of contacting ELMO, GDP/GTP exchange factor and a test substance, and then estimating the level of formation of association of ELMO and GDP/GTP exchange factor ("13"); a method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor, comprising the steps of contacting N terminus domain of ELMO, GDP/GTP exchange factor and a test substance, and then estimating the level of formation of association of N terminus domain of ELMO and GDP/GTP exchange factor ("14"); the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to "13" or "14", wherein ELMO or its N terminus domain and/or GDP/GTP exchange factor is fused with another peptide ("15"); the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to any one of "13" to "15", wherein an antibody against ELMO or its N terminus domain is acted to a GDP/GTP exchange factor fractionated by an antibody against GDP/GTP exchange factor or by an antibody against another peptide fused with GDP/GTP exchange factor, and the level of formation of association is estimated ("16"); the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to any one of "13" to "16", wherein the level of formation of association is estimated by detecting GTP-binding form of activated-Rac ("17"); the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to anyone of "13" to "17", wherein the substance interfering in the association of ELMO and GDP/GTP exchange factor is a substance promoting or suppressing the function of regulating lymphocyte migration ("18"); the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to any one of "13" to "17", wherein the substance interfering in the association of ELMO and GDP/GTP exchange factor is a substance inhibiting the binding of ELMO and GDP/GTP exchange factor ("19"); the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to any one of "13" to "19", wherein ELMO is an ELMO bound with DOCK2 ("20"); the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to any one of "13" to "20", wherein ELMO is ELMO1 ("21"); the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to any one of "13" to "21", wherein the GDP/GTP exchange factor is a Rac-specific GDP/GTP exchange factor ("22"); the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to "22", wherein the Rac-specific GDP/GTP exchange factor is Tiam1 ("23"); a method for searching a therapeutic agent for immune related diseases such as allergy, autoimmune diseases, GvH, and graft rejection, wherein the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to any one of "13" to "23" is used ("24"); and a method for searching a therapeutic agent for diseases caused by the suppression of lymphocyte migration, which promotes cytoskeletal reorganization by activating Rac, wherein the method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor according to any one of "13" to "23" is used ("25").
[0011]Furthermore, the present invention relates to a method for screening a substance for promoting or suppressing Rac activation, comprising the steps of contacting DOCK2, ELMO, GDP/GTP exchange factor and a test substance, and then estimating the level of formation of association of DOCK2 and ELMO, or the level of formation of association of ELMO and GDP/GTP exchange factor ("26"); a method for screening a substance for promoting or suppressing Rac activation, comprising the steps of contacting SH3 domain of DOCK2, ELMO, GDP/GTP exchange factor and a test substance and then estimating the level of formation of association of SH3 domain of DOCK2 and ELMO, or the level of formation of association of ELMO and GDP/GTP exchange factor ("27"); the method for screening a substance for promoting or suppressing Rac activation according to "26" or "27", wherein the level of formation of association is estimated by detecting GTP-binding form of activated-Rac ("28"); the method for screening a substance for promoting or suppressing Rac activation according to any one of "26" to "28", wherein ELMO is an ELMO bound with DOCK2 ("29"); the method for screening a substance for promoting or suppressing Rac activation according to any one of "26" to "29", wherein ELMO is ELMO1 ("30"); the method for screening a substance for promoting or suppressing Rac activation according to any one of "26" to "30", wherein the GDP/GTP exchange factor is a Rac-specific GDP/GTP exchange factor ("31"); the method for screening a substance for promoting or suppressing Rac activation according to "31", wherein the Rac-specific GDP/GTP exchange factor is Tiam1 ("32"); a method for searching a substance for promoting or suppressing the function of regulating lymphocyte migration, wherein the method for screening a substance promoting or suppressing Rac activation according to any one of "26" to "32" is used ("33"); a method for searching a therapeutic agent for immune related diseases such as allergy, autoimmune diseases, GvH, and graft rejection, wherein the method for screening a substance for promoting or suppressing Rac activation according to any one of "26" to "32" is used ("34"); and a method for searching a therapeutic agent for diseases caused by the suppression of lymphocyte migration, which promotes reconstruction of cytoskeleton by activating Rac, wherein the method for screening a substance for promoting or suppressing Rac activation according to any one of "26" to "32" is used ("35"); a therapeutic agent for immune related diseases such as allergy, autoimmune diseases, GvH and graft rejection, obtained by the searching method according to "11", "24" or "34" ("36"); a therapeutic agent for diseases caused by the suppression of lymphocyte migration, promoting cytoskeletal reorganization by activating Rac, obtained by the searching method according to "12", "25" or "35" ("37"); a method for screening a substance inhibiting DOCK2-function, by targeting N terminus domain of DOCK2 including SH3 domain, comprising the steps of contacting SH3 domain of DOCK2, the SH3 domain-binding protein and a test substance, and then estimating the level of formation of association of DOCK2 and SH3 domain-binding protein ("38"); and a method for screening a substance inhibiting DOCK2-function, by using a transgenic cell line expressing full-length DOCK2 and DOCK2-deleted mutants, comprising the steps of measuring and estimating the level of Rac activation in these cell lines, identifying the functional domain of DOCK2, searching a molecule associated with functional domain that associates with the functional domain, contacting the functional domain of DOCK2, the molecule associated with functional domain and a test substance, and estimating the level of formation of association of functional domain of DOCK2 and molecule associated with functional domain of DOCK2 ("39").
BRIEF DESCRIPTION OF DRAWINGS
[0012]FIG. 1 is a figure showing that DOCK2 binds with ELMO1 at its N terminus domain.
A is a view showing a frame format of the structure of DOCK2 and DOCK2-deleted mutants. In the figure, the black-colored part is the SH3 domain.B is a figure showing the analysis of the binding with ELMO1 by immunoprecipitation and Western Blot method, by transfecting genes encoding DOCK2 or DOCK2-deleted mutants to 293T cells with PcDNA ELMO1-V5 and by collecting the cells 48 hours later. Types of samples used for analysis, antibodies used for immunoprecipitation and Western Blot are shown on the left side.
[0013]FIG. 2 is a set of pictures showing that the Rac-activating ability is significantly decreased and that actin polymerization cannot be induced in DOCK2ΔN lacking N terminus domain essential for the binding with ELMO1.
A is a picture showing the analysis of the expression of DOCK2 or DOCK2ΔN in BEα16-3, N3-5, and transgenic cell lines (17-11, 84-3) by Western Blot with the use of polyclonal antibody against DOCK2. In the figures, NS means non-specific band. B is a picture that activated Rac is detected by pulling-down cell extract of 84-3, 17-11, BEα16-3 with GST fusion protein of PAK1 Rac-binding domain, and by staining with anti-Rac antibody.C is a picture showing the investigation of cell polarization and actin polymerization by staining BEα16-3, 17-11, 84-3 with propidium iodide and phalloidin.
[0014]FIG. 3 is a picture showing that DOCK 2 associates with ELMO1 via its SH3 domain.
A is a figure showing the amino acid sequence 10-89 including DOCK2 SH3 domain. Amino acid residues substituted to glutamic acid are shown in bold letter.B is a figure showing the analysis of the binding of DOCK2 with ELMO1 by immunoprecipitation and Western Blot method, by transfecting genes encoding DOCK2 or DOCK2 SH3-deleted mutants to 293T cells with PcDNA ELMO1-V5 and by collecting the cells 48 hours later. Types of samples used for analysis, antibodies used for immunoprecipitation and Western Blot are shown on the left side.
[0015]FIG. 4 is a figure showing that ELMO1 is bound with DOCK2 at its C terminus domain.
A is a view showing a frame format of the structure of ELMO1 and of ELMO1-deleted mutants used in this experiment.B is a figure showing the analysis of the binding of ELMO1 with DOCK2 by immunoprecipitation and Western Blot method, by transfecting genes encoding ELMO1 or ELMO1-deleted mutants to 293T cells with PcDNA DOCK2-HA or a control vector and by collecting the cells 48 hours later. Types of samples used for analysis, antibodies used for immunoprecipitation and Western Blot are shown on the left side.
[0016]FIG. 5 is a figure showing that ELMO1 is bound to Tiam1 at its N terminus domain.
A is a view showing a frame format of the structure of ELMO1 and of ELMO1-deleted mutants used in this experiment. B is a figure showing the analysis of the binding with Tiam1 by immunoprecipitation and Western Blot method, by transfecting genes encoding ELMO1 or ELMO1-deleted mutants to 293T cells with PCI Tiam1-HA or a control vector and by collecting the cells 48 hours later. Types of samples used for analysis, antibodies used for immunoprecipitation and Western Blot are shown on the left side.
[0017]FIG. 6 is a schematical view of the Rac-activating mechanism by DOCK2.
[0018]It is a figure showing that DOCK2 activated Rac via ELMO1 by recruiting Tiam1 functioning as GEF of Rac.
BEST MODE OF CARRYING OUT THE INVENTION
[0019]As for the method for screening a substance interfering in the association of DOCK2 and ELMO of the present invention, there is no specific limitation as long as it is a method comprising the steps of contacting DOCK2, ELMO and a test substance, and then estimating the level of formation of the association of DOCK2 and ELMO; a method comprising the steps of contacting SH3 domain DOCK2, ELMO and a test substance, and then estimating the level of formation of the association of SH3 domain of DOCK2 and ELMO; a method comprising the steps of contacting DOCK2, C terminus domain of ELMO and a test substance, and then estimating the level of formation of the association of DOCK2, C terminus domain of ELMO; a method comprising the steps of contacting SH3 domain of DOCK2, C terminus domain of ELMO, and a test substance, and then estimating the level of formation of the association of SH3 domain of DOCK2, C terminus of ELMO. Moreover, as for the above-mentioned DOCK2 or its SH3 domain and/or ELMO or its C terminus domain, a fusion protein or a fusion peptide wherein these and marker protein and/or peptide tag are bound can be used. Moreover, as for the above ELMO, ELMO1, ELMO2, ELMO3 can be specifically exemplified, and ELMO1 can be preferably exemplified.
[0020]As for the above SH3 domain of DOCK2, a DOCK2 mutant having a function to associate with ELMO, and that is a peptide containing a whole or a part of SH3 domain of DOCK2 can be exemplified, and specific examples include DOCK2N comprising amino acid residue 1-502 of DOCK2 and DOCK2ΔC comprising amino acid residue 1-1311 of DOCK2. Furthermore, as for the above C terminus domain of ELMO, a mutant of ELMO having the function to associate with SH3 domain of DOCK2, and that is a peptide containing a whole or a part of C terminus domain of ELMO can be exemplified, and specific examples include ELMO1-del1 comprising amino acid residue 147-727 of ELMO1, and ELMO1-del8 comprising amino acid residue 345-727 of ELMO1. Hereinafter, DOCK2 and the above SH3 domain of DOCK2 can be referred together to as "DOCK2 and the like", and ELMO such as ELMO1 and the above C terminus domain of ELMO can be referred together to as "ELMO and the like".
[0021]The above DOCK2 mutant or ELMO mutant can be prepared by modifying DOCK2 genes or ELMO genes according to a common procedure. As for DOCK2 genes, Hch (mouse DOCK2) genes (GenBank Accession No. AY027438; Nature, Vol 412, 23 August, 826-831, 2001) and human DOCK2 genes (XM--047961; DNA Res. 3, 321-329) can be specifically exemplified, but the origin of DOCK2 genes is not limited to mouse, human and the like. Moreover, as for ELMO genes such as ELMO1, besides mouse ELMO1 genes (AF398883; Cell, Vol. 107 (1), 27-41, 2001) and human ELMO1 genes (AF398885; Cell, Vol. 107(1) 27-41, 2001), ELMO2 genes (human AF398886, mouse AF398884), ELMO3 genes (human NM--024712) can be specifically exemplified. However, the origin of DOCK2 and ELMO genes is not limited to mouse, human and the like. Additionally, the amino acid sequence of mouse DOCK2, human DOCK2, mouse ELMO1, and human ELMO1 are shown as Seq. ID Nos. 1, 2, 3 and 4, respectively.
[0022]As for a marker protein in a fusion protein or fusion peptide wherein the above DOCK2 and the like or ELMO and the like are bound with a marker protein and/or peptide tag, there is no specific limitation as long it is a marker protein conventionally known, and alkaline phosphatase, Fc domain of an antibody, HRP, and GFP can be exemplified. Moreover, as for a peptide tag, examples include peptide tags conventionally known, including epitope tags such as HA, FLAG and Myc; affinity tag such as GST, maltose-binding protein, biotinylated peptide and oligo-histidine. The fusion protein or fusion peptide can be prepared by a common procedure, and can separate/fractionate fusion protein or fusion peptide with DOCK2 and the like, ELMO1 and the like and HA-tag, by using specific antibody against HA tag.
[0023]In the method for screening a substance interfering in the association of DOCK2 and ELMO such as ELMO1, as for a method for contacting DOCK2 and the like, ELMO and the like, and a test substance, there is no specific limitation as long as it is a contacting method that can evaluate the level of the formation of the association of DOCK2 and the like and ELMO and the like, and examples include a method for contacting DOCK2 and the like and ELMO and the like, in the presence of a test substance in a cell-free system; a method for introducing an expression vector integrated with ELMO and the like or genes encoding ELMO and the like, in a cell expressing DOCK2 and the like together with a test substance; a method for introducing an expression vector integrated with DOCK2 and the like or genes encoding DOCK2 and the like, in a cell expressing ELMO and the like together with a test substance; or a method for introducing an expression vector integrated with DOCK2 and the like or genes encoding DOCK2 and the like, an expression vector integrated with ELMO and the like or genes encoding ELMO and the like, and a test substance, in a cell not expressing DOCK2 and the like nor ELMO and the like.
[0024]As for cells used for contacting with the above test substance, bacterial prokaryotic cells such as E. Coli, streptomyces, Bacillus subtilis, Streptococcus and Staphylococcus; eukaryotic cells such as yeast and Aspergillus; insect cells such as Drosophila S2 and Spodoptera Sf9; plant and animal cells such as L cells, CHO cells, COS cells, HeLa cells, C127 cells and BALB/c3T3 cells (including mutant strain lacking dihydrofolate reductase or thymidine kinase), BHK21 cells, HEK293 cells, Bowes melanoma cells and oocytes can be exemplified, and animal cells are preferable. Moreover, as for the method for introducing DOCK2 and the like or ELMO and the like in these cells, besides the above methods for introducing genes, a noncytotoxcic reagent such as Chariot (Active Motif) that can form a non-covalent binding with an enormous molecule, change the structure of an enormous molecule such as protein, and that can deliver the enormous molecule such as protein into the cells, can be used.
[0025]As for the above expression vector, expression vector for animal cells are preferable, and examples of the expression vector for animal cells include: expression system derived from chromosome, episome, and virus; for example vectors derived from bacterial plasmid, yeast plasmid, papovavirus such as SV40, vaccinia virus, adenovirus, fowl poxvirus, pseudorabies virus, lentivirus, and retrovirus; vectors derived from bacteriophage, transposon, or from combination thereof, for example those derived from plasmid and bacteriophage elements, such as cosmids and phagemids. These expression systems can include regulatory sequences that not only induce expression but also regulate expression. Moreover, liposome can be used in place of expression vectors for animal cells. Further, the introduction of the expression vectors for animal cells into cells can be performed by a method described in various standard laboratory manuals such as Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and examples include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electropolation, transduction, scrape loading, ballistic introduction, and infection.
[0026]In the method for screening a substance interfering the association of DOCK2 and ELMO such as ELMO1 of the present invention, as for the method for estimating the level of formation of the association of DOCK2 and the like and ELMO and the like, a method for measuring/estimating immunochemically the level of formation of the association of DOCK2 and the like and ELMO and the like, by acting an antibody against ELMO and the like to DOCK2 and the like being separated/fractionated, or by acting an antibody against DOCK2 and the like to ELMO and the like being separated/fractionated, can be exemplified. To separate/fractionate DOCK2 and the like or ELMO and the like, specific antibodies against DOCK2 and the like or ELMO and the like or tag-specific antibodies can be used. Moreover, yeast two hybrid system that can detect protein-protein interaction by using a minute amount of protein and without labeling; or a biosensor using the surface plasmon resonance reaction that can observe at real time as a surface plasmon resonance signal; or a method for measuring/estimating the level of formation of the association by using NMR method that can detect the change of tertiary structure, can be also exemplified. Moreover, publicly known methods for searching interacting protein, such as far western method using E. coli expression system and a method using affinity chromatography can be exemplified.
[0027]In the method for screening a substance interfering in the association of DOCK2 and ELMO of the present invention, as another method to estimate the level of formation of the association of DOCK2 and the like and ELMO and the like, an estimation method by detecting a GTP-binding form of activated Rac can be exemplified. To detect activated Rac, a pull-down method using GST fusion protein of PAK1 Rac-binding domain can be used.
[0028]As for samples to be tested in the method for screening a substance interfering in the association of DOCK2 and ELMO of the present invention, for example, peptides, proteins, synthesized compounds, microbial fermented materials, marine organism extracts, plant extracts, prokaryotic cells extract, eukaryotic unicellular extract, animal cells extract or library thereof can be exemplified. Furthermore, in the method for screening a substance interfering in the association of DOCK2 and ELMO of the present invention, control experiment can be carried out simultaneously. As for control, negative control that does not affect the formation of association of DOCK2 and the like and ELMO and the like, and/or positive control that affect the formation of association of DOCK2 and the like and ELMO and the like can be used.
[0029]As for the above substances interfering in the association of DOCK2 and ELMO, substances promoting or suppressing the function of regulating lymphocyte migration, particularly a substance suppressing the function of regulating lymphocyte migration such as substances inhibiting the binding of DOCK2 and ELMO. As for the function of regulating lymphocyte migration, there is no specific limitation as long as it is a function regulating the mobility of lymphocytes based on the expression of DOCK2 genes. Examples include a function promoting cytoskeletal reorganization, in particular actin polymerization in lymphocytes by activating Rac and making a Rac-GTP binding; a function of migrating lymphocytes in response to stimulation of chemokines such as SLC, SDF-1, and BLC; homing function to a secondary lymphoid organ such as spleen, lymph nodes, payer's notch and the like; function of transferring mature thymus T cells to peripheral blood in response to ELC chemokine stimulation; or a function of migrating CD4+CD8+ immature thymus cells in response to SDF-1 chemokine stimulation.
[0030]The present invention relates also to a method for screening a substance interfering in the association of ELMO and GDP/GTP exchange factor (GEF), or to a method for screening a substance promoting or suppressing Rac activation. As for the method for screening a substance interfering in the association of ELMO and GEF, there is no specific limitation as long as it is a method comprising the steps of contacting ELMO, GEF, and a test substance, and then estimating the level of formation of association of ELMO and GEF; or a method comprising the steps of contacting N terminus domain of ELMO, GEF and a test substance, and then estimating the level of formation of association of N terminus domain of ELMO and GEF. Moreover, as for the method for screening a substance promoting or suppressing Rac activation, there is no specific limitation as long as it is a method comprising the steps of contacting DOCK2, ELMO, GEF and a test substance, or by contacting SH3 domain of DOCK2, ELMO, GEF and a test substance, and then estimating the level of formation of association of DOCK2 and ELMO, or the level of formation of association of ELMO and GEF. Further, as for the above ELMO, ELMO bound with DOCK2 can be used.
[0031]As for the above ELMO, examples include ELMO1, ELMO2, ELMO3, and among these, ELMO1 can be preferably exemplified. Moreover, as for the above GEF, Rac-specific GDP/GTP exchange factors such as Tiam1, Tiam2, Vav1, Vav2, Vav3, Trio, STEF, P-Rex1 are preferable, and among these, Tiam1 can be preferably exemplified. As for the above Tiam1 gene, mouse Tiam1 gene (NM--009384; Cell Vol. 77(4), 537-549, 1994), human Tiam1 gene (NM--003253; Oncogene Vol. 10(7), 1371-1376, 1995) can be exemplified, but the origin of Tiam1 gene is not limited to mouse, human and the like. Amino acid sequences of mouse Tiam1, human Tiam1 are shown in Seq. ID. Nos. 5 and 6, respectively.
[0032]Methods used for the above method for screening a substance interfering in the association of DOCK2 and ELMO, including the above method for screening a substance interfering in the association of ELMO and GEF, or a method for estimating the level of formation of association of ELMO and GEF, a method for estimating the level of formation of association of DOCK2 and ELMO, a method of using ELMO fused with other peptides, or its N terminus, and GEF, in the method for screening a substance for promoting or suppressing Rac activity, can be applied accordingly.
[0033]By using the method for screening a substance interfering in the association of DOCK2, ELMO such as ELMO1, the method for screening a substance interfering in the association of ELMO and GEF, the method for screening a substance promoting or suppressing Rac activation of the present invention, particularly the method for screening a substance promoting or suppressing the function of regulating lymphocyte migration, screening of preventive/therapeutic agents of immune related diseases such as allergy, autoimmune diseases, GvH, graft rejection targeting DOCK2 can be possible. As it can be anticipated that substances suppressing the function of regulating lymphocyte migration obtained by the method for screening a substance promoting or suppressing the function of regulating lymphocyte migration, such as anti-DOCK2 SH3 domain antibody, DOCK2 SH3 domain-binding molecule (including low molecular compounds), antisense strand of DOCK2 gene, antibodies recognizing specifically the DOCK2 SH3 domain-binding site of C terminus domain of ELMO such as ELMO1, molecules binding to the DOCK2 SH2 domain-binding site of C terminus domain of ELMO such as ELMO1 (including low molecular compounds), antibodies recognizing specifically GEF-binding site such as Tiam1 of N terminus domain of ELMO such as ELMO1, molecules binding to GEF-binding site such as Tiam1 of N terminus domain of ELMO such as ELMO1 (including low molecular compound), or antisense strand of ELMO such as ELMO1, can suppress artificially lymphocyte mobility, the possibility for these suppressive substances to be a therapeutic agent against immune-related diseases such as allergy, autoimmune diseases, GvH, graft rejection is high. When the therapeutic agent is used as drugs, various prescribed compounds such as pharmaceutically acceptable normal carrier, bonding agent, stabilizing agent, excipient, diluent, pH buffer agent, disintegrator, solubilizer, dissolving adjuvant, isotonic agent can be added, and can be administered by an administration form used generally, for example orally in formulation form such as powder, granule, capsule, syrup, and suspending agent, or parenterally in form of injection those formulated in form of solution, emulsion, suspending solution and the like.
[0034]Moreover, when using the method for screening a substance interfering in the association of DOCK2 and ELMO1, the method for screening a substance interfering in the association of ELMO1 and Tiam1, the method for screening a substance promoting or suppressing Rac activity of the present invention, in particular the method for screening a substance promoting the function of regulating lymphocyte migration, cytoskeletal reorganization is promoted by activating Rac, and thus, screening of preventive/therapeutic agents against diseases caused by suppression of lymphocyte migration, such as various cancers, or immunodeficiency caused by drugs/irradiation, can be possible.
[0035]Furthermore, as for the method for screening a substance inhibiting DOCK2 function of the present invention, examples include a method making the N terminus domain of DOCK2 including SH3 domain as target, comprising the steps of contacting SH3 domain of DOCK2 and the SH3 domain-binding protein and a test substance, and then estimating the level of formation of association of DOCK2 and SH3 domain-binding protein; and a method by using transgenic cell line expressing full length DOCK2 and DOCK2-deleted mutant, measuring/estimating the level of Rac activation in these cell lines, identifying the functional domain of DOCK2, searching a molecule associated with functional domain that associates with the functional domain, contacting the functional domain of DOCK2, the molecule associated with functional domain and a test substance, and estimating the level of formation of association of functional domain of DOCK2 and molecule associated with the functional domain. As for the method for contacting with a test substance, the method for estimating the level of formation of association, or the method for measuring the level of Rac activation, the methods mentioned above can be used. As for the method for identifying the functional domain of DOCK2, or for the preparation of transgenic cell line expressing full length DOCK2 and DOCK2-deleted mutant, methods described in the following examples can be used.
[0036]In the following, the present invention will be explained in detail by reference to the examples, while the technical scope of the present invention is not limited to these examples.
Example 1
Binding of N Terminus Domain of DOCK2 and ELMO 1
[0037]Recently, CED-12 has been identified as a molecule that associates with CED-5 and regulates cytoskeleton in nematodes, and ELMO1 has been reported as its mammal homologue (Cell 107, 27-41, 2001). Therefore, in order to investigate whether DOCK2 binds with ELMO1 or not, by using PcDNA/His max vector (Invitrogen), gene constructs encoding full length DOCK2 or various DOCK2-deleted mutants in which HA tag (YPYDVPDYA: Seq. ID No. 7) is introduced at the C terminus (PcDNA DOCK2-HA, PcDNA DOCK2 N-HA, PcDNA DOCK2ΔC-HA, PcDNA DOCK2ΔN-HA), were constructed. Then, the gene constructs were introduced into 293T cells (provided by Dr. Shinji Hatakeyama, Kyushu University) together with a gene in which ELMO1 cDNA is introduced into PcDNA V5-His vector (Invitrogen) (PcDNA ELMO1-V5). DOCK2 construct was prepared from genes isolated by the present inventors (Nature, 412, 826-831, 2001), and ELMO1 construct was prepared from mouse tissue cDNA by PCR according to a common method. The genes encoding the used DOCK2-deleted mutant are as follows, and they are shown schematically in FIG. 1.
1) PcDNA DOCK2 N-HA; genes encoding amino acid residue 1-502 of DOCK22) PcDNA DOCK2ΔC-HA; genes encoding amino acid residue 1-1311 of DOCK23) PcDNA DOCK2ΔN-HA; genes encoding amino acid residue 505-1828 of DOCK2
[0038]The cells were collected 48 hours after gene introduction, dissolved with Lysis buffer (Cell signaling), and analysed by Western Blot method using anti-V5 antibody (Invistrogen) to immunoprecipitants by total cell lysate and anti-HA antibody (Roche). For each of total cell lysate, a band of approximately 100-KD corresponding to ELMO1 was detected for anti-V5 antibody (FIG. 1B; top). However, for the immunoprecipitants, a band corresponding to ELMO1 was detected, when genes encoding full length DOCK2, DOCK2ΔC and DOCK2 N, while no band was detected when DOCK2ΔN lacking amino acid residues from N terminus to 504 of DOCK2 (FIG. 1B; lower figure of the middle line). From these results, it has been clarified that DOCK2 associates with ELMO1 in the domain of amino acid residues from its N terminus to 502.
Example 2
Rac Activation in DOCK2ΔN Lacking the N Terminus Domain
[0039]To clarify the influence of the association with ELMO1 to the function of DOCK 2, gene constructs encoding full length DOCK2 and a mutant lacking 504 amino acid residues of the N terminus of DOCK2 (DOCK2ΔN) were constructed by using PBJ1 vector. Then, a stable transgenic cell strain was established by introducing the gene constructs into the T cell strain, BEα16-3 (provided from National Jewish Center, Dr. Philppa Marrack), wherein the expression of DOCK 2 gene is deleted. N3-5 is a wild-type T cell strain expressing DOCK2, and 17-11 (Nature, 412, 826-831, 2001) and 84-3 are transgenic cell strains expressing full length DOCK2 and DOCK2ΔN, respectively, that the present inventors have established. In the Western Blot analysis using anti-DOCK2 polyclonal antibody that the present inventors have prepared, the expression of DOCK2 and DOCK2ΔN was approximately the same in 17-11 and 84-3 (FIG. 2A). Therefore, by targeting to 17-11 and 84-3, Rac activity in these cell strains was compared and analyzed by pull-down method using GST fusion protein of PAK1 Rac binding domain. In 17-11 expressing full length DOCK2, GTP-binding form of activated Rac was easily detected, whereas in 84-3 expressing DOCK2ΔN lacking the binding site with ELMO 1, Rac activating ability was significantly decreased (FIG. 2B). From the nuclear stain of 17-11 and 84-3 with PI (propidium iodide), it has been revealed that in any case, the nucleus is eccentrically located, in other words, that cell polarization is performed, which is different from BEα16-3, the parent cell strain (FIG. 2C; top). On the contrary, when these cells are stained with phalloidin, which is a probe for F-actin, actin polymerization was observed only for 17-11, and not in 84-3, as in the case of Beα16-3, wherein the DOCK2 expression is deleted (FIG. 2C; bottom). From these results, the association of DOCK2 and ELMO1 has been suggested to be extremely crucial to the full activation of Rac as well as to cytoskeletal reorganization, relating thereof. From the above, it has been clarified that in DOCK2ΔN, lacking N terminus domain being essential for the binding with ELMO1, the Rac-activating ability is significantly decreased, and that actin polymerization cannot be induced.
Example 3
Association with ELMO1, Via SH3 Domain of DOCK2
[0040]SH (Src-homolgy)3 domain known to be related with the protein-protein interaction is encoded at the N-terminus of DOCK2. As it was found that 502 amino acid residues at the N terminus of DOCK2 are crucial for the association with ELMO1, it was investigated if it is mediated by SH3 domain. Amino acid residues commonly conserved exist in the SH3 domain. Therefore, gene constructs encoding various DOCK2 SH3 mutants wherein HA tag is introduced into C terminus by using PcDNA/His max vector, were constructed. Then, these were introduced into 293T cells with PcDNA ELMO1-V5, and were analyzed in the same manner as in FIG. 1B. Genes encoding DOCK2 SH3 mutant are as follows:
1) PcDNA L27E-HA; gene encoding mutant wherein leucin at the 27 position of DOCK2 is substituted to glutamic acid2) PcDNA G32E-HA; gene encoding mutant wherein glycine at the 32 position of DOCK2 is substituted to glutamic acid3) PcDNA P60E-HA; gene encoding mutant wherein poline at the 60 position of DOCK2 is substituted to glutamic acid4) PcDNA F63E-HA; gene encoding mutant wherein phenylalanine at the 63 position of DOCK2 is substituted to glutamic acid
[0041]Amino acid sequence from 10-89 including DOCK2 SH3 domain is shown in FIG. 3A. For each of total cell lysate, an approximately 100-KD band corresponding to ELMO1 for anti-V5 antibody was detected (FIG. 3B; top). However, when targeting to immunoprecipitants using anti-HA antibody, the band corresponding to ELMO1 was not detected except for those introduced with PcDNA DOCK2-HA and PcDNA L27E-HA (FIG. 3B; middle). On the other hand, when any one of the genes has been introduced, DOCK2 and DOCK2 SH3 mutant expressions were almost of the same level (FIG. 3B; lower). The above results show that the association of DOCK2 and ELMO1 is completely inhibited by substituting a single amino acid of SH3 domain. Therefore, it has been clarified that DOCK2 is bound to ELMO1 via its SH3 domain.
Example 4
Binding of C Terminus Domain of ELMO1 and DOCK2
[0042]Next, to identify the functional domain of ELMO1 binding with DOCK2, gene constructs encoding various ELMO1-deleted mutants were constructed by using PcDNA V5H is vector, and were analyzed by introducing these into 293T cells with PcDNA DOCK2-HA. Genes herein used, encoding ELMO1-deleted mutants are as follows, which are shown schematically in FIG. 4A.
1) PcDNA ELMO1-del1-V5; gene encoding amino acid residues at the position 147-727 of ELMO12) PcDNA ELMO1-del8-V5; gene encoding amino acid residues at the position 345-727 of ELMO13) PcDNA ELMO1-del10-V5; gene encoding amino acid residues at the position 1-613 of ELMO1
[0043]For each of the total cell lysate, band corresponding to ELMO1 or its deleted mutant was detected with anti-V5 antibody (FIG. 4B; top). However, as for immunoprecipitants with anti-HA antibody, bands reacting to anti-V5 antibody were observed when genes encoding full length ELMO1, ELMO1-del1 and ELMO1-del8 were introduced, but not when PcDNA ELMO1-del10 lacking amino acid residues at the position 614-727 of ELMO1, was expressed (FIG. 4B; middle, bottom). From these, C terminus domain including amino acid residues at the position 614-727 of ELMO1 was revealed to be crucial for the association of DOCK2 SH3 domain. From these results, it has been clarified that ELMO1 was bound with DOCK2 in its C terminus domain.
Example 5
Binding of N Terminus Domain of ELMO1 and Tiam1
[0044]Tiam 1 has been identified as a molecule that determines the invasion of thymoma cell lines, and is known to function as Rac-specific GDP/GTP exchange factor (GEF) (Cell 77, 537-549, 1994; Nature 375, 338-340, 1995). As the association of DOCK2 and ELMO1 is necessary for the full activation of Rac, it has been estimated that DOCK2 might recruit Tiam1 via ELMO1. To investigate this assumption, from a Tiam1 gene amplified by PCR method from cDNA derived from mouse organs, a construct encoding Tiam1 wherein HA tag was introduced at its C terminus (PCI Tiam1-HA) was constructed with the use of PCI vector (Promega), introduced into 293T cells with genes encoding full length or various ELMO1-deleted mutants (PcDNA ELMO1-V5, PcDNA ELMO1-delPH-V5, PcDNA ELMO1-del8-V5, PcDNA ELMO1-del1), and was then analyzed. PcDNA ELMO1-delPH-V5 is a gene encoding amino acid residues at the position 1-565 and 695-727 of ELMO1. ELMO1-deleted mutants herein used are shown schematically in FIG. 5A. For each of the total cell lysate, a band corresponding to ELMO1 or its deleted mutant was detected with anti-V5 antibody (FIG. 5B; top). In immunoprecipitants with anti-HA antibody, when PcDNA ELMO1-V5 and PcDNA ELMO1-delPH-V5 were introduced, bands reacting to anti-V5 antibody were detected (FIG. 5B; middle, bottom). This shows that Tiam1 binds with ELMO1. However, as for mutants lacking amino acid residues from N terminus to 146, or to 344, of ELMO1, such binding was not observed (FIG. 5B; middle, bottom). From these results, it has been revealed that ELMO1 is associated with Tiam1 at its N terminus.
[0045]From the above, the following has been revealed:
1) DOCK2 binds to the C terminus domain of ELMO1 via SH3 domain2) ELMO1 binds with Tiam1 via its N terminus domain3) Rac-activating ability is significantly decreased in DOCK2 mutants that cannot bind with ELMO1.
[0046]Therefore, it has been shown that DOCK2 activates Rac by recruiting Tiam1 that functions as GEF of Rac, via ELMO1 (FIG. 6).
[0047]As autoimmune diseases and graft rejection are induced when lymphocytes invade into the target tissues, DOCK2 signaling should be the excellent target to treat or prevent these diseases or pathologic conditions. The finding of the invention shows that interaction between molecules such as DOCK2, ELMO1 and Tiam1 regulate Rac activation that is essential for cell mobility. Therefore, it can be thought that by blocking the intermolecular interaction, the invasion of lymphocytes can be inhibited. Therefore, these intermolecular interactions are anticipated to be the target of drug discovery heading to the development of method for treating or preventing autoimmune diseases or graft rejection.
INDUSTRIAL APPLICABILITY
[0048]According to the present invention, it is possible to elucidate the interaction between molecules of DOCK2, and to provide a substance controlling lymphocyte migration and a method to regulate lymphocyte migration targeting DOCK2. Moreover, according to the present invention, it is possible to provide preventive or therapeutic agents of autoimmune diseases or graft rejections after implantation.
Sequence CWU
1
811828PRTMus musculus 1Met Ala Pro Trp Arg Lys Thr Asp Lys Glu Arg His Gly
Val Ala Ile 1 5 10 15Tyr
Asn Phe Gln Gly Ser Glu Ala Gln His Leu Thr Leu Gln Ile Gly
20 25 30Asp Val Val Arg Ile Gln Glu Thr
Cys Gly Asp Trp Tyr Arg Gly Tyr 35 40
45Leu Ile Lys His Lys Leu Ser Gln Gly Ile Phe Pro Thr Ser Phe Ile
50 55 60His Leu Lys Glu Val Thr Val
Glu Lys Arg Arg Asn Ile Glu Asn Ile 65 70
75 80Ile Pro Ala Glu Ile Pro Leu Ala Gln Glu Val Thr
Thr Thr Leu Trp 85 90
95Glu Trp Gly Ser Ile Trp Lys Gln Leu Tyr Val Ala Ser Lys Lys Glu
100 105 110Arg Phe Leu Gln Val Gln Ser
Met Met Tyr Asp Leu Met Glu Trp Arg 115 120
125Ser Gln Leu Leu Ser Gly Thr Leu Pro Lys Asp Glu Leu Lys Glu
Leu 130 135 140Lys Gln Lys Val Thr Ser
Lys Ile Asp Tyr Gly Asn Lys Ile Leu Glu145 150
155 160Leu Asp Leu Ile Val Arg Asp Glu Asp Gly Asn
Ile Leu Asp Pro Asp 165 170
175Lys Thr Ser Val Ile Ser Leu Phe His Ala His Glu Glu Ala Thr Tyr
180 185 190Lys Ile Thr Glu Arg Ile
Lys Glu Glu Met Ser Lys Asp Gln Pro Asp 195 200
205Tyr Gly Val Tyr Ser Arg Ile Ser Ser Ser Pro Thr His Ser
Leu Tyr 210 215 220Val Phe Val Arg Asn
Phe Val Cys Arg Ile Gly Glu Asp Ala Glu Leu225 230
235 240Phe Met Ser Leu Tyr Asp Pro His Lys Gln
Thr Val Ile Ser Glu Asn 245 250
255Tyr Leu Val Arg Trp Gly Ser Lys Gly Phe Pro Lys Glu Ile Glu Met
260 265 270Leu Asn Asn Leu Lys
Val Val Phe Thr Asp Leu Gly Asn Lys Asp Leu 275
280 285Asn Arg Asp Lys Ile Phe Leu Ile Cys Gln Ile Val
Arg Ile Gly Lys 290 295 300Met Asp Leu
Lys Asp Ile Asn Ala Lys Lys Cys Thr Gln Gly Leu Arg305
310 315 320Arg Pro Phe Gly Val Ala Val
Met Asp Ile Thr Asp Ile Ile Lys Gly 325
330 335Lys Ala Glu Ser Asp Glu Glu Lys Gln His Phe Ile
Pro Phe His Pro 340 345 350Val
Ser Ala Glu Asn Asp Phe Leu His Ser Leu Leu Gly Lys Val Ile 355
360 365Ala Ser Lys Gly Asp Ser Gly Gly Gln
Gly Leu Trp Val Thr Met Lys 370 375
380Met Leu Val Gly Asp Ile Ile Gln Ile Arg Lys Asp Tyr Pro His Leu385
390 395 400Val Asp Arg Thr
Thr Val Val Ala Arg Lys Leu Gly Phe Pro Glu Ile 405
410 415Ile Met Pro Gly Asp Val Arg Asn Asp Ile
Tyr Ile Thr Leu Leu Gln 420 425
430Gly Asp Phe Asp Lys Tyr Thr Lys Thr Thr Gln Arg Asn Val Glu Val
435 440 445Ile Met Cys Val Cys Thr Glu
Asp Gly Lys Val Leu Pro Asn Ala Ile 450 455
460Cys Val Gly Ala Gly Asp Lys Ala Met Asn Glu Tyr His Ser Val
Val465 470 475 480Tyr Tyr
Gln Val Lys Gln Pro Arg Trp Met Glu Thr Val Lys Val Ala
485 490 495Val Pro Ile Glu Asp Met Gln
Arg Ile His Leu Arg Phe Met Phe Arg 500 505
510His Arg Ser Ser Leu Glu Ser Lys Asp Lys Gly Glu Lys Asn
Phe Ala 515 520 525Met Ser Tyr Val
Lys Leu Met Lys Glu Asp Gly Thr Thr Leu His Asp 530
535 540Gly Tyr His Glu Leu Val Val Leu Lys Gly Asp Ser
Lys Lys Met Glu545 550 555
560Asp Ala Ser Ala Tyr Leu Thr Leu Pro Ser Tyr Arg His Pro Val Glu
565 570 575Asn Lys Gly Ala Thr
Leu Ser Arg Ser Ser Ser Ser Val Gly Gly Leu 580
585 590Ser Val Ser Ser Arg Asp Val Phe Ser Ile Ser Thr
Leu Val Cys Ser 595 600 605Thr Lys
Leu Thr Gln Asn Val Gly Leu Leu Gly Leu Leu Lys Trp Arg 610
615 620Met Lys Pro Gln Leu Leu Gln Glu Asn Leu Glu
Lys Leu Lys Ile Val625 630 635
640Asp Gly Glu Glu Val Val Lys Phe Leu Gln Asp Thr Leu Asp Ala Leu
645 650 655Phe Asn Ile Met
Met Glu His Ser Gln Ser Asn Glu Tyr Asp Ile Leu 660
665 670Val Phe Asp Ala Leu Ile Tyr Ile Ile Gly Leu
Ile Ala Asp Arg Lys 675 680 685Phe
Gln His Phe Asn Thr Val Leu Glu Ala Tyr Ile Gln Gln His Phe 690
695 700Ser Ala Thr Leu Ala Tyr Lys Lys Leu Met
Thr Val Leu Lys Thr Tyr705 710 715
720Leu Asp Thr Ser Ser Arg Gly Glu Gln Cys Glu Pro Ile Leu Arg
Thr 725 730 735Leu Lys Ala
Leu Glu Tyr Val Phe Lys Phe Ile Val Arg Ser Arg Thr 740
745 750Leu Phe Ser Gln Leu Tyr Glu Gly Lys Glu
Gln Met Glu Phe Glu Glu 755 760
765Ser Met Arg Arg Leu Phe Glu Ser Ile Asn Asn Leu Met Lys Ser Gln 770
775 780Tyr Lys Thr Thr Ile Leu Leu Gln
Val Ala Ala Leu Lys Tyr Ile Pro785 790
795 800Ser Val Leu His Asp Val Glu Thr Val Phe Asp Ala
Lys Leu Leu Ser 805 810
815Gln Leu Leu Tyr Glu Phe Tyr Thr Cys Ile Pro Pro Val Lys Leu Gln
820 825 830Lys Gln Lys Val Gln Ser
Met Asn Glu Ile Val Gln Ser Asn Leu Phe 835 840
845Lys Lys Gln Glu Cys Arg Asp Ile Leu Leu Pro Val Ile Thr
Lys Glu 850 855 860Leu Lys Glu Leu Leu
Glu Gln Arg Asp Asp Gly Gln His Gln Ala Glu865 870
875 880Lys Lys His Cys Val Glu Leu Leu Asn Ser
Ile Leu Glu Val Leu Ser 885 890
895Cys Gln Asp Ala Ala Phe Thr Tyr Asp His Ile Gln Glu Ile Met Val
900 905 910Gln Leu Leu Arg Thr
Val Asn Arg Thr Val Ile Thr Met Gly Arg Asp 915
920 925His Ala Leu Ile Ser His Phe Glu Ala Cys Met Thr
Ala Ile Leu Asp 930 935 940Gln Met Gly
Asp Gln His Tyr Ser Phe Tyr Ile Glu Thr Phe Gln Thr945
950 955 960Ser Ser Asp Leu Val Asp Phe
Leu Met Glu Thr Phe Ile Met Phe Lys 965
970 975Asp Leu Ile Gly Lys Asn Val Tyr Pro Gly Asp Trp
Met Ala Met Ser 980 985 990Met
Val Gln Asn Arg Val Phe Leu Arg Ala Ile Asn Lys Phe Ala Glu 995
1000 1005Thr Met Asn Gln Lys Phe Leu Glu His
Thr Ser Phe Glu Phe Gln Leu 1010 1015
1020Trp Asn Asn Tyr Phe His Leu Ala Val Ala Phe Ile Thr Gln Asp Ser1025
1030 1035 1040Leu Gln Leu Glu
Gln Phe Thr His Ala Lys Tyr Asn Lys Ile Leu Asn 1045
1050 1055Lys Tyr Gly Asp Met Arg Arg Leu Ile Gly
Phe Ser Ile Arg Asp Met 1060 1065
1070Trp Tyr Lys Leu Gly Gln Asn Lys Ile Cys Phe Ile Pro Gly Met Val
1075 1080 1085Gly Pro Ile Leu Glu Met Thr
Leu Ile Pro Glu Ala Glu Leu Arg Lys 1090 1095
1100Ala Thr Ile Pro Ile Phe Phe Asp Met Met Leu Cys Glu Tyr Gln
Arg1105 1110 1115 1120Thr Gly
Ala Phe Lys Lys Phe Glu Asn Glu Ile Ile Leu Lys Leu Asp
1125 1130 1135His Glu Val Glu Gly Gly Arg
Gly Asp Glu Gln Tyr Met Gln Leu Leu 1140 1145
1150Glu Ser Ile Leu Met Glu Cys Thr Ala Glu His Pro Thr Ile
Ala Lys 1155 1160 1165Ser Val Glu Asn
Phe Val Ser Leu Val Lys Gly Leu Leu Glu Lys Leu 1170
1175 1180Leu Asp Tyr Arg Gly Val Met Thr Asp Glu Ser Lys
Asp Asn Arg Met1185 1190 1195
1200Ser Cys Thr Val Asn Leu Leu Asn Phe Tyr Lys Asp Asn Asn Arg Glu
1205 1210 1215Glu Met Tyr Ile Arg
Tyr Leu Tyr Lys Leu Arg Asp Leu His Leu Asp 1220
1225 1230Cys Glu Asn Tyr Thr Glu Ala Ala Tyr Thr Leu Leu
Leu His Thr Trp 1235 1240 1245Leu Leu
Lys Trp Ser Asp Glu Gln Cys Ala Ser Gln Val Met Gln Thr 1250
1255 1260Gly Gln Gln His Pro Gln Thr His Arg Gln Leu
Lys Glu Thr Leu Tyr1265 1270 1275
1280Glu Thr Ile Ile Gly Tyr Phe Asp Lys Gly Lys Met Trp Glu Glu Ala
1285 1290 1295Ile Ser Leu Cys
Lys Glu Leu Ala Glu Gln Tyr Glu Met Glu Ile Phe 1300
1305 1310Asp Tyr Glu Leu Leu Ser Gln Asn Leu Thr Gln
Gln Ala Lys Phe Tyr 1315 1320 1325Glu
Asn Ile Met Lys Ile Leu Arg Thr Lys Pro Asp Tyr Phe Ala Val 1330
1335 1340Gly Tyr Tyr Gly Gln Gly Phe Pro Ser Phe
Leu Arg Asn Lys Val Phe1345 1350 1355
1360Ile Tyr Arg Gly Lys Glu Tyr Glu Arg Arg Glu Asp Phe Gln Met
Gln 1365 1370 1375Leu Leu Ser
Gln Phe Pro Asn Ala Glu Lys Met Asn Thr Thr Ser Ala 1380
1385 1390Pro Gly Asp Asp Val Arg Asn Ala Pro Gly
Gln Tyr Ile Gln Cys Phe 1395 1400
1405Thr Val Gln Pro Val Leu Asp Glu His Pro Arg Phe Lys Asn Lys Pro
1410 1415 1420Val Pro Asp Gln Ile Ile Asn
Phe Tyr Lys Ser Asn Tyr Val Gln Lys1425 1430
1435 1440Phe His Tyr Ser Arg Pro Val Arg Arg Gly Lys Val
Asp Pro Glu Asn 1445 1450
1455Glu Phe Ala Ser Met Trp Ile Glu Arg Thr Ser Phe Leu Thr Ala Tyr
1460 1465 1470Lys Leu Pro Gly Ile Leu
Arg Trp Phe Glu Val Val His Met Ser Gln 1475 1480
1485Thr Thr Ile Ser Pro Leu Glu Asn Ala Ile Glu Thr Met Ser
Thr Val 1490 1495 1500Asn Glu Lys Ile Leu
Met Met Ile Asn Gln Tyr Gln Ser Asp Glu Ser1505 1510
1515 1520Leu Pro Ile Asn Pro Leu Ser Met Leu Leu
Asn Gly Ile Val Asp Pro 1525 1530
1535Ala Val Met Gly Gly Phe Ala Lys Tyr Glu Lys Ala Phe Phe Thr Glu
1540 1545 1550Glu Tyr Ser Arg Glu
His Pro Glu Asp Gln Asp Lys Leu Ser His Leu 1555
1560 1565Lys Asp Leu Ile Ala Trp Gln Ile Pro Phe Leu Gly
Ala Gly Ile Lys 1570 1575 1580Ile His Glu
Lys Arg Val Ser Asp Asn Leu Arg Pro Phe His Asp Arg1585
1590 1595 1600Met Glu Glu Cys Phe Lys Asn
Leu Lys Met Lys Val Glu Lys Glu Tyr 1605
1610 1615Gly Val Arg Glu Met Pro Asp Phe Glu Asp Arg Arg
Val Gly Arg Pro 1620 1625 1630Arg
Ser Met Leu Arg Ser Tyr Arg Gln Met Ser Val Ile Ser Leu Ala 1635
1640 1645Ser Met His Ser Asp Cys Ser Thr Pro
Ser Lys Val Pro Ala Glu Ser 1650 1655
1660Phe Asp Leu Glu Ser Ala Pro Pro Lys Thr Pro Lys Val Glu Glu Glu1665
1670 1675 1680Pro Ile Ser Pro
Gly Ser Thr Leu Pro Glu Val Lys Leu Arg Arg Ser 1685
1690 1695Lys Lys Arg Thr Lys Arg Ser Ser Val Val
Phe Ala Asp Glu Lys Ala 1700 1705
1710Ala Thr Glu Ser Asp Leu Lys Arg Leu Ser Arg Lys Gln Glu Phe Met
1715 1720 1725Ser Asp Thr Asn Leu Ser Glu
His Ala Ala Ile Pro Ala Arg Val Ser 1730 1735
1740Ile Leu Ser Gln Met Ser Phe Ala Ser Gln Ser Met Pro Thr Ile
Pro1745 1750 1755 1760Ala Leu
Thr Leu Ser Val Ala Gly Val Pro Gly Leu Asp Glu Ala Asn
1765 1770 1775Thr Ser Pro Arg Leu Ser Gln
Thr Phe Phe Gln Val Ser Asp Gly Asp 1780 1785
1790Lys Lys Thr Leu Lys Lys Lys Lys Val Asn Gln Phe Phe Lys
Thr Met 1795 1800 1805Leu Ala Ser Lys
Ser Ser Glu Glu Ser Lys Gln Ile Pro Asp Phe Leu 1810
1815 1820Ser Thr Asn Met182521830PRTHomo sapiens 2Met Ala
Pro Trp Arg Lys Ala Asp Lys Glu Arg His Gly Val Ala Ile 1
5 10 15Tyr Asn Phe Gln Gly Ser Gly Ala
Pro Gln Leu Ser Leu Gln Ile Gly 20 25
30Asp Val Val Arg Ile Gln Glu Thr Cys Gly Asp Trp Tyr Arg Gly
Tyr 35 40 45Leu Ile Lys His Lys
Met Leu Gln Gly Ile Phe Pro Lys Ser Phe Ile 50 55
60His Ile Lys Glu Val Thr Val Glu Lys Arg Arg Asn Thr Glu
Asn Ile 65 70 75 80Ile
Pro Ala Glu Ile Pro Leu Ala Gln Glu Val Thr Thr Thr Leu Trp
85 90 95Glu Trp Gly Ser Ile Trp Lys
Gln Leu Tyr Val Ala Ser Lys Lys Glu 100 105
110Arg Phe Leu Gln Val Gln Ser Met Met Tyr Asp Leu Met Glu
Trp Arg 115 120 125Ser Gln Leu Leu
Ser Gly Thr Leu Pro Lys Asp Glu Leu Lys Glu Leu 130
135 140Lys Gln Lys Val Thr Ser Lys Ile Asp Tyr Gly Asn
Lys Ile Leu Glu145 150 155
160Leu Asp Leu Ile Val Arg Asp Glu Asp Gly Asn Ile Leu Asp Pro Asp
165 170 175Asn Thr Ser Val Ile
Ser Leu Phe His Ala His Glu Glu Ala Thr Asp 180
185 190Lys Ile Thr Glu Arg Ile Lys Glu Glu Met Ser Lys
Asp Gln Pro Asp 195 200 205Tyr Ala
Met Tyr Ser Arg Ile Ser Ser Ser Pro Thr His Ser Leu Tyr 210
215 220Val Phe Val Arg Asn Phe Val Cys Arg Ile Gly
Glu Asp Ala Glu Leu225 230 235
240Phe Met Ser Leu Tyr Asp Pro Asn Lys Gln Thr Val Ile Ser Glu Asn
245 250 255Tyr Leu Val Arg
Trp Gly Ser Arg Gly Phe Pro Lys Glu Ile Glu Met 260
265 270Leu Asn Asn Leu Lys Val Val Phe Thr Asp Leu
Gly Asn Lys Asp Leu 275 280 285Asn
Arg Asp Lys Ile Tyr Leu Ile Cys Gln Ile Val Arg Val Gly Lys 290
295 300Met Asp Leu Lys Asp Thr Gly Ala Lys Lys
Cys Thr Gln Gly Leu Arg305 310 315
320Arg Pro Phe Gly Val Ala Val Met Asp Ile Thr Asp Ile Ile Lys
Gly 325 330 335Lys Ala Glu
Ser Asp Glu Glu Lys Gln His Phe Ile Pro Phe His Pro 340
345 350Val Thr Ala Glu Asn Asp Phe Leu His Ser
Leu Leu Gly Lys Val Ile 355 360
365Ala Ser Lys Gly Asp Ser Gly Gly Gln Gly Leu Trp Val Thr Met Lys 370
375 380Met Leu Val Gly Asp Ile Ile Gln
Ile Arg Lys Asp Tyr Pro His Leu385 390
395 400Val Asp Arg Thr Thr Val Val Ala Arg Lys Leu Gly
Phe Pro Glu Ile 405 410
415Ile Met Pro Gly Asp Val Arg Asn Asp Ile Tyr Ile Thr Leu Leu Gln
420 425 430Gly Asp Phe Asp Lys Tyr
Asn Lys Thr Thr Gln Arg Asn Val Glu Val 435 440
445Ile Met Cys Val Cys Ala Glu Asp Gly Lys Thr Leu Pro Asn
Ala Ile 450 455 460Cys Val Gly Ala Gly
Asp Lys Pro Met Asn Glu Tyr Arg Ser Val Val465 470
475 480Tyr Tyr Gln Val Lys Gln Pro Arg Trp Met
Glu Thr Val Lys Val Ala 485 490
495Val Pro Ile Glu Asp Met Gln Arg Ile His Leu Arg Phe Met Phe Arg
500 505 510His Arg Ser Ser Leu
Glu Ser Lys Asp Lys Gly Glu Lys Asn Phe Ala 515
520 525Met Ser Tyr Val Lys Leu Met Lys Glu Asp Gly Thr
Thr Leu His Asp 530 535 540Gly Phe His
Asp Leu Val Val Leu Lys Gly Asp Ser Lys Lys Met Glu545
550 555 560Asp Ala Ser Ala Tyr Leu Thr
Leu Pro Ser Tyr Arg His His Val Glu 565
570 575Asn Lys Gly Ala Thr Leu Ser Arg Ser Ser Ser Ser
Val Gly Gly Leu 580 585 590Ser
Val Ser Ser Arg Asp Val Phe Ser Ile Ser Thr Leu Val Cys Ser 595
600 605Thr Lys Leu Thr Gln Asn Val Gly Leu
Leu Gly Leu Leu Lys Trp Arg 610 615
620Met Lys Pro Gln Leu Leu Gln Glu Asn Leu Glu Lys Leu Lys Ile Val625
630 635 640Asp Gly Glu Glu
Val Val Lys Phe Leu Gln Asp Thr Leu Asp Ala Leu 645
650 655Phe Asn Ile Met Met Glu His Ser Gln Ser
Asp Glu Tyr Asp Ile Leu 660 665
670Val Phe Asp Ala Leu Ile Tyr Ile Ile Gly Leu Ile Ala Asp Arg Lys
675 680 685Phe Gln His Phe Asn Thr Val
Leu Glu Ala Tyr Ile Gln Gln His Phe 690 695
700Ser Ala Thr Leu Ala Tyr Lys Lys Leu Met Thr Val Leu Lys Thr
Tyr705 710 715 720Leu Asp
Thr Ser Ser Arg Gly Glu Gln Cys Glu Pro Ile Leu Arg Thr
725 730 735Leu Lys Ala Leu Glu Tyr Val
Phe Lys Phe Ile Val Arg Ser Arg Thr 740 745
750Leu Phe Ser Gln Leu Tyr Glu Gly Lys Glu Gln Met Glu Phe
Glu Glu 755 760 765Ser Met Arg Arg
Leu Phe Glu Ser Ile Asn Asn Leu Met Lys Ser Gln 770
775 780Tyr Lys Thr Thr Ile Leu Leu Gln Val Ala Ala Leu
Lys Tyr Ile Pro785 790 795
800Ser Val Leu His Asp Val Glu Met Val Phe Asp Ala Lys Leu Leu Ser
805 810 815Gln Leu Leu Tyr Glu
Phe Tyr Thr Cys Ile Pro Pro Val Lys Leu Gln 820
825 830Lys Gln Lys Val Gln Ser Met Asn Glu Ile Val Gln
Ser Asn Leu Phe 835 840 845Lys Lys
Gln Glu Cys Arg Asp Ile Leu Leu Pro Val Ile Thr Lys Glu 850
855 860Leu Lys Glu Leu Leu Glu Gln Lys Asp Asp Met
Gln His Gln Val Leu865 870 875
880Glu Arg Lys Tyr Cys Val Glu Leu Leu Asn Ser Ile Leu Glu Val Leu
885 890 895Ser Tyr Gln Asp
Ala Ala Phe Thr Tyr His His Ile Gln Glu Ile Met 900
905 910Val Gln Leu Leu Arg Thr Val Asn Arg Thr Val
Ile Thr Met Gly Arg 915 920 925Asp
His Ile Leu Ile Ser His Phe Val Ala Cys Met Thr Ala Ile Leu 930
935 940Asn Gln Met Gly Asp Gln His Tyr Ser Phe
Tyr Ile Glu Thr Phe Gln945 950 955
960Thr Ser Ser Glu Leu Val Asp Phe Leu Met Glu Thr Phe Ile Met
Phe 965 970 975Lys Asp Leu
Ile Gly Lys Asn Val Tyr Pro Gly Asp Trp Met Ala Met 980
985 990Ser Met Val Gln Asn Arg Val Phe Leu Arg
Ala Ile Asn Lys Phe Ala 995 1000
1005Glu Thr Met Asn Gln Lys Phe Leu Glu His Thr Asn Phe Glu Phe Gln
1010 1015 1020Leu Trp Asn Asn Tyr Phe His
Leu Ala Val Ala Phe Ile Thr Gln Asp1025 1030
1035 1040Ser Leu Gln Leu Glu Gln Phe Ser His Ala Lys Tyr
Asn Lys Ile Leu 1045 1050
1055Asn Lys Tyr Gly Asp Met Arg Arg Leu Ile Gly Phe Ser Ile Arg Asp
1060 1065 1070Met Trp Tyr Lys Leu Gly
Gln Asn Lys Ile Cys Phe Ile Pro Gly Met 1075 1080
1085Val Gly Pro Ile Leu Glu Met Thr Leu Ile Pro Glu Ala Glu
Leu Arg 1090 1095 1100Lys Ala Thr Ile Pro
Ile Phe Phe Asp Met Met Leu Cys Glu Tyr Gln1105 1110
1115 1120Arg Ser Gly Asp Phe Lys Lys Phe Glu Asn
Glu Ile Ile Leu Lys Leu 1125 1130
1135Asp His Glu Val Glu Gly Gly Arg Gly Asp Glu Gln Tyr Met Gln Leu
1140 1145 1150Leu Glu Ser Ile Leu
Met Glu Cys Ala Ala Glu His Pro Thr Ile Ala 1155
1160 1165Lys Ser Val Glu Asn Phe Val Asn Leu Val Lys Gly
Leu Leu Glu Lys 1170 1175 1180Leu Leu Asp
Tyr Arg Gly Val Met Thr Asp Glu Ser Lys Asp Asn Arg1185
1190 1195 1200Met Ser Cys Thr Val Asn Leu
Leu Asn Phe Tyr Lys Asp Asn Asn Arg 1205
1210 1215Glu Glu Met Tyr Ile Arg Tyr Leu Tyr Lys Leu Arg
Asp Leu His Leu 1220 1225 1230Asp
Cys Asp Asn Tyr Thr Glu Ala Ala Tyr Thr Leu Leu Leu His Thr 1235
1240 1245Trp Leu Leu Lys Trp Ser Asp Glu Gln
Cys Ala Ser Gln Val Met Gln 1250 1255
1260Thr Gly Gln Gln His Pro Gln Thr His Arg Gln Leu Lys Glu Thr Leu1265
1270 1275 1280Tyr Glu Thr Ile
Ile Gly Tyr Phe Asp Lys Gly Lys Met Trp Glu Glu 1285
1290 1295Ala Ile Ser Leu Cys Lys Glu Leu Ala Glu
Gln Tyr Glu Met Glu Ile 1300 1305
1310Phe Asp Tyr Glu Leu Leu Ser Gln Asn Leu Ile Gln Gln Ala Lys Phe
1315 1320 1325Tyr Glu Ser Ile Met Lys Ile
Leu Arg Pro Lys Pro Asp Tyr Phe Ala 1330 1335
1340Val Gly Tyr Tyr Gly Gln Gly Phe Pro Ser Phe Leu Arg Asn Lys
Val1345 1350 1355 1360Phe Ile
Tyr Arg Gly Lys Glu Tyr Glu Arg Arg Glu Asp Phe Gln Met
1365 1370 1375Gln Leu Met Thr Gln Phe Pro
Asn Ala Glu Lys Met Asn Thr Thr Ser 1380 1385
1390Ala Pro Gly Asp Asp Val Lys Asn Ala Pro Gly Gln Tyr Ile
Gln Cys 1395 1400 1405Phe Thr Val Gln
Pro Val Leu Asp Glu His Pro Arg Phe Lys Asn Lys 1410
1415 1420Pro Val Pro Asp Gln Ile Ile Asn Phe Tyr Lys Ser
Asn Tyr Val Gln1425 1430 1435
1440Arg Phe His Tyr Ser Arg Pro Val Arg Arg Gly Thr Val Asp Pro Glu
1445 1450 1455Asn Glu Phe Ala Ser
Met Trp Ile Glu Arg Thr Ser Phe Val Thr Ala 1460
1465 1470Tyr Lys Leu Pro Gly Ile Leu Arg Trp Phe Glu Val
Val His Met Ser 1475 1480 1485Gln Thr
Thr Ile Ser Pro Leu Glu Asn Ala Ile Glu Thr Met Ser Thr 1490
1495 1500Ala Asn Glu Lys Ile Leu Met Met Ile Asn Gln
Tyr Gln Ser Asp Glu1505 1510 1515
1520Thr Leu Pro Ile Asn Pro Leu Ser Met Leu Leu Asn Gly Ile Val Asp
1525 1530 1535Pro Ala Val Met
Gly Gly Phe Ala Lys Tyr Glu Lys Ala Phe Phe Thr 1540
1545 1550Glu Glu Tyr Val Arg Asp His Pro Glu Asp Gln
Asp Lys Leu Thr His 1555 1560 1565Leu
Lys Asp Leu Ile Ala Trp Gln Ile Pro Phe Leu Gly Ala Gly Ile 1570
1575 1580Lys Ile His Glu Lys Arg Val Ser Asp Asn
Leu Arg Pro Phe His Asp1585 1590 1595
1600Arg Met Glu Glu Cys Phe Lys Asn Leu Lys Met Lys Val Glu Lys
Glu 1605 1610 1615Tyr Gly Val
Arg Glu Met Pro Asp Phe Asp Asp Arg Arg Val Gly Arg 1620
1625 1630Pro Arg Ser Met Leu Arg Ser Tyr Arg Gln
Met Ser Ile Ile Ser Leu 1635 1640
1645Ala Ser Met Asn Ser Asp Cys Ser Thr Pro Ser Lys Pro Thr Ser Glu
1650 1655 1660Ser Phe Asp Leu Glu Leu Ala
Ser Pro Lys Thr Pro Arg Val Glu Gln1665 1670
1675 1680Glu Glu Pro Ile Ser Pro Gly Ser Thr Leu Pro Glu
Val Lys Leu Arg 1685 1690
1695Arg Ser Lys Lys Arg Thr Lys Arg Ser Ser Val Val Phe Ala Asp Glu
1700 1705 1710Lys Ala Ala Ala Glu Ser
Asp Leu Lys Arg Leu Ser Arg Lys His Glu 1715 1720
1725Phe Met Ser Asp Thr Asn Leu Ser Glu His Ala Ala Ile Pro
Leu Lys 1730 1735 1740Ala Ser Val Leu Ser
Gln Met Ser Phe Ala Ser Gln Ser Met Pro Thr1745 1750
1755 1760Ile Pro Ala Leu Ala Leu Ser Val Ala Gly
Ile Pro Gly Leu Asp Glu 1765 1770
1775Ala Asn Thr Ser Pro Arg Leu Ser Gln Thr Phe Leu Gln Leu Ser Asp
1780 1785 1790Gly Asp Lys Lys Thr
Leu Thr Arg Lys Lys Val Asn Gln Phe Phe Lys 1795
1800 1805Thr Met Leu Ala Ser Lys Ser Ala Glu Glu Gly Lys
Gln Ile Pro Asp 1810 1815 1820Ser Leu Ser
Thr Asp Leu1825 18303727PRTMus musculus 3Met Pro Pro Pro
Ser Asp Ile Val Lys Val Ala Ile Glu Trp Pro Gly 1 5
10 15Ala Tyr Pro Lys Leu Met Glu Ile Asp Gln
Lys Lys Pro Leu Ser Ala 20 25
30Ile Ile Lys Glu Val Cys Asp Gly Trp Ser Leu Ala Asn His Glu Tyr
35 40 45Phe Ala Leu Gln His Ala Asp
Ser Ser Asn Phe Tyr Ile Thr Glu Lys 50 55
60Asn Arg Asn Glu Ile Lys Asn Gly Thr Ile Leu Arg Leu Thr Thr Ser
65 70 75 80Pro Ala Gln
Asn Ala Gln Gln Leu His Glu Arg Ile Gln Ser Ser Ser 85
90 95Met Asp Ala Lys Leu Glu Ala Leu Lys
Asp Leu Ala Ser Leu Ser Arg 100 105
110Asp Val Thr Phe Ala Gln Glu Phe Ile Asn Leu Asp Gly Ile Ser Leu
115 120 125Leu Thr Gln Met Val Glu
Ser Gly Thr Glu Arg Tyr Gln Lys Leu Gln 130 135
140Lys Ile Met Lys Pro Cys Phe Gly Asp Met Leu Ser Phe Thr Leu
Thr145 150 155 160Ala Phe
Val Glu Leu Met Asp His Gly Ile Val Ser Trp Asp Thr Phe
165 170 175Ser Val Ala Phe Ile Lys Lys
Ile Ala Ser Phe Val Asn Lys Ser Ala 180 185
190Ile Asp Ile Ser Ile Leu Gln Arg Ser Leu Ala Ile Leu Glu
Ser Met 195 200 205Val Leu Asn Ser
His Asp Leu Tyr Gln Lys Val Ala Gln Glu Ile Thr 210
215 220Ile Gly Gln Leu Ile Pro His Leu Gln Gly Thr Asp
Gln Glu Ile Gln225 230 235
240Thr Tyr Thr Ile Ala Val Ile Asn Ala Leu Phe Leu Lys Ala Pro Asp
245 250 255Glu Arg Arg Gln Glu
Met Ala Asn Ile Leu Ala Gln Lys Gln Leu Arg 260
265 270Tyr Ile Ile Leu Thr His Val Ile Arg Ala Gln Arg
Ala Ile Asn Asn 275 280 285Glu Met
Ala His Gln Leu Tyr Val Leu Gln Val Leu Thr Phe Asn Leu 290
295 300Leu Glu Asp Arg Met Met Thr Lys Met Asp Pro
Gln Asp Gln Ala Gln305 310 315
320Arg Asp Ile Ile Phe Glu Leu Arg Arg Ile Ala Phe Asp Ala Glu Ser
325 330 335Glu Pro Asn Asn
Ser Ser Gly Ser Met Glu Lys Arg Lys Ser Met Tyr 340
345 350Thr Arg Asp Tyr Lys Lys Leu Gly Phe Ile Asn
His Val Asn Pro Ala 355 360 365Met
Asp Phe Thr Gln Thr Pro Pro Gly Met Leu Ala Leu Asp Asn Met 370
375 380Leu Tyr Phe Ala Lys His His Gln Asp Ala
Tyr Ile Arg Ile Val Leu385 390 395
400Glu Asn Ser Ser Arg Glu Asp Lys His Glu Cys Pro Phe Gly Arg
Ser 405 410 415Ser Ile Glu
Leu Thr Lys Met Leu Cys Glu Ile Leu Lys Val Gly Glu 420
425 430Leu Pro Ser Glu Thr Cys Asn Asp Phe His
Pro Met Phe Phe Thr His 435 440
445Asp Arg Ser Phe Glu Glu Phe Phe Cys Ile Cys Ile Gln Leu Leu Asn 450
455 460Lys Thr Trp Lys Glu Met Arg Ala
Thr Ser Glu Asp Phe Asn Lys Val465 470
475 480Met Gln Val Val Lys Glu Gln Val Met Arg Ala Leu
Thr Thr Lys Pro 485 490
495Ser Ser Leu Asp Gln Phe Lys Ser Lys Leu Gln Asn Leu Ser Tyr Thr
500 505 510Glu Ile Leu Lys Ile Arg
Gln Ser Glu Arg Met Asn Gln Glu Asp Phe 515 520
525Gln Ser Arg Pro Ile Leu Glu Leu Lys Glu Lys Ile Gln Pro
Glu Ile 530 535 540Leu Glu Leu Ile Lys
Gln Gln Arg Leu Asn Arg Leu Val Glu Gly Thr545 550
555 560Cys Phe Arg Lys Leu Asn Ala Arg Arg Arg
Gln Asp Lys Phe Trp Tyr 565 570
575Cys Arg Leu Ser Pro Asn His Lys Val Leu His Tyr Gly Asp Leu Glu
580 585 590Glu Ser Pro Gln Gly
Glu Val Pro His Asp Ser Leu Gln Asp Lys Leu 595
600 605Pro Val Ala Asp Ile Lys Ala Val Val Thr Gly Lys
Asp Cys Pro His 610 615 620Met Lys Glu
Lys Gly Ala Leu Lys Gln Asn Lys Glu Val Leu Glu Leu625
630 635 640Ala Phe Ser Ile Leu Tyr Asp
Ser Asn Cys Gln Leu Asn Phe Ile Ala 645
650 655Pro Asp Lys His Glu Tyr Cys Ile Trp Thr Asp Gly
Leu Asn Ala Leu 660 665 670Leu
Gly Lys Asp Met Met Ser Asp Leu Thr Arg Asn Asp Leu Asp Thr 675
680 685Leu Leu Ser Met Glu Ile Lys Leu Arg
Leu Leu Asp Leu Glu Asn Ile 690 695
700Gln Ile Pro Asp Ala Pro Pro Pro Ile Pro Lys Glu Pro Ser Asn Tyr705
710 715 720Asp Phe Val Tyr
Asp Cys Asn 7254727PRTHomo sapiens 4Met Pro Pro Pro Ala
Asp Ile Val Lys Val Ala Ile Glu Trp Pro Gly 1 5
10 15Ala Tyr Pro Lys Leu Met Glu Ile Asp Gln Lys
Lys Pro Leu Ser Ala 20 25
30Ile Ile Lys Glu Val Cys Asp Gly Trp Ser Leu Ala Asn His Glu Tyr
35 40 45Phe Ala Leu Gln His Ala Asp Ser
Ser Asn Phe Tyr Ile Thr Glu Lys 50 55
60Asn Arg Asn Glu Ile Lys Asn Gly Thr Ile Leu Arg Leu Thr Thr Ser 65
70 75 80Pro Ala Gln Asn
Ala Gln Gln Leu His Glu Arg Ile Gln Ser Ser Ser 85
90 95Met Asp Ala Lys Leu Glu Ala Leu Lys Asp
Leu Ala Ser Leu Ser Arg 100 105
110Asp Val Thr Phe Ala Gln Glu Phe Ile Asn Leu Asp Gly Ile Ser Leu
115 120 125Leu Thr Gln Met Val Glu Ser
Gly Thr Glu Arg Tyr Gln Lys Leu Gln 130 135
140Lys Ile Met Lys Pro Cys Phe Gly Asp Met Leu Ser Phe Thr Leu
Thr145 150 155 160Ala Phe
Val Glu Leu Met Asp His Gly Ile Val Ser Trp Asp Thr Phe
165 170 175Ser Val Ala Phe Ile Lys Lys
Ile Ala Ser Phe Val Asn Lys Ser Ala 180 185
190Ile Asp Ile Ser Ile Leu Gln Arg Ser Leu Ala Ile Leu Glu
Ser Met 195 200 205Val Leu Asn Ser
His Asp Leu Tyr Gln Lys Val Ala Gln Glu Ile Thr 210
215 220Ile Gly Gln Leu Ile Pro His Leu Gln Gly Ser Asp
Gln Glu Ile Gln225 230 235
240Thr Tyr Thr Ile Ala Val Ile Asn Ala Leu Phe Leu Lys Ala Pro Asp
245 250 255Glu Arg Arg Gln Glu
Met Ala Asn Ile Leu Ala Gln Lys Gln Leu Arg 260
265 270Ser Ile Ile Leu Thr His Val Ile Arg Ala Gln Arg
Ala Ile Asn Asn 275 280 285Glu Met
Ala His Gln Leu Tyr Val Leu Gln Val Leu Thr Phe Asn Leu 290
295 300Leu Glu Asp Arg Met Met Thr Lys Met Asp Pro
Gln Asp Gln Ala Gln305 310 315
320Arg Asp Ile Ile Phe Glu Leu Arg Arg Ile Ala Phe Asp Ala Glu Ser
325 330 335Glu Pro Asn Asn
Ser Ser Gly Ser Met Glu Lys Arg Lys Ser Met Tyr 340
345 350Thr Arg Asp Tyr Lys Lys Leu Gly Phe Ile Asn
His Val Asn Pro Ala 355 360 365Met
Asp Phe Thr Gln Thr Pro Pro Gly Met Leu Ala Leu Asp Asn Met 370
375 380Leu Tyr Phe Ala Lys His His Gln Asp Ala
Tyr Ile Arg Ile Val Leu385 390 395
400Glu Asn Ser Ser Arg Glu Asp Lys His Glu Cys Pro Phe Gly Arg
Ser 405 410 415Ser Ile Glu
Leu Thr Lys Met Leu Cys Glu Ile Leu Lys Val Gly Glu 420
425 430Leu Pro Ser Glu Thr Cys Asn Asp Phe His
Pro Met Phe Phe Thr His 435 440
445Asp Arg Ser Phe Glu Glu Phe Phe Cys Ile Cys Ile Gln Leu Leu Asn 450
455 460Lys Thr Trp Lys Glu Met Arg Ala
Thr Ser Glu Asp Phe Asn Lys Val465 470
475 480Met Gln Val Val Lys Glu Gln Val Met Arg Ala Leu
Thr Thr Lys Pro 485 490
495Ser Ser Leu Asp Gln Phe Lys Ser Lys Leu Gln Asn Leu Ser Tyr Thr
500 505 510Glu Ile Leu Lys Ile Arg
Gln Ser Glu Arg Met Asn Gln Glu Asp Phe 515 520
525Gln Ser Arg Pro Ile Leu Glu Leu Lys Glu Lys Ile Gln Pro
Glu Ile 530 535 540Leu Glu Leu Ile Lys
Gln Gln Arg Leu Asn Arg Leu Val Glu Gly Thr545 550
555 560Cys Phe Arg Lys Leu Asn Ala Arg Arg Arg
Gln Asp Lys Phe Trp Tyr 565 570
575Cys Arg Leu Ser Pro Asn His Lys Val Leu His Tyr Gly Asp Leu Glu
580 585 590Glu Ser Pro Gln Gly
Glu Val Pro His Asp Ser Leu Gln Asp Lys Leu 595
600 605Pro Val Ala Asp Ile Lys Ala Val Val Thr Gly Lys
Asp Cys Pro His 610 615 620Met Lys Glu
Lys Gly Ala Leu Lys Gln Asn Lys Glu Val Leu Glu Leu625
630 635 640Ala Phe Ser Ile Leu Tyr Asp
Ser Asn Cys Gln Leu Asn Phe Ile Ala 645
650 655Pro Asp Lys His Glu Tyr Cys Ile Trp Thr Asp Gly
Leu Asn Ala Leu 660 665 670Leu
Gly Lys Asp Met Met Ser Asp Leu Thr Arg Asn Asp Leu Asp Thr 675
680 685Leu Leu Ser Met Glu Ile Lys Leu Arg
Leu Leu Asp Leu Glu Asn Ile 690 695
700Gln Ile Pro Asp Ala Pro Pro Pro Ile Pro Lys Glu Pro Ser Asn Tyr705
710 715 720Asp Phe Val Tyr
Asp Cys Asn 72551591PRTMus musculus 5Met Gly Asn Ala Glu
Ser Gln Asn Val Asp His Glu Phe Tyr Gly Glu 1 5
10 15Lys His Ala Ser Leu Gly Arg Lys His Thr Ser
Arg Ser Leu Arg Leu 20 25
30Ser His Lys Thr Arg Arg Thr Arg His Ala Ser Ser Gly Lys Ala Ile
35 40 45His Arg Asn Ser Glu Val Ser Thr
Arg Ser Ser Ser Thr Pro Ser Ile 50 55
60Pro Gln Ser Leu Ala Glu Asn Gly Leu Glu Pro Phe Ser Gln Glu Gly 65
70 75 80Ala Leu Asp Asp
Phe Gly Asp Pro Ile Trp Val Asp Arg Val Asp Met 85
90 95Gly Leu Arg Pro Val Ser Tyr Thr Asp Ser
Ser Val Thr Pro Ser Val 100 105
110Asp Gly Ser Ile Val Leu Thr Ala Ala Ser Val Gln Ser Met Pro Asp
115 120 125Ser Glu Glu Ser Arg Leu Tyr
Gly Asp Asp Ala Thr Tyr Leu Ala Glu 130 135
140Gly Gly Arg Arg Gln Cys Pro Tyr Thr Ser Asn Gly Pro Thr Phe
Met145 150 155 160Glu Thr
Ala Ser Phe Lys Lys Lys Arg Ser Lys Ser Ala Asp Ile Trp
165 170 175Arg Glu Asp Ser Leu Glu Phe
Ser Leu Ser Asp Leu Ser Gln Glu His 180 185
190Leu Thr Ser Asn Glu Glu Ile Leu Gly Ser Ala Glu Glu Lys
Asp Cys 195 200 205Glu Glu Ala Arg
Gly Met Glu Thr Glu Ala Ser Pro Arg Gln Leu Ser 210
215 220Thr Cys Gln Arg Ala Asn Ser Leu Gly Asp Leu Tyr
Ala Gln Lys Asn225 230 235
240Ser Gly Val Lys Ala Asn Gly Gly Pro Arg Asn Arg Phe Ser Ser Tyr
245 250 255Cys Arg Asn Leu Val
Ser Asp Ile Pro Asp Leu Ala Lys His Lys Met 260
265 270Pro Pro Ala Ala Ala Glu Glu Thr Pro Pro Tyr Ser
Asn Tyr Asn Thr 275 280 285Leu Pro
Cys Arg Lys Ser His Cys Leu Ser Glu Gly Ala Thr Asn Pro 290
295 300Gln Ile Ser Leu Ser Lys Ser Met Gln Gly Arg
Arg Ala Lys Thr Thr305 310 315
320Gln Asp Val Asn Thr Gly Glu Gly Ser Glu Phe Ala Asp Ser Gly Ile
325 330 335Glu Gly Ala Thr
Thr Asp Thr Asp Leu Leu Ser Arg Arg Ser Asn Ala 340
345 350Thr Asn Ser Ser Tyr Ser Pro Pro Thr Gly Arg
Ala Phe Val Gly Ser 355 360 365Asp
Ser Gly Ser Ser Ser Thr Gly Asp Arg Ala Arg Gln Gly Val Tyr 370
375 380Glu Asn Phe Arg Arg Glu Leu Glu Met Ser
Thr Thr Asn Ser Glu Ser385 390 395
400Leu Glu Glu Ala Gly Ser Ala His Ser Asp Glu Gln Ser Ser Gly
Thr 405 410 415Leu Ser Ser
Pro Gly Gln Ser Asp Ile Leu Leu Thr Ala Ala Gln Gly 420
425 430Thr Val Arg Lys Ala Gly Ala Leu Ala Val
Lys Asn Phe Leu Val His 435 440
445Lys Lys Asn Lys Lys Val Glu Ser Ala Thr Arg Arg Lys Trp Lys His 450
455 460Tyr Trp Val Ser Leu Lys Gly Cys
Thr Leu Phe Phe Tyr Glu Thr Asp465 470
475 480Gly Arg Ser Gly Ile Asp His Asn Ser Val Pro Lys
His Ala Val Trp 485 490
495Val Glu Asn Ser Ile Val Gln Ala Val Pro Glu His Pro Lys Lys Asp
500 505 510Phe Val Phe Cys Leu Ser
Asn Ser Leu Gly Asp Ala Phe Leu Phe Gln 515 520
525Thr Thr Ser Gln Thr Glu Leu Glu Asn Trp Ile Thr Ala Ile
His Ser 530 535 540Ala Cys Ala Ala Ala
Val Ala Arg His His His Lys Glu Asp Thr Leu545 550
555 560Arg Leu Leu Lys Ser Glu Ile Lys Lys Leu
Glu Gln Lys Ile Asp Met 565 570
575Asp Glu Lys Met Lys Lys Met Gly Glu Met Gln Leu Ser Ser Val Thr
580 585 590Asp Ser Lys Lys Lys
Lys Thr Ile Leu Asp Gln Ile Phe Val Trp Glu 595
600 605Gln Asn Leu Glu Gln Phe Gln Met Asp Leu Phe Arg
Phe Arg Cys Tyr 610 615 620Leu Ala Ser
Leu Gln Gly Gly Glu Leu Pro Asn Pro Lys Arg Leu Leu625
630 635 640Ala Phe Ala Ser Arg Pro Thr
Lys Val Ala Met Gly Arg Leu Gly Ile 645
650 655Phe Ser Val Ser Ser Phe His Ala Leu Val Ala Ala
Arg Thr Gly Glu 660 665 670Ile
Gly Val Arg Arg Arg Thr Gln Ala Met Ser Arg Ser Ala Ser Lys 675
680 685Arg Arg Ser Arg Phe Ser Ser Leu Trp
Gly Leu Asp Thr Thr Ser Lys 690 695
700Lys Lys Gln Gly Arg Pro Thr Ile Asn Gln Val Phe Gly Glu Gly Thr705
710 715 720Asp Ala Val Lys
Arg Ser Leu Glu Gly Ile Phe Asp Asp Thr Val Pro 725
730 735Asp Gly Lys Arg Glu Lys Glu Val Val Leu
Pro Ser Val His Gln His 740 745
750Asn Pro Asp Cys Asp Ile Trp Val His Glu Tyr Phe Thr Pro Ser Trp
755 760 765Phe Cys Leu Pro Asn Asn Gln
Pro Ala Leu Thr Val Val Arg Pro Gly 770 775
780Asp Thr Ala Arg Asp Thr Leu Glu Leu Ile Cys Lys Thr His Gln
Leu785 790 795 800Asp His
Ser Ala His Tyr Leu Arg Leu Lys Phe Leu Met Glu Asn Arg
805 810 815Val Gln Phe Tyr Ile Pro Gln
Pro Glu Glu Asp Ile Tyr Glu Leu Leu 820 825
830Tyr Lys Glu Ile Glu Ile Cys Pro Lys Val Thr Gln Asn Ile
His Ile 835 840 845Glu Lys Ser Asp
Ala Ala Ala Asp Asn Tyr Gly Phe Leu Leu Ser Ser 850
855 860Val Asp Glu Asp Gly Ile Arg Arg Leu Tyr Val Asn
Ser Val Lys Glu865 870 875
880Thr Gly Leu Ala Ser Lys Lys Gly Leu Lys Ala Gly Asp Glu Ile Leu
885 890 895Glu Ile Asn Asn Arg
Ala Ala Gly Thr Leu Asn Ser Ser Met Leu Lys 900
905 910Asp Phe Leu Ser Gln Pro Ser Leu Gly Leu Leu Val
Arg Thr Tyr Pro 915 920 925Glu Pro
Glu Gly Gly Val Glu Leu Leu Glu Asn Pro Pro His Arg Val 930
935 940Asp Gly Pro Val Asp Leu Gly Glu Ser Pro Leu
Ala Phe Leu Thr Ser945 950 955
960Asn Pro Gly His Ser Leu Ser Ser Glu Gln Gly Ser Ser Ala Glu Thr
965 970 975Ala Pro Glu Glu
Gly Glu Gly Pro Asp Leu Glu Ser Ser Asp Glu Thr 980
985 990Asp His Ser Ser Lys Ser Thr Glu Gln Val Ala
Ala Phe Cys Arg Ser 995 1000 1005Leu
His Glu Met Ser Pro Ser Asp Ser Ser Pro Ser Pro Gln Asp Ala 1010
1015 1020Thr Ser Pro Gln Leu Ala Thr Thr Arg Gln
Leu Ser Asp Ala Asp Lys1025 1030 1035
1040Leu Arg Lys Val Ile Cys Glu Leu Leu Glu Thr Glu Arg Thr Tyr
Val 1045 1050 1055Lys Asp Leu
Asn Cys Leu Met Glu Arg Tyr Leu Lys Pro Leu Gln Lys 1060
1065 1070Glu Thr Phe Leu Thr Gln Asp Glu Leu Asp
Val Leu Phe Gly Asn Leu 1075 1080
1085Thr Glu Met Val Glu Phe Gln Val Glu Phe Leu Lys Thr Leu Glu Asp
1090 1095 1100Gly Val Arg Leu Val Pro Asp
Leu Glu Lys Leu Glu Lys Val Asp Gln1105 1110
1115 1120Phe Lys Lys Val Leu Phe Ser Leu Gly Gly Ser Phe
Leu Tyr Tyr Ala 1125 1130
1135Asp Arg Phe Lys Leu Tyr Ser Ala Phe Cys Ala Ser His Thr Lys Val
1140 1145 1150Pro Lys Val Leu Val Lys
Ala Lys Thr Asp Thr Ala Phe Lys Ala Phe 1155 1160
1165Leu Asp Ala Gln Asn Pro Arg Gln Gln His Ser Ser Thr Leu
Glu Ser 1170 1175 1180Tyr Leu Ile Lys Pro
Ile Gln Arg Val Leu Lys Tyr Pro Leu Leu Leu1185 1190
1195 1200Arg Glu Leu Phe Ala Leu Thr Asp Ala Glu
Ser Glu Glu His Tyr His 1205 1210
1215Leu Asp Val Ala Ile Lys Thr Met Asn Lys Val Ala Ser His Ile Asn
1220 1225 1230Glu Met Gln Lys Ile
His Glu Glu Phe Gly Ala Val Phe Asp Gln Leu 1235
1240 1245Ile Ala Glu Gln Thr Gly Glu Lys Lys Glu Val Ala
Asp Leu Ser Met 1250 1255 1260Gly Asp Leu
Leu Leu His Thr Ser Val Ile Trp Leu Asn Pro Pro Ala1265
1270 1275 1280Ser Leu Gly Lys Trp Lys Lys
Glu Pro Glu Leu Ala Ala Phe Val Phe 1285
1290 1295Lys Thr Ala Val Val Leu Val Tyr Lys Asp Gly Ser
Lys Gln Lys Lys 1300 1305 1310Lys
Leu Val Gly Ser His Arg Leu Ser Ile Tyr Glu Glu Trp Asp Pro 1315
1320 1325Phe Arg Phe Arg His Met Ile Pro Thr
Glu Ala Leu Gln Val Arg Ala 1330 1335
1340Leu Pro Ser Ala Asp Ala Glu Ala Asn Ala Val Cys Glu Ile Val His1345
1350 1355 1360Val Lys Ser Glu
Ser Glu Gly Arg Pro Glu Arg Val Phe His Leu Cys 1365
1370 1375Cys Ser Ser Pro Glu Ser Arg Lys Asp Phe
Leu Lys Ser Val His Ser 1380 1385
1390Ile Leu Arg Asp Lys His Arg Arg Gln Leu Leu Lys Thr Glu Ser Leu
1395 1400 1405Pro Ser Ala Gln Gln Tyr Val
Pro Phe Gly Gly Lys Arg Leu Cys Ala 1410 1415
1420Leu Lys Gly Ala Arg Pro Ala Met Ser Arg Ala Val Ser Ala Pro
Ser1425 1430 1435 1440Lys Ser
Leu Gly Arg Arg Arg Arg Arg Leu Ala Arg Asn Arg Phe Thr
1445 1450 1455Ile Asp Ser Asp Ala Ile Ser
Ala Ser Ser Pro Glu Lys Glu Pro Gln 1460 1465
1470Gln Pro Ala Gly Gly Gly Asp Thr Asp Arg Trp Val Glu Glu
Gln Phe 1475 1480 1485Asp Leu Ala Gln
Tyr Glu Glu Gln Asp Asp Ile Lys Glu Thr Asp Ile 1490
1495 1500Leu Ser Asp Asp Asp Glu Phe Cys Glu Ser Leu Lys
Gly Ala Ser Val1505 1510 1515
1520Asp Arg Asp Leu Gln Glu Gln Leu Gln Ala Ala Ser Ile Ser Gln Arg
1525 1530 1535Ala Arg Gly Arg Arg
Thr Leu Asp Ser His Ala Ser Arg Met Thr Gln 1540
1545 1550Leu Lys Lys Gln Ala Ala Leu Ser Gly Ile Asn Gly
Gly Leu Glu Ser 1555 1560 1565Ala Ser
Glu Glu Val Ile Trp Val Arg Arg Glu Asp Phe Ala Pro Ser 1570
1575 1580Arg Lys Leu Asn Thr Glu Ile1585
159061591PRTHomo sapiens 6Met Gly Asn Ala Glu Ser Gln His Val Glu His Glu
Phe Tyr Gly Glu 1 5 10
15Lys His Ala Ser Leu Gly Arg Asn Asp Thr Ser Arg Ser Leu Arg Leu
20 25 30Ser His Lys Thr Arg Arg Thr
Arg His Ala Ser Ser Gly Lys Val Ile 35 40
45His Arg Asn Ser Glu Val Ser Thr Arg Ser Ser Ser Thr Pro Ser
Ile 50 55 60Pro Gln Ser Leu Ala Glu
Asn Gly Leu Glu Pro Phe Ser Gln Asp Gly 65 70
75 80Thr Leu Glu Asp Phe Gly Ser Pro Ile Trp Val
Asp Arg Val Asp Met 85 90
95Gly Leu Arg Pro Val Ser Tyr Thr Asp Ser Ser Val Thr Pro Ser Val
100 105 110Asp Ser Ser Ile Val Leu
Thr Ala Ala Ser Val Gln Ser Met Pro Asp 115 120
125Thr Glu Glu Ser Arg Leu Tyr Gly Asp Asp Ala Thr Tyr Leu
Ala Glu 130 135 140Gly Gly Arg Arg Gln
His Ser Tyr Thr Ser Asn Gly Pro Thr Phe Met145 150
155 160Glu Thr Ala Ser Phe Lys Lys Lys Arg Ser
Lys Ser Ala Asp Ile Trp 165 170
175Arg Glu Asp Ser Leu Glu Phe Ser Leu Ser Asp Leu Ser Gln Glu His
180 185 190Leu Thr Ser Asn Glu
Glu Ile Leu Gly Ser Ala Glu Glu Lys Asp Cys 195
200 205Glu Glu Ala Arg Gly Met Glu Thr Arg Ala Ser Pro
Arg Gln Leu Ser 210 215 220Thr Cys Gln
Arg Ala Asn Ser Leu Gly Asp Leu Tyr Ala Gln Lys Asn225
230 235 240Ser Gly Val Thr Ala Asn Met
Gly Pro Gly Ser Lys Phe Ala Gly Tyr 245
250 255Cys Arg Asn Leu Val Ser Asp Ile Pro Asn Leu Ala
Asn His Lys Met 260 265 270Pro
Pro Ala Ala Ala Glu Glu Thr Pro Pro Tyr Ser Asn Tyr Asn Thr 275
280 285Leu Pro Cys Arg Lys Ser His Cys Leu
Ser Glu Gly Ala Thr Asn Pro 290 295
300Gln Ile Ser His Ser Asn Ser Met Gln Gly Arg Arg Ala Lys Thr Thr305
310 315 320Gln Asp Val Asn
Ala Gly Glu Gly Ser Glu Phe Ala Asp Ser Gly Ile 325
330 335Glu Gly Ala Thr Thr Asp Thr Asp Leu Leu
Ser Arg Arg Ser Asn Ala 340 345
350Thr Asn Ser Ser Tyr Ser Pro Thr Thr Gly Arg Ala Phe Val Gly Ser
355 360 365Asp Ser Gly Ser Ser Ser Thr
Gly Asp Ala Ala Arg Gln Gly Val Tyr 370 375
380Glu Asn Phe Arg Arg Glu Leu Glu Met Ser Thr Thr Asn Ser Glu
Ser385 390 395 400Leu Glu
Glu Ala Gly Ser Ala His Ser Asp Glu Gln Ser Ser Gly Thr
405 410 415Leu Ser Ser Pro Gly Gln Ser
Asp Ile Leu Leu Thr Ala Ala Gln Gly 420 425
430Thr Val Arg Lys Ala Gly Ala Leu Ala Val Lys Asn Phe Leu
Val His 435 440 445Lys Lys Asn Lys
Lys Val Glu Ser Ala Thr Arg Arg Lys Trp Lys His 450
455 460Tyr Trp Val Ser Leu Lys Gly Cys Thr Leu Phe Phe
Tyr Glu Ser Asp465 470 475
480Gly Arg Ser Gly Ile Asp His Asn Ser Ile Pro Lys His Ala Val Trp
485 490 495Val Glu Asn Ser Ile
Val Gln Ala Val Pro Glu His Pro Lys Lys Asp 500
505 510Phe Val Phe Cys Leu Ser Asn Ser Leu Gly Asp Ala
Phe Leu Phe Gln 515 520 525Thr Thr
Ser Gln Thr Glu Leu Glu Asn Trp Ile Thr Ala Ile His Ser 530
535 540Ala Cys Ala Thr Ala Val Ala Arg His His His
Lys Glu Asp Thr Leu545 550 555
560Arg Leu Leu Lys Ser Glu Ile Lys Lys Leu Glu Gln Lys Ile Asp Met
565 570 575Asp Glu Lys Met
Lys Lys Met Gly Glu Met Gln Leu Ser Ser Val Thr 580
585 590Asp Ser Lys Lys Lys Lys Thr Ile Leu Asp Gln
Ile Phe Val Trp Glu 595 600 605Gln
Asn Leu Glu Gln Phe Gln Met Asp Leu Phe Arg Phe Arg Cys Tyr 610
615 620Leu Ala Ser Leu Gln Gly Gly Glu Leu Pro
Asn Pro Lys Arg Leu Leu625 630 635
640Ala Phe Ala Ser Arg Pro Thr Lys Val Ala Met Gly Arg Leu Gly
Ile 645 650 655Phe Ser Val
Ser Ser Phe His Ala Leu Val Ala Ala Arg Thr Gly Glu 660
665 670Thr Gly Val Arg Arg Arg Thr Gln Ala Met
Ser Arg Ser Ala Ser Lys 675 680
685Arg Arg Ser Arg Phe Ser Ser Leu Trp Gly Leu Asp Thr Thr Ser Lys 690
695 700Lys Lys Gln Gly Arg Pro Ser Ile
Asn Gln Val Phe Gly Glu Gly Thr705 710
715 720Glu Ala Val Lys Lys Ser Leu Glu Gly Ile Phe Asp
Asp Ile Val Pro 725 730
735Asp Gly Lys Arg Glu Lys Glu Val Val Leu Pro Asn Val His Gln His
740 745 750Asn Pro Asp Cys Asp Ile
Trp Val His Glu Tyr Phe Thr Pro Ser Trp 755 760
765Phe Cys Leu Pro Asn Asn Gln Pro Ala Leu Thr Val Val Arg
Pro Gly 770 775 780Asp Thr Ala Arg Asp
Thr Leu Glu Leu Ile Cys Lys Thr His Gln Leu785 790
795 800Asp His Ser Ala His Tyr Leu Arg Leu Lys
Phe Leu Ile Glu Asn Lys 805 810
815Met Gln Leu Tyr Val Pro Gln Pro Glu Glu Asp Ile Tyr Glu Leu Leu
820 825 830Tyr Lys Glu Ile Glu
Ile Cys Pro Lys Val Thr His Ser Ile His Ile 835
840 845Glu Lys Ser Asp Thr Ala Ala Asp Thr Tyr Gly Phe
Ser Leu Ser Ser 850 855 860Val Glu Glu
Asp Gly Ile Arg Arg Leu Tyr Val Asn Ser Val Lys Glu865
870 875 880Thr Gly Leu Ala Ser Lys Lys
Gly Leu Lys Ala Gly Asp Glu Ile Leu 885
890 895Glu Ile Asn Asn Arg Ala Ala Asp Ala Leu Asn Ser
Ser Met Leu Lys 900 905 910Asp
Phe Leu Ser Gln Pro Ser Leu Gly Leu Leu Val Arg Thr Tyr Pro 915
920 925Glu Leu Glu Glu Gly Val Glu Leu Leu
Glu Ser Pro Pro His Arg Val 930 935
940Asp Gly Pro Ala Asp Leu Asp Glu Ser Pro Leu Ala Phe Leu Thr Ser945
950 955 960Asn Pro Gly His
Ser Leu Cys Ser Glu Gln Gly Ser Ser Ala Glu Thr 965
970 975Ala Pro Glu Glu Thr Glu Gly Pro Asp Leu
Glu Ser Ser Asp Glu Thr 980 985
990Asp His Ser Ser Lys Ser Thr Glu Gln Val Ala Ala Phe Cys Arg Ser
995 1000 1005Leu His Glu Met Asn Pro Ser
Asp Gln Asn Pro Ser Pro Gln Asp Ser 1010 1015
1020Thr Gly Pro Gln Leu Ala Thr Met Arg Gln Leu Ser Asp Ala Asp
Asn1025 1030 1035 1040Val Arg
Lys Val Ile Cys Glu Leu Leu Glu Thr Glu Arg Thr Tyr Val
1045 1050 1055Lys Asp Leu Asn Cys Leu Met
Glu Arg Tyr Leu Lys Pro Leu Gln Lys 1060 1065
1070Glu Thr Phe Leu Thr Gln Asp Glu Leu Asp Val Leu Phe Gly
Asn Leu 1075 1080 1085Thr Glu Met Val
Glu Phe Gln Val Glu Phe Leu Lys Thr Leu Glu Asp 1090
1095 1100Gly Val Arg Leu Val Pro Asp Leu Glu Lys Leu Glu
Lys Val Asp Gln1105 1110 1115
1120Phe Lys Lys Val Leu Phe Ser Leu Gly Gly Ser Phe Leu Tyr Tyr Ala
1125 1130 1135Asp Arg Phe Lys Leu
Tyr Ser Ala Phe Cys Ala Ile His Thr Lys Val 1140
1145 1150Pro Lys Val Leu Val Lys Ala Lys Thr Asp Thr Ala
Phe Lys Ala Phe 1155 1160 1165Leu Asp
Ala Gln Asn Pro Lys Gln Gln His Ser Ser Thr Leu Glu Ser 1170
1175 1180Tyr Leu Ile Lys Pro Ile Gln Arg Ile Leu Lys
Tyr Pro Leu Leu Leu1185 1190 1195
1200Arg Glu Leu Phe Ala Leu Thr Asp Ala Glu Ser Glu Glu His Tyr His
1205 1210 1215Leu Asp Val Ala
Ile Lys Thr Met Asn Lys Val Ala Ser His Ile Asn 1220
1225 1230Glu Met Gln Lys Ile His Glu Glu Phe Gly Ala
Val Phe Asp Gln Leu 1235 1240 1245Ile
Ala Glu Gln Thr Gly Glu Lys Lys Glu Val Ala Asp Leu Ser Met 1250
1255 1260Gly Asp Leu Leu Leu His Thr Thr Val Ile
Trp Leu Asn Pro Pro Ala1265 1270 1275
1280Ser Leu Gly Lys Trp Lys Lys Glu Pro Glu Leu Ala Ala Phe Val
Phe 1285 1290 1295Lys Thr Ala
Val Val Leu Val Tyr Lys Asp Gly Ser Lys Gln Lys Lys 1300
1305 1310Lys Leu Val Gly Ser His Arg Leu Ser Ile
Tyr Glu Asp Trp Asp Pro 1315 1320
1325Phe Arg Phe Arg His Met Ile Pro Thr Glu Ala Leu Gln Val Arg Ala
1330 1335 1340Leu Ala Ser Ala Asp Ala Glu
Ala Asn Ala Val Cys Glu Ile Val His1345 1350
1355 1360Val Lys Ser Glu Ser Glu Gly Arg Pro Glu Arg Val
Phe His Leu Cys 1365 1370
1375Cys Ser Ser Pro Glu Ser Arg Lys Asp Phe Leu Lys Ala Val His Ser
1380 1385 1390Ile Leu Arg Asp Lys His
Arg Arg Gln Leu Leu Lys Thr Glu Ser Leu 1395 1400
1405Pro Ser Ser Gln Gln Tyr Val Pro Phe Gly Gly Lys Arg Leu
Cys Ala 1410 1415 1420Leu Lys Gly Ala Arg
Pro Ala Met Ser Arg Ala Val Ser Ala Pro Ser1425 1430
1435 1440Lys Ser Leu Gly Arg Arg Arg Arg Arg Leu
Ala Arg Asn Arg Phe Thr 1445 1450
1455Ile Asp Ser Asp Ala Val Ser Ala Ser Ser Pro Glu Lys Glu Ser Gln
1460 1465 1470Gln Pro Pro Gly Gly
Gly Asp Thr Asp Arg Trp Val Glu Glu Gln Phe 1475
1480 1485Asp Leu Ala Gln Tyr Glu Glu Gln Asp Asp Ile Lys
Glu Thr Asp Ile 1490 1495 1500Leu Ser Asp
Asp Asp Glu Phe Cys Glu Ser Val Lys Gly Ala Ser Val1505
1510 1515 1520Asp Arg Asp Leu Gln Glu Arg
Leu Gln Ala Thr Ser Ile Ser Gln Arg 1525
1530 1535Glu Arg Gly Arg Lys Thr Leu Asp Ser His Ala Ser
Arg Met Ala Gln 1540 1545 1550Leu
Lys Lys Gln Ala Ala Leu Ser Gly Ile Asn Gly Gly Leu Glu Ser 1555
1560 1565Ala Ser Glu Glu Val Ile Trp Val Arg
Arg Glu Asp Phe Ala Pro Ser 1570 1575
1580Arg Lys Leu Asn Thr Glu Ile1585 159079PRTArtificial
SequenceDescription of Artificial Sequence Synthetic HA-tag sequence
7Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5880PRTMus musculus
8Glu Arg His Gly Val Ala Phe Tyr Asn Phe Gly Gly Ser Glu Ala Gln 1
5 10 15His Leu Thr Leu Gln Ile
Gly Asp Val Val Arg Ile Gln Glu Thr Gly 20
25 30Gly Asp Trp Tyr Arg Gly Tyr Leu Ile Lys His Lys Leu
Ser Gln Gly 35 40 45Ile Phe Pro
Thr Ser Phe Ile His Leu Lys Glu Val Thr Val Glu Lys 50
55 60Arg Arg Asn Ile Glu Asn Ile Ile Pro Ala Glu Ile
Pro Leu Ala Gln 65 70 75
80
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