CN101315385A - A drug target for inflammation and blood clots, and drugs against the target - Google Patents

Abstract

The present invention provides the use of protein Naf1 and/or PSGL-1 as a target for screening drugs for treating inflammatory and/or thrombotic diseases and as a drug target for inflammatory and/or thrombotic disease drugs, protein Naf1 and/or PSGL -1 specific antibody and drugs containing specific antibodies to protein Naf1 and/or PSGL-1, as well as drugs targeting protein Naf1 and/or PSGL-1 for the treatment of inflammatory and/or thrombotic diseases It also provides a peptide segment capable of blocking the combination of Naf1 and the intracellular region of PSGL-1 and the application of the above peptide segment for inflammation and/or thrombosis disease drugs. The peptides provided by the invention can weaken the adhesion of white blood cells, and can inhibit the inflammatory response in the body, and at the same time, provide new methods for the treatment of diseases such as immune injury, inflammation, thrombosis, and tumor metastasis.

Description

A drug target for inflammation and blood clots, and drugs targeting it

technical field

The present invention relates to the field of biomedicine, in particular, the present invention relates to a drug target for inflammation and thrombus, and the drug for the target.

Background technique

In recent years, cell adhesion molecules and their research have become a hot spot, and they regulate the interaction between cells and extracellular matrix, as well as their physiological and biological functions in cell growth and development, immune response, inflammatory response, blood coagulation, tissue repair and tumor metastasis. The important role in the pathological process is also given high attention.

As a family of cell adhesion molecules, selectins include P-selectin, E-selectin and L-selectin, whose main function is to mediate the adhesion and interaction of activated platelets, endothelial cells and leukocytes, tumor cells, etc. And it is closely related to immune injury, inflammation, thrombosis and tumor metastasis. P-selectin (P-selectin) is a member of the selectin family, a membrane glycoprotein with a molecular weight of 140kDa, located in the alpha granules of platelets and the endothelial cell rod tubular (Weibel-palade) bodies. The full length consists of 789 amino acid residues, 730 amino acids at the N-terminal constitute the extracellular region, 24 amino acids at the C-terminal constitute the transmembrane region, and 35 amino acids constitute the short cytoplasmic tail. The extracellular region includes a lectin-like region, an epidermal growth factor-like region (EGF region), and nine shorter repeats (SCR regions) known as complement regulatory proteins. Among them, the lectin-like region can bind certain carbohydrates, which is the site of selectin binding ligand. The cytoplasmic domain may be associated with the cytoskeleton. P-selectin recognizes some oligosaccharide groups, mainly sialylated Lewis oligosaccharides (sialyl Lweisx, sLex, CD15s) or similar structural molecules.

P-selectin glycoprotein ligand 1 (P-selectin glycoprotein ligand 1, PSGL-1, CD162) is a transmembrane glycoprotein with a homodimer structure, expressed on the surface of almost all leukocytes, and is the most The most detailed list of selectin ligands was obtained. PSGL-1 has a high affinity with P-selectin and is also a ligand of L-selectin and E-selectin. PSGL-1 is a highly sialylated mucin, the relative molecular weight of each monomer is about 120kDa, and its protein backbone has 1-3 N-linked glycans and a large number of sialylated O-glycans. Sialylation (sialyl kx) and O-glycans (O-glycans) structure play an important role in the function of PSGL-1.

After activation of platelets and endothelial cells, P-selectin is transported to the cell membrane (Blann AD, Nadar SK, Lip GY. Eur Heart. J. 2003; 24: 2166-2179; McEver RP. Selectins. Curr. Opin. Immunol. 1994;6:75-84). P-selectin and its ligand P-selectin ligand PSGL-1 on leukocytes combine with each other to initiate the adhesion process between leukocytes and endothelial cells or platelets.

Although the adhesion between leukocytes and endothelial cells is an important part of the body's barrier, excessive adhesion of leukocytes to endothelial cells is harmful. For example, in ischemia/reperfusion injury, increased cell adhesion after reperfusion triggers increased inflammatory exudation, causing greater tissue and organ damage than ischemia. The occurrence and development of thrombus involves many complex pathophysiological reactions in the body. Thrombosis is not only related to some coagulation factors in blood plasma, but also closely related to the adhesion between leukocytes, endothelial cells and platelets mediated by P-selectin (Li N, Hu H, LindqvlstM, eta1.ArterioselerThromb Vasc Biol.2000.20: 2702～2708). Adhesion molecule P-selectin-mediated cell adhesion between platelets and endothelial cells, and between these cells and leukocytes is a basic phenomenon and a common process in the pathophysiological process of inflammation and thrombosis. Inhibition and regulation of P-selectin and its mediated cell adhesion and action through various pathways and drugs have proved to be an effective means of prevention and treatment of inflammatory and thrombotic diseases.

The most direct anti-cell adhesion is to block the binding between receptor ligands. Monoclonal antibodies against P-selectin accelerated thrombus lysis in a primate model of thrombosis and reduced thrombosis and ischemic injury in a model of coronary endothelial injury. In addition, in the rat model of venous thrombosis, the monoclonal antibody against P-selectin not only has effective antithrombotic effect, but also can reduce the infiltration of inflammatory cells. At present, anti-P-selectin antibodies can be used for localized diagnosis and targeted therapy of thrombus and/or inflammation through introduction, and have been successfully used in preclinical models. Antibodies against P-selectin and humanized monoclonal antibodies have also been researched and developed, such as recombinant soluble P-selectin glycoprotein ligand-1 and small molecule selectin inhibitors have entered clinical trials, anti-human P-selectin agglutination The monoclonal antibody of the protein-epidermal growth factor functional domain has also applied for a Chinese patent (application number 200310108522.5).

Since selectins can recognize the sialylated Lewis sugar x (sLex) structure, sLex mimics are rapidly developed due to their high affinity, such as TBC1269, with a molecular weight of less than 1000, which has entered II as a candidate drug for the treatment of asthma and psoriasis Phase clinical. Non-carbohydrate molecules such as OC229648 have stronger inhibitory effects than TBC1269 in vitro and have been successfully applied to animal models of inflammation. Therefore, anti-adhesion therapy will be a prevention and treatment method along with the in-depth and elucidated research on P-selectin and inflammatory and thrombotic diseases, and has good clinical application value and prospects.

In the initial adhesion process, PSGL-1 can not only establish the connection between leukocytes and endothelial cells through the interaction with selectin, but also act as a signal receptor to transmit signals into cells to cause the activation of leukocytes. P-selectin combined with PSGL-1 can activate integrin α _M β ₂ (Ma YQ, Plow EF, Geng JG. Blood. 2004; 104: 2549-2556), but the specific molecular mechanism of this activation pathway is still unclear.

Integrins are a large class of transmembrane heterodimeric adhesion molecules expressed on the surface of almost all eukaryotic cells and play an important role in cell-to-cell and cell-matrix interactions. The β ₂ family of integrins is often called leukocyte integrins, including four members, consisting of four different α subunits (α _M , α _L , α _X and α _D ) and a common β ₂ subunit , wherein the β ₂ integrins expressed on neutrophils are mainly α _M β ₂ (CD11bCD18, Mac-1) and α _L β ₂ (CD11cCD18, LFA-1). These two integrins bind a variety of ligands, including fibrinogen, intercellular adhesion molecules (ICAM-1, CD54), coagulation factor X, complement pathway product C3bi, and several extracellular matrix proteins. The binding of Mac-1 and LFA-1 to the ligand depends on the increase of leukocyte affinity to the ligand and the increased affinity of its own aggregation when the leukocyte is activated, and also depends on the distribution of the molecules stored in the cell to the cell surface . The activation of _β2 integrin plays a key role in the adhesion of leukocytes on the surface of vascular endothelium in inflammatory and thrombotic diseases.

Studying the pathway and mechanism of PSGL-1 transmitting signals into cells can find new mediators and molecular targets that are closely related to the occurrence and development of inflammatory diseases and thrombotic diseases.

Contents of the invention

The inventors of the present invention have studied the signal transduction mechanism and in vivo function of P-selectin and its ligand PSGL-1, and the research results show that:

1. Phosphatidylinositol 3-position kinase (PI3K) is involved in the signaling pathway in the activation process. After PSGL-1 binds P-selectin, the p85 subunit of PI3K can bind to the intracellular region of PSGL-1. And can enhance the kinase activity of PI3K;

2. After the combination of Naf1 and the intracellular region of PSGL-1, the tyrosine kinase Src is activated under the action of P-selectin, and then phosphorylates Naf1, and aggregates the p85 subunit of PI3K to bind to Naf1;

3. Naf1 can act as an adapter protein, linking the intracellular region of PSGL-1 and the p85 subunit of PI3K.

Because, P-selectin-mediated cell adhesion between platelets and endothelial cells, and between these cells and leukocytes is a basic phenomenon and common process in the pathophysiological process of inflammation and thrombosis. Inhibition and regulation of P-selectin and its mediated cell adhesion and action through various pathways and drugs have proved to be an effective means of prevention and treatment of inflammatory and thrombotic diseases. At the same time, anti-adhesion therapy, as a preventive method, has good clinical application value and prospect. Moreover, the activation of _β2 integrins plays a key role in the adhesion of leukocytes to the vascular endothelial surface in inflammatory and thrombotic diseases.

Therefore, the inventors of the present invention designed a peptide PSGL-1 intracellular region peptide that can inhibit the activation of _β2 integrin caused by P-selectin according to the above research results, and verified the peptide It is determined that the peptide can block P-selectin from activating _β2 integrin, so it can be further used as a medicine for inflammation and thrombosis.

One object of the present invention is to provide the use of proteins Naf1 and PSGL-1 as targets for screening drugs for treating inflammatory and/or thrombotic diseases and as drug targets for drugs for inflammatory and/or thrombotic diseases.

Another object of the present invention is to provide a specific antibody for protein Naf1 and/or PSGL-1 and a specific antibody containing protein Naf1 and/or PSGL-1 for treating inflammation and/or thrombosis Medicines for diseases.

Another object of the present invention is to provide a drug for treating inflammatory and/or thrombotic diseases with the protein Naf1 and/or PSGL-1 as the drug target.

Another object of the present invention is to provide a peptide that can block the binding of the intracellular region of Naf1 and PSGL-1 and the application of the above peptide for inflammatory and/or thrombotic diseases.

Another object of the present invention is to provide a polypeptide derived from the 17th exon of Naf1.

The present invention studies the signal transduction mechanism and in vivo function of P-selectin combined with its ligand PSGL-1. Aiming at this signal transduction pathway, Naf1 and/or PSGL-1 can be used as targets for development of inflammation and thrombosis Drugs for diseases; specific antibodies to Naf1 or PSGL-1, and drugs for inflammatory and thrombotic diseases containing the specific antibodies; the peptide Tat-PSGL-1 provided can block Naf1 and PSGL-1 At the same time of combination, it can also weaken the adhesion of white blood cells and inhibit the inflammatory response in the body, thus providing a new method for the treatment of diseases such as immune injury, inflammation, thrombosis and tumor metastasis.

Description of drawings

Figure 1 shows the mutual association of Naf1α and the intracellular domain of PSGL-1 in yeast Y190. Among them, Figure 1A is the result of the β-galactosidase reporter gene; Figure 1B is the result of the His reporter gene.

Figure 2 shows that Naf1 cDNA was obtained by yeast two-hybrid screening, and Naf1 antibody was prepared.

Among them, Figure 2A is the amino acid sequence of the Naf1 gene and a schematic diagram of the constructed yeast two-hybrid "bait" (bait); Figure 2B is the result of Western blot of the anti-Naf1 polyclonal antibody, respectively detecting HL-60 lysate and neutrophils The lysate and the 293 lysate transfected with pCMV-Naf1 were simultaneously detected with the anti-c-myc monoclonal antibody 9E10 to detect the 293 lysate transfected with pCMV-Naf1 to recognize the same size band, which proved that the polyclonal antibody specifically recognized Naf1.

Figure 3 shows the specific binding between Naf1 and PSGL-1 intracellular region, and the binding site where Naf1 and PSGL-1 bind to each other.

Among them: Figure 3A is the result of incubation of human neutrophil lysate supernatant with glutathione-sepharose pre-loaded with GST protein or fusion protein GST-Naf1 respectively; The fusion protein GST-Naf1 and GST protein on the matrix were eluted and quantified by Coomassie brilliant blue staining; Figure 3C is the co-transfection of PSGL-1 expression plasmid pCDNA3.1-PSGL-1 and Naf1 with c-myc tag in 293 cells The results of expressing plasmid pCMV-Naf1 or single transfection of pCMV-Naf1; Figure 3D is the result of co-immunoprecipitation of the lysed supernatant of HL-60 cells with anti-PSGL-1 polyclonal antibody or rabbit IgG before immunization; Figure 3E is the result of HL- The results of 60 cells stimulated with human IgG or P-Rg; Figure 3F is a schematic diagram of the amino acid sequence of the intracellular region of PSGL-1 and the deletion mutant; Figure 3G is the result of co-transfection of PSGL-1 deletion mutant and Naf1 in 293 cells; Figure 3H is a schematic diagram of point mutations in the intracellular region of PSLG-1, the amino acids in bold (R, N, Y, S, P, T, E) are mutated to alanine (A); Figure 3I is the cotransfection of PSGL-1 in 93 cells The results of deletion mutants and Naf1 or PSGL-1 wild type and Naf1; Figure 3J is a schematic diagram of Naf1 C-terminal deletion mutation; Figure 3K is the result of Naf1 C-terminal deletion mutation and PSGL-1 co-immunoprecipitation.

Figure 4 shows that Naf1 mediates the binding of PSGL-1 and p85.

Wherein, Figure 4A is the result of 293 cells being co-transformed into the vector expressing PSGL-1, p85 and Naf1 or the control empty vector pCMV; Figure 4B is the result of 293 cells being co-transfected into the vector expressing p85 and Naf1 or the control empty vector pCMV; Figure 4C is the result of 293 cells being cotransfected into the vector expressing p85 and Naf1Y552F, Naf1 or the control empty vector pCMV; Figure 4D is the result of 293 cells being cotransfected into the vector expressing PSGL-1, p85 and Naf1 or Naf1Y552F or the control empty vector pCMV result.

Figure 5 shows that PSGL-1/Naf1/p85 forms a complex in human neutrophils, and P-Rg stimulation can activate Src, thereby phosphorylating Naf1 and enhancing PI3K kinase activity.

Wherein, Fig. 5A is human neutrophils respectively incubated with control buffer PBS (-), human IgG (hIgG) or P-selectin Rg (P-Rg) at 10 μg/ml for 20 minutes, and western blot detection results; Fig. 5B After stimulating human neutrophils with human IgG (hIgG) or P-selectin Rg (P-Rg), the results of immunodetection with 3B8; Co-immunoprecipitation of PI3K in the supernatant of leukocyte cell lysate, and then use the immunoprecipitate to decatalyze the substrate phosphatidylinositol (PI) and ³² P-labeled ATP, and the autoradiography of the product, the arrow in the figure is marked as PIP; Figure 5D is the relative value of the spot intensity, and the results of three independent experiments were averaged; Figure 5E is the immunoprecipitation of 3B8 and 5C4 after incubation and lysis of white blood cells and PP2 at room temperature with DMSO, and immunoprecipitation of Naf1 with Naf1 antibody Detection results; Figure 5F shows the treatment of leukocytes is the same as that of 5E, and other experiments are the same as in Figure 5A; Figure 5G shows the results of leukocyte lysis directly after stimulation, and the lysate was immunodetected with Src antibody and anti-Src416 tyrosine phosphorylation antibody.

Fig. 6 shows the result of P-selectin activating α _M β ₂ integrin through Naf1 and p85 pathway.

Among them, Fig. 6A is the detection result of adherent cells of human neutrophils or HL-60 cells; Fig. 6B is the result of immunoblotting detection of anti-Naf1 polyclonal antibodies after electroporation of HL-60 cells with annealed S1 and S4; Co-immunoprecipitation of HL-60 cells transfected with S1 and S4 to detect the combination of PSGL-1 and p85; Figure 6D shows the results of cell adhesion of HL-60 cells transfected with S1 and S4.

Figure 7 shows the results of mutant Naf1Y552F blocking the binding of PSGL-1 and p85 and blocking the signaling pathway of P-selectin activation of αMβ2 integrin.

Among them, Figure 7A is the result of immunodetection with anti-Naf1 antibody after virus particles with Naf1Y552F or LacZ gene infected HL-60; Figure 7B shows the co-immunoprecipitation of virus particles with Naf1Y552F or LacZ gene after infection with HL-60 The result of detecting the combination of PSGL-1 and p85; FIG. 7C is the result of cell adhesion detection after virus particles with Naf1Y552F or LacZ gene infected HL-60.

Figure 8 shows the results of the Tat fusion polypeptide in the intracellular region of PSGL-1 being phagocytosed by cells and blocking the combination of PSGL-1 and Naf1.

Among them, Figure 8A is a schematic diagram of the constructed fusion polypeptide; Figure 8B is the result of confocal microscopy after the fusion polypeptide labeled FITC was incubated with mouse neutrophils; Figure 8C and D are the Ni beads combined with the fusion polypeptide and Human (C) or mouse (D) neutrophil lysate was incubated, and the combined Naf1 was detected by Naf1 polyclonal antibody Western immunoassay; The result of Western detection.

Fig. 9 is a schematic diagram showing that peptides in the intracellular region of PSGL-1 block P-selectin-activated integrins in vitro and in vivo.

Among them, Figure 9A shows the results of adhesion experiments stimulated with hIgG and P-Rg after human neutrophils were incubated with fusion peptides for 30 minutes; Figure 9B shows the results of repeat A using mouse neutrophils and mouse fibrinogen Schematic diagram of the results; Figure 9C is the results of adhesion experiments stimulated with hIgG and P-Rg after human neutrophils were incubated with fusion peptides or antibodies; The results of observing the rolling and adhering white blood cells in the microvessels on the cremaster muscle; Figure 9E is the result of acute peritonitis experiment after the mice were pre-injected intravenously with relevant reagents.

Figure 10 shows that after HL-60 cells express the corresponding virus, the expression of α _M subunit (A), the expression of PSGL-1 (B) and the combination of P-Rg (C )the result of.

Figure 11 is the separation of neutrophils from mice, and after incubation with peptides, the expression and expression of αM subunit (A), PSGL-1 (B) and small Binding of murine P-Rg (C) Results.

Figure 12 shows the results that inhibiting the expression of endogenous Naf1 in HL-60 cells does not affect the expression of PSGL-1 and αM subunit on the cell surface and the combination of P-Rg and PSGL-1.

Among them, Figure 12A is the result of electroporation of S1 or S4 HL-60 cells incubated with the antibody 44a against the αM subunit, and mouse IgG was used as the control; Figure 12B is the control of rabbit IgG, and the fluorescence intensity of the cell surface was measured by flow cytometry Figure 12C is the result of measuring the fluorescence intensity of the cell surface by flow cytometry after incubation of P-Rg and electroporated HL-60 cells with human IgG as a control.

Figure 13 is a schematic diagram of the effect of P-selectin on deep vein thrombosis in P-selectin knockout mice, wherein, C57 is the experimental result of direct ligation of C57 mice, and PKO is the direct ligation of P-selectin KO mice mP-Rg is the experimental result of P-selectin KO mice intravenously injected with mouse P-Rg, 12CA5 is the experimental result of P-selectin KO mice intravenously injected with 12CA5, ^* means p<0.05, ^** means p<0.01 .

Figure 14 shows the results of in vivo injection of Tat-PSGL-1 to inhibit deep vein thrombosis in mice, wherein mice injected with Tat-PSGL-1 Mut or saline were used as controls.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention.

Experimental methods not specified in the following examples are generally carried out according to conventional conditions, such as the conditions and methods described in "Molecular Cloning Experiment Guide (Third Edition)" (J. Sambrook et al., 2003).

Materials used in the present invention:

Transformed human embryonic kidney cell line 293 (CRL-1573) and 293T, and myeloid leukemia cell line HL-60 (CRL-240) were purchased from American Tissue Culture Collection Center (ATCC). The above cell lines were respectively treated with DMEM culture medium (293 and 293T cells) or RPMI-1640 culture medium (HL -60 cells), cultured at 37°C in a 5% CO ₂ incubator.

Anti-human α-Tubulin monoclonal antibody anti-α-Tubulin (clone B 5-1-2), human and mouse IgG were purchased from Sigma (St.Louis, MO). KPL-1 (anti-PSGL-1 monoclonal antibody), CBRM1/5 (αM subunit blocking monoclonal antibody), 2PH1 (mouse PSGL-1 blocking monoclonal antibody), M1/70 (mouse Mac- 1 blocking monoclonal antibody), M17/4 (mouse LFA-1 blocking monoclonal antibody), RB40.34 (mouse P-selectin blocking monoclonal antibody) and subtype control antibodies were purchased from BDPharmingen Company (San Diego, CA). Mab 38 was purchased from Axxora (San Diego, CA). Anti-p85 polyclonal antibody was purchased from Upstate Company (Lake Placid, NY). 9E10 (anti-c-myc monoclonal antibody), IB4 (blocking monoclonal antibody of β2 subunit), OKM1 (non-blocking monoclonal antibody of αM subunit) and 44a (blocking monoclonal antibody of αM subunit) all purchased from the American Tissue Culture Collection Center (ATCC). Human fibrinogen was purchased from Enzyme Research Laboratories (South Bend, IN), the pCDNA3.1/Hygro vector was purchased from Invitrogen, the pCMV-Tag 3B vector was purchased from Stratagene, and other reagents were purchased from Sigma.

Protein 12CA5 (purchased from American Tissue Culture Collection Center, ATCC), which is a HA monoclonal antibody, has the same Fc fragment as mouse P-Rg (mP-Rg), and can be used as a control for mP-Rg in the experiment.

.P.J.Cell Biol.1997；137：743-754.)所述的方法；pCEP4F载体见文献(ZhuX，Mancini M A，Chang K H，Liu C Y，Chen C F，Shan B，Jones D，Yang-Feng T L，Lee W H.Mol Cell Biol.1995；15：5017-5029)。Human P-selectin and receptor globulin fusion protein (P-selectin Rg, P-Rg), G1 (blocking monoclonal antibody for P-selectin to leukocyte adhesion), PS1 (P-selectin to leukocyte adhesion Attached non-blocking monoclonal antibody) see literature (Ma L, Raycroft L, Asa D, Anderson DC, Geng JG.J Biol Chem.1994; 269:27739-27746; Geng JG, Bevilacqua MP, Moore KL, et al. Nature.1990; 343:757-760.); the preparation of rabbit IgG and anti-human PSGL-1 peptide rabbit polyclonal antibody before immunization, using literature (JG, Raub TJ, Baker CA, Sawada GA, Ma L, and Elhammer

.PJCell Biol.1997; 137:743-754.) described method; pCEP4F vector sees literature (ZhuX, Mancini M A, Chang K H, Liu C Y, Chen C F, Shan B, Jones D, Yang-Feng T L, Lee W H. Mol Cell Biol. 1995; 15:5017-5029).

In the following examples of the present invention, P-selectin knockout mice (PKO, purchased from Jackson Laboratory) and control C57 mice (purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences), and other mice not specifically mentioned They were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences and raised at SPF level. See literature for p110δ ^D910A/D910A mutant mice (Okkenhaug K, Bilancio A, Farjot G, Priddle H, Sancho S, PeskettE, Pearce W, Meek SE, Salpekar A, Waterfield MD, Smith AJ, Vanhaesebroeck B. Science. 2002; 297 : 1031-4).

The experimental method adopted in the present invention is specifically as follows:

1. Fusion protein expression and antibody preparation

Referring to the operating manual "GST Gene Fusion System" of Amersham Pharmacia Biotech, the Naf1 cDNA fragment (encoding amino acids 394-629) obtained by yeast two-hybrid was cloned into the vector pGEX-4T-2 (AmershamPharmacia Biotech) to prepare GST fusion protein.

Rabbits were immunized with the constructed GST fusion protein as an immunogen to prepare polyclonal antibodies against Naf1. The specificity of the polyclonal antibodies was confirmed by Western blotting of eukaryotic expression of Naf1α with c-myc-tag.

The specific anti-Y552 tyrosine phosphorylated monoclonal antibody 3B8 of Naf1 and the control monoclonal antibody 5C4 were prepared by immunizing mice with specific peptides and then screened by ELISA method.

In this process, the amino acid sequence of the peptide used is as follows: HLCGAYPYAUPPMPAMVPHH (where U is pY, which is phosphorylated tyrosine), while the amino acid corresponding to U in the control peptide is unphosphorylated tyrosine, Its sequence is as follows: HLCGAYPYAYPPMPAMVPHH.

2. GST pull-down experiment

Isolate human neutrophils, see the literature for the separation method (Ma YQ, Plow EF, Geng JG.Blood.2004; 104: 2549-2556); then separate the human neutrophils obtained in CHAPS lysate in an ice bath ( 3% CHAPS, 20mM Tris, pH 7.4, 150mM NaCl, 0.1% BSA, 1mM PMSF, 2μg/ml aprotinin, 2μg/ml leupeptin, 1μg/ml pepstanin (A), after lysate for 1 hour, the lysate was centrifuged at 12,000g, 4℃ After 10 minutes, collect the lysed supernatant.

At 4°C, the lysed supernatant containing about 10 ⁷ leukocytes was taken and incubated with 10 μl of glutathione-sepharose beads (Amersham Pharmacia Biotech) preloaded with the same amount of GST or GST fusion protein for 4 hours.

After incubation, the glutathione-sepharose beads were washed 3 times with washing solution (0.05% Triton X-100, 20mM Tris, pH7.4, 150mM NaCl), then boiled and eluted with non-reducing 1×SDS loading buffer, and then passed Western blot for detection.

Wherein, the GST or GST fusion protein used is quantified by Coomassie Blue staining after expression and purification.

3. Co-immunoprecipitation and Western blotting

293 cells were co-transfected into the following vector by the calcium phosphate method (Chen C., and Okayama H.. Mol. Cell. Biol. 1987; 7: 2745-2752): pCMV-Naf1 vector, encoding PSGL-1 wild-type or mutant type of vector or the corresponding expression vector.

After 48 hours of transfection, the cells were washed with 1×PBS in an ice bath and incubated in an ice bath lysis buffer (1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl, 0.5 mM EGTA, 10% glycerol ), 1mM PMSF, 2μg/ml aprotinin, 2μg/ml leupeptin, 1μg/ml pepstaninA,) (in the phosphorylation experiments, phosphatase inhibitors 10mM NaF and 1mM Na ₃ VO ₄ were added to the lysate) Lyse for 1 hour, then centrifuge the lysate at 12,000g, 4°C for 10 minutes, and collect the centrifuged supernatant. Supernatant, corresponding antibody and protein A-sepharose beads (Amersham Pharmacia Biotech), incubated at 4°C for 6-8 hours, and then washed with washing buffer (0.05% Triton X-100, 20mM Tris, pH7.4, 150mM NaCl) After washing 3 times, mix the immunoprecipitation complex or the supernatant of the corresponding cell lysate with the reducing 1×SDS loading buffer, boil for 5 minutes, and after electrophoresis by SDS-PAGE, transfer to the PVDF membrane and use the corresponding The primary antibody and HRP-labeled secondary antibody were used for detection, and finally the ECL chemiluminescence kit (Shanghai Pufei Biotechnology Co., Ltd.) was used for color development.

4. Determination of Kinase Activity

The isolated human neutrophils were starved for 2 hours in M199 medium containing 2% fetal bovine serum, and then treated with 10 μg/ml human IgG (hIgG) or P-selectin Rg (P-Rg) at 22°C After 15 minutes of stimulation, the cells were lysed in the lysate (recipe is the same as described in the method "co-immunoprecipitation and Western blotting"), and then the phospholipids were co-immunoprecipitated with anti-PSGL-1 polyclonal antibody (the control used rabbit IgG before immunization) Acyl inositol 3-position kinases (PI3Ks), PI3K kinase activity determination reference literature (Wallasch C., Weiss F.U., Niederfellner G., Jallal B., Issing W., and Ullrich A.EMBO.J.1995; 14:4267 -75) in the method.

5. siRNA inhibition experiment

5.1. siRNA transfection of HL-60

The Nucleofector Device (program T-19) and the Cell Line Specific Nucleofector Kit V (Amaxa, Cologne, Germany) from Amaxa were used for transfection. Wherein, the transfection concentration of annealed dsRNAioligonucleotides (purchased from Ambion, Austin, TX) is 1 μ M, and its sequences are respectively:

S1: 5'-GGAGAAUUCCCGGCUGAAGTT-3'/3'-TTCCUCUUAAGGGCCGACUUC-5';

S4: 5'-GCUUUUGGAAGAGUCCCAGTT-3'/3'-CTCGAAAACCUUCUCAGGGUC-5'

Among them, S4 can inhibit the expression of endogenous Naf1, and S1 as a control cannot inhibit the expression of endogenous Naf1.

Twenty-four hours after transfection, cells were harvested for co-immunoprecipitation and Western blotting, adhesion assays, and flow cytometric analysis.

5.2, 293T cell transfection

Use LIPOFECTAMINE 2000 Reagent (Invitrogen Company) to carry out transfection. Wherein, the transfection concentration of the annealed dsRNAioligonucleotides is 100nM (the sequences are the same as those in 5.1, respectively S1 and S4).

48 hours after transfection, cells were harvested for Western blotting.

6. Cell adhesion experiment

Human neutrophils or HL-60 cells were stimulated with 10 μg/ml hIgG or P-Rg with or without compound (DMSO was used as control), and then the cells were added to the pre-plated fibrinogen ( fibrinogen) in a 96-well plate at 37° C. in a 5% CO ₂ incubator for 20 min.

When inhibited by antibodies, take 10μg P-Rg first and 20μg G1F(ab') ₂ or 20μg PS1F(ab') ₂ in 30μl PBS buffer (1×PBS, 1mM CaCl ₂ , 1mM MgCl ₂ ) Place at room temperature for 15 minutes, and then add to human neutrophils; or human neutrophils were pre-incubated with 10 μg/ml of 44a and its subtype matching antibody 9E10.

When inhibited by compounds, human neutrophils were incubated with 0.1% DMSO, 25 nMPP2, 150 nM Wortmannin or 10 μM LY294002 for 5 min at room temperature before being stimulated with P-Rg. For the operation of cell adhesion, refer to the method described in the literature (MaYQ, Plow EF, Geng JG. Blood. 2004; 104: 2549-2556).

7. Flow cytometry analysis

)。Collect HL-60 cells or transfected HL-60 cells, wash once with 1×PBS, resuspend in PBS buffer (1×PBS, 0.5% BSA, 1mM CaCl ₂ , 1mM MgCl ₂ ), and add 10μg P-Rg/ml or the corresponding antibody at 2 μg/ml; after incubating at 4°C for 1 hour, wash with PBS buffer, resuspend in PBS buffer, and add 2 μg/ml of the corresponding FITC-labeled secondary antibody; Then incubate at 4°C for 1 hour, wash with PBS buffer, collect the cells and resuspend in PBS buffer, and use the cells for flow cytometry analysis (FACScan of BD Company)

).

8. Expression and purification of TAT fusion protein

The intracellular region of PSGL-1 is obtained by PCR amplification using the full-length cDNA of human PSGL-1 as a template and the following primer pairs as primers, wherein the sequences of the primer pairs used are:

5'-CTTATAATAGGATCCCGCCTCTCCCGCAAGGGCC-3';

5'-TGGACCTGCAAGCTTCTAAGGGAGGAAGCTGTGC-3';

The full-length cDNA of human PSGL-1 is shown in SEQ ID NO:2.

The obtained PCR product was double-digested with BamHI and HindIII, and cloned into the pET30a(+) vector (Novagen) that had been double-digested with BamHI and HindIII in advance, and the constructed vector was used to express fusion with 6×His PSGL-1 intracellular peptide (PSGL-1P), PSGL-1P has the amino acid sequence shown in SEQ ID NO:4.

Referring to the product manual, use the MutanBEST Kit (TaKaRa) to insert the Tat sequence (YGRKKRRQRRR) into pET30a(+)-PSGL-1-tail, and the sequences of the primer pairs used are:

5'-AGACAGCGACGAAGAGACGACAAGGCCATGGCTGATA-3';

5'-CCGCTTCTTCCTGCCATACACCAGACCAGAAGAATGATGA-3';

The constructed vector is used to express the intracellular region of PSGL-1 (Tat-PSGL-1) fused with Tat and 6×His, and Tat-PSGL-1 has the amino acid sequence shown in SEQ ID NO:5.

The mutant of PSGL-1 intracellular region fused with Tat and 6×His (Tat-PSGL-1Mut) (among them, R345A, N346A, Y347A, P349A is the mutation site) was also constructed with MutanBEST Kit (TaKaRa), and the primer pair used The sequences are:

5'-GCTGCTGCCTCCGCCACCGAGATGGTCTGCATCTCA-3';

5'-CACGGGGTACATGTGGCCCTTGCGGGAGAGGCG-3'.

Tat-PSGL-1Mut has an amino acid sequence as shown in SEQ ID NO:6.

The constructed vector was verified by sequencing to ensure that the construction result was correct.

The expression and purification of the fusion polypeptide were performed according to the BD company operating manual (BD TALON ^TM Metal Affinity Resins User Manual) and literature (Franken KL, HSHiemstra, KEVan Meijgaarden, Y.Subronto, J.Den Hartigh, THOttenhoff, and JWDrijfhout.J.Exp.Med .2003; 197:63-75) method.

Confocal microscopy was used to detect the uptake of fusion polypeptides by cells. After incubation of isolated mouse neutrophils with FITC-labeled PSGL-1P, Tat-PSGL-1 or Tat-PSGL-1Mut at 37°C for 30 min, the cells were fixed and immediately analyzed by confocal microscopy.

9. Microscopic experiments in vivo

Male C57 (body weight is about 25 g/only), intravenously inject PBS or corresponding antibody or protein, 30 minutes later with 125 mg/kg ketamine (ketamine), 12.5 mg/kg methylthiazine (xylazine) and 25 μg/kg atropine sulfate (atropine sulfate) anesthetized, put it on a 37°C insulation board, peel off the cremaster muscle, fix it with a pin, and then observe it with an inverted microscope (CK40-F200, Olympus) (20× objective lens, 10× eyepiece ). The observation method is as follows:

Select venules with a diameter of 20-40 μm for observation. Rolling leukocytes whose flow rate is slower than red blood cells in blood are defined as "rolling leukocytes", and the number of rolling leukocytes in the field of view within 10 minutes is divided by 10 to obtain the average number of rolling leukocytes per minute for each mouse.

Leukocytes adhered for more than 30 seconds were defined as "adhered leukocytes", and the total number of cells adhered to blood vessels with a length of about 150 μm within 10 minutes was counted.

Count 3-5 fields per mouse.

10. Establishment of mouse acute peritonitis model

Balb/c mice (6-8 weeks, 20g body weight) used 1ml 3% thioglycolic acid medium or normal saline (model control) to induce acute peritonitis by intraperitoneal injection of mice, and killed the mice after 2 hours. K.K., DornerB.G., Merkel U., Ryffel B., Schutt C., Golenbock D., Freeman M.W., and Jack R.S.mice.J.Immunol.2002, 169:4475-4480; Ajuebor M.N., Gibbs L., Flower R.J., DasA.M., and Perretti M.Br.J.Pharmacol.1998, 125:319-326) report method, count the total number of leukocytes exuded in the peritoneal cavity.

In the peptide inhibition or antibody control experiment, 30 minutes before the injection of thioglycolic acid, the mice were injected with peptide (30 μg/mouse) or antibody (10 μg/mouse) by tail vein injection, and mice P-Rg (20 μg /Only).

Example 1. Yeast two-hybrid screening for intracellular proteins that bind to the intracellular region of human PSGL-1

Referring to the operation manual of Clontech Company, the cDNA fragment encoding the intracellular region 334-402 amino acids (sequence shown in SEQ ID NO: 3) of PSGL-1 was cloned into vector pAS2-1 (Clontech Company) to construct recombinant plasmid pAS2 -1-PSGL-1CT.

Using the fusion protein of the intracellular region and the DNA binding domain of GAL4 as bait, the yeast two-hybrid system (Matchmaker Two-Hybrid System) of Clontech Company was used to screen the cDNA library of human leukocytes to obtain the protein that binds to the intracellular region of human PSGL-1. of intracellular proteins.

According to the results of activating the β-galactosidase reporter gene and the His reporter gene, some positive clones were obtained, and then the above-mentioned positive clones were sequenced and analyzed. According to the sequencing results, one of the clones has a partial sequence encoding Naf1 protein.

The above-mentioned reporter gene of yeast Y190 was used to co-transform an empty vector and an irrelevant protein as a control experiment, and the results are shown in FIG. 1 .

According to the results in Fig. 1, Naf1 and PSGL-1 have specific effects.

Embodiment 2 , preparation Naf1

Using the Human Leokocyte MATCHMAKER cDNA Library (Clontech) as a template and Naf1 forward and Naf1 reverse as primers, the full-length Naf1 cDNA sequence was prepared by PCR.

The sequences of primers Naf1 forward and Naf1 reverse are as follows:

Naf1 forward: 5'-TTACGGATCCATGGAAGGGAGAGGACCGTAC-3';

Naf1 reverse: 5'-CGCCAAGCTTAAATGACACAATCTGGTCTCACTG-3';

The obtained PCR product was digested with BamHI and HindIII, and then connected to the pCMV-Tag 3B (Stratagene) vector, which was also digested with BamHI and HindIII, to construct the Naf1 eukaryotic expression vector pCMV- with c-myc-tag. Naf1.

By sequencing, it was confirmed that there was no mutation in the Naf1 cDNA sequence.

The full-length Naf1 cDNA sequence (sequence is SEQ ID NO: 1) was ligated into the pCEP4F vector to construct the eukaryotic expression vector pCEP4F-Naf1.

We expressed the full-length Naf1 and His-tag fusion protein with bacteria, and then used the protein as an antigen to immunize rabbits to obtain a polyclonal antibody against Naf1, which was used to detect HL-60 cells and transfect Naf1 (with c- myc) 293 cell lysates, and the anti-c-myc monoclonal antibody 9E10 was used to simultaneously detect the 293 cell lysates transfected with Naf1, the results are shown in Figure 2B.

According to the results in Figure 2B, the antibody specifically recognizes transfected Naf1, and HL-60 cells express Naf1 endogenously. At the same time, human neutrophil lysates were detected with this polyclonal antibody, and human neutrophils expressed Naf1.

We expressed the cloned Naf1 in 293 cells, first immunoprecipitated the protein from the cell lysate with anti-FLAG antibody, and then detected the immunoprecipitated protein by immunoblotting with anti-Naf1 polyclonal antibody, the results are shown in Figure 2C.

According to the results in Figure 2C, the protein Naf1 can be expressed in 293 cells, and under reducing conditions, Naf1 is about 80kDa.

Embodiment 3 , the function of Naf1

3.1, the binding site of Naf1 and PSGL-1

3.1.1 The binding between Naf1 and PSGL-1 intracellular region is specific

The pull-down experiment was performed using the GST fusion protein containing the Naf1 sequence, and the results are shown in Figure 3A and 3B. According to the results in Figure 3A and 3B, the beads bound to GST-Naf1 can bind PSGL-1 from leukocyte lysates, but the control GST protein cannot.

This result shows that the binding between Naf1 and the intracellular region of PSGL-1 is specific.

3.1.2. The intracellular region of Naf1 and PSGL-1 can bind to each other in transiently expressed mammalian cells

293 cells were co-transfected to express PSGL-1 and Naf1 or only transfected to express Naf1, and the corresponding antibodies were used to perform co-immunoprecipitation experiments. When co-transfected to express PSGL-1 and Naf1, the antibodies used were rabbit IgG and α-PSGL -1, when only Naf1 was expressed by transfection, the antibodies used were α-PSGL-1 and 9E10, and the results are shown in Figure 3C.

According to the results in Figure 3C, the anti-PSGL-1 antibody can immunoprecipitate Naf1, but the control antibody cannot. Cells only transfected with Naf1 are immunoprecipitated with anti-PSGL-1 antibody and 9E10 as a co-immunoprecipitation control , antibodies against PSGL-1 could not co-immunoprecipitate Naf1 from cells transfected with Naf1 alone.

The above results prove that the intracellular region of Naf1 and PSGL-1 can bind to each other in transiently expressed mammalian cells.

3.1.3 Interaction between Naf1 and PSGL-1 in normal physiological state

The lysed extract of HL-60 cells was prepared, and then the lysed extract of HL-60 cells was immunoprecipitated with anti-PSGL-1 rabbit polyclonal antibody and rabbit IgG before immunization, and analyzed by Western. The results are shown in Figure 3D.

According to the results in Figure 3D, the anti-PSGL-1 polyclonal antibody can co-immunoprecipitate endogenous PSGL-1 and endogenous Naf1, but the control rabbit IgG before immunization cannot.

Further, co-immunoprecipitation was performed under the condition of stimulation with P-Rg, and the results are shown in Figure 3E.

According to the results in Figure 3E, the binding of Naf1 and PSGL-1 was not enhanced compared to the human IgG control.

The above results indicate that under normal physiological conditions, Naf1 and PSGL-1 can combine with each other.

3.1.4. The binding sequence of PSGL-1 intracellular region and Naf1

The intracellular region of PSGL-1 was deleted and mutated, and the intracellular region of PSGL-1 was deleted from the C-terminal to the N-terminal. A total of five deletion mutants (M1, M2, M3, M4, M5) were constructed. The results As shown in Figure 3F.

These deletion mutants were co-transfected with Naf1 into 293 cells, and then co-immunoprecipitation experiments were performed, and the results are shown in Figure 3G.

According to the results in Figure 3G, M1, M2, and M3 can combine Naf1 immunoprecipitation, while M4 and M5 cannot.

Based on the above results, it can be deduced that the RNYSPTE sequence from position 345 to position 351 in the intracellular region of PSGL-1 is important for the binding of PSGL-1 and Naf1.

3.1.5. The key site for the binding of PSGL-1 intracellular region and Naf1

Point mutations were further performed on the sequence, and seven amino acids from positions 345 to 351 were mutated to alanine respectively, and seven point mutants were constructed. The results are shown in Figure 3H.

Then, point mutants were co-transfected with Naf1 for co-immunoprecipitation experiments, and the results are shown in Figure 3I.

According to the results in Figure 3I, the mutants R345A, N346A, Y347A, and P349A cannot combine Naf1 co-immunoprecipitation.

According to the results, amino acids 345, 346, 347, and 349 in the intracellular region of PSGL-1 are the key sites for the combination of PSGL-1 and Naf1.

3.1.6. The section on Naf1 that is critical for the binding of PSGL-1 and Naf1

Deletion mutations were performed on the C-terminus of Naf1, and the results are shown in Figure 3J.

The results of co-immunoprecipitation of these deletion mutations and PSGL-1 are shown in Figure 3K.

According to the results in Figure 3K, C3, C4, and C5 mutants could not be co-immunoprecipitated by PSGL-1 polyclonal antibodies, which indicated that the 17th exon played a key role in the combination of PSGL-1 and Naf1.

3.2. The key domain in Naf1 that mediates the binding of PSGL-1 and the p85 subunit of PI3K

3.2.1. Naf1 mediates the binding of PSGL-1 and p85 subunit of PI3K in vitro

In 293 cells, the plasmid vectors expressing PSGL-1, p85 and Naf1 or the corresponding control empty vector pCMV were co-transfected, and the cells were lysed after 48 hours. Take the supernatant of the cell lysate and immunoprecipitate with anti-PSGL-1 polyclonal antibody or rabbit IgG before immunization respectively. The precipitate is detected with anti-p85 polyclonal antibody immunoblotting, and the immunoprecipitated PSGL-1 is detected with KPL-1. The p85 in the supernatant of the cell lysate was detected by immunoblotting with anti-p85 multiple antibodies as a control, and the results are shown in Figure 4A.

According to the results in Figure 4A, in the 293 cells co-transfected into the plasmid vectors expressing PSGL-1, p85 and Naf1, the anti-PSGL-1 polyclonal antibody could immunoprecipitate p85, while the control rabbit IgG could not; In the 293 cells expressing PSGL-1, p85 and the control empty vector pCMV, the anti-PSGL-1 polyclonal antibody could not immunoprecipitate p85; detected by KPL-1, the anti-PSGL-1 polyclonal antibody could immunoprecipitate PSGL-1 down, but the control rabbit IgG before immunization could not.

On the other hand, 293 cells were co-transfected into a plasmid vector expressing p85 and Naf1 or the corresponding control empty vector pCMV, the supernatant of the cell lysate was used for co-immunoprecipitation with 9E10 or control mouse IgG, and the immunoprecipitates were respectively treated with anti-p85 multi- Antibody and 9E10 detection, the results are shown in Figure 4B.

According to the results in Figure 4B, 9E10 could immunoprecipitate p85, but the control mouse IgG could not. At the same time, 9E10 could not immunoprecipitate p85 in 293 cells co-transfected with p85 and control empty vector pCMV. Naf1 could be detected in the immunoprecipitate of 9E10 with 9E10, while the control mouse IgG could not immunoprecipitate Naf1. The supernatant of the cell lysate was directly used as a control to detect the expression of p85 with anti-p85 polyclonal antibody, and there was no significant difference in the results among the groups.

The above results directly prove that Naf1 can act as an adapter protein to mediate the combination of PSGL-1 and p85 subunit of PI3K.

3.2.2 The key domain of Naf1 binding to p85

The 552nd tyrosine (Y) of Naf1 was mutated to F (phenylalanine) to construct a Naf1 mutant named Naf1Y552F.

The mutant and p85 were co-transfected into 293 and detected by co-immunoprecipitation. The results are shown in Figure 4C.

According to the results in Figure 4C, the mutation at position 552 of Naf1 made Naf1 lose the ability to bind to p85.

The mutant Naf1Y552F, p85, and PSGL-1 were co-transfected into 293 cells, and detected by immunoblotting with anti-PSGL-1 polyclonal antibody. The results are shown in Figure 4D.

According to the results in Figure 4D, the mutation at position 552 made Naf1 lose its ability to mediate the binding of PSGL-1 and p85 as an adapter protein.

The Naf1Y552F mutant prevents the phosphorylation of Y 552, so the phosphorylation of this site is critical for the binding of Naf1 to p85.

3.3. In human neutrophils, tyrosine phosphorylation at position 552 of Naf1 induces the binding of PSGL-1 and p85 of PI3K

3.3.1. In human neutrophils, Naf1 mediates the binding of PSGL-1 and p85

After separating human neutrophils, incubate human neutrophils with control buffer PBS(-), 10 μg/ml human IgG (hIgG) or P-selectin Rg (P-Rg) at 37°C for 20 minutes, Lyse with cell lysis buffer. The supernatant of cell lysate was taken, and co-immunoprecipitated with anti-PSGL-1 polyclonal antibody, and the precipitate was detected with anti-p85 polyclonal antibody, and the results are shown in Figure 5A.

According to the results in Figure 5A, p85 can only be co-immunoprecipitated from neutrophils stimulated by P-Rg (it can be seen from the figure that the amount of combined p85 is not much, which is combined with p85 and many proteins in the cells consistent), but could not be co-immunoprecipitated from neutrophils added with PBS or human IgG. As a control, PSGL-1 immunoprecipitated by the anti-PSGL-1 polyclonal antibody can be detected by KPL-1 immunoblotting, and the cell lysed supernatant is directly detected by anti-p85 polyantibody immunoblotting as a control.

When the above experiment was repeated using the myeloid leukemia cell line HL-60, the results of the above experiment could be completely repeated.

According to the above experimental results, PSGL-1, which is the ligand of P-selectin, binds to P-Rg, transmits the activation signal to the cell, gathers and binds the p85 subunit of PI3K to its intracellular region, that is to say, P-selectin It can trigger the formation of PSGL-1/Naf1/p85 complex in human neutrophils, that is, in human neutrophils, Naf1 acts as an adapter protein to mediate the combination of PSGL-1 and p85.

3.3.2. Tyrosine phosphorylation at position 552 of Naf1 induces the binding of PSGL-1 and p85

Using the same conditions as in Example 3.3.1, stimulate human neutrophils with human IgG (hIgG) or P-selectin Rg (P-Rg) respectively, then directly boil the cells with reducing loading buffer for 5 minutes, and then The specific monoclonal antibody 3B8 and the control monoclonal antibody 5C4 against phosphorylated tyrosine 552 of Naf1 were used for immunodetection, and the results are shown in Figure 5B.

According to the results in Figure 5B, the tyrosine phosphorylation at position 552 of Naf1 was significantly enhanced.

After stimulating human neutrophils with human IgG or P-Rg for 20 minutes, use anti-PSGL-1 polyclonal antibody or rabbit IgG before immunization to immunoprecipitate PI3K from the supernatant of the cell lysate, and then detect the kinase of the immunoprecipitate activity, the results are shown in Figure 5C.

According to the results in Figure 5C, when stimulated by P-Rg, the kinase activity of the precipitate immunoprecipitated with anti-PSGL-1 polyclonal antibody was enhanced compared with that stimulated with human IgG, that is, P-selectin can enhance PI3K kinase in human neutrophils activity.

Fig. 5D is the average of three experimental results based on the results of Fig. 5C, and the standard deviation is also marked (tested by t-test, wherein * means p<0.05).

The specific inhibitor of Src kinase, PP2, was first incubated with the cells at room temperature for 15 minutes, and then neutrophils were stimulated with P-Rg for 10 minutes. The solvent DMSO was used as a control, and then Naf1 was immunoprecipitated with Naf1 antibody. The specific monoclonal antibody 3B8 against phosphorylated tyrosine 552 of Naf1 was used for immunodetection, and the same membrane was immunodetected with control antibody 5C4. The results are shown in Figure 5E.

According to the results in Figure 5E, PP2 can inhibit the phosphorylation of Naf1 tyrosine 552.

The leukocytes were treated in the same way as above. After the cells were lysed, they were co-immunoprecipitated with anti-PSGL-1 polyclonal antibody, and the precipitate was detected with anti-p85 polyclonal antibody. The results are shown in Figure 5F.

According to the results in Figure 5F, when PP2 was used to inhibit the phosphorylation of tyrosine 552 of Naf1, it could also inhibit the binding of PSGL-1 and p85 subunit induced by P-selectin.

The leukocytes were directly lysed after stimulation, and the lysates were immunodetected with Src antibody and anti-Src416 tyrosine phosphorylation antibody (which can recognize Hck in neutrophils, Lyn 416 tyrosine phosphorylation), the results are shown in Figure 5G Show.

According to the results in Fig. 5G, P-Rg stimulation can activate Src kinase.

The above experimental results in Example 3.3 show that in human neutrophils, phosphorylation of tyrosine at position 552 of Naf1 induces the binding of PSGL-1 and p85 of PI3K.

Example 4 , P-selectin binds to PSGL-1, activates p85 through Naf1, and then activates α _M β ₂ integrin

4.1. PI3K is involved in the signaling pathway from P-selectin to α _M β ₂ activation

Combine P-Rg with G1F(ab') ₂ (G1 is a blocking monomer for P-selectin adhesion to leukocytes) or PS1 F(ab') ₂ (PS1 is a non-blocking monomer for P-selectin adhesion to leukocytes) in advance. After incubating in PBS buffer (1xPBS, 1mM CaCl ₂ , 1mM MgCl ₂ ) at room temperature for 15 minutes, the cell adhesion experiment was carried out.

According to the results of cell adhesion experiments, G1 can neutralize the activation induced by P-selectin Rg (adhesion enhancement of white blood cells), while PS1 has no effect on the activation induced by P-selectin Rg, indicating that the activation is caused by P-selectin Rg and PSGL -1 combined with a specific guide.

Human neutrophils were pre-treated with 44a (44a is the adhesion blocking monoclonal antibody of _β2 subunit) or 9E10 (as the subtype control antibody of 44a) in PBS buffer (1xPBS, 1mM CaCl ₂ , 1mM MgCl ₂ ) after incubating at room temperature for 15 minutes, the cell adhesion experiment was performed.

According to the results of cell adhesion experiments, 44a can block the enhancement of human neutrophils' adhesion to solidified fibrinogen, while 9E10 has no effect, proving that α _M β ₂ has specificity in this process.

When the cells were pre-incubated with PI3K-specific inhibitors wortmannin, LY294002 and PP2, the P-Rg-induced enhancement of human neutrophil adhesion to immobilized fibrinogen was inhibited, and the control DMSO had no effect, indicating that PI3K was involved Signaling pathway from P-selectin to α _M β ₂ activation. The above experimental results are shown in Figure 6A.

According to the above experimental results, P-selectin Rg can increase the adhesion of human neutrophils or HL-60 to solidified fibrinogen (fibrinogen), while the control human IgG cannot.

4.2. Naf1 participates in the signaling pathway of P-selectin activation of α _M β ₂ integrin

In HL-60 cells, after transfection of S1 and S4, the corresponding antibodies were used to detect Naf1, PSGL-1, α _M , p85 and tubulin by immunoblotting, and the results are shown in Figure 6B.

According to the results in Figure 6B, when the expression of endogenous Naf1 was suppressed, the expressions of PSGL-1, p85, and _αM were not affected.

It was verified by flow cytometry experiments, and the results are shown in Figure 12.

According to the results in Figure 12, when the expression of endogenous Naf1 is suppressed, the expression of PSGL-1 and α _M subunit on the surface of HL-60 cells and the combination of P-Rg and PSGL-1 are not affected.

At the same time, the same inhibitory results as in HL-60 were obtained in 293T cells with S1 and S4, and the results are shown in Figure 6B.

When the expression of endogenous Naf1 was suppressed, the combination of PSGL-1 and p85 subunit was detected by co-immunoprecipitation, and the results are shown in Figure 6C.

According to the results in Figure 6C, when the expression of endogenous Naf1 was suppressed, the combination of PSGL-1 and p85 subunits was significantly weakened compared with the control under P-Rg stimulation conditions, which was consistent with the results in Figure 4A, It shows that Naf1 plays the role of adapter protein in the combination of PSGL-1 and p85 subunit.

HL-60 cells were transfected with S1 and S4 respectively, and the cell adhesion experiment was carried out under the conditions of no stimulation, P-Rg stimulation and hIgG stimulation respectively, and the results are shown in Fig. 6D.

According to the results in Figure 6D, when endogenous Naf1 in HL-60 cells was suppressed by transfection of S4, P-Rg stimulation could not activate α _M β ₂ , while cells transfected with control S1 were not affected.

After the S4 and Naf1 expression plasmids were co-transfected into HL-60, the cell adhesion experiment was carried out.

The results showed that after inhibition of Naf1 relative to single transfection of S4, the number of adherent cells recovered to some extent and increased.

Based on the above experimental results, the following conclusions were drawn: Naf1, as an adapter protein, mediates the combination of PSGL-1 and p85 subunits, transmits the intracellular activation signal from PSGL-1 to p85, and activates α _M β during P-selectin activation. ₂ Integrin signaling pathway plays an important role.

Example 5. The phosphorylation of tyrosine 552 in Naf1 plays a key role in transmitting the signal of P-selectin activation of α _M β ₂ integrin

Refer to the operation manual, use ViraPower lentiviral expression systems (invitrogen company), construct mutant Naf1Y552F into virus particles, and construct virus particles with LacZ gene as a control. HL-60 cells were infected with the virus particles carrying Naf1Y552F, and its expression was detected, the results are shown in Figure 7A.

According to the results in Fig. 7A, the expression of PSGL-1, p85, _αM was not affected by the overexpression of Naf1Y552F.

And it was detected by flow cytometry experiment, and the result is shown in Fig. 10 .

According to the results in Figure 10, when Naf1Y552F was overexpressed, the expression of PSGL-1 and α _M subunit on the surface of HL-60 cells and the combination of P-Rg and PSGL-1 were not affected.

When Naf1Y552F was overexpressed in HL-60 cells, the combination of PSGL-1 and p85 was detected by co-immunoprecipitation, and the results are shown in Figure 7B.

According to the results in Figure 7B, the overexpression of Naf1Y552F can block the combination of PSGL-1 and p85. The results indicated that the mutant Naf1Y552F had a dominant-negative effect.

The activation of α _M β ₂ integrin on HL-60 cells overexpressing Naf1Y552F was detected by cell adhesion assay, and the results are shown in Figure 7C.

According to the structure of Fig. 7C, in the cells overexpressing Naf1Y552F, P-Rg stimulation could not increase the adhesion of the cells to the fibrinogen plate, and the HL-60 cells overexpressing the LacZ gene were the same as normal cells. The results indicated that overexpression of Naf1Y552F blocked the signaling pathway of P-selectin activation of α _M β ₂ integrin.

The above experimental results prove that the mutant Naf1Y552F can block the signaling pathway of P-selectin activating α _M β ₂ integrin, and the phosphorylation of Naf1 tyrosine 552 plays a key role in this pathway.

Based on the results of Examples 1-5, a new signaling pathway is revealed: P-selectin binding to PSGL-1 causes the activation of _β2 integrin. The signal transmission of PSGL-1 to the cell depends on the constitutive combination of its intracellular region and Naf1. After P-selectin binds to its ligand PSGL-1, Src kinase is activated, and Src kinase phosphorylates the 552nd on the YPPM domain of Naf1. Tyrosine, the phosphorylated YPPM domain can bind the p85 subunit of PI3K, and finally transmit signals from the inside to the outside through PI3K to activate the integrin α _M β ₂ and α _L β ₂ .

Because, P-selectin-mediated cell adhesion between platelets and endothelial cells, and between these cells and leukocytes is a basic phenomenon and common process in the pathophysiological process of inflammation and thrombosis. Inhibition and regulation of P-selectin and its mediated cell adhesion and action through various pathways and drugs have proved to be an effective means of prevention and treatment of inflammatory and thrombotic diseases. At the same time, anti-adhesion therapy can be regarded as a preventive method, which has good clinical application value and prospect. Moreover, the activation of _β2 integrins plays a key role in the adhesion of leukocytes to the vascular endothelial surface in inflammatory and thrombotic diseases. Therefore, according to the results of Examples 1-5, the proteins Naf1 and PSGL-1 can be used as targets for screening drugs for the treatment of inflammatory and/or thrombotic diseases and as drug targets for drugs for inflammatory and/or thrombotic diseases. Screening Specific antibodies and drugs containing specific antibodies to protein Naf1 and/or PSGL-1 for treating inflammatory and/or thrombotic diseases, and drugs targeting protein Naf1 and/or PSGL-1 for treating Drugs for inflammatory and/or thrombotic disorders.

Designing peptides targeting Naf1 and/or PSGL-1 to block this signaling pathway may inhibit the activation of _β2 integrin caused by P-selectin. The following experiments verify this assumption.

Example 6. Construction and expression of PSGL-1 intracellular peptide

Expressed and purified the 6×His fusion polypeptide (PSGL-1P) of the intracellular region of PSGL-1, the fusion polypeptide (Tat-PSGL-1) of the intracellular region with HIV virus Tat sequence and the fusion polypeptide with R345A, N346A, Y347A, P349A mutant intracellular region fusion polypeptide (Tat-PSGL-1Mut), the schematic diagram of the above polypeptide is shown in Figure 8A.

The above polypeptides were labeled with FITC, and then incubated with isolated mouse neutrophils, and observed with a co-polymer cross microscope, the results are shown in Figure 8B.

According to the results in Fig. 8B, the cells can swallow the fusion polypeptide with Tat sequence into the cells.

The above polypeptides were then used to bind Naf1 in human and mouse neutrophil lysates, and the results are shown in Figures 8C, 8D, and 8E.

According to the results in Fig. 8C and 8D, PSGL-1P and Tat-PSGL-1 can bind Naf1, but the mutant Tat-PSGL-1Mut cannot. According to the results in FIG. 8E , compared with Tat-PSGL-1Mut, PSGL-1P and Tat-PSGL-1 can also compete for the binding of endogenous PSGL-1 and Naf1 in leukocyte lysate.

The above experimental results show that the expressed fusion polypeptide Tat-PSGL-1 has the function of entering cells and binding to Naf1.

Example 7 , PSGL-1 Intracellular Peptides Can Block P-selectin Activation of _β2 Integrin in Vitro

The above peptides (PSGL-1P, Tat-PSGL-1 and Tat-PSGL-1Mut) were pre-incubated with isolated leukocytes, and then cell adhesion experiments were performed, the results are shown in Figure 9A.

According to the results in Figure 9A, Tat-PSGL-1 can significantly inhibit the adhesion of human neutrophils to the fibrinogen plate induced by P-Rg, while PSGL-1P has no inhibitory effect because it cannot enter the cells, and Tat-PSGL-1Mut Although it can enter cells, it cannot block the binding of endogenous PSGL-1 and Naf1 without inhibitory effect.

The above experiments were repeated on plates of mouse neutrophils and mouse fibrinogen, and the same results were obtained, as shown in Figure 9B.

As a control, the incubation of polypeptides and cells did not affect the expression of PSGL-1 and α _M subunit on the surface of mouse neutrophils and the combination of mouse P-Rg and PSGL-1, the results are shown in Figure 11A, B, C Show.

According to the results in Figure 11A, B, C, P-Rg stimulation can enhance the adhesion of human neutrophils to the ICAM-1 plate, and this enhancement can be completely blocked by the blocking antibody IB4 of the β2 subunit, α _M The blocking antibodies CBRM1/5 and 38 of α L and α _L can only partially block, and can be completely blocked when the two antibodies work together, while the antibody subtype control has no effect. The above results indicated that P-Rg can activate both α _M β ₂ and α _L β ₂ integrins.

Tat-PSGL-1 can completely inhibit the P-Rg-induced adhesion of human neutrophils to the ICAM-1 plate, while the control PSGL-1P and Tat-PSGL-1Mut have no effect, the above results are shown in Figure 9C .

The above experimental results show that the PSGL-1 signaling pathway plays a role in the activation of both α _M β ₂ and α _L β ₂ .

Example 8 , PSGL-1 Intracellular Peptides Can Block P-selectin Activation of _β2 Integrin in Vivo

The fusion polypeptide of PSGL-1 intracellular region was intravenously injected into the mice in advance, and the rolling and adhesion of leukocytes on the blood vessel wall caused by the operation were observed 30 minutes later, and the blocking antibodies M1/70 and α _M and α _L were used M17/4 was used as a control, and the results are shown in Figure 9D.

According to the results in Figure 9D, compared with the control mice, the fusion polypeptide did not affect the rolling of leukocytes, while Tat-PSGL-1 could significantly reduce the number of leukocytes adhered to the blood vessel wall, PSGL-1P and the mutant Tat-PSGL-1Mut Adherent leukocytes are not affected. The blocking antibodies M1/70 and M17/4 of α _M and α _L do not affect the rolling of leukocytes, but have certain effects on adhesion, and the binding inhibition effect of the two is more obvious. The blocking monoclonal antibody RB40.34 Both rolling and adhesion were significantly inhibited, while the isotype control antibody had no effect.

According to the above experimental results, the rolling and adhesion of leukocytes caused by surgery are dependent on P-selectin, and in addition to mediating the function of leukocyte rolling, PSGL-1 can also mediate signaling pathways, thereby activating integrins, so in important functions in the body.

Example 9. PSGL-1 Intracellular Peptides Can Block P-selectin Activation of _β2 Integrin in the Mouse Model of Acute Peritonitis

The mouse model experiment of acute peritonitis was carried out, and the results are shown in Figure 9E.

According to the results in Figure 9E, in the mouse acute peritonitis model, compared with the control PSGL-1P and the mutant Tat-PSGL-1Mut, Tat-PSGL-1 can significantly reduce the infiltration of leukocytes into the peritoneal cavity. As controls, M1/70 and Co-injection of M17/4 and RB40.34 also significantly reduced leukocyte infiltration, while subtype control antibody had no effect.

The above experimental results show that the infiltration of leukocytes into the peritoneal cavity depends on αM, αL and P-selectin.

According to the above experimental results, the peptide Tat-PSGL-1 specifically inhibits the combination of PSGL-1 and Naf1, thereby blocking the activation of integrins mediated by PSGL-1, which can inhibit the inflammatory response in vivo.

Example 10. P-selectin restores deep vein thrombosis in P-selectin knockout mice

The P-selectin knockout mice and control C57 mice were ligated respectively to create deep vein thrombus, and the length and weight of the thrombus were measured 2 days later, and plotted according to the mass-length ratio of the thrombus. The results are shown in Figure 13.

According to the results in Fig. 13, deep vein thrombosis induced in P-selectin knockout mice was lighter than that in control mice C57.

Then, the mouse P-Rg and the control protein 12CA5 were injected intravenously into the P-selectin knockout mice, and then the inferior vena cava of the mice was ligated to create deep vein thrombosis, and the length of the thrombus was detected 2 days later and weight, and according to the mass-length ratio of the thrombus, the results are shown in Figure 13.

According to the results in FIG. 13 , after intravenous injection of mP-Rg into P-selectin knockout mice, the deep vein thrombosis of the mice was recovered, while injection of the control protein 12CA5 had no recovery effect.

The above results indicate that P-selectin plays an important role in deep vein thrombosis, and it is possible to inhibit deep vein thrombosis by targeting its signaling pathway.

Example 11. Blocking polypeptide inhibits deep vein thrombosis in mice

The polypeptide Tat-PSGL-1 and the mutant polypeptide Tat-PSGL-1Mut were injected intravenously into the mice respectively, and then the inferior vena cava of the mice was ligated to create deep vein thrombosis. The length and weight of the thrombus were detected 2 days later, and at the same time , taking the mice injected with normal saline as the control, and plotting according to the mass-length ratio of the thrombus, the results are shown in Figure 14.

The results in Fig. 14 show that the in vivo injection of the peptide Tat-PSGL-1 can significantly inhibit deep vein thrombosis in mice, while the polypeptide Tat-PSGL-1Mut has no inhibitory effect on deep vein thrombosis in mice.

Based on the results of Examples 6-10, after blocking the combination of PSGL-1 and Naf1, it can block the integrin activation induced by P-selectin both in vivo and in vitro, and at the same time, it can also weaken the inflammatory response in vivo and inhibit The formation of deep vein thrombosis. This signaling pathway has important physiological functions. Key proteins in this signaling pathway, such as PSGL-1 and Naf1, can be used as targets for screening antagonists of this signaling pathway, so as to develop drugs for inflammatory and/or thrombotic diseases.

In addition, the 17th exon on Naf1 is critical for the binding of PSGL-1 and Naf1, so it would be obvious to those skilled in the art to design blocking proteins targeting this exon, for example, in Naf1 Add a protein transduction sequence (HIV Tat) from the HIV virus transcription transactor on the basis of the amino acids at positions 594-626, so it is also claimed by the present invention.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> A target for an inflammatory and thrombotic drug, and a drug against that target

<130>P5081028

<160>6

<170>PatentIn version 3.1

<210>1

<211>2801

<212>DNA

<213> Human

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agtgagccga ggagccttca tagggacagc cgcccctggt gcacacaccc tcgtattctc 60

ctgcccttcc ccagccaggc gggcaccacg gcaggggctg agctacccctc atggaaggga 120

gaggaccgta ccggatctac gaccctgggg gcagcgtgcc ctcaggagag gcatccgcag 180

cttttgagcg cctagtgaag gagaattccc ggctgaagga aaaaatgcaa gggataaaga 240

tgttagggga gcttttggaa gagtcccaga tggaagcgac caggctccgg cagaaggcag 300

aggagctagt gaaggacaac gagctgctcc caccaccttc tccctccttg ggctccttcg 360

accccctggc tgagctcaca ggaaaggact caaatgtcac agcatctccc acagcccctg 420

catgccccag tgacaagcca gcaccagtcc agaagcctcc atccagtggc acctcctctg 480

aatttgaagt ggtcactcct gaggagcaga attcaccaga gagcagcagc catgccaatg 540

cgatggcgct gggccccctg ccccgtgagg acggcaacct gatgctgcac ctgcagcgcc 600

tggagaccac gctgagtgtg tgtgccgagg agccggacca cggccagctc ttcacccacc 660

tgggccgcat ggccctggag ttcaaccgac tggcatccaa ggtgcacaag aatgagcagc 720

gcacctccat tctgcagacc ctgtgtgagc agcttcggaa ggagaacgag gctctgaagg 780

ccaagttgga taagggcctg gaacagcggg atcaggctgc cgagaggctg cgggaggaaa 840

attggagct caagaagttg ttgatgagca atggcaacaa agagggtgcg tctgggcggc 900

caggctcacc gaagatggaa gggacaggca agaaggcagt ggctggacag cagcaggcta 960

gtgtgacggc aggtaaggtc ccagaggtgg tggccttggg cgcagccgag aagaaggtga 1020

agatgctgga gcagcagcgc agtgagctgc tggaagtgaa caagcagtgg gaccagcatt 1080

tccggtccat gaagcagcag tatgagcaga agatcactga gctgcgtcag aagctggctg 1140

atttgcagaa gcaggtgact gacctggagg ccgagcggga gcagaagcag cgtgactttg 1200

accgcaagct cctcctggcc aagtccaaga ttgaaatgga ggagaccgac aaggagcagc 1260

tgacagcaga ggccaaggag ctgcgccaaa aggtcaagta cctgcaggat cagctgagcc 1320

cactcacccg acagcgtgag taccaggaaa aggagatcca gcggctcaac aaggccctgg 1380

aggaagcact gagcatccaa accccgccat catctccacc aacagcattt gggagcccag 1440

aaggagcagg ggccctccta aggaaacagg agctggtcac gcagaatgag ttgctgaaac 1500

agcaggtgaa gatcttcgag gaggacttcc agagggagcg cagtgatcgt gagcgcatga 1560

atgaggagaa ggaagagctg aagaagcaag tggagaagct gcaggcccag gtcaccctgt 1620

caaatgccca gctaaaagca ttcaaagatg aggagaaggc aagagaagcc ctcagacagc 1680

agaagaggaa agcaaaggcc tcaggagagc gttaccatgt ggagccccac ccagaacatc 1740

tctgcggggc ctacccctac gcctacccgc ccatgccagc catggtgcca caccatggct 1800

tcgaggactg gtcccagatc cgctaccccc ctccccccat ggccatggag cacccgcccc 1860

cactccccaa ctcgcgcctc ttccatctgc cggaatacac ctggcgtcta ccctgtggag 1920

gggttcgaaa tccaaatcag agctcccaag tgatggaccc tccccacagcc aggcctacag 1980

aaccagagtc tccaaaaaat gaccgtgagg ggcctcagtg agaccagatt gtgtcatttg 2040

gctccacctt catcttgcag agccagctga tctcagattg ccaagaaact agaagccact 2100

tgcacggtgt ggccagagcc tcagctggat gagaggctga gatgggtggc cagcttgtat 2160

accagtccct gaactgagct gtttacagga ctggggaggc tccaccaga aggctttcat 2220

ttgtactctg ctgggagtga ctgggaaaaa ctccttccct gctgctgagt ggagagaggc 2280

ctcatccggc tttgacccac catccgttgc agaagcctcc aggagcagca atcctaagag 2340

tgggaggcag ccaagacccc cttccttcaa aacctcccgg aagtggtttc aggccctcta 2400

gttgccatga ccaatttgtg tgtgtgttta atttttgctt caagctctgt agcaggacct 2460

gccccacgca cacccctacc cctctgtgag gagctgtggg aagtgtgggt ttgtctccag 2520

aacagaagag aatgatggat attctggctc tggggccctc tccaccacca ctcacagtag 2580

ccttgctgaa gccatcacag atgggagaag gccatgccag ccacgtccgc cgaggggcgc 2640

cagcctgaag ctgccaggcc ctgaggttca gaccctggac cccatagctg gaggcctgtg 2700

gtgccagaag cccagattag ggtggctgtc catccctgga tagctatttg cacgaatcat 2760

ggacataaat ccaagttgaa gaagaaaaaa aaaaaaaaaa a 2801

<210>2

<211>2550

<212>DNA

<213> Human

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acacacagcc attgggggtt gctcggatcc gggactgccg cagggggtgc cacagcagtg 60

cctggcagcg tgggctggga ccttgtcact aaagcagaga agccacttct tctgggccca 120

cgaggcagct gtcccatgct ctgctgagca cggtggtgcc atgcctctgc aactcctcct 180

gttgctgatc ctactgggcc ctggcaacag cttgcagctg tgggaacacct gggcagatga 240

agccgagaaa gccttgggtc ccctgcttgc ccgggaccgg agacaggcca ccgaatatga 300

gtacctagat tatgatttcc tgccagaaac ggagcctcca gaaatgctga ggaacagcac 360

tgacaccact cctctgactg ggcctggaac ccctgagtct accactgtgg agcctgctgc 420

aaggcgttct actggcctgg atgcaggagg ggcagtcaca gagctgacca cggagctggc 480

caacatgggg aacctgtcca cggattcagc agctatggag atacagacca ctcaaccagc 540

agccacggag gcacagacca ctccactggc agccacagag gcacagacaa ctcgactgac 600

ggccacggag gcacagacca ctccactggc agccacagag gcacagacca ctccaccagc 660

agccacggaa gcacagacca ctcaacccac aggcctggag gcacagacca ctgcaccagc 720

agccatggag gcacagacca ctgcaccagc agccatggaa gcacagacca ctccaccagc 780

agccatggag gcacagacca ctcaaaccac agccatggag gcacagacca ctgcaccaga 840

agccacggag gcacagacca ctcaacccac agccacggag gcacagacca ctccactggc 900

agccatggag gccctgtcca cagaacccag tgccacagag gccctgtcca tggaacctac 960

taccaaaaga ggtctgttca tacccttttc tgtgtcctct gttactcaca agggcattcc 1020

catggcagcc agcaatttgt ccgtcaacta cccagtgggg gccccagacc acatctctgt 1080

gaagcagtgc ctgctggcca tcctaatctt ggcgctggtg gccactatct tcttcgtgtg 1140

cactgtggtg ctggcggtcc gcctctcccg caagggccac atgtaccccg tgcgtaatta 1200

ctcccccacc gagatggtct gcatctcatc cctgttgcct gatgggggtg aggggccctc 1260

tgccacagcc aatgggggcc tgtccaaggc caagagcccg ggcctgacgc cagagcccag 1320

ggaggaccgt gagggggatg acctcaccct gcacagcttc ctcccttagc tcactctgcc 1380

atctgttttg gcaagacccc acctccacgg gctctcctgg gccacccctg agtgcccaga 1440

ccccattcca cagctctggg cttcctcgga gacccctggg gatggggatc ttcagggaag 1500

gaactctggc cacccaaaca ggacaagagc agcctggggc caagcagacg ggcaagtgga 1560

gccacctctt tcctccctcc gcggatgaag cccagccaca tttcagccga ggtccaaggc 1620

aggaggccat ttacttgaga cagattctct cctttttcct gtcccccatc ttctctgggt 1680

ccctctaaca tctcccatgg ctctccccgc ttctcctggt cactggagtc tcctccccat 1740

gtacccaagg aagatggagc tcccccatcc cacacgcact gcactgccat tgtcttttgg 1800

ttgccatggt caccaaacag gaagtggaca ttctaaggga ggagtactga agagtgacgg 1860

acttctgagg ctgtttcctg ctgctcctct gacttggggc agcttgggtc ttcttgggca 1920

cctctctggg aaaacccagg gtgaggttca gcctgtgagg gctgggatgg gtttcgtggg 1980

cccaagggca gacctttctt tgggactgtg tggaccaagg agcttccatc tagtgacaag 2040

tgacccccag ctatcgcctc ttgccttccc ctgtggccac tttccagggt ggactctgtc 2100

ttgttcactg cagtatccca actgcaggtc cagtgcaggc aataaatatg tgatggacaa 2160

acgatagcgg aatccttcaa ggtttcaagg ctgtctcctt caggcagcct tcccggaatt 2220

ctccatccct cagtgcagga tgggggctgg tcctcagctg tctgccctca gcccctggcc 2280

ccccaggaag cctctttcat gggctgttag gttgacttca gttttgcctc ttggacaaca 2340

gggggtcttg tacatccttg ggtgaccagg aaaagttcag gctatggggg gccaaaggga 2400

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<212>PRT

<213> Human

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20 25 30

Pro Ser Ala Thr Ala Asn Gly GIy Leu Ser Lys Ala Lys Ser Pro GIy

35 40 45

Leu Thr Pro Glu Pro Arg GIu Asp Arg Glu Gly Asp Asp Leu Thr Leu

50 55 60

His Ser Phe Leu Pro

65

<210>4

<211>119

<212>PRT

<213> Human

<400>4

Met His His His His His His His Ser Ser Ser Gly Leu Val Pro Arg Gly Ser

1 5 10 15

Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp

20 25 30

Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile

35 40 45

Gly Ser Arg Leu Ser Arg Lys Gly His Met Tyr Pro Val Arg Asn Tyr

50 55 60

Ser Pro Thr GIu Met Val Cys Ile Ser Ser Leu Leu Pro Asp Gly Gly

65 70 75 80

Glu Gly Pro Ser Ala Thr Ala Asn Gly Gly Leu Ser Lys Ala Lys Ser

85 90 95

Pro Gly Leu Thr Pro Glu Pro Arg Glu Asp Arg Glu Gly Asp Asp Leu

100 105 110

Thr Leu His Ser Phe Leu Pro

115

<210>5

<211>102

<212>PRT

<213> Human

<400>5

Met His His His His His His His Ser Ser Ser Gly Leu Val Tyr Gly Arg Lys

1 5 10 15

Lys Arg Arg Gln Arg Arg Arg Asp Asp Lys Ala Met Ala Asp Ile Gly

20 25 30

Ser Arg Leu Ser Arg Lys Gly His Met Tyr Pro Val Arg Asn Tyr Ser

35 40 45

Pro Thr Glu Met Val Cys Ile Ser Ser Leu Leu Pro Asp Gly Gly Glu

50 55 60

Gly Pro Ser Ala Thr Ala Asn Gly Gly Leu Ser Lys Ala Lys Ser Pro

65 70 75 80

Gly Leu Thr Pro Glu Pro Arg Glu Asp Arg Glu Gly Asp Asp Leu Thr

85 90 95

Leu His Ser Phe Leu Pro

100

<210>6

<211>102

<212>PRT

<213> Human

<400>6

Met His His His His His His His Ser Ser Ser Gly Leu Val Tyr Gly Arg Lys

1 5 10 15

Lys Arg Arg Gln Arg Arg Arg Asp Asp Lys Ala Met Ala Asp Ile Gly

20 25 30

Ser Arg Leu Ser Arg Lys Gly His Met Tyr Pro Val Ala Ala Ala Ser

35 40 45

Ala Thr Glu Met Val Cys Ile Ser Ser Leu Leu Pro Asp Gly Gly Glu

50 55 60

Gly Pro Ser Ala Thr Ala Asn Gly Gly Leu Ser Lys Ala Lys Ser Pro

65 70 75 80

Gly Leu Thr Pro Glu Pro Arg Glu Asp Arg Glu Gly Asp Asp Leu Thr

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Leu His Ser Phe Leu Pro

100

Claims (24)

1. The application of protein Naf1, characterized in that it is used as a target for screening drugs for treating inflammation and/or thrombosis diseases. 2. The use of the protein Naf1, which is characterized in that it is used as a drug target for inflammatory and/or thrombotic disease drugs. 3. The application of the protein PSGL-1, characterized in that it is used as a target for screening drugs for treating inflammation and/or thrombosis diseases. 4. The use of the protein PSGL-1, characterized in that it is used as a drug target for inflammatory and/or thrombotic disease drugs. 5. The use according to any one of claims 1-4, characterized in that the drug blocks the p85 subunit of phosphatidylinositol 3-kinase PI3K from binding to the intracellular region of PSGL-1. 6. The use according to any one of claims 1-4, characterized in that the drug blocks the phosphorylation of tyrosine at position 552 on Naf1. 7. The use according to any one of claims 1-4, characterized in that the drug blocks the binding of Naf1 and the intracellular region of PSGL-1. 8. A specific antibody for protein Naf1. 9. A medicament for treating inflammatory and/or thrombotic diseases, characterized in that the medicament contains the antibody as claimed in claim 8. 10. An antibody specific for the protein PSGL-1. 11. A medicament for treating inflammatory and/or thrombotic diseases, characterized in that the medicament contains the antibody as claimed in claim 10. 12. A drug for treating inflammatory and/or thrombotic diseases, characterized in that the drug targets protein Naf1. 13. A drug for treating inflammatory and/or thrombotic diseases, characterized in that the drug targets protein PSGL-1. 14. A peptide capable of blocking the binding of Naf1 and the intracellular region of PSGL-1, characterized in that the peptide has the amino acid sequence shown in SEQ ID NO:3. 15. The peptide according to claim 14, wherein said peptide further blocks the activation of integrin _β2 induced by P-selectin in vivo. 16. The peptide according to claim 14 or 15, wherein the peptide inhibits P-selectin and its mediated cell adhesion and action in vivo. 17. Use of the peptide according to any one of claims 14-16 for the preparation of a medicament for treating inflammatory and/or thrombotic diseases. 18. The use according to claim 17, characterized in that said peptide blocks the binding between Naf1 and the intracellular region of PSGL-1. 19. The use according to claim 18, characterized in that said peptide segment further blocks the activation of integrin _β2 induced by P-selectin in vivo. 20. The use according to any one of claims 17-19, characterized in that the peptide segment inhibits P-selectin and its mediated cell adhesion and action. 21. A peptide segment derived from the 17th exon of Naf1, characterized in that the peptide segment comprises the 594-626 amino acid sequence of SEQ ID NO:1. 22. The peptide segment according to claim 21, characterized in that, the peptide segment may further comprise a transducing sequence. 23. The peptide according to claim 22, wherein the transduction sequence is HIV Tat. 24. The peptide according to any one of claims 21-23, wherein said peptide blocks the binding of PSGL-1 and Naf1.

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