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• W2050510894 abstract "The plasminogen receptors responsible for enhancing cell surface-dependent plasminogen activation expose COOH-terminal lysines on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). We treated U937 cells with CpB, then subjected membrane fractions to two-dimensional gel electrophoresis followed by ligand blotting with125I-plasminogen. A 54-kDa protein lost the ability to bind 125I-plasminogen after treatment of intact cells and was purified by two-dimensional gel electrophoresis and then sequenced by mass spectrometry. Two separate amino acid sequences were obtained and were identical to sequences contained within human and rat TIP49a. The cDNA for the 54-kDa protein matched the human TIP49a sequence, and encoded a COOH-terminal lysine, consistent with susceptibility to CpB. Antibodies against rat TIP49a recognized the plasminogen-binding protein on two-dimensional Western blots of U937 cell membranes. Human 125I-Glu-plasminogen bound specifically to TIP49a protein, and binding was inhibited by ε-aminocaproic acid. A single class of binding sites was detected, and a Kd of 0.57 ± 0.14 μm was determined. TIP49a enhanced plasminogen activation 8-fold compared with the BSA control, and this was equivalent to the enhancement mediated by plasmin-treated fibrinogen. These results suggest that TIP49a is a previously unrecognized plasminogen-binding protein on the U937 cell surface. The plasminogen receptors responsible for enhancing cell surface-dependent plasminogen activation expose COOH-terminal lysines on the cell surface and are sensitive to proteolysis by carboxypeptidase B (CpB). We treated U937 cells with CpB, then subjected membrane fractions to two-dimensional gel electrophoresis followed by ligand blotting with125I-plasminogen. A 54-kDa protein lost the ability to bind 125I-plasminogen after treatment of intact cells and was purified by two-dimensional gel electrophoresis and then sequenced by mass spectrometry. Two separate amino acid sequences were obtained and were identical to sequences contained within human and rat TIP49a. The cDNA for the 54-kDa protein matched the human TIP49a sequence, and encoded a COOH-terminal lysine, consistent with susceptibility to CpB. Antibodies against rat TIP49a recognized the plasminogen-binding protein on two-dimensional Western blots of U937 cell membranes. Human 125I-Glu-plasminogen bound specifically to TIP49a protein, and binding was inhibited by ε-aminocaproic acid. A single class of binding sites was detected, and a Kd of 0.57 ± 0.14 μm was determined. TIP49a enhanced plasminogen activation 8-fold compared with the BSA control, and this was equivalent to the enhancement mediated by plasmin-treated fibrinogen. These results suggest that TIP49a is a previously unrecognized plasminogen-binding protein on the U937 cell surface. carboxypeptidase B two-dimensional polyacrylamide gel electrophoresis bovine serum albumin Hanks' balanced salt solution supplemented with 20 mM HEPES horseradish peroxidase isoelectric focusing PBS containing BSA, EDTA, and heat-inactivated human serum phosphate-buffered saline PBS containing 0.025% Tween 80 polyacrylamide gel electrophoresis Tris-buffered saline with 0.1% Tween 20 TATA-binding protein polymerase chain reaction expressed sequence tag tentative human consensus 3-(cyclohexylamino)propanesulfonic acid tissue plasminogen activator Assembly of fibrinolytic molecules on the cell surface promotes plasminogen activation and the association of plasmin with cell surfaces (reviewed by Plow et al. in Ref. 1Plow E.F. Herren T. Redlitz A. Miles L.A. Hoover-Plow J.L. FASEB J. 1995; 9: 939-945Crossref PubMed Scopus (379) Google Scholar). This proteolytic activity on the cell surface participates in physiological processes in which cells must degrade extracellular matrices to migrate. The binding of plasminogen to cell surfaces is required for enhanced plasminogen activation (2Miles L.A. Plow E.F. J. Biol. Chem. 1985; 260: 4303-4311Abstract Full Text PDF PubMed Google Scholar, 3Hajjar K.A. Harpel P.C. Jaffe E.A. Nachman R.L. J. Biol. Chem. 1986; 261: 11656-11662Abstract Full Text PDF PubMed Google Scholar, 4Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar, 5Felez J. Miles L.A. Fabregas P. Jardi M. Plow E.F. Lijnen R.H. Thromb. Haemost. 1996; 76: 577-584Crossref PubMed Scopus (83) Google Scholar), and inactivation of plasminogen binding sites eliminates the cell-dependent enhancement of activation (4Ellis V. Behrendt N. Dano K. J. Biol. Chem. 1991; 266: 12752-12758Abstract Full Text PDF PubMed Google Scholar, 6Felez J. Miles L.A. Plow E.F. Fabregas P. Lijnen H.R. Thromb. Haemost. 1993; 69 (abstr.): 1232Google Scholar). Monocytoid U937 cells have a high capacity for plasminogen (∼1.6 × 107 sites/cell; Ref. 7Plow E.F. Miles L.A. Cell Diff. Dev. 1990; 32: 293-298Crossref PubMed Scopus (48) Google Scholar), and no single molecule can account for the entire capacity to bind plasminogen. Both protein and nonprotein (gangliosides; Refs. 8Miles L.A. Dahlberg C.M. Levin E.G. Plow E.F. Biochemistry. 1989; 28: 9337-9343Crossref PubMed Scopus (79) Google Scholar and 9Liepkalns V.A. Burtin M.C. Correc P. Durand H. Maunory M.T. J. Recept. Res. 1990; 10: 333-351Crossref PubMed Scopus (6) Google Scholar) molecules have been identified as plasminogen-binding molecules present on cell surfaces. Treatment of cells with carboxypeptidase B (CpB)1 reduces plasminogen binding to proteinaceous receptors on the cell surface (5Felez J. Miles L.A. Fabregas P. Jardi M. Plow E.F. Lijnen R.H. Thromb. Haemost. 1996; 76: 577-584Crossref PubMed Scopus (83) Google Scholar, 10Miles L.A. Dahlberg C.M. Plescia J. Felez J. Kato K. Plow E.F. Biochemistry. 1991; 30: 1682-1691Crossref PubMed Scopus (481) Google Scholar, 11Kim S.O. Plow E.F. Miles L.A. J. Biol. Chem. 1996; 271: 23761-23767Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 12Camacho M. Fondaneche M.C. Burtin P. FEBS Lett. 1989; 245: 21-24Crossref PubMed Scopus (28) Google Scholar). COOH-terminal lysyl, rather than arginyl, residues are implicated as plasminogen binding sites because plasminogen has a much higher affinity for lysine than arginine. Although CpB treatment reduces plasminogen binding to U937 monocytoid cells by ∼60%, the cell surface-dependent enhancement of plasminogen activation is reduced by >95% (5Felez J. Miles L.A. Fabregas P. Jardi M. Plow E.F. Lijnen R.H. Thromb. Haemost. 1996; 76: 577-584Crossref PubMed Scopus (83) Google Scholar). This suggests that the class of plasminogen binding sites with COOH-terminal lysine residues accessible to CpB is predominantly responsible for the cell surface-dependent enhancement of plasminogen activation. α-Enolase is a candidate monocytoid cell plasminogen receptor with a COOH-terminal lysine (10Miles L.A. Dahlberg C.M. Plescia J. Felez J. Kato K. Plow E.F. Biochemistry. 1991; 30: 1682-1691Crossref PubMed Scopus (481) Google Scholar, 13Redlitz A. Fowler B.J. Plow E.F. Miles L.A. Eur. J. Biochem. 1995; 227: 407-415Crossref PubMed Scopus (208) Google Scholar). However, the number of molecules of α-enolase present on the surface of U937 cells is only 10% of the number of plasminogen binding sites. In the current study, we have utilized susceptibility to CpB treatment as a means to identify a previously unrecognized plasminogen-binding protein that exposes a COOH-terminal lysine in an accessible orientation on the cell surface. To discriminate between such proteins and proteins with inaccessible COOH-terminal lysines, whole cells were treated with CpB to remove exposed COOH-terminal lysines. Cell membranes were subsequently prepared and analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by ligand blotting with125I-plasminogen. Comparison of ligand blots of membranes of untreated intact cells with membranes of CpB-treated intact cells revealed the plasminogen-binding proteins that exposed COOH-terminal lysines on the cell surface, i.e. the class of plasminogen-binding proteins that is predominantly responsible for the cell surface-dependent enhancement of plasminogen activation. Using this methodology we have purified and identified a previously unrecognized plasminogen-binding protein present on the surface of U937 monocytoid cells. We obtained its cDNA sequence, which was identical to human TIP49a. TIP49a was characterized previously as a nuclear protein that has single-stranded DNA-stimulated ATPase and ATP-dependent DNA helicase activity (14Makino Y. Kanemaki M. Kurokawa Y. Koji T. Tamura T. J. Biol. Chem. 1999; 274: 15329-15335Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and is present in the cytosol as well (15Holzmann K. Gerner C. Korosec T. Poltl A. Grimm R. Sauermann G. Biochem. Biophys. Res. Commun. 1998; 252: 39-45Crossref PubMed Scopus (39) Google Scholar). Here, we identify a new subcellular location for TIP49a and demonstrate that TIP49a binds plasminogen and enhances plasminogen activation. Glu-plasminogen was obtained from plasma prepared from fresh human blood collected into 3 mm EDTA, 3 mm benzamidine, 100 KIU/ml Trasylol (Miles, Kankakee, IL), and 100 μg/ml soybean trypsin inhibitor (Sigma). Glu-plasminogen was purified from the plasma by affinity chromatography on lysine-Sepharose (16Deutsch D.G. Mertz E.T. Science. 1970; 170: 1095-1096Crossref PubMed Scopus (1668) Google Scholar) in 0.01 m sodium phosphate, pH 7.3, 0.15 m NaCl (phosphate-buffered saline (PBS)) containing 1 mm benzamidine, 3 mm EDTA, and 0.02% NaN3, followed by gel filtration on Bio-Gel A-5m (Bio-Rad) as described previously (17Miles L.A. Dahlberg C.M. Plow E.F. J. Biol. Chem. 1988; 263: 11928-11934Abstract Full Text PDF PubMed Google Scholar). The concentration of purified Glu-plasminogen was determined at 280 nm using an extinction coefficient of 1.61 E1 cm1% (18Wallen P. Wiman B. Biochim. Biophys. Acta. 1970; 221: 20-30Crossref PubMed Scopus (108) Google Scholar). Glu-plasminogen was iodinated to specific activities of 0.34–0.45 μCi/μg using the IODOGEN method (19Fraker P.J. Speck Jr., J.C. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3613) Google Scholar). Recombinant rat TIP49a was expressed as a fusion protein containing an NH2-terminal histidine tag in BL21(DE3)pLysS Escherichia coli by isopropyl-1-thio-β-d-galactopyranoside induction (20Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5981) Google Scholar). The TIP49a protein was purified from bacterial lysates by nickel-agarose and Mono-Q chromatography as described (21Kanemaki M. Makino Y. Yoshida T. Kishimoto T. Koga A. Yamamoto K. Yamamoto M. Moncollin V. Egly J.M. Muramatsu M. Tamura T. Biochem. Biophys. Res. Commun. 1997; 235: 64-68Crossref PubMed Scopus (97) Google Scholar, 22Makino Y. Mimori T. Koike C. Kanemaki M. Kurokawa Y. Inoue S. Kishimoto T. Tamura T. Biochem. Biophys. Res. Commun. 1998; 245: 819-823Crossref PubMed Scopus (40) Google Scholar). A polyclonal antibody against the recombinant rat TIP49a was generated by injection of protein recovered from sodium dodecyl sulfate (SDS)-polyacrylamide gels as described (21Kanemaki M. Makino Y. Yoshida T. Kishimoto T. Koga A. Yamamoto K. Yamamoto M. Moncollin V. Egly J.M. Muramatsu M. Tamura T. Biochem. Biophys. Res. Commun. 1997; 235: 64-68Crossref PubMed Scopus (97) Google Scholar). U937 cells were cultured in RPMI 1640 supplemented with 5% fetal calf serum, 2 mm l-glutamine, 100 units/ml penicillin G, and 100 μg/ml streptomycin. To prepare peripheral blood monocytes, mononuclear cells first were isolated from fresh human blood collected into acid citrate dextrose. The blood was diluted with two volumes of RPMI 1640 medium without fetal calf serum, and 35-ml aliquots were layered onto 15 ml of Histopaque-1077 (Sigma), then centrifuged at 400 × g for 40 min at 20 °C. The upper layer was removed, and the interface containing the mononuclear cells was carefully aspirated. Contaminating erythrocytes were lysed by resuspending the mononuclear fraction in three volumes of H2O for 20 s, and then quenched with one volume of 0.6m KCl. The mononuclear cells were washed with PBS, then washed twice with PBS containing 2 mm EDTA. Monocytes were isolated from mononuclear cells using the MACS monocyte isolation kit (no. 533-01; Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. For every 107 cells, the cell pellet was resuspended in 160 μl of PBS containing 0.5% bovine serum albumin (BSA), 2 mm EDTA, and 10% heat-inactivated human serum (MACS buffer). 20 μl of FcR blocking reagent and 20 μl of hapten-antibody mixture were added, and the cells were incubated for 5 min on ice. The cells were washed twice, and then 20 μl of FcR blocking reagent and 20 μl of MACS anti-Hapten microbeads/107 cells were added, and the cells incubated for 15 min on ice. The cells were washed once in a 10-fold volume of MACS buffer and resuspended at a density of 2 × 108cells/ml in MACS buffer. The suspension was passed over a CS column in a VarioMACS magnetic separator, and the monocytes were collected as the unbound fraction. For CpB treatment of intact cells, the cells were washed three times in Hanks' balanced salt solution supplemented with 20 mmHEPES (HBSS). The cells were resuspended at a final concentration of 3 × 107 cells/ml, and CpB (Roche Molecular Biochemicals) was added to a final concentration of 100 units/ml. The cells were incubated for 30 min at 37 °C with gentle agitation every 5 min. Control cells were incubated with an equivalent volume of PBS. The cells retained >95% viability as determined by trypan blue exclusion. Prior to subcellular fractionation, cells were washed three times in HBSS, and resuspended at 3 × 107 cells/ml in 20 mm HEPES, pH 7.2, containing the protease inhibitors, 2 μm leupeptin (Calbiochem), 1.5 μm pepstatin A (Calbiochem), 50 KIU/ml Trasylol, 8 μm2-guanidinoethylmercaptosuccinic acid (Calbiochem), and 2 mm phenylmethylsulfonyl fluoride. The cells were chilled on ice for 5 min and then sheared in a 7-ml Dounce homogenizer (Kontes, Vineland, NJ). After homogenization, an equivalent volume of 20 mm HEPES containing 0.5 m sucrose, 10 mm MgCl2, 0.1 m KCl, 2 mm CaCl2, and the protease inhibitors above was added to the cell homogenate. Cell debris was pelleted by centrifugation at 500 × g, and the supernatant was then centrifuged at 100,000 × g for 1 h. The membrane pellet was washed by centrifugation three times with 20 mm HEPES containing 0.25 m sucrose, 5 mm MgCl2, 0.2 m KCl, 1 mm CaCl2, and the protease inhibitors above. Protein concentrations were determined by the BCA protein assay (Bio-Rad) using BSA as a standard. Denaturing first dimension isoelectric focusing (IEF) was performed in a vertical slab format. The IEF gel mixture contained 2% Triton X-100 and 9m urea with either 2.0% Bio-Lytes, pH 6–8 (Bio-Rad), or 2.0% Ampholine, pH 6–8 (Amersham Pharmacia Biotech), unless indicated otherwise, in a 4% polyacrylamide gel with piperazine diacrylamide (Bio-Rad) as cross-linker. Either 100 μg of membrane proteins or 10 μg of cytoplasmic proteins in 0.25% SDS, 2% Triton X-100, 9m urea, and 2% Ampholines were loaded onto the IEF gels, and focused under constant voltage using a stepped-voltage gradient from 50 to 250 V for a total of 4000 V-h, with the current never exceeding 15 mA. The IEF gels were fixed in 12% trichloroacetic acid and washed six times with 50 ml of H2O. Individual lanes were excised from the gels and soaked in reduced sample buffer to resolubilize the proteins. Each lane was placed on the second dimension gel and overlaid with molten 0.5% agarose in 120 mm Tris, pH 6.8. Second dimension SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Samples were subjected to 2D-PAGE and transferred to Immobilon P (Millipore, Bedford, MA) at 100 mA for 18 h in either 25 mm Tris-HCl containing 192 mm glycine and 10% methanol or 10 mm CAPS buffer, pH 11, with 10% methanol. Blots were blocked for 2 h in 2% BSA in PBS containing 0.1% Tween 20 (PBS-BSA), then incubated with 50 nm125I-plasminogen in PBS-BSA for 2 h at 22 °C. The blots were washed seven times with PBS-BSA containing 0.5 m NaCl, then dried and subjected to autoradiography. Control blots were incubated with 50 nm125I-plasminogen in the presence of 0.1 mε-aminocaproic acid. Autoradiography was performed using BioMax MS film (Eastman Kodak Co.). Western blotting was performed on the Immobilon P membranes after probing as ligand blots with125I-plasminogen. The membranes were blocked for 1 h in bovine lactotransfer technique optimizer (5% dry nonfat milk in PBS with 0.03% antifoam A and 2.5 mm phenylmethylsulfonyl fluoride (BLOTTO)) (24Johnson D.A. Gautsch J.W. Sportsman J.R. Elder J.H. Gene Anal. Tech. 1984; 1: 3-8Crossref Scopus (1169) Google Scholar). The primary antibody was added for 1 h, and the membranes were washed three times in Tris-buffered saline (0.01m Tris-HCl, pH 7.2, 0.15 m NaCl) containing 0.1% Tween 20 (TBST). Goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate (Pierce) was added for 1 h in TBST. The blots were washed three times with TBST and then subjected to chemiluminescent detection using either ECL (Amersham Pharmacia Biotech) or Supersignal CL-HRP (Pierce). Nunc Maxisorp 96-well plates were coated with either 5 μg/ml TIP49a or 10 mg/ml BSA in 0.1m NaHCO3, pH 8.5, for 2 h at 37 °C. All wells were postcoated with 10 mg/ml BSA in PBS containing 0.025% Tween 80 (PBST-80) for 1 h at 22 °C, and then washed three times with 1 mg/ml BSA in PBST-80. The immobilized TIP49a or the control BSA-coated wells were incubated with varying concentrations of125I-plasminogen diluted into PBST-80 containing 1 mg/ml BSA for 4 h at 22 °C. The wells were washed four times with PBST-80, the bound 125I-plasminogen was solubilized with 10% SDS, and the radioactivity was quantitated by counting in a γ-counter (Iso-Data Inc., Palatine, IL). Nonspecific binding was determined as the amount of 125I-plasminogen bound to BSA, and specific binding was calculated by subtracting nonspecific binding from total binding. The binding kinetics were analyzed by weighted least squares regression using the program LIGAND (25Munson P.J. Methods Enzymol. 1983; 92: 543-576Crossref PubMed Scopus (275) Google Scholar). Nunc Maxisorp 96-well plates were coated with either 5 μg/ml TIP49a or 10 mg/ml BSA in 0.1m NaHCO3, pH 8.5, for 2 h at 37 °C, or with 10 μg/ml fibrinogen in PBS for 90 min at 37 °C. The fibrinogen-coated wells were washed once with 50 mmTris·Cl, pH 7.5, containing 150 mm NaCl, 0.01% Tween 80, 1 mg/ml BSA, 1 mg/ml gelatin, and 50 units/ml Trasylol, and treated with 60 ng/ml plasmin (Kabi Pharmacia Inc., Franklin, OH) in PBS for 30 min at 37 °C. The plasmin was inactivated with 0.1 mm p-nitrophenyl-p′-guanidine benzoate and 0.5 mm 4-(2-aminoethyl)benzenesulfonyl fluoride in PBS for 20 min at 25 °C. All wells were postcoated with 10 mg/ml BSA in PBST-80 for 1 h at 22 °C and washed three times with 1 mg/ml BSA in PBST-80. The immobilized TIP49a and plasmin-treated fibrinogen were incubated with varying concentrations of 125I-plasminogen diluted into PBST-80 containing 1 mg/ml BSA for 4 h at 22 °C. The wells were then washed four times with PBST-80. A duplicate 96-well plate was included to determine the amount of125I-plasminogen bound to TIP49a and plasmin-treated fibrinogen. The 125I-plasminogen bound in these duplicate wells was solubilized with 10% SDS, and the radioactivity was quantified by counting in a γ-counter. An amount of125I-plasminogen equivalent to the amount bound to the TIP49a-coated wells was added to the BSA-coated wells so that the effects of soluble plasminogen could be compared with plasminogen bound to either TIP49a or plasmin-treated fibrinogen. The plasmin substrate, S-2251 (DiaPharma Group Inc., Franklin, OH), was added to all wells to a final concentration of 0.5 mm and t-PA was added to 15 nm, then A405 was monitored as a measure of plasmin generation. Screening of expressed sequence tag (EST) clone 185194 by PCR was performed using 200 μm each dNTP, 300 nm each primer, 200 ng of plasmid template, and 0.75 unit of Expand Taq (Roche Molecular Biochemicals) in a 25-μl reaction containing 15 mm MgCl2. The reactions were denatured for 2 min at 94 °C, then subjected to 10 cycles of denaturation at 94 °C (30 s), hybridization at 55 °C (30 s), and extension at 68 °C (1 min), followed by 15 cycles where the extension was lengthened by 20 s each cycle. The forward primer was 5′-ATCAACGGATCCGCACTGTCCTAGCTGCTGGT-3′, and the reverse primer was 5′-GGAATGGAATTCTGCAGACCCACGCCTGAATG-3′. The anti-α-enolase antibody was a rabbit polyclonal antibody raised against a peptide corresponding to the COOH-terminal nine amino acid residues of human α-enolase (13Redlitz A. Fowler B.J. Plow E.F. Miles L.A. Eur. J. Biochem. 1995; 227: 407-415Crossref PubMed Scopus (208) Google Scholar). The specificity of this antibody was established by comparison with an α-enolase-specific polyclonal antibody that was a kind gift of Dr. Kanefusa Kato (Institute for Developmental Research, Aichi, Japan) (26Kato K. Asai R. Shimizu A. Suzuki F. Ariyoshi Y. Clin. Chim. Acta. 1983; 127: 353-363Crossref PubMed Scopus (111) Google Scholar) and was specific for α-enolase when used for Western blotting. The anti-cytokeratin 8 antibody was clone M20 (Sigma). We examined U937 membrane-associated proteins for the presence of COOH-terminal lysines exposed to the extracellular environment. Intact U937 cells were incubated in either the presence or absence of 100 units/ml CpB prior to preparing membrane and cytoplasmic fractions as described under “Experimental Procedures.” The fractions were subjected to 2D-PAGE and ligand blotted with125I-plasminogen. Comparison of ligand blots from membrane fractions of intact control cells (Fig.1, panel A) with ligand blots of membrane fractions from intact cells treated with CpB (Fig. 1, panel C), showed that a prominent plasminogen-binding protein (mass ∼ 54 kDa, pI 6.5) was undetectable after CpB treatment of intact cells (arrow), indicating that it lost the ability to bind plasminogen. Cytoplasmic proteins did not show changes in plasminogen binding following CpB treatment of intact cells (Fig. 1, compare panels B and D), suggesting that proteolysis by CpB did not occur in the interior of intact cells. (One plasminogen-binding protein at ∼28 kDa was present in both membrane (panel A) and cytoplasmic (panel B) fractions and did show a decrease in 125I-plasminogen binding in the membrane fraction, and an anomalous decrease in125I-plasminogen binding in the cytoplasmic fraction following CpB treatment. Since only this single protein showed a decrease in 125I-plasminogen binding following CpB treatment in both membrane and cytoplasmic fractions, it most likely represents a contamination of the cytoplasmic preparation by this membrane protein). We examined whether the 54-kDa CpB-sensitive plasminogen-binding protein corresponded to either α-enolase or cytokeratin 8, since these known plasminogen-binding proteins are in the size range of the 54-kDa protein. Western blotting with an anti-α-enolase antibody was performed on the same blots used for ligand blotting of control membranes and cytosol. Two spots reacting with anti-α-enolase were observed on the Western blots at a similar size as the 54-kDa protein, but at different pI values (Fig. 1,panels E and F), consistent with the reported distribution of multiple α-enolase isoforms with distinct pI values (27Heydorn W.E. Creed G.J. Marangos P.J. Jacobowitz D.M. J. Neurochem. 1985; 44: 201-209Crossref PubMed Scopus (26) Google Scholar). Cytokeratin 8 was not detected in U937 membranes or cytosol by Western blotting (data not shown), consistent with its reported pattern of expression (28Moll R. Franke W.W. Schiller D.L. Geiger B. Krepler R. Cell. 1982; 31: 11-24Abstract Full Text PDF PubMed Scopus (4474) Google Scholar, 29Leube R.E. Bosch F.X. Romano V. Zimbelmann R. Hofler H. Franke W.W. Differentiation. 1986; 33: 69-85Crossref PubMed Scopus (82) Google Scholar). These data suggest that a previously unrecognized 54-kDa membrane-associated plasminogen-binding protein exposed a COOH-terminal lysine in an accessible orientation on the extracellular face of the cell. We examined whether the 54-kDa plasminogen-binding protein was up-regulated during cell death. Cells were treated with 10 μg/ml cycloheximide for 18 h, resulting in an increase in the percentage of dead cells from 2% to 23% (as determined by propidium iodide incorporation measured by fluorescence-activated cell sorting). The125I-plasminogen ligand blotting intensity of the 54-kDa protein increased ∼4-fold following the cycloheximide treatment (data not shown). To purify and identify the 54-kDa plasminogen-binding protein, we first examined whether the protein could be concentrated by solubilization of the U937 membranes in Triton X-100 containing protease inhibitors. The Triton X-100 insoluble material was subsequently solubilized in 0.5% SDS, and both soluble and insoluble fractions were subjected to two-dimensional ligand blotting with125I-plasminogen. The 54-kDa protein was present in the Triton X-100 insoluble fraction in an ∼3-fold higher concentration than in the Triton X-100-soluble fraction (Fig.2). The concentration by Triton X-100 solubilization was sufficient to allow purification of the novel plasminogen-binding protein directly from two-dimensional gels. We sought to purify the 54-kDa plasminogen-binding protein directly from two-dimensional gels. 300 μg of the Triton X-100-insoluble fraction from U937 membranes were subjected to 2D-PAGE and replicate gels were either ligand-blotted with 125I-plasminogen (Fig.3, panel A) or stained with colloidal Coomassie (Fig. 3, panel B). Two replicate colloidal Coomassie-stained gels were aligned with the 125I-plasminogen ligand blot and the 54-kDa plasminogen-binding protein was excised from the gels. The protein was eluted from the gels and digested with trypsin. A capillary reverse-phase chromatograph coupled to the electrospray ionization source of a Finnigan LCQ quadrupole ion trap mass spectrometer was used to obtain two peptide sequences, 11 and 18 residues, from the tryptic digest of the protein (Table I). The exact peptides were found in a rat protein, TATA-binding protein-interacting protein (TIP49a) by searching the nonredundant protein sequence data base (30Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (58771) Google Scholar). When we obtained these sequences, they were not present in the National Center for Biotechnology Information nonredundant human protein data base.Table IAlignment of internal sequences of the 54-kDa plasminogen-binding protein with human TIP49aFragment 1L*D P S I*F*E S L*Q*K*Human TIP49aL D P S I F E S L Q KFragment 2R V E A G D V I*Y I*E A N S G A V K*Human TIP49aR V E A G D V I Y I E A N S G A V KAmino acid sequences of the peptides obtained by trypsin digestion of the purified 54 kDa plasminogen-binding protein are aligned with the cDNA-derived protein sequence of human TIP49a from Makino et al. (22Makino Y. Mimori T. Koike C. Kanemaki M. Kurokawa Y. Inoue S. Kishimoto T. Tamura T. Biochem. Biophys. Res. Commun. 1998; 245: 819-823Crossref PubMed Scopus (40) Google Scholar). Isobaric residues are marked with an asterisk. Open table in a new tab Amino acid sequences of the peptides obtained by trypsin digestion of the purified 54 kDa plasminogen-binding protein are aligned with the cDNA-derived protein sequence of human TIP49a from Makino et al. (22Makino Y. Mimori T. Koike C. Kanemaki M. Kurokawa Y. Inoue S. Kishimoto T. Tamura T. Biochem. Biophys. Res. Commun. 1998; 245: 819-823Crossref PubMed Scopus (40) Google Scholar). Isobaric residues are marked with an asterisk. BLAST searches of the nonredundant human EST data base yielded three ESTs whose calculated translation products were an exact match to the 11-residue peptide sequence from the 54-kDa protein, and one EST whose calculated translation product was an exact match to the 18-residue peptide sequence. The EST that matched the 18-residue peptide sequence also contained cDNA sequence encoding the 11-residue peptide sequence in frame with the other peptide sequence. These ESTs were used to search the TIGR Human Gene Index data base (available via the World Wide Web) for tentative human consensus (THC) sequences (31Adams M.D. Kerlavage A.R. Fleischmann R.D. Fuldner R.A. Bult C.J. Lee N.H. Kirkness E.F. Weinstock K.G. Gocayne J.D. White O. et al.Nature. 1995; 377: 3-174PubMed Google Scholar). The ESTs used to assemble the THC sequence were then used to search GenBank for additional ESTs with significant sequence identity. Alignment of the ESTs revealed that there were significant amounts of sequence not contained within the original THC, and that the aligned ESTs extended over two THC sequences, THC 171358 and THC 153501 (Fig.4). All ESTs mapped to this alignment, suggesting that only one molecule could account for the peptide sequences generated. A consensus sequence was generated from the EST alignment, and the predicted translation product of the consensus sequence was identical to the rat TIP49a protein except for five amino acid substitutions (Fig. 5).Figure 5Protein alignments. Figure shows alignment of the predicted translation products of the cloned human TIP49a cDNA, the rat TIP49a cDNA, and the consensus sequence from our EST alignment, with the region of protein sequenced by mass spectrometry shown underlined. Diff" @default.
• W2050510894 created "2016-06-24" @default.
• W2050510894 creator A5014757822 @default.
• W2050510894 creator A5083982323 @default.
• W2050510894 creator A5085639432 @default.
• W2050510894 date "2001-01-01" @default.
• W2050510894 modified "2023-10-13" @default.
• W2050510894 title "Purification, Cloning, and Characterization of a Profibrinolytic Plasminogen-binding Protein, TIP49a" @default.
• W2050510894 cites W1511842456 @default.
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