Identification of Annexin II Heterotetramer as a Plasmin Reductase
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m111219200
ISSN1083-351X
AutoresMijung Kwon, Jennifer F. Caplan, Nolan R. Filipenko, Kyu‐Sil Choi, Sandra L. Fitzpatrick, Libo Zhang, David M. Waisman,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoAnnexin II heterotetramer (AIIt) is a Ca2+- and phospholipid-binding protein that consists of two copies of a p36 and p11 subunit. AIIt regulates the production and autoproteolysis of plasmin at the cell surface. In addition to its role as a key cellular protease, plasmin also plays a role in angiogenesis as the precursor for antiangiogenic proteins. Recently we demonstrated that the primary antiangiogenic plasmin fragment, called A61 (Lys78-Lys468) was released from cultured cells. In the present study we report for the first time that AIIt possesses an intrinsic plasmin reductase activity. AIIt stimulated the reduction of the plasmin Cys462-Cys541 bond in a time- and concentration-dependent manner, which resulted in the release of A61 from plasmin. Mutagenesis of p36 C334S and either p11 C61S or p11 C82S inactivated the plasmin reductase activity of the isolated subunits, suggesting that specific cysteinyl residues participated in the plasmin reductase activity of each subunit. Furthermore, we demonstrated that the loss of AIIt from the cell surface of HT1080 cells transduced with a retroviral vector encoding p11 antisense dramatically reduced the cellular production of A61 from plasminogen. This is the first demonstration that AIIt regulates the cellular production of the antiangiogenic plasminogen fragment, A61. Annexin II heterotetramer (AIIt) is a Ca2+- and phospholipid-binding protein that consists of two copies of a p36 and p11 subunit. AIIt regulates the production and autoproteolysis of plasmin at the cell surface. In addition to its role as a key cellular protease, plasmin also plays a role in angiogenesis as the precursor for antiangiogenic proteins. Recently we demonstrated that the primary antiangiogenic plasmin fragment, called A61 (Lys78-Lys468) was released from cultured cells. In the present study we report for the first time that AIIt possesses an intrinsic plasmin reductase activity. AIIt stimulated the reduction of the plasmin Cys462-Cys541 bond in a time- and concentration-dependent manner, which resulted in the release of A61 from plasmin. Mutagenesis of p36 C334S and either p11 C61S or p11 C82S inactivated the plasmin reductase activity of the isolated subunits, suggesting that specific cysteinyl residues participated in the plasmin reductase activity of each subunit. Furthermore, we demonstrated that the loss of AIIt from the cell surface of HT1080 cells transduced with a retroviral vector encoding p11 antisense dramatically reduced the cellular production of A61 from plasminogen. This is the first demonstration that AIIt regulates the cellular production of the antiangiogenic plasminogen fragment, A61. Angiostatin was originally identified in the urine of mice bearing Lewis lung carcinoma as a 38-kDa proteolytically derived fragment of plasminogen which encompassed the first four kringle domains (Lys78-Ala440). Angiostatin was shown to be a potent antiangiogenic protein that inhibited the growth of human and murine carcinomas and also induced dormancy in their metastases. Angiostatin was also characterized as a specific antiangiogenic protein that blocked microvascular endothelial cell proliferation but not the proliferation of nonendothelial cells (1.O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Cao Y. Moses M. Lane W.S. Sage E.H. Folkman J. Cold Spring Harbor Symp. Quant. Biol. 1994; 59: 471-482Crossref PubMed Scopus (183) Google Scholar). It is now apparent that angiostatin is a member of a family of antiangiogenic plasminogen fragments (AAPFs). 1The abbreviations used are: AAPF(s)antiangiogenic plasminogen fragment(s)A61plasminogen fragment defined by the amino acid sequence Lys78-Lys468AIItannexin II heterotetramerDMEMDulbecco's modified Eagle's mediumHRPhorseradish peroxidaseMPB3-(N-maleimidopropionyl)biocytinPBSphosphate buffered salineu-PAurokinase-type plasminogen activator Physiologically relevant AAPFs include a 38-kDa AAPF isolated from the conditioned media of tumor-infiltrating macrophages (2.Dong Z. Kumar R. Yang X. Fidler I.J. Cell. 1997; 88: 801-810Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar) and AAPFs of 48, 42, and 50 kDa present in macrophage-conditioned media (3.Falcone D.J. Khan K.F. Layne T. Fernandes L. J. Biol. Chem. 1998; 273: 31480-31485Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Other AAPFs include a 50-kDa AAPF isolated from the conditioned media of human prostate carcinoma PC-3 cells (4.Gately S. Twardowski P. Stack M.S. Patrick M. Boggio L. Cundiff D.L. Schnaper H.W. Madison L. Volpert O. Bouck N. Enghild J. Kwaan H.C. Soff G.A. Cancer Res. 1996; 56: 4887-4890PubMed Google Scholar, 5.Gately S. Twardowski P. Stack M.S. Cundiff D.L. Grella D. Castellino F.J. Enghild J. Kwaan H.C. Lee F. Kramer R.A. Volpert O. Bouck N. Soff G.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10868-10872Crossref PubMed Scopus (277) Google Scholar) and AAPFs of 66, 60, and 57 kDa detected in the conditioned media of HT1080 and Chinese hamster ovary cells (6.Stathakis P. Lay A.J. Fitzgerald M. Schlieker C. Matthias L.J. Hogg P.J. J. Biol. Chem. 1999; 274: 8910-8916Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Because the carboxyl terminus of most of these AAPFs was not determined, the exact primary sequence of most of the AAPFs is not known. antiangiogenic plasminogen fragment(s) plasminogen fragment defined by the amino acid sequence Lys78-Lys468 annexin II heterotetramer Dulbecco's modified Eagle's medium horseradish peroxidase 3-(N-maleimidopropionyl)biocytin phosphate buffered saline urokinase-type plasminogen activator Two distinct pathways for the formation of AAPFs have been identified. First, certain proteinases can directly cleave plasminogen into AAPFs. These proteinases include metalloelastase, gelatinase B (matrix metalloproteinase-9), stromelysin-1 (matrix metalloproteinase-3), matrilysin (matrix metalloproteinase-7), cathepsin D, and prostate-specific antigen (7.Patterson B.C. Sang Q.A. J. Biol. Chem. 1997; 272: 28823-28825Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 8.Cornelius L.A. Nehring L.C. Harding E. Bolanowski M. Welgus H.G. Kobayashi D.K. Pierce R.A. Shapiro S.D. J. Immunol. 1998; 161: 6845-6852PubMed Google Scholar, 9.Lijnen H.R. Ugwu F. Bini A. Collen D. Biochemistry. 1998; 37: 4699-4702Crossref PubMed Scopus (180) Google Scholar, 10.Morikawa W. Yamamoto K. Ishikawa S. Takemoto S. Ono M. Fukushi J. Naito S. Nozaki C. Iwanaga S. Kuwano M. J. Biol. Chem. 2000; 275: 38912-38920Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 11.Heidtmann H.H. Nettelbeck D.M. Mingels A. Jager R. Welker H.G. Kontermann R.E. Br. J. Cancer. 1999; 81: 1269-1273Crossref PubMed Scopus (123) Google Scholar). The source of these proteinases may be tumor-infiltrating macrophages (2.Dong Z. Kumar R. Yang X. Fidler I.J. Cell. 1997; 88: 801-810Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar) or the cancer cells themselves. For example, the conversion of plasminogen to angiostatin by macrophages is dependent on the release of metalloelastase from these cells. In comparison, Lewis lung carcinoma cells release matrix metalloproteinase-2, which also cleaves plasminogen to angiostatin (12.O'Reilly M.S. Wiederschain D. Stetler-Stevenson W.G. Folkman J. Moses M.A. J. Biol. Chem. 1999; 274: 29568-29571Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Second, AAPFs are also generated by a three-step mechanism that involves the conversion of plasminogen to plasmin by urokinase-type plasminogen activator (u-PA), the autoproteolytic cleavage of plasmin, and the release of the resultant plasmin fragment by cleavage of disulfide bonds. The cleavage of the plasmin disulfide bonds can be accomplished by free sulfhydryl group donors such as glutathione or by hydroxyl ions at alkaline pH (4.Gately S. Twardowski P. Stack M.S. Patrick M. Boggio L. Cundiff D.L. Schnaper H.W. Madison L. Volpert O. Bouck N. Enghild J. Kwaan H.C. Soff G.A. Cancer Res. 1996; 56: 4887-4890PubMed Google Scholar, 5.Gately S. Twardowski P. Stack M.S. Cundiff D.L. Grella D. Castellino F.J. Enghild J. Kwaan H.C. Lee F. Kramer R.A. Volpert O. Bouck N. Soff G.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10868-10872Crossref PubMed Scopus (277) Google Scholar, 13.Wu H.L. Shi G.Y. Wohl R.C. Bender M.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8793-8795Crossref PubMed Scopus (48) Google Scholar, 14.Kassam G. Kwon M. Yoon C.-S. Graham K.S. Young M.K. Gluck S. Waisman D.M. J. Biol. Chem. 2001; 276: 8924-8933Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Alternatively, the plasmin disulfide bonds can be cleaved enzymatically by a plasmin reductase such as phosphoglycerate kinase (15.Stathakis P. Fitzgerald M. Matthias L.J. Chesterman C.N. Hogg P.J. J. Biol. Chem. 1997; 272: 20641-20645Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 16.Lay A.J. Jiang X.M. Kisker O. Flynn E. Underwood A. Condron R. Hogg P.J. Nature. 2000; 408: 869-873Crossref PubMed Scopus (237) Google Scholar). Our laboratory has shown that the primary AAPF present in mouse and human blood has a molecular mass of 61 kDa (14.Kassam G. Kwon M. Yoon C.-S. Graham K.S. Young M.K. Gluck S. Waisman D.M. J. Biol. Chem. 2001; 276: 8924-8933Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We have produced this AAPF, called A61, in a cell-free system consisting of u-PA and plasminogen. A61 was shown to be a novel four-kringle-containing plasminogen fragment consisting of the amino acid sequence Lys78-Lys468 (14.Kassam G. Kwon M. Yoon C.-S. Graham K.S. Young M.K. Gluck S. Waisman D.M. J. Biol. Chem. 2001; 276: 8924-8933Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The release of A61 from plasmin required cleavage of the Lys468-Gly469 bond by plasmin autoproteolysis and also cleavage of the Cys462-Cys541disulfide. Because A61 was generated in our cell-free system from plasmin at alkaline pH in the absence of sulfhydryl donors, we concluded that cleavage of the Cys462-Cys541disulfide was catalyzed by hydroxyl ions in vitro. In contrast, at physiological pH, we observed that the conversion of plasminogen to A61 was very slow. These results contrasted with our observation that at physiological pH, HT1080 fibrosarcoma and bovine capillary endothelial cells stimulated the rapid formation of A61. The mechanism by which these cells stimulated plasmin reduction and the release of A61 from plasmin was unclear, but it was reasonable to suspect that the cells utilized a plasminogen-binding protein on the cell surface to facilitate the formation of A61. Annexin II tetramer (AIIt) is a Ca2+-binding protein that binds plasminogen and plasmin and stimulates both the formation and autoproteolysis of plasmin at the cell surface (17–19; for review see Ref. 20.Kang H.M. Choi K.S. Kassam G. Fitzpatrick S.L. Kwon M. Waisman D.M. Trends Cardiovasc. Med. 1999; 9: 92-102Crossref PubMed Scopus (52) Google Scholar). The protein consists of two copies of a 36-kDa subunit (p36) called annexin II and two copies of a 11-kDa subunit (p11) called S100A10. Previous work from our laboratory has shown that the carboxyl-terminal lysines of the p11 subunit play a key role in plasminogen binding and activation (18.Kassam G. Le B.H. Choi K.S. Kang H.M. Fitzpatrick S.L. Louie P. Waisman D.M. Biochemistry. 1998; 37: 16958-16966Crossref PubMed Scopus (116) Google Scholar). In the present report we show that AIIt stimulates the conversion of plasminogen to A61 in vitro. We also report for the first time that AIIt possesses an intrinsic plasmin reductase activity and that the cysteinyl residues of both subunits of AIIt participate in the reduction of plasmin. Among the seven annexins tested, only annexin II (p36) and AIIt act as plasmin reductase. Additionally, the antisense nucleotide-directed down-regulation of AIIt on the surface of HT1080 fibrosarcoma cells greatly diminishes A61 production by these cancer cells. Our results therefore show for the first time that AIIt may play a role in angiogenesis by regulating the cellular release of an antiangiogenic protein. Two-chain u-PA was a generous gift from Dr. H. Stack (Abbott Laboratories). Glu-plasminogen and plasmin were purchased from American Diagnostica. A61 was purified as outlined previously (14.Kassam G. Kwon M. Yoon C.-S. Graham K.S. Young M.K. Gluck S. Waisman D.M. J. Biol. Chem. 2001; 276: 8924-8933Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). AIIt and other annexins were purified from bovine lung as described in by Khanna et al. (21.Khanna N.C. Helwig E.D. Ikebuchi N.W. Fitzpatrick S. Bajwa R. Waisman D.M. Biochemistry. 1990; 29: 4852-4862Crossref PubMed Scopus (66) Google Scholar). Monoclonal anti-human plasminogen kringle 1–3 antibody was purchased from Enzyme Research Laboratories, Inc. Monoclonal anti-annexin II and anti-annexin II light chain antibodies were purchased from Transduction Laboratories. Anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody was purchased from Santa Cruz Biotechnology. HRP-conjugated streptavidin and protein disulfide isomerase were purchased from Calbiochem. Anti-mouse R-phycoerythrin-conjugated secondary antibody was purchased from Caltag Laboratories, Inc. 3-(N-Maleimidopropionyl)biocytin (MPB) was purchased from Molecular Probes. Reduced glutathione, iodoacetamide, iodoacetic acid, diisopropylfluorophosphate, and thioredoxin were purchased from Sigma. l-Lys-Sepharose was purchased from Amersham Biosciences, Inc. IODO-BEADS were purchased from Pierce. Phosphoglycerate kinase was a generous gift from Dr. P. J. Hogg (Center for Thrombosis and Vascular Research, University of New South Wales, Sydney, Australia). Stably transfected HeLa cells expressing p11 antisense or sense mRNA were a generous gift from Dr. J. H. Shelhamer (Critical Care Medicine Department, National Institutes of Health). HT1080 fibrosarcoma cells were obtained from American Type Culture Collection. Dulbecco's modified Eagle's medium (DMEM) was purchased from Invitrogen. Bacterial expression vectors containing the wild-type sequence for annexin II (pAED4.91-annexin II) and p11 (pAED4.91-p11) were mutated using the QuikChange™ site-directed mutagenesis kit (Stratagene). Briefly, mutagenic primers were synthesized which introduced Cys → Ser mutations at positions 8 and 334 of annexin II and at positions 61 and 82 of p11. All of the mutations introduced were verified by DNA sequence analysis. These various plasmids were then transformed into Escherichia coli BL21 (DE3) and grown as described previously (22.Filipenko N.R. Waisman D.M. J. Biol. Chem. 2001; 276: 5310-5315Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). After a 4-h induction with isopropyl-1-thio-β-d-galactopyranoside, bacteria were collected by low speed centrifugation. The cells were subsequently sonicated in lysis buffer (10 mm imidazole, pH 7.5, 150 mm NaCl, 2 mm EGTA, 1 mmdithiothreitol + protease inhibitors) and centrifuged at 100,000 × g for 1 h at 4 °C. Both mutant annexin II proteins were purified in the same manner as wild-type annexin II via hydroxyapatite, heparin-Sepharose affinity, and gel permeation chromatography as reported previously (22.Filipenko N.R. Waisman D.M. J. Biol. Chem. 2001; 276: 5310-5315Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The elution profiles of the recombinant wild-type and mutant annexin II on hydroxyapatite, heparin affinity, and gel permeation chromatography were indistinguishable. In addition, the circular dichroism spectra of each of the proteins were very similar, indicating little secondary structure perturbation. After 4 h of induction with isopropyl-1-thio-β-d-galactopyranoside, bacteria were collected by low speed centrifugation. The cells were subsequently sonicated in lysis buffer (100 mm Tris-HCl, pH 7.5, 200 mm NaCl, 10 mm MgCl2, 2 mm dithiothreitol + protease inhibitors) and centrifuged at 100,000 × g for 1 h at 4 °C. Both p11 mutants were purified in the same manner as wild-type p11 (23.Ayala-Sanmartin J. Vincent M. Sopkova J. Gallay J. Biochemistry. 2000; 39: 15179-15189Crossref PubMed Scopus (23) Google Scholar). Briefly, the cell lysis supernatant was precipitated with 50% (NH4)2SO4, and the supernatant was applied to a butyl-Sepharose column equilibrated in lysis buffer containing 50% (NH4)2SO4. The p11 was eluted with a linear gradient of (NH4)2SO4 from 50 to 0%, and peak fractions containing p11 were pooled and dialyzed against 10 mm imidazole, pH 7.4, 1 mm EGTA, 0.5 mm dithiothreitol, and 0.1 mm EDTA. The dialyzed fractions were subsequently applied to a DEAE-Sepharose column equilibrated in the same buffer. The p11 was eluted with a linear NaCl gradient, concentrated to 4 ml, and applied to a Sephacryl S-100 column equilibrated in 40 mm Tris-HCl, pH 7.4, 0.1 mmEGTA, and 0.1 mm dithiothreitol. A single protein peak was recovered at the expected molecular weight based on gel filtration standards. Sense and antisense p11 expression vector were produced by cloning the full-length human p11 cDNA into the pLin retroviral vector in the sense (pLin-p11S) or antisense orientation (pLin-p11AS) as reported (24.Ghahary A. Tredget E.E. Chang L.J. Scott P.G. Shen Q. J. Invest. Dermatol. 1998; 110: 800-805Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Control cells transduced with the vector alone (pLin-V) were also established. The pLin vector carries the Moloney murine leukemia virus 5′-long terminal repeat enhancer/promoter region to promote strong, constitutive expression of the cloned p11 inserts and neomycin phosphotransferase gene in mammalian cells. The pLin constructs were propagated in a PA317 retroviral packaging cell line. Packaging cells were selected in 300 μg/ml neomycin, and conditioned media that contained high titers of the virus were used to transduce the HT1080 fibrosarcoma cells. After viral transduction the neomycin-resistant HT1080 fibrosarcoma cells were cloned and permanent cell lines established. 2K. S. Choi, D. K. Fogg, and D. M. Waisman, manuscript in preparation. After purification or reconstitution, AIIt, p11, p36 (annexin II), other annexins, thioredoxin, protein disulfide isomerase, and phosphoglycerate kinase were dialyzed against 20 mm Tris, pH 7.5, and 140 mm NaCl under argon gas to prevent possible oxidation. 4 μm Glu-plasminogen was incubated with 0.075 μm u-PA and a candidate plasmin reductase protein (4 μm, unless described) in a buffer containing 20 mm Tris, pH 7.5, and 140 mm NaCl at 37 °C for 2 h. A portion of reaction mixture was diluted with SDS-PAGE sample buffer and subjected to nonreduced SDS-PAGE followed by Coomassie Blue staining. To label any free thiol groups of produced protein(s), the reaction mixture was incubated with 100 μm MPB at room temperature for 30 min. The reaction mixture was then treated with 200 μm reduced glutathione at room temperature for 10 min to quench the unreacted MPB. The unreacted glutathione and other free thiols in the reaction mixture were blocked with 400 μm iodoacetamide at room temperature for 10 min. Then the reaction mixture was incubated withl-Lys-Sepharose at room temperature for 30 min to purify the kringle-containing, plasminogen-derived proteins. The matrix was washed extensively with PBS (137 mm NaCl, 8 mmNa2HPO4, 1.4 mm KH2PO4, 2.7 mm KCl, pH 8.0), and the bound proteins were eluted by boiling the resin with SDS-PAGE sample buffer. Each sample was subjected to nonreduced SDS-PAGE followed by Western blot with HRP-conjugated streptavidin (streptavidin-HRP) as indicated below. 2 μm AIIt, p11, or p36 was incubated with 100 μm MPB in a buffer containing 20 mm Tris, pH 7.5, and 140 mm NaCl at room temperature for 30 min. After incubation, 200 μmreduced glutathione and 400 μm iodoacetamide were added sequentially, and the reaction mixture was subjected to reduced SDS-PAGE followed by either Coomassie Blue staining or Western blot with streptavidin-HRP as indicated below. Transduced HT1080 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mml-glutamine, 10 units/ml penicillin G, 10 μmstreptomycin sulfate, and 300 μg/ml neomycin. Approximately 1 × 105 cells were added to each well of 24-well tissue culture plates and incubated at 37 °C for 24 h. The cell monolayers were then washed three times with DMEM, and 2 μmGlu-plasminogen, plasmin, or diisopropylfluorophosphate-treated plasmin in DMEM was added to each well. The conditioned medium was removed at indicated times, diluted with SDS-PAGE sample buffer with or without β-mercaptoethanol, and subjected to SDS-PAGE followed by Western blot with monoclonal anti-human plasminogen kringle 1–3 antibody as indicated below. Samples were diluted with SDS-PAGE sample buffer and subjected to SDS-PAGE and electrophoretically transferred to nitrocellulose membrane (0.45-μm pore size) at 4 °C for 1 h. The membrane was blocked in PBS containing 0.1% Tween 20 (TPBS) (137 mm NaCl, 8 mm Na2HPO4, 1.4 mmKH2PO4, 2.7 mm KCl, pH 8.0, and 0.1% Tween 20) with 5% skim milk at room temperature for 1 h and incubated at 4 °C overnight with a 0.4 μg/ml monoclonal anti-human plasminogen kringle 1–3 antibody in TPBS with 5% skim milk. The blot was washed extensively with TPBS at room temperature and then incubated at room temperature for 1 h with a 0.16 μg/ml HRP-conjugated goat anti-mouse secondary antibody in TPBS with 5% skim milk. In the case of MPB-reacted protein samples, the membrane was blocked and incubated at room temperature for 1 h with a 0.1 μg/ml streptavidin-HRP in TPBS with 5% skim milk. The membrane was washed extensively with TPBS and visualized by enhanced chemiluminescence (Pierce). The purified A61 was iodinated according to manufacturer's procedures. Iodinated A61 retained biological activity as determined by the endothelial cell proliferation assay (14.Kassam G. Kwon M. Yoon C.-S. Graham K.S. Young M.K. Gluck S. Waisman D.M. J. Biol. Chem. 2001; 276: 8924-8933Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 96-well Immulon-1 Removawell strips (Dynex Technologies) were coated with a phospholipid mixture containing a 3:1 ratio of phosphatidylserine to phosphatidylcholine and air dried. The coated strips were blocked with 1% fatty acid-free bovine serum albumin in a buffer containing 20 mm Hepes, pH 7.4, 140 mm NaCl, and 2 mm CaCl2 (buffer A) at room temperature for 2 h. The strips were washed with buffer A and incubated with 1 μm AIIt in buffer A at room temperature for 4 h. The strips were then washed and incubated with 0.008–5 μmiodinated A61 with or without 30-fold molar excess of cold A61 or bovine serum albumin at 4 °C. After overnight incubation, the strips were washed five times with PBS, and individual wells were detached and measured for radioactivity with a γ-counter. The data shown (n = 6) are the average of two separate experiments. Transduced HT1080 cells were harvested, and 1 × 106 cells in PBS were divided into each tube. The cells were fixed with 4% paraformaldehyde in PBS at room temperature for 20 min and washed twice with PBS. The cells were then incubated with 1 μg of monoclonal anti-annexin II or anti-annexin II light chain antibody at room temperature for 30 min. For the control staining, 1 μg of mouse Ig G was used. The cells were washed and incubated with 2 μg/ml anti-mouse R-phycoerythrin-conjugated secondary antibody at room temperature for 30 min. The cells were washed and subjected to flow cytometric analysis using FACScan (Beckton Dickinson) and analyzed by the FlowJo program. The data shown are a representative of three separate experiments. A61 is an internal fragment of plasminogen which encompasses the sequence Lys78-Lys468. For A61 to be released from plasmin, the Cys462-Cys541disulfide of plasmin must be reduced. The release of A61will therefore result in the generation of a free sulfhydryl residue at Cys462. Because plasminogen and plasmin contain only disulfides, A61 can be discriminated from these proteins on the basis of its reactivity with free sulfhydryl-reactive reagents such as MPB. The reaction of free sulfhydryl-containing proteins with MPB results in the biotinylation of the protein, which allows easy detection with streptavidin-HRP. As shown in Fig. 1A, lane 2, the incubation of u-PA with plasminogen resulted in the generation of plasmin. As expected, the plasmin generated by this reaction did not contain a free cysteinyl residue and therefore did not react with MPB (Fig. 1B, lane 2). However, the addition of AIIt to the u-PA-plasminogen reaction resulted in the appearance of A61 (which displays a single major band and two minor bands of about 50 kDa on nonreduced SDS-PAGE) and disappearance of plasmin (Fig. 1A). Furthermore, the A61 generated in these reactions reacted with MPB, confirming the presence of a free sulfhydryl in A61 (Fig. 1B). AIIt stimulated the dose- and time-dependent conversion of plasminogen to A61 (Fig. 1, A–D). The maximal conversion of plasminogen to A61 occurred at approximately equimolar concentrations of AIIt and plasminogen (Fig. 1, A and B). At equimolar concentrations the half-maximal conversion of plasminogen to A61 occurred between 30 and 60 min (Fig. 1, C and D). Because AIIt stimulated the generation of A61 in the absence of sulfhydryl donors, our data suggest that AIIt promoted the cleavage of a plasmin disulfide, presumably the Cys462-Cys541disulfide, resulting in the release of A61 from plasmin and the generation of a free cysteine (Cys462) in A61. Interestingly, because AIIt loses reactivity to MPB during the reaction (data not shown) it is likely that AIIt becomes oxidized during plasmin reduction. AIIt is composed of two copies of a p36 and two copies of a p11 subunit. The p11 subunit plays a key role in plasminogen binding and stimulation of the plasminogen activator-catalyzed conversion of plasminogen to plasmin (18.Kassam G. Le B.H. Choi K.S. Kang H.M. Fitzpatrick S.L. Louie P. Waisman D.M. Biochemistry. 1998; 37: 16958-16966Crossref PubMed Scopus (116) Google Scholar). We therefore attempted to identify which subunit of AIIt was responsible for the plasmin reductase activity of AIIt. As shown in Fig. 2, A and B, the incubation of either the p36 or p11 subunit with u-PA and plasminogen stimulated the formation of A61. However, AIIt was a more potent plasmin reductase than either subunit, suggesting that the interaction of the subunits potentiated the plasmin reductase activity of either subunit. Theoretically, either the disulfides or cysteinyl residues of AIIt could participate in the reduction of plasmin. It is possible that upon plasmin(ogen) binding the disulfide of annexin II is reduced by the thiols of annexin II, and the newly formed thiols participate in reduction of plasmin. Because the p11 subunit does not contain disulfides we suspected that the thiols of this subunit were important for its plasmin reductase activity. p11 contains two cysteinyl residues: Cys61, which plays a critical role in the binding of p36, and Cys82, which is a free thiol (25.Johnsson N. Weber K. J. Biol. Chem. 1990; 265: 14464-14468Abstract Full Text PDF PubMed Google Scholar). We prepared two recombinant forms of this subunit in which individual cysteinyl residues were mutated to serine. This conservative mutation results in the substitution of a thiol group for a hydroxyl group. As shown in Fig. 2B, substitution of either of these cysteine residues resulted in a loss of plasmin reductase activity of the p11 subunit. This suggests that both cysteinyl residues of p11 are required to sustain the plasmin reductase activity of the protein. Human p36 contains two thiol-containing cysteines, Cys8 and Cys334. As shown in Fig. 2B, substitution of Cys334, but not Cys8, with serine blocked the plasmin reductase activity of p36. This result suggests that the Cys334 thiol is critical for plasmin reductase activity of the p36 subunit. To identify the reactive thiols of AIIt we incubated AIIt and its individual subunits with MPB and resolved the mixture with SDS-PAGE followed by Coomassie Blue staining (Fig. 3A) or Western blot with streptavidin-HRP (Fig. 3B). As shown in Fig. 3B, the p11 subunit and both C61S and C82S p11 mutants were labeled with MBP. In contrast, although p36 and the C334S p36 mutant were labeled with MPB, the C8S mutant was not labeled. This suggests that the thiols of the isolated p11 are accessible to MPB, whereas the Cys334 of p36 is not accessible. Interestingly, both the p36 and p11 subunits within AIIt were labeled with MPB. However, preincubation of AIIt with iodoacetic acid or MPB resulted in only a small decrease in the plasmin reductase activity of AIIt (Fig. 3C). This suggests that the Cys334 thiol of the p36 subunit of AIIt is inaccessible to the solvent. The Cys334 residue is highly conserved among many of the annexins. Because our data suggested that this residue participated in the plasmin reductase activity of p36, we suspected that other annexins that possess this cysteine might also have plasmin reductase activity. We therefore examined seven different annexins for plasmin reductase activity. As shown in Fig. 4, only annexin II (p36) and AIIt possessed plasmin reductase activity. This result establishes that plasmin reductase activity is not a common feature of the annexins. Previous data from our laboratory have established that AIIt binds both plasminogen and plasmin and is present at discrete regions of the extracellular surface (17.Kassam G. Choi K.S. Ghuman J. Kang H.M. Fitzpatrick S.L. Zackson T. Zackson S. Toba M. Shinomiya A. Waisman D.M. J. Biol. Chem. 1998; 273: 4790-4799Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 20.Kang H.M. Choi K.S. Kassam G. Fitzpatrick S.L. Kwon M. Waisman D.M. Trends Cardiova
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