Plasmin Reduction by Phosphoglycerate Kinase Is a Thiol-independent Process
2002; Elsevier BV; Volume: 277; Issue: 11 Linguagem: Inglês
10.1074/jbc.m111531200
ISSN1083-351X
AutoresAngelina J. Lay, Xing‐Mai Jiang, Elise B. Daly, Lisa Sun, Philip J. Hogg,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoPhosphoglycerate kinase (PGK) is secreted by tumor cells and facilitates reduction of disulfide bond(s) in plasmin (Lay, A. J., Jiang, X.-M., Kisker, O., Flynn, E., Underwood, A., Condron, R., and Hogg, P. J. (2000) Nature 408, 869–873). The angiogenesis inhibitor, angiostatin, is cleaved from the reduced plasmin by a combination of serine- and metalloproteinases. The chemistry of protein reductants is typically mediated by a pair of closely spaced Cys residues. There are seven Cys in human PGK, and mutation of all seven to Ala did not appreciably affect plasmin reductase activity, although some of the mutations perturbed the tertiary structure of the protein. Cys-379 and Cys-380 are close to the hinge that links the N- and C-terminal domains of PGK. Alkylation/oxidation of Cys-379 and -380 by four different thiol-reactive compounds reduced plasmin reductase activity to 7–35% of control. Binding of 3-phosphoglycerate and/or MgATP to the N- and C-terminal domains of PGK, respectively, triggers a hinge bending conformational change in the enzyme. Incubation of PGK with 3-phosphoglycerate and/or MgATP ablated plasmin reductase activity, with half-maximal inhibitory effects at ∼1 mmconcentration. In summary, reduction of plasmin by PGK is a thiol-independent process, although either alkylation/oxidation of the fast-reacting Cys near the hinge or hinge bending conformational change in PGK perturbs plasmin reduction by PGK, perhaps by obstructing the interaction of plasmin with PGK or perturbing conformational changes in PGK required for plasmin reduction. Phosphoglycerate kinase (PGK) is secreted by tumor cells and facilitates reduction of disulfide bond(s) in plasmin (Lay, A. J., Jiang, X.-M., Kisker, O., Flynn, E., Underwood, A., Condron, R., and Hogg, P. J. (2000) Nature 408, 869–873). The angiogenesis inhibitor, angiostatin, is cleaved from the reduced plasmin by a combination of serine- and metalloproteinases. The chemistry of protein reductants is typically mediated by a pair of closely spaced Cys residues. There are seven Cys in human PGK, and mutation of all seven to Ala did not appreciably affect plasmin reductase activity, although some of the mutations perturbed the tertiary structure of the protein. Cys-379 and Cys-380 are close to the hinge that links the N- and C-terminal domains of PGK. Alkylation/oxidation of Cys-379 and -380 by four different thiol-reactive compounds reduced plasmin reductase activity to 7–35% of control. Binding of 3-phosphoglycerate and/or MgATP to the N- and C-terminal domains of PGK, respectively, triggers a hinge bending conformational change in the enzyme. Incubation of PGK with 3-phosphoglycerate and/or MgATP ablated plasmin reductase activity, with half-maximal inhibitory effects at ∼1 mmconcentration. In summary, reduction of plasmin by PGK is a thiol-independent process, although either alkylation/oxidation of the fast-reacting Cys near the hinge or hinge bending conformational change in PGK perturbs plasmin reduction by PGK, perhaps by obstructing the interaction of plasmin with PGK or perturbing conformational changes in PGK required for plasmin reduction. phosphoglycerate kinase dibromobimane 5,5′-dithiobis(2-nitrobenzoic acid) reduced glutathione oxidized glutathione 3-(N-maleimidylpropionyl)biocytin 3-phosphoglycerate N-ethylmaleimide tetrathionate wild type 4-morpholineethanesulfonic acid Disulfide bonds of certain cell-surface proteins can interchange between the oxidized and reduced state (1Jiang X.-M. Fitzgerald M. Grant C.M. Hogg P.J. J. Biol. Chem. 1999; 274: 2416-2423Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 2Lawrence D.A. Song R. Weber P. J. Leuk. Biol. 1996; 60: 611-618Crossref PubMed Scopus (86) Google Scholar, 3Täger M. Kröning H. Thiel U. Ansorage S. Exp. Hematol. 1997; 25: 601-607PubMed Google Scholar). These observations suggest that the function of some secreted proteins may be controlled by interchange of one or more disulfide bonds (1Jiang X.-M. Fitzgerald M. Grant C.M. Hogg P.J. J. Biol. Chem. 1999; 274: 2416-2423Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The reduction of disulfide bonds in plasmin by a tumor cell-derived protein was the first example of disulfide exchange in a secreted soluble protein (4Stathakis 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, 5Stathakis 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, 6Lay 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). A second example is disulfide exchange in von Willebrand Factor, which is facilitated by thrombospondin-1 (7Xie L. Chesterman C.N. Hogg P.J. J. Exp. Med. 2001; 193: 1341-1349Crossref PubMed Scopus (112) Google Scholar). Plasmin reduction is the first step in formation of the tumor angiogenesis inhibitor, angiostatin. Tumor expansion and metastasis is dependent on tumor neovascularization, or angiogenesis (8Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7218) Google Scholar). Angiogenesis is balanced by several protein activators and inhibitors (9Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6091) Google Scholar). One such inhibitor is angiostatin (10O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3172) Google Scholar), which is an internal fragment of the plasma zymogen, plasminogen. Plasminogen contains five consecutive kringle domains followed by a serine proteinase module. Urokinase- or tissue-plasminogen activator converts plasminogen to plasmin by hydrolysis of a single peptide bond in the serine proteinase module. Plasmin is processed in the conditioned medium of tumor cells, producing angiostatin fragments consisting of kringle domains 1–4½, 1–4, and 1–3 (Ref. 6Lay 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 and references therein). Plasmin proteolysis occurs in three stages. First, the Cys-462–Cys-541 and Cys-512–Cys-536 disulfide bonds in kringle 5 of plasmin are reduced by a plasmin reductase (5Stathakis 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). Second, reduction of the kringle 5 disulfide bonds triggers cleavage at Arg-530–Lys-531 in kringle 5 and also at 2 other positions C- terminal of Cys-462, by a serine proteinase (5Stathakis 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). Autoproteolysis can account for the cleavage (5Stathakis 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, 11Gately 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), although another serine proteinase is responsible in human fibrosarcoma cell-conditioned medium (5Stathakis 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). Third, the kringle 1–4½ fragments are cleaved by matrix metalloproteinases to produce either kringle 1–4 or 1–3 (12Dong Z. Kumar R. Yang X. Fidler I.J. Cell. 1997; 88: 801-810Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 13Cornelius 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, 14O'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). All three kringle-containing fragments have been shown to inhibit endothelial cell proliferation in vitro and angiogenesis in vivo (Ref. 15Cao R. Wu H.L. Veitonmaki N. Linden P. Farnebo J. Shi G.Y. Cao Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5728-5733Crossref PubMed Scopus (184) Google Scholar and references therein). The plasmin reductase was recently purified from human fibrosarcoma cell-conditioned medium and shown to be the glycolytic enzyme, phosphoglycerate kinase (PGK1; ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) (6Lay 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). Plasma of mice bearing fibrosarcoma tumors contained severalfold more PGK than mice without tumors and administration of PGK to tumor-bearing mice caused an increase in plasma levels of angiostatin and a decrease in tumor vascularity and rate of tumor growth. Solid tumors employ PGK and other glycolytic enzymes to facilitate anaerobic production of ATP. These findings indicate that PGK plays an additional role in tumorigenesis by initiating an extracellular formation of angiostatin from plasmin. PGK is the sixth enzyme of the glycolytic pathway where it catalyzes the high energy phosphoryl transfer reaction from the acid anhydride bond of 1,3-bisphosphoglycerate to the β-phosphate of MgADP. PGK also influences DNA replication and repair in mammalian cell nuclei (16Vishwanatha J.K. Jindal H.K. Davis R.G. J. Cell Sci. 1992; 101: 25-34Crossref PubMed Google Scholar,17Popanda O. Fox G. Thielmann H.W. Biochim. Biophys. Acta. 1998; 1397: 102-117Crossref PubMed Scopus (109) Google Scholar), stimulates viral mRNA synthesis in the cytosol (18Ogino T. Iwama M. Kinouchi J. Shibagaki Y. Tsukamoto T. Mizumoto K. J. Biol. Chem. 1999; 274: 35999-36008Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and extends through the cell wall of Candida albicans (19Alloush H.M. López-Ribot J.L. Masten B.J. Chaffin W.L. Microbiology (Reading). 1997; 143: 321-330Crossref PubMed Scopus (88) Google Scholar). The human enzyme has a molecular mass of ∼45 kDa and consists of a single polypeptide chain of 418 residues. Crystallographic studies of the yeast (20Banks R.D. Blake C.C.F. Evans P.R. Haser R. Rice D.W. Hardy G.W. Merrett M. Phillips A.W. Nature. 1979; 279: 773-777Crossref PubMed Scopus (391) Google Scholar, 21Watson H.C. Walker N.P.C. Shaw P.J. Bryant T.N. Wendell P.L. Fothergill L.A. Perkins R.E. Conroy S.C. Dobson M.J. Tuite M.F. Kingsman A.J. Kingsman S.M. EMBO J. 1982; 1: 1635-1640Crossref PubMed Scopus (347) Google Scholar), horse (22Blake C.C.F. Evans P.R. J. Mol. Biol. 1974; 84: 585-601Crossref PubMed Scopus (155) Google Scholar), and pig (23Harlos K. Vas M. Blake C.C.F. Proteins. 1992; 12: 133-144Crossref PubMed Scopus (118) Google Scholar, 24May A. Vas M. Harlos K. Blake C. Proteins. 1996; 24: 292-303Crossref PubMed Scopus (45) Google Scholar, 25Szilágyi A.N. Ghosh M. Garman E. Vas M. J. Mol. Biol. 2001; 306: 499-511Crossref PubMed Scopus (57) Google Scholar) enzyme revealed that the molecule is composed of two domains of similar size, which corresponds to the N- and C-terminal halves of the chain separated by a hinge region. Mammalian PGKs contain seven Cys, and only two of the seven are nearby in the primary or tertiary structure (26Minard P. Desmadril M. Ballery N. Perahia D. Mouawad L. Eur. J. Biochem. 1989; 185: 419-423Crossref PubMed Scopus (31) Google Scholar). Cys-379 and -380 are close to the hinge region and have been referred to as “fast-reacting” as they are amenable to alkylation by several thiol-reactive compounds (27Cserpán I. Vas M. Eur. J. Biochem. 1983; 131: 157-162Crossref PubMed Scopus (31) Google Scholar). The role of all seven Cys and in particular the two fast-reacting Cys in reduction of the plasmin disulfide bonds by PGK has been explored in this study. We show that none of the PGK Cys residues are directly involved in plasmin reduction but that alkylation/oxidation of the fast-reacting Cys or conformational changes in the same region of the protein inhibit reductase activity. 3-(N-Maleimidylpropionyl)biocytin (MPB) and dibromobimane (bBBr) were from Molecular Probes, Eugene, OR. HgCl2, sodium tetrathionate (TT), reduced (GSH) and oxidized glutathione (GSSG), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), ATP, and 3-phosphoglycerate (3-PG) were from Sigma. Plasminogen was purified from fresh frozen human plasma and separated into its two carbohydrate variants according to Castellino and Powell (28Castellino F.J. Powell J.R. Methods Enzymol. 1981; 80: 365-378Crossref PubMed Scopus (180) Google Scholar). Glu-1-plasminogen was used in the experiments described herein. Urokinase plasminogen activator was a gift from Serono Australia. Plasmin was generated by incubating plasminogen (20 μm) with urokinase plasminogen activator (20 nm) for 30 min in 20 mm HEPES, 0.14m NaCl, pH 7.4, buffer at 37 °C. A 1.33-kilobase human PGK cDNA was isolated by reverse transcriptase-PCR from total RNA extracted from HT1080 cells as described previously (6Lay 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). The Cys residues at positions 379 and 380 in wild-type PGK were mutated to Ala by replacing the T at positions 1214 and 1217 to G and the G at positions 1215 and 1218 to C using the following primers and the QuikChange site-directed mutagenesis kit from Stratagene, La Jolla, CA: 5′-GACACTGCCACTGCCGCTGCCAAATGGAACAC-3′ (forward, positions 1202–1233) and 5′-GTGTTCCATTTGGCAGCGGCAGTGGCAGTGTC-3′ (reverse, positions 1233–1202). The derived mutant was called C379A,C380A. Subsequently, Cys-367, -316, -108, -99, and -50 were cumulatively mutated to Ala using the same approach as for C379A,C380A. A single Cys-Ala mutation at position 50 was also generated using wt PGK as the template. The integrity of the mutant DNA was confirmed by automated DNA sequencing. The PGK DNAs were sub-cloned into the plasmid vector, pET11a, which was then transfected intoEscherichia coli strain, BL21 (DE3) (Novagen, Madison, WI). Expression of recombinant protein was induced by 0.5 mmisopropyl-β-d-thiogalactopyranoside for 5 h at 37 °C. The BL21 cells were collected by centrifugation at 5000 × g for 15 min and resuspended in B-PER bacterial extraction reagent (Pierce) at a ratio of 60 ml.liter−1 of culture. The cells were lysed by sonication in the presence of 0.5% Triton X-100, and the lysate was clarified by centrifugation at 14,000 × g for 30 min. Solid (NH4)2SO4 was added slowly to the clarified supernatant to a concentration of 70% and stirred for 20 min at 4 °C. The pellet was collected by centrifugation at 12,000 × g for 20 min, and additional (NH4)2SO4 was added to the supernatant to a final concentration of 90–100% and stirred for 20 min at 4 °C. The pellet was collected by centrifugation at 12,000 × g for 20 min, dissolved in 20 mmHEPES, 0.05 m NaCl, 1 mm EDTA, 0.02% NaN3, pH 7.4, buffer and dialyzed extensively against the same buffer. The protein was applied to an 80-ml (2.5 × 17 cm) column of Cibachron Blue-Sepharose (Amersham Biosciences, Inc.) equilibrated with the same HEPES buffer. The column was washed with 3 bed volumes of the HEPES buffer at a flow rate of 1 ml·min−1 to elute unbound proteins and developed with a 240-ml linear NaCl gradient from 0.05 to 2 m in the HEPES buffer. PGK eluted at ∼1 m NaCl. The eluate was dialyzed against 20 mm Tris-HCl, 150 mm NaCl, 0.5 mm EDTA, 1 mm dithiothreitol, pH 7.9, buffer and applied to a 5-ml column of heparin-Sepharose (Amersham Biosciences, Inc.) equilibrated with the same buffer. The column was washed with 3 bed volumes of the Tris buffer at a flow rate of 0.5 ml·min−1 to elute unbound proteins and developed with a 40-ml linear NaCl gradient from 0.15 to 1 m in the Tris buffer. PGK eluted at ∼0.8 m NaCl. The purified PGK was dialyzed against 20 mm HEPES, 0.14 m NaCl, 1 mm EDTA, pH 7.4, buffer and stored at −80 °C until use. Plasmin (2 μg.ml−1) was incubated with wt or mutant PGKs (1–40 μg.ml−1) in 20 mm HEPES, 0.14 m NaCl, 0.05% Tween 20, pH 7.4, buffer (HEPES/Tween) for 30 min at temperatures between 20 and 50 °C. On some occasions, the incubation buffer was 50 mm MES, 0.125 m NaCl, 2 mm EDTA, pH 6.0, 50 mm HEPES, 0.125 m NaCl, 2 mm EDTA, pH 7.0 or 50 mm HEPES, 0.125m NaCl, 2 mm EDTA, pH 8.0. On other occasions, the PGK was incubated with 3-PG (0–13 mm) and/or ATP (0–13 mm) and 1 mm MgCl2 in HEPES/Tween before the addition of plasmin. The control reactions were plasmin or PGK incubated alone in HEPES/Tween. Free thiols generated in plasmin/angiostatin were labeled with MPB (100 μm) for 30 min at 37 °C followed by quenching of the unreacted MPB with GSH (200 μm) for 10 min at 37 °C. Unreacted GSH and other free sulfhydryls in the system were blocked with NEM (400 μm) for 10 min at 37 °C. The plasmin kringle products were collected on 50 μl of packed lysine-Sepharose (Amersham Biosciences, Inc.) beads by incubation on a rotating wheel for 1 h at room temperature, washed three times with HEPES/Tween, and eluted with 50 μl of 50 mm ε-amino-caproic acid in HEPES/Tween. Two assays for plasmin reduction were employed. In one assay, the MPB-labeled plasmin/angiostatin fragments were immobilized on wells coated with the murine anti-angiostatin monoclonal antibody, 8.19, and detected using streptavidin-peroxidase (6Lay 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). The 8.19 antibody (100 μl of 5 μg.ml−1 in 0.1 m NaHCO3, 0.02% NaN3, pH 8.3 buffer) was adsorbed to PolySorp 96-well plates (Nunc, Roskilde, Denmark) overnight at 4 °C in a humid environment. Wells were washed once with HEPES/Tween, and nonspecific binding sites were blocked by adding 200 μl of 5% nonfat milk powder in 20 mm HEPES, 0.14 m NaCl, 0.02% NaN3, pH 7.4, buffer and incubating for 90 min at 37 °C and then washed two times with HEPES/Tween. MPB-labeled plasmin/angiostatin fragments were diluted 1:8 in HEPES/Tween, and 100-μl aliquots were added to antibody-coated wells and incubated for 30 min at room temperature with orbital shaking. Wells were washed three times with HEPES/Tween, and 100 μl of 1:100 dilution of StreptABComplex/horseradish peroxidase (Dako, Carpinteria, CA) in HEPES/Tween was added and incubated for 30 min at room temperature with orbital shaking. Wells were washed three times, and the bound peroxidase was detected as described previously (5Stathakis 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). In the other assay, the MPB-labeled plasmin/angiostatin fragments were resolved on 8–16% SDS-PAGE under non-reducing conditions, transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA), blotted with a 1:2000 dilution of streptavidin horseradish peroxidase (Molecular Probes), then developed and visualized using chemiluminescence (PerkinElmer Life Sciences). wt PGK (10 μm) was incubated with a 1-, 2-, or 4-fold molar excess of HgCl2, bBBr, or TT in 0.1 m HEPES, 0.3 m NaCl, 1 mm EDTA, pH 7.0, buffer for 1 h at room temperature. wt PGK (90 μm) was also incubated with a 1,000-fold molar excess of GSSG in the HEPES buffer for 2 h at room temperature, and the unreacted GSSG was removed by dialysis against the HEPES buffer. The untreated and alkylated PGKs (10 μm) were incubated with DTNB (200 μm) in 0.1 m HEPES, 0.3 m NaCl, 1 mm EDTA, pH 7.0, buffer for 30 min at room temperature. The absorbance at 412 nm due to the formation of the 5-thio-2-nitrobenzoic acid dianion was measured using a Molecular Devices Thermomax Plus (Palo Alto, CA) microplate reader. The extinction coefficient for the 5-thio-2-nitrobenzoic acid dianion at pH 7.0 is 14,150 m−1 cm−1 at 412 nm (29Riddles P.W. Blakeley R.L. Zerner B. Methods Enzymol. 1983; 91: 49-60Crossref PubMed Scopus (1068) Google Scholar). The seven Cys in wt PGK were mutated to Ala in a cumulative fashion. The two fast-reacting Cys, Cys-379 and -380, were mutated first, and the resulting C379A,C380A PGK cDNA was then used to mutate Cys-367. The corresponding C379A,C380A,C367A PGK cDNA was then used to mutate Cys-316 and so forth. The Cys were mutated in the order, Cys-379, -380, -367, -316, -108, -99, and -50. The C50A PGK mutant was also made. The wt and mutant PGKs were expressed in E. coli and extracted with lysozyme. The extract was clarified by ammonium sulfate precipitation, and the protein was purified by affinity chromatography on Cibachron Blue-Sepharose and heparin-Sepharose. The wt and mutant PGKs were homogeneous by SDS-PAGE (Fig. 1). Different concentrations of the wt and mutant PGKs were incubated with plasmin in HEPES/Tween-buffered saline for 30 min at 37 °C, and the thiols in the reduced plasmin/angiostatin were labeled with MPB. The plasmin/angiostatin fragments were immobilized on antibody-coated wells, and the MPB was detected using streptavidin-peroxidase. The control reactions were the incubations of PGK or plasmin alone. These controls tested for any confounding effects of labeling of the existing free thiols in plasmin or PGK by MPB. Both reactions resulted in negligible signals. The specific plasmin reductase activity of the wt and mutant PGKs were similar, with the exception of the Cys-less PGK (C379A,C380A,C367A,C316A,C108A,C99A,C50A PGK) (Fig. 2 A). This result suggested that Cys-50, which was the last Cys to be mutated, was required for plasmin reductase activity. To test this hypothesis, the C50A PGK mutant was made and assayed for plasmin reductase activity. This C50A mutant had similar specific activity as wt PGK (Fig.2 A). The same qualitative results were observed for all the PGK mutants when plasmin reduction was measured by resolving the MPB-labeled proteins on SDS-PAGE and blotting with streptavidin-peroxidase to detect the labeled angiostatin fragments (Fig. 2 B). These results indicated that neither Cys-50 nor any of the other PGK Cys were required for plasmin reduction. We hypothesized that the Cys-less PGK had lost plasmin reductase activity due to secondary effects of the mutations on the integrity of the PGK tertiary structure. This theory was tested by examining the susceptibility of the mutant PGKs to proteolysis by plasmin. wt or mutant PGKs were incubated with plasmin, and proteolysis of the PGKs was examined by SDS-PAGE. wt PGK and the C379A,C380A,C379A, C380S,C367A,C316A, and C50A PGK mutants were resistant to plasmin proteolysis (Fig.3). In contrast, the C379A,C380A, C367A, C379A,C380A,C367A,C316A,C108A, C379A,C380A,C367A,C316A,C108A,C99A, and C379A,C380A,C367A,C316A, C108A,C99A,C50A PGK mutants were proteolysed by plasmin to different extents. In particular, the 6 and 7 Cys to Ala mutants were completely degraded by plasmin during the incubation. The proteolysis was plasmin-dependent, as all the PGKs remained intact after incubation with plasmin that had been inactivated with Val-Phe-Lys-chloromethyl ketone (not shown). The involvement of the PGK fast-reacting thiols in plasmin reduction was tested by modifying them with various alkylating/oxidizing reagents. Four different alkylating/oxidizing agents were employed; they were HgCl2, bBBr, TT, and GSSG. HgCl2 and bBBr have been used to alkylate the fast-reacting Cys in pig muscle PGK (30Vas M. Csanády G. Eur. J. Biochem. 1987; 163: 365-368Crossref PubMed Scopus (13) Google Scholar). Reaction of PGK with all four alkylating/oxidizing agents reduced the number of reactive thiols per mol of PGK to ∼0.2–0.5 (Fig. 4, A–D). Equimolar concentrations of HgCl2, bBBr, and TT were maximally effective because a 4-fold molar excess of these agents did not change the number of residual thiols in PGK. Incubation of PGK with a 1,000-fold molar excess of GSSG reduced the number of reactive thiols per mol of PGK to ∼0.4. The alkylated/oxidized PGKs were tested for plasmin reductase activity. The PGKs were incubated with plasmin in HEPES/Tween-buffered saline for 30 min at 37 °C, and the thiols in the reduced plasmin/angiostatin were labeled with MPB. The plasmin/angiostatin fragments were immobilized on antibody-coated wells, and the MPB was detected using streptavidin-peroxidase. The activity of the modified proteins were reduced to 7–35% of control (Fig. 4 E). PGK was incubated with plasmin in different pH buffers for discrete times at 37 °C (Fig.5 A) with increasing concentrations of NaCl in pH 7.4 buffer for 30 min (Fig. 5 B) or at different temperatures in pH 7.4 buffer for 30 min (Fig.5 C). The thiols in the reduced plasmin/angiostatin were labeled with MPB and detected using streptavidin-peroxidase. The plasmin reductase activity of PGK was optimal at pH 7 at early time points of incubation. There was no obvious effect, however, of pH on plasmin reduction after 60 min of incubation. Increasing NaCl concentrations reduced plasmin reduction, although the effects were relatively modest. Reductase activity was reduced by 73% when the NaCl concentration was increased from 0 to 2 m. There was no substantial effect of temperature on plasmin reductase activity of PGK between 20 and 50 °C. The reactivity of the fast-reacting Cys in pig muscle PGK are reduced upon binding of 3-PG and/or MgATP to PGK (31Tompa P. Hong P.T. Vas M. Eur. J. Biochem. 1986; 154: 643-649Crossref PubMed Scopus (33) Google Scholar). This is a result of conformational changes in the enzyme induced by substrate binding (21Watson H.C. Walker N.P.C. Shaw P.J. Bryant T.N. Wendell P.L. Fothergill L.A. Perkins R.E. Conroy S.C. Dobson M.J. Tuite M.F. Kingsman A.J. Kingsman S.M. EMBO J. 1982; 1: 1635-1640Crossref PubMed Scopus (347) Google Scholar). The consequences of substrate-induced conformational changes in PGK for plasmin reductase activity was tested. PGK was incubated with plasmin in the absence or presence of 3-PG and/or ATP and 1 mm MgCl2 in HEPES/Tween-buffered saline for 30 min at 37 °C. The thiols in the reduced plasmin/angiostatin were labeled with MPB and detected using streptavidin-peroxidase. Incubation of PGK with 1 mm 3-PG or MgATP reduced plasmin reductase activity by ∼60%, whereas incubation with 1 mm3-PG and MgATP reduced activity by ∼90% (Fig.6 A). Titration of the inhibitory effects of 3-PG and MgATP on plasmin reductase activity is shown in Figs. 6, B and C. The half-maximal inhibitory effects of 3-PG and MgATP on plasmin reductase activity were ∼1 mm. Gallic and ellagic acid competitively inhibit PGK kinase activity (32Joao H.C. Williams R.J.P. Littlechild J.A. Nagasuma R. Watson H.C. Eur. J. Biochem. 1992; 205: 1077-1088Crossref PubMed Scopus (8) Google Scholar,33Hickey M.J. Coutts I.G.C. Tsang-Tan L.L. Pogson C.I. Biochem. Soc. Trans. 1995; 23: 607Crossref Scopus (5) Google Scholar). Gallic acid appears to bind to the same site as MgATP (32Joao H.C. Williams R.J.P. Littlechild J.A. Nagasuma R. Watson H.C. Eur. J. Biochem. 1992; 205: 1077-1088Crossref PubMed Scopus (8) Google Scholar). Both gallic and ellagic acid at 100 μm concentration inhibited plasmin reduction by PGK by >80% (not shown). NAD(H), another adenine nucleotide, also inhibited plasmin reduction by >60% at 10 mm concentration, presumably through binding to the same site on PGK as MgATP (not shown). Protein reductant active sites typically contain a redox active dithiol/disulfide with the sequence, Cys-Gly-X-Cys (34Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (656) Google Scholar). The Cys thiols cycle between the reduced dithiol and oxidized disulfide bond in coordination with a dithiol or disulfide of a protein substrate. This can result in reduction, formation, or interchange of disulfide bonds in the protein substrate. There are seven Cys in PGK, none of which are involved in disulfide bonds, and only two of the seven are nearby in the primary or tertiary structure (22Blake C.C.F. Evans P.R. J. Mol. Biol. 1974; 84: 585-601Crossref PubMed Scopus (155) Google Scholar, 23Harlos K. Vas M. Blake C.C.F. Proteins. 1992; 12: 133-144Crossref PubMed Scopus (118) Google Scholar, 24May A. Vas M. Harlos K. Blake C. Proteins. 1996; 24: 292-303Crossref PubMed Scopus (45) Google Scholar, 25Szilágyi A.N. Ghosh M. Garman E. Vas M. J. Mol. Biol. 2001; 306: 499-511Crossref PubMed Scopus (57) Google Scholar). This suggests that the mechanism by which PGK reduces disulfide bonds in plasmin is unconventional. We have reported that the plasmin reductase activity of PGK is inhibited by NEM and iodoacetamide (6Lay 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), which implies a role for one or more of the PGK Cys residues in plasmin reduction. In this study we have explored the role of all seven PGK Cys and, in particular, the two fast-reacting Cys in reduction of plasmin disulfide bonds. The 7 Cys in PGK were mutated to Ala in a cumulative fashion in the order, Cys-379, -380, -367, -316, -108, -99, and -50. The two fast-reacting Cys, Cys-379 and -380, were mutated first, and the resulting cDNA was then used to mutate Cys-367 and so forth. The specific plasmin reductase activity of the mutant PGKs, with the exception of the Cys-less PGK, was similar to that of the wild-type protein. Some mutations were shown to change the tertiary structure of PGK, as measured by susceptibility to proteolysis by plasmin. For instance, the Cys-less PGK was rapidly degraded by plasmin, which probably accounted for the loss of plasmin reductase activity. These results implied that PGK Cys residues were not directly involved in plasmin reduction. The question remained, therefore, why alkylation of Cys-379 and -380 inhibited plasmin reduction. This question was explored by reacting the Cys residues with different alkylating/oxidizing agents and examining the consequences for plasmin reductase activity. Carboxymethylation, but not methylation, of the fast-reacting Cys of pig PGK inactivates the kinase activity (35Dékány K. Vas M. Eur. J. Biochem. 1984; 139: 125-130Crossref PubMed Scopus (17) Google Scholar). Reaction of the pig enzyme with DTNB also inactivates the kinase activity (27Cserpán I. Vas M. Eur. J. Biochem. 1983; 131: 157-162Crossref PubMed Scopus (31) Google Scholar), whereas reaction with HgCl2 or bBBr reduces kinase activity by up to 80% (30Vas M. Csanády G. Eur. J. Biochem. 1987; 163: 365-368Crossref PubMed Scopus (13) Google Scholar). HgCl2 reacts with free thiols and can facilitate oxidation of a dithiol to a disulfide bond. bBBr is a homobifunctional alkylating agent that can cross-link thiols in close proximity. The fast-reacting thiols of PGK were also reacted with TT and GSSG. Accessible thiols react with these compounds to form mixed disulfides with thiosulfate and glutathione, respectively. Reaction of PGK with all four alkylating/oxidizing agents reduced the number of reactive thiols per mol of PGK to ∼0.2–0.5. The plasmin reductase activity of the modified proteins was reduced to 7–35% of control. These results indicate that alkylation of the fast-reacting thiols perturb plasmin reductase activity, which is consistent with our earlier report of inhibition of plasmin reductase activity by NEM and iodoacetamide (6Lay 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). Neither changes in pH, ionic strength, nor temperature markedly affected plasmin reductase activity, which implies that the reaction of PGK and plasmin involved predominantly hydrophobic interactions. Kinase substrate binding studies show that MgATP and MgADP bind to the inner surface of the C-domain (20Banks R.D. Blake C.C.F. Evans P.R. Haser R. Rice D.W. Hardy G.W. Merrett M. Phillips A.W. Nature. 1979; 279: 773-777Crossref PubMed Scopus (391) Google Scholar, 21Watson H.C. Walker N.P.C. Shaw P.J. Bryant T.N. Wendell P.L. Fothergill L.A. Perkins R.E. Conroy S.C. Dobson M.J. Tuite M.F. Kingsman A.J. Kingsman S.M. EMBO J. 1982; 1: 1635-1640Crossref PubMed Scopus (347) Google Scholar, 25Szilágyi A.N. Ghosh M. Garman E. Vas M. J. Mol. Biol. 2001; 306: 499-511Crossref PubMed Scopus (57) Google Scholar, 36Davies G.J. Gamblin S.J. Littlechild J.A. Dauter Z. Wilson K.S. Watson H.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 202-209Crossref PubMed Scopus (93) Google Scholar), whereas 3-PG binds to the inner surface of the N-domain (23Harlos K. Vas M. Blake C.C.F. Proteins. 1992; 12: 133-144Crossref PubMed Scopus (118) Google Scholar, 24May A. Vas M. Harlos K. Blake C. Proteins. 1996; 24: 292-303Crossref PubMed Scopus (45) Google Scholar, 25Szilágyi A.N. Ghosh M. Garman E. Vas M. J. Mol. Biol. 2001; 306: 499-511Crossref PubMed Scopus (57) Google Scholar). The bound substrates are ≥10 Å from each other, too large a distance for direct in-line phosphoryl transfer. The enzyme overcomes this distance by undergoing a hinge bending conformational change that brings the two substrates closer together. The reactivity of both the fast-reacting thiols is reduced by binding of 3-PG and/or MgADP and MgATP to PGK (31Tompa P. Hong P.T. Vas M. Eur. J. Biochem. 1986; 154: 643-649Crossref PubMed Scopus (33) Google Scholar). Moreover, carboxyamidomethylation of the two fast-reacting Cys in pig PGK blocks the substrate-induced hinge bending conformational change in the enzyme (37Sinev M.A. Razgulyaev O.I. Vas M. Timchenko A.A. Ptitsyn O.B. Eur. J. Biochem. 1989; 180: 61-66Crossref PubMed Scopus (45) Google Scholar). These observations indicate that alkylation of the fast-reacting thiols in PGK can perturb the conformational changes required for kinase activity. We tested the effect of 3-PG/MgADP-induced conformational changes in PGK for plasmin reductase activity. Incubation of PGK with 1 mm 3-PG or MgATP reduced plasmin reductase activity by ∼60%, whereas incubation with 1 mm 3-PG and MgATP reduced activity by ∼90%. The half-maximal effects of 3-PG and MgATP on plasmin reductase activity were ∼1 mm, which is in the range of the Michaelis constants for these substrates in the kinase reaction (38McHarg J. Kelly S.M. Price N.C. Cooper A. Littlechild J.A. Eur. J. Biochem. 1999; 259: 939-945Crossref PubMed Scopus (30) Google Scholar). It is noteworthy that the effects of 3-PG and MgATP were additive, which may reflect cooperativity between 3-PG and MgATP in the conformational change in PGK. These findings indicate that hinge bending conformational change in PGK negates the plasmin reductase activity of the protein. A model of the molecular events facilitated by PGK in plasmin kringle 5 is shown in Fig. 7. We have previously proposed that both the Cys-462–Cys-541 and Cys-512–Cys-536 disulfide bonds in plasmin kringle 5 are cleaved, which renders kringle 5 susceptible to proteolysis at either the Arg-530–Lys-531 peptide bond or two other unidentified peptide bonds (5Stathakis 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). The other peptide bonds are likely to be Arg-474–Val-475 (5Stathakis 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) and Lys-468–Gly-469 (39Kassam 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). Plasmin undergoes autoproteolysis in alkaline pH, producing a catalytically active microplasmin fragment with a Lys-531 N terminus. Wu et al. (40Wu H.-L. Shi G.-Y. Bender M.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8292-8295Crossref PubMed Scopus (46) Google Scholar, 41Wu 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) noticed that both the Cys-462–Cys-541 and Cys-512–Cys-536 disulfide bonds in K5 must have been cleaved to release microplasmin from K1–4, and they proposed that the increased−OH ion concentration at alkaline pH is responsible for cleaving the Cys-462–Cys-541 disulfide bond. We have suggested that the mechanism of plasmin proteolysis at alkaline pH is the same as the mechanism of proteolysis facilitated by PGK at neutral pH (5Stathakis 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). Calculation of the dihedral strain energies (42Katz B.A. Kossiakoff A. J. Biol. Chem. 1986; 261: 15480-15485Abstract Full Text PDF PubMed Google Scholar, 43Weiner S.J. Kollman P.A. Case D.A. Singh U.C. Ghio C. Alagona G. Profeta Jr., S. Weiner P. J. Am. Chem. Soc. 1984; 106: 765-784Crossref Scopus (4885) Google Scholar) of the kringle 5 disulfide bonds from the crystal structure described by Chang et al. (44Chang Y. Mochalkin I. McCance S.G. Cheng B. Tulinsky A. Castellino F.J. Biochemistry. 1998; 37: 3258-3271Crossref PubMed Scopus (88) Google Scholar) reveal that the left-handed Cys-512–Cys-536 bond has a high strain energy (2.63 kcal.mol−1) compared with the right-handed Cys-462–Cys-541 (1.54 kcal.mol−1) and left-handed Cys-483–Cys-524 (0.98 kcal.mol−1) disulfide bonds. The average dihedral strain energies for left- and right-handed disulfide bonds are 1.68 and 3.19 kcal.mol−1, respectively (42Katz B.A. Kossiakoff A. J. Biol. Chem. 1986; 261: 15480-15485Abstract Full Text PDF PubMed Google Scholar, 43Weiner S.J. Kollman P.A. Case D.A. Singh U.C. Ghio C. Alagona G. Profeta Jr., S. Weiner P. J. Am. Chem. Soc. 1984; 106: 765-784Crossref Scopus (4885) Google Scholar). A high dihedral strain energy correlates with ease of cleavage of the disulfide bond (42Katz B.A. Kossiakoff A. J. Biol. Chem. 1986; 261: 15480-15485Abstract Full Text PDF PubMed Google Scholar). We propose, therefore, that PGK facilitates cleavage of the Cys-512–Cys-536 disulfide bond by −OH, which results in formation of a sulfenic acid at position 512 and a free thiol at Cys-536. The Cys-536 thiol is then available to exchange with the Cys-462–Cys-541 disulfide bond, resulting in formation of a new disulfide at Cys-536–Cys-541 and a free thiol at Cys-462. Kringle 5 is then susceptible to proteolysis at Arg-530–Lys-531, Arg-474–Val-475, and/or Lys-468–Gly-469. This is the simplest sequence of events that can explain all the available data. For instance, cleavage of only the Cys-512–Cys-536 would not enable release of the kringle 1–4 angiostatin fragments from plasmin. The free thiol that is labeled by MPB in the three angiostatin fragments would be Cys-462. We do not exclude cleavage of the Cys-483–Cys-524 disulfide bond, although this is not required to explain the experimental observations. PGK presumably binds to plasmin and induces a conformational change in kringle 5 that facilitates the −OH attack on the Cys-512–Cys-536 disulfide bond. This suggests that other molecules that interact with plasmin might also facilitate cleavage of the kringle 5 disulfides. There is recent evidence to support this hypothesis. Interaction of a truncated porcine plasminogen activator inhibitor-1 (residues 80–265), but not full-length protein, with plasmin has been shown to result in generation of kringle-containing angiostatin fragments (45Mulligan-Kehoe M.J. Wagner R. Wieland C. Powell R. J. Biol. Chem. 2001; 276: 8588-8596Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). It is possible that the truncated protein facilitates the same sequence of events in plasmin that are achieved by PGK.
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