Acid-induced Conformational Changes in Phosphoglucose Isomerase Result in Its Increased Cell Surface Association and Deposition on Fibronectin Fibrils
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m304778200
ISSN1083-351X
AutoresMohammad Amraei, Zongjian Jia, Pascal Reboul, Ivan R. Nabi,
Tópico(s)Cellular transport and secretion
ResumoPhosphoglucose isomerase (PGI) is a glycolytic enzyme that exhibits extracellular cytokine activity as autocrine motility factor, neuroleukin, and maturation factor and that has been recently implicated as an autoantigen in rheumatoid arthritis. In contrast to its receptor-mediated endocytosis at neutral pH, addition of 25 μg/ml of either Alexa 568- or FITC-conjugated PGI to NIH-3T3 cells at progressively acid pH results in its quantitatively increased association with cell surface fibrillar structures that is particularly evident at pH 5. A similar pH-dependent cell surface association of PGI is observed for first passage human chondrocytes obtained from osteoarthritic joints. At acid pH, PGI colocalizes with fibronectin fibrils, and this association occurs directly upon addition of PGI to the cells. In contrast to the receptor-mediated endocytosis of PGI, fibril association of 25 μg/ml PGI at pH 5 is not competed with an excess (2 mg/ml) of unlabeled PGI. PGI binding at acid pH is therefore neither saturable nor mediated by its receptor. PGI is enzymatically active as a dimer and we show here by non-denaturing gel electrophoresis as well as by glutaraldehyde cross-linking that it exists at neutral pH in a tetrameric form. Increasingly acid pH results in the appearance of PGI monomers that correlates directly with its enhanced cell surface association. However, glutaraldehyde cross-linked PGI is endocytosed at neutral pH and still exhibits enhanced cell surface binding at pH 5. Circular dichroism analysis revealed pH-dependent changes in the near but not the far UV spectra indicating that the tertiary structure of the protein is specifically altered at pH 5. Conformational changes of PGI and exposure of the monomer-monomer interface under acidic conditions, such as those encountered in the synovial fluid of arthritic joints, could therefore result in its deposition on the surface of joints and the induction of an autoimmune response. Phosphoglucose isomerase (PGI) is a glycolytic enzyme that exhibits extracellular cytokine activity as autocrine motility factor, neuroleukin, and maturation factor and that has been recently implicated as an autoantigen in rheumatoid arthritis. In contrast to its receptor-mediated endocytosis at neutral pH, addition of 25 μg/ml of either Alexa 568- or FITC-conjugated PGI to NIH-3T3 cells at progressively acid pH results in its quantitatively increased association with cell surface fibrillar structures that is particularly evident at pH 5. A similar pH-dependent cell surface association of PGI is observed for first passage human chondrocytes obtained from osteoarthritic joints. At acid pH, PGI colocalizes with fibronectin fibrils, and this association occurs directly upon addition of PGI to the cells. In contrast to the receptor-mediated endocytosis of PGI, fibril association of 25 μg/ml PGI at pH 5 is not competed with an excess (2 mg/ml) of unlabeled PGI. PGI binding at acid pH is therefore neither saturable nor mediated by its receptor. PGI is enzymatically active as a dimer and we show here by non-denaturing gel electrophoresis as well as by glutaraldehyde cross-linking that it exists at neutral pH in a tetrameric form. Increasingly acid pH results in the appearance of PGI monomers that correlates directly with its enhanced cell surface association. However, glutaraldehyde cross-linked PGI is endocytosed at neutral pH and still exhibits enhanced cell surface binding at pH 5. Circular dichroism analysis revealed pH-dependent changes in the near but not the far UV spectra indicating that the tertiary structure of the protein is specifically altered at pH 5. Conformational changes of PGI and exposure of the monomer-monomer interface under acidic conditions, such as those encountered in the synovial fluid of arthritic joints, could therefore result in its deposition on the surface of joints and the induction of an autoimmune response. Glucose-6-phosphate isomerase or phosphoglucose isomerase (PGI) 1The abbreviations used are: PGI, phosphoglucose isomerase; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; AMF, autocrine motility factor; ER, endoplasmic reticulum; CD, circular dichroism. is a glycolytic enzyme essential for neoglucogenesis that is equivalent to the autocrine motility factor (AMF)/neuroleukin/maturation factor (MF) cytokine (1Watanabe H. Takehana K. Date M. Shinozaki T. Raz A. Cancer Res. 1996; 56: 2960-2963PubMed Google Scholar, 2Chaput M. Claes V. Portetelle D. Cludts I. Cravador A. Burny A. Gras H. Tartar A. Nature. 1988; 332: 454-457Crossref PubMed Scopus (225) Google Scholar, 3Faik P. Walker J.I.H. Redmill A.A.M. Morgan M.J. Nature. 1988; 332: 455-457Crossref PubMed Scopus (164) Google Scholar, 4Xu W. Seiter K. Feldman E. Ahmed T. Chiao J.W. Blood. 1996; 87: 4502-4506Crossref PubMed Google Scholar). PGI is therefore a cytosolic enzyme that upon release from the cell acquires a de novo function as a neurokine, lymphokine, and tumor cell cytokine (4Xu W. Seiter K. Feldman E. Ahmed T. Chiao J.W. Blood. 1996; 87: 4502-4506Crossref PubMed Google Scholar, 5Gurney M.E. Apatoff B.R. Heinrich S.P. J. Cell Biol. 1986; 102: 2264-2272Crossref PubMed Scopus (28) Google Scholar, 6Gurney M.E. Apatoff B.R. Spear G.T. Baumel M.J. Antel J.P. Brown Bania M. Reder A.T. Science. 1986; 234: 574-581Crossref PubMed Scopus (169) Google Scholar, 7Nabi I.R. Watanabe H. Raz A. Cancer Res. 1990; 50: 409-414PubMed Google Scholar, 8Silletti S. Watanabe H. Hogan V. Nabi I.R. Raz A. Cancer Res. 1991; 51: 3301-3311Google Scholar). While the mechanism of release of PGI remains uncertain, enhanced secretion of PGI following overexpression of PGI by stable transfection of NIH-3T3 cells induces cellular transformation and tumorigenicity (9Tsutsumi S. Hogan V. Nabi I.R. Raz A. Cancer Res. 2003; 63: 242-249PubMed Google Scholar). Serum PGI activity has long been reported and is associated with tumor expression (10Bodansky O. Cancer. 1954; 7: 1200-1226Crossref PubMed Scopus (41) Google Scholar, 11Schwartz M.K. Clin. Chem. 1973; 19: 10-22Crossref PubMed Scopus (66) Google Scholar) indicating that this protein is actively released from both normal and tumor cells. PGI exists as a dimer and enzyme dimerization is necessary for its enzymatic activity (12Pon N.G. Schnackerz K.D. Blackburn M.N. Chatterjee G.C. Noltmann E.A. Biochem. 1970; 9: 1506-1514Crossref PubMed Scopus (35) Google Scholar, 13Blackburn M.N. Noltmann E.A. J. Biol. Chem. 1972; 247: 5668-5674Abstract Full Text PDF PubMed Google Scholar, 14Bruch P. Schnackerz K.D. Gracy R.W. Eur. J. Biochem. 1976; 68: 153-158Crossref PubMed Scopus (23) Google Scholar, 15Dyson J.E. Noltmann E.A. J. Biol. Chem. 1968; 243: 1401-1414Abstract Full Text PDF PubMed Google Scholar). The active site of the enzyme has been characterized by x-ray crystallography and is localized to the cleft between the two PGI monomers (16Arsenieva D. Hardre R. Salmon L. Jeffery C.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5872-5877Crossref PubMed Scopus (45) Google Scholar, 17Sun Y.J. Chou C.C. Chen W.S. Wu R.T. Meng M. Hsiao C.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5412-5417Crossref PubMed Scopus (105) Google Scholar, 18Jeffery C.J. Bahnson B.J. Chien W. Ringe D. Petsko G.A. Biochem. 2000; 39: 955-964Crossref PubMed Scopus (109) Google Scholar, 19Read J. Pearce J. Li X. Muirhead H. Chirgwin J. Davies C. J. Mol. Biol. 2001; 309: 447-463Crossref PubMed Scopus (89) Google Scholar). The motifs responsible for PGI cytokine activity remain to be determined. Inhibitors of PGI isomerase activity block its cytokine activity (1Watanabe H. Takehana K. Date M. Shinozaki T. Raz A. Cancer Res. 1996; 56: 2960-2963PubMed Google Scholar, 20Funasaka T. Haga A. Raz A. Nagase H. Biochem. Biophys. Res. Commun. 2001; 285: 118-128Crossref PubMed Scopus (47) Google Scholar, 21Zhi J. Sommerfeldt D.W. Rubin C.T. Hadjiargyrou M. J. Bone Miner. Res. 2001; 16: 1994-2004Crossref PubMed Scopus (31) Google Scholar). Reports that the bacterial form of PGI, whose sequence homology with the mammalian enzyme is limited to residues in the enzymatic active site, presents cytokine activity further supported a role for the active site in PGI cytokine activity (17Sun Y.J. Chou C.C. Chen W.S. Wu R.T. Meng M. Hsiao C.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5412-5417Crossref PubMed Scopus (105) Google Scholar, 22Chou C.-C. Sun Y.-J. Meng M. Hsiao C.-D. J. Biol. Chem. 2000; 275: 23154-23160Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). However, neither cytokine activity or receptor binding of the bacterial or yeast forms of the enzyme were detected in NIH-3T3 cells (23Amraei M. Nabi I.R. FEBS Lett. 2002; 525: 151-155Crossref PubMed Scopus (23) Google Scholar) and punctual mutations in the PGI sequence that disrupt its enzymatic activity do not affect its cytokine function (24Tsutsumi S. Gupta S.K. Hogan V. Tanaka N. Nakamura K.T. Nabi I.R. Raz A. FEBS Lett. 2003; 534: 49-53Crossref PubMed Scopus (24) Google Scholar). The latter studies argue that motifs implicated in receptor binding include regions of the protein that present differences between the mammalian and bacterial forms of the enzyme including the N-terminal, C-terminal, and internal hook domains (18Jeffery C.J. Bahnson B.J. Chien W. Ringe D. Petsko G.A. Biochem. 2000; 39: 955-964Crossref PubMed Scopus (109) Google Scholar). The PGI receptor, gp78 or AMF-R, is a seven-transmembrane domain G protein-coupled receptor (25Shimizu K. Tani M. Watanabe H. Nagamachi Y. Niinaka Y. Shiroishi T. Ohwada S. Raz A. Yokota J. FEBS Lett. 1999; 456: 295-300Crossref PubMed Scopus (105) Google Scholar). AMF-R expression is significantly increased in neoplastic tissue and its expression is correlated with tumor malignancy and poor survival and prognosis of patients with gastric, colorectal, bladder and esophogeal carcinomas, cutaneous malignant melanoma, and pulmonary adenocarcinoma (26Hirono Y. Fushida S. Yonemura Y. Yamamoto H. Watanabe H. Raz A. Br. J. Cancer. 1996; 74: 2003-2007Crossref PubMed Scopus (55) Google Scholar, 27Nakamori S. Watanabe H. Kameyama M. Imaoka S. Furukawa H. Ishikawa O. Sasaki Y. Kabuto T. Raz A. Cancer. 1994; 74: 1855-1862Crossref PubMed Scopus (75) Google Scholar, 28Otto T. Birchmeier W. Schmidt U. Hinke A. Schipper J. Rübben H. Raz A. Cancer Res. 1994; 54: 3120-3123PubMed Google Scholar, 29Maruyama K. Watanabe H. Hitoshi S. Takayama T. Gofuku J. Yano H. Inoue M. Tamura S. Raz A. Monden M. Int. J. Cancer. 1995; 64: 316-321Crossref PubMed Scopus (52) Google Scholar, 30Nagai Y. Ishikawa O. Miyachi Y. Watanabe H. Dermatology. 1996; 192: 8-11Crossref PubMed Scopus (20) Google Scholar, 31Takanami I. Takeuchi K. Naruke M. Kodaira S. Tanaka F. Watanabe H. Raz A. Tumour Biol. 1998; 19: 384-389Crossref PubMed Scopus (28) Google Scholar, 32Taniguchi K. Yonemura Y. Nojima N. Hirono Y. Fushida S. Fujimura T. Miwa K. Endo Y. Yamamoto H. Watanabe H. Cancer. 1998; 82: 2112-2122Crossref PubMed Scopus (101) Google Scholar). In normal brain, AMF-R expression is increased during development and associated with learning and development implicating PGI cytokine activity in normal cellular activity (33Leclerc N. Vallée A. Nabi I.R. J. Neurosci. Res. 2000; 60: 602-612Crossref PubMed Scopus (25) Google Scholar, 34Luo Y. Long J.M. Lu C. Chan S.L. Spangler E.L. Mascarucci P. Raz A. Longo D.L. Mattson M.P. Ingram D.K. Weng N.P. J. Neurochem. 2002; 80: 354-361Crossref PubMed Scopus (31) Google Scholar). AMF-R is expressed both at the cell surface where it associates with caveolae as well as within a smooth domain of the endoplasmic reticulum (35Benlimame N. Le P.U. Nabi I.R. Mol. Biol. Cell. 1998; 9: 1773-1786Crossref PubMed Scopus (104) Google Scholar, 36Benlimame N. Simard D. Nabi I.R. J. Cell Biol. 1995; 129: 459-471Crossref PubMed Scopus (46) Google Scholar, 37Wang H.-J. Guay G. Pogan L. Sauve R. Nabi I.R. J. Cell Biol. 2000; 150: 1489-1498Crossref PubMed Scopus (143) Google Scholar, 38Wang H.-J. Benlimame N. Nabi I.R. J. Cell Sci. 1997; 110: 3043-3053PubMed Google Scholar, 39Accola M.A. Huang B. Al Masri A. McNiven M.A. J. Biol. Chem. 2002; : M201641200Google Scholar). AMF-R internalizes its ligand via both caveolae/raft-dependent endocytosis to the smooth ER and clathrin-dependent endocytosis to multivesicular bodies (35Benlimame N. Le P.U. Nabi I.R. Mol. Biol. Cell. 1998; 9: 1773-1786Crossref PubMed Scopus (104) Google Scholar, 40Le P.U. Benlimame N. Lagana A. Raz A. Nabi I.R. J. Cell Sci. 2000; 113: 3227-3240PubMed Google Scholar, 41Le P.U. Guay G. Altschuler Y. Nabi I.R. J. Biol. Chem. 2002; 277: 3371-3379Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 42Le P.U. Nabi I.R. J. Cell Sci. 2003; 116: 1059-1071Crossref PubMed Scopus (171) Google Scholar, 43Nabi I.R. Le P.U. J. Cell Biol. 2003; 161: 673-677Crossref PubMed Scopus (602) Google Scholar). The latter pathway is associated with the recycling of AMF/PGI to cell surface fibronectin fibrils (40Le P.U. Benlimame N. Lagana A. Raz A. Nabi I.R. J. Cell Sci. 2000; 113: 3227-3240PubMed Google Scholar). Interestingly, PGI has recently been identified as an autoantigen implicated in rheumatoid arthritis (RA) in the K/BxN mouse model as well as in humans (44Matsumoto I. Staub A. Benoist C. Mathis D. Science. 1999; 286: 1732-1735Crossref PubMed Scopus (498) Google Scholar, 45Schaller M. Burton D.R. Ditzel H.J. Nat. Immunol. 2001; 2: 746-753Crossref PubMed Scopus (159) Google Scholar). PGI and anti-PGI are specifically localized to the articular surface of joints (45Schaller M. Burton D.R. Ditzel H.J. Nat. Immunol. 2001; 2: 746-753Crossref PubMed Scopus (159) Google Scholar, 46Matsumoto I. Maccioni M. Lee D.M. Maurice M. Simmons B. Brenner M. Mathis D. Benoist C. Nat. Immunol. 2002; 3: 360-365Crossref PubMed Scopus (296) Google Scholar, 47Wipke B.T. Wang Z. Kim J. McCarthy T.J. Allen P.M. Nat. Immunol. 2002; 3: 366-372Crossref PubMed Scopus (110) Google Scholar). However, the basis for the selective binding of this protein to the surface of the synovial lining as well as for the generation of an immune response against this ubiquitous self-antigen remains a paradox (46Matsumoto I. Maccioni M. Lee D.M. Maurice M. Simmons B. Brenner M. Mathis D. Benoist C. Nat. Immunol. 2002; 3: 360-365Crossref PubMed Scopus (296) Google Scholar, 48Maccioni M. Zeder-Lutz G. Huang H. Ebel C. Gerber P. Hergueux J. Marchal P. Duchatelle V. Degott C. van Regenmortel M. Benoist C. Mathis D. J. Exp. Med. 2002; 195: 1071-1077Crossref PubMed Scopus (187) Google Scholar). Indeed, the extent to which PGI autoantibodies are prevalent in the sera of RA patients remains controversial. While earlier reports indicated that 64% of sera from RA patients contain antibodies to PGI (45Schaller M. Burton D.R. Ditzel H.J. Nat. Immunol. 2001; 2: 746-753Crossref PubMed Scopus (159) Google Scholar), more recent studies have questioned the prevalence and specificity of the PGI autoimmune response in RA (49Herve C.A. Wait R. Venables P.J. Rheumatology (Oxford). 2003; 212: 986-988Crossref Scopus (28) Google Scholar, 50Matsumoto I. Lee D.M. Goldbach-Mansky R. Sumida T. Hitchon C.A. Schur P.H. Anderson R.J. Coblyn J.S. Weinblatt M.E. Brenner M. Duclos B. Pasquali J.L. El-Gabalawy H. Mathis D. Benoist C. Arthritis Rheum. 2003; 48: 944-954Crossref PubMed Scopus (105) Google Scholar, 51Kassahn D. Kolb C. Solomon S. Bochtler P. Illges H. Nat. Immunol. 2002; 3 (discussion 412-413): 411-412Crossref PubMed Scopus (13) Google Scholar). Localization of the autoimmune response to PGI to lymph nodes adjacent to the affected joints in the K/BxN mouse led the authors to suggest that PGI in the joint is different in form or quantity than that circulating in other regions of the body (52Mandik-Nayak L. Wipke B.T. Shih F.F. Unanue E.R. Allen P.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14368-14373Crossref PubMed Scopus (60) Google Scholar). We demonstrate here the dramatically increased binding of PGI to fibronectin fibrils at acid pH that corresponds directly to PGI denaturation and, more specifically, to changes in PGI tertiary structure. Localized acidosis in synovial fluid could therefore enable denaturation and consequent binding of circulating PGI to synovial cell extracellular matrix permitting the generation of an autoimmune response against exposed non-native PGI epitopes. Cells and Materials—NIH-3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, vitamins, non-essential amino acids, glutamine, and penicillin-streptomycin antibiotics (Canadian Life Technologies) at 37 °C in a humidified 5% CO2/95% air incubator as previously described (35Benlimame N. Le P.U. Nabi I.R. Mol. Biol. Cell. 1998; 9: 1773-1786Crossref PubMed Scopus (104) Google Scholar). Human chondrocytes were obtained from articular cartilage (femoral condyles and tibial plateaus) of patients (aged 58 ± 8, mean ± S.D.) who had undergone total knee arthroplasty. All osteoarthritis (OA) patients were evaluated by a certified rheumatologist and were diagnosed as having OA based on the criteria developed by the American College of Rheumatology Diagnostic Subcommittee for OA (53Altman R. Asch E. Bloch D. Bole G. Borenstein D. Brandt K. Christy W. Cooke T.D. Greenwald R. Hochberg M. et al.Arthritis Rheum. 1986; 29: 1039-1049Crossref PubMed Scopus (5352) Google Scholar). Chondrocytes were released from the articular cartilage by sequential enzymatic digestion at 37 °C as previously described (54Tardif G. Pelletier J.P. Dupuis M. Geng C. Cloutier J.M. Martel-Pelletier J. Arthritis Rheum. 1999; 42: 1147-1158Crossref PubMed Scopus (72) Google Scholar), and cultured in DMEM supplemented with 10% fetal bovine serum (Canadian Life Technologies) and penicillin-streptomycin antibiotics (Canadian Life Technologies) at 37 °C in a humidified atmosphere of 5% CO2/95% air. Chondrocytes were used at first passage on Labtek plastic cover slips. Rabbit PGI (P9544) was purchased from Sigma Chemical Co. (Oakville, Ontario). Mouse anti-fibronectin was purchased from Transduction Laboratories (Mississuaga, Ontario) and Alexa 488-conjugated anti-mouse secondary antibody from Molecular Probes (Eugene, OR). Nondenatured protein molecular weight markers were purchased from Sigma and Kaleidoscope SDS molecular weight markers from BioRad. The Alexa Fluor 568 protein labeling kit and the FluoReporter® FITC protein labeling kit were purchased from Molecular Probes, and Alexa 568 and FITC-PGI conjugates were prepared according to the manufacturer's instructions. Briefly, PGI was incubated with either the succinimidyl ester of Alexa Fluor 568 carboxylic acid or fluorescein isothiocyanate in bicarbonate buffer (pH 8.3-9) for 1 h at room temperature. Fluorescent PGI conjugates were separated from free dye by size exclusion chromatography using either a column (Alexa 568) or spin column (FITC). Immunofluorescence Labeling—60,000 NIH-3T3 cells were plated for 2 days on cover slips and incubated at 37 °C for 5 or 30 min with Alexa 568 or FITC-conjugated PGI (25 μg/ml) in bicarbonate containing medium in a CO2 incubator (controls) or in bicarbonate-free medium supplemented with 100 mm HEPES at pH 7.5, 7.0, and 6.5 or with 100 mm MES at pH 6.0, 5.5, and 5.0 in a CO2-free incubator. Cells were then washed with PBS-CM and fixed with 3% paraformaldehyde. Cell associated fluorescence was then quantified using a Wallac VictorV fluorescence plate reader (PerkinElmer, Montreal, Quebec) and appropriate filter sets. Alternatively, labeled cells were visualized by confocal microscopy using a Leica TCS SP-1 confocal microscope. Where indicated, cells incubated with Alexa 568-PGI were labeled with anti-fibronectin antibodies and Alexa 488-conjugated anti-mouse secondary antibodies. Non-denaturing Gel Electrophoresis—Lyophilized rabbit PGI was dissolved in water and protein concentration determined with the BCA protein assay (Pierce). 1.5 μg of mammalian PGI was loaded on non-denaturating 8% acrylamide gels prepared with an upper gel using Laemmli buffers without SDS (55Laemmli U. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207478) Google Scholar). To a solution of 1.5 μg of PGI, HEPES or MES buffers at the indicated pH were added to a final concentration of 250 mm and then sample buffer consisting of glycerol and bromphenol blue was added. Protein bands and non-denatured protein markers (Jack bean urease: 272 kDa; bovine serum albumin: 132 and 66 kDa; chicken egg albumin: 45 kDa; carbonic anhydrase: 29 kDa) were revealed by silver staining. PGI Cross-linking—Cross-linking of rabbit PGI with glutaraldehyde (MECALAB LTD. Montreal, Quebec, Canada) was carried out as previously described (56Darawshe S. Tsafadyah Y. Daniel E. Biochem. J. 1987; 242: 689-694Crossref PubMed Scopus (41) Google Scholar). Briefly, 0.5 mg/ml PGI was incubated with 0.1% (v/v) glutaraldehyde in 75 μl of PBS for different times at room temperature. The reaction was stopped by addition of SDS sample buffer. Samples and SDS molecular weight markers were boiled and reduced, separated in 8% SDS-polyacrylamide gels, and protein bands revealed by staining with Coomassie Blue. Alternatively, Alexa 568 and FITC-PGI were cross-linked, diluted with 2 ml of 100 mm glycine for at least 30 min to quench the reaction and then concentrated using Amicon filter units and added to cells at 25 μg/ml for fluorescent visualization as described previously. Circular Dichroism—Circular dichroism (CD) analysis of PGI was performed using a Jasco J-710 spectropolarimeter (Dept. of Chemistry and Biochemistry, Concordia University, Montreal, Quebec). Spectra were recorded in a 0.1-cm quartz cuvette at room temperature in MES- or HEPES-based buffers at pH 7.5, 6, 5.5, and 5 and background signal obtained from parallel scans of the buffer alone were subtracted from the measurements. Far UV spectra of 0.1 μg/ml PGI were recorded from 260 to 190 nm at a speed of 100 nm/min in 0.2-nm steps and with a signal averaging time of 0.25 s. Near UV spectra of 0.5 μg/ml PGI were recorded from 320 to 250 nm at a speed of 20 nm/min in 0.2-nm steps and with a signal averaging time of 2 s. The presented spectra are representative of at least three separate scans. Increased Cell Surface Fibrillar Binding of PGI Binding at Acid pH—PGI is an intracellular glycolytic enzyme however its ability to associate with the articular surface of joints (45Schaller M. Burton D.R. Ditzel H.J. Nat. Immunol. 2001; 2: 746-753Crossref PubMed Scopus (159) Google Scholar, 46Matsumoto I. Maccioni M. Lee D.M. Maurice M. Simmons B. Brenner M. Mathis D. Benoist C. Nat. Immunol. 2002; 3: 360-365Crossref PubMed Scopus (296) Google Scholar, 47Wipke B.T. Wang Z. Kim J. McCarthy T.J. Allen P.M. Nat. Immunol. 2002; 3: 366-372Crossref PubMed Scopus (110) Google Scholar) implicates the binding of exogenous PGI to the surface of cells. Incubation of NIH-3T3 cells with Alexa 568-conjugated PGI and fixation with paraformaldehyde results in the predominant visualization of its expression in multivesicular bodies (MVBs) as well as to more faintly labeled fibrils (40Le P.U. Benlimame N. Lagana A. Raz A. Nabi I.R. J. Cell Sci. 2000; 113: 3227-3240PubMed Google Scholar), a distribution still observed upon pH reduction of the medium to pH 6.5 (Fig. 1). At pH 6.0, fewer MVBs are labeled, likely due to inhibition of clathrin-dependent endocytosis at low pH (57Heuser J. J. Cell Biol. 1989; 108: 401-411Crossref PubMed Scopus (173) Google Scholar). Further reduction to pH 5.5 and 5.0 results in the progressive accumulation of fibril-associated PGI such that at pH 5.0 the extent of cell-associated PGI is dramatically increased (Fig. 1). At pH 5.0, Alexa 568-PGI also exhibits significant association with cell-free regions of the substrate. Identical results were obtained using FITC-conjugated PGI although increased recycling of FITC-PGI to cell surface fibrils was observed at neutral pH compared with Alexa 568-PGI (not shown). Quantification of the binding of both Alexa 568 and FITC-PGI as a function of pH shows a dramatic increase in fluorescence at pH 5 (Fig. 2). In particular, a significant increase in binding is seen between labeling at pH 5 and pH 5.5. A similar pH-dependent increase in binding of Alexa 568-PGI to human chondrocytes obtained from patients with osteoarthritis was also observed (Fig. 3). As for NIH-3T3 cells, a dramatic increase in binding at pH 5 was observed compared with pH 5.5.Fig. 2Quantification of the acid-dependent cellular association of PGI. NIH-3T3 cells were incubated for 30 min with either 25 μg/ml Alexa 568-PGI (upper graph) or FITC-PGI (lower graph) in bicarbonate-free medium containing 100 mm HEPES at pH 7.5 or 6.5 or 100 mm MES at pH 6.0, 5.5, or 5.0, as indicated. After fixation with 3% paraformaldehyde, and several washes with PBS-CM, cell-associated fluorescence was quantified using a fluorescent plate reader and the appropriate filters. The values represent the average and S.E. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3PGI also binds to osteoarthritic chondrocytes at acid pH. Primary cultures of chondrocytes obtained from cartilage of a patient with osteoarthritis were incubated with 25 μg/ml Alexa 568-PGI for 30 min in bicarbonate-free medium containing 100 mm HEPES at pH 7.5 or 100 mm MES at pH 5.5 or 5.0, as indicated. All images were acquired with the same confocal settings, and a similar dramatic increase in cell binding was observed at pH 5 for cells obtained from four different patients.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PGI labeling of NIH-3T3 cells at pH 5.0 colocalizes with fibronectin fibrils and is also observed to associate with non-fibrillar cell surface domains and the cell-free substrate (Fig. 4). Fibrillar association of PGI at pH 5.0 can be observed as early as 5 min after incubation with PGI (Fig. 4) indicating that PGI association with cell surface fibronectin occurs directly and not following PGI endocytosis and recycling (40Le P.U. Benlimame N. Lagana A. Raz A. Nabi I.R. J. Cell Sci. 2000; 113: 3227-3240PubMed Google Scholar). The enhanced binding of PGI to the cells at acid pH was not due to loss of cell integrity as subsequent incubation of the cells at neutral pH resulted in the internalization of cell associated PGI (Fig. 5, A and B). While 2 mg/ml unlabeled PGI inhibits the endocytosis of 25 μg/ml Alexa 568-labeled PGI at pH 7.5, the same concentration of unlabeled PGI had only a minimal effect on the fibrillar association of PGI at pH 5.0 (Fig. 5, C-F). Therefore, in contrast to the limited, saturable number of PGI binding sites at neutral pH (23Amraei M. Nabi I.R. FEBS Lett. 2002; 525: 151-155Crossref PubMed Scopus (23) Google Scholar, 40Le P.U. Benlimame N. Lagana A. Raz A. Nabi I.R. J. Cell Sci. 2000; 113: 3227-3240PubMed Google Scholar, 58Niinaka Y. Haga A. Negishi A. Yoshimasu H. Raz A. Amagasa T. Oral Oncol. 2002; 38: 49-55Crossref PubMed Scopus (15) Google Scholar), the association of PGI with cell surface fibronectin fibrils at pH 5.0 is not saturable, potentially nonspecific, and apparently not mediated by the PGI receptor, AMF-R.Fig. 5Binding of PGI to fibronectin fibrils at acid pH is not due to loss of cell integrity and is not saturable. NIH-3T3 cells were incubated for 30 min at 37 °C with 25 μ g/ml Alexa568-PGI in bicarbonate-free medium supplemented with either 100 mm MES, pH 5.0 (A, B, E, F) or 100 mm HEPES pH 7.5 (C and D). In B, incubation at pH 5 was followed by a subsequent 30 min incubation in bicarbonate-free medium supplemented with 100 mm HEPES pH 7.5 that resulted in the endocytosis of cell-associated PGI demonstrating the viability of the cells. In D and F, incubation with Alexa 568-AMF was performed in the presence of 2 mg/ml unlabeled PGI. While excess PGI competed efficiently for PGI endocytosis at neutral pH (C and D) it did not prevent PGI fibril association at acid pH (E and F). The cells were fixed with paraformaldehyde and PGI distribution visualized by confocal microscopy.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Acid pH Induces Changes in PGI Conformation—PGI is active as a dimer of two monomers of ∼66 kDa each (12Pon N.G. Schnackerz K.D. Blackburn M.N. Chatterjee G.C. Noltmann E.A. Biochem. 1970; 9: 1506-1514Crossref PubMed Scopus (35) Google Scholar, 13Blackburn M.N. Noltmann E.A. J. Biol. Chem. 1972; 247: 5668-5674Abstract Full Text PDF PubMed Google Scholar, 14Bruch P. Schnackerz K.D. Gracy R.W. Eur. J. Biochem. 1976; 68: 153-158Crossref PubMed Scopus (23) Google Scholar, 15Dyson J.E. Noltmann E.A. J. Biol. Chem. 1968; 243: 1401-1414Abstract Full Text PDF PubMed Google Scholar). However, in non-denaturing gels, PGI equilibrated at pH 7.5 before loading migrates at 270 ± 14 kDa (Fig. 6A). In the presence of buffers at pH 6 and below, a band of 73 ± 4 kDa is detected corresponding to the monomeric form of the protein. While this band represents only a minor form at pH 6, increasing amounts of this band are detected at pH 5.5 and pH 5 such that at pH 5.0, only the monomeric form of the protein is present (Fig. 6A). The extent of formation of the monomeric form correlates directly with the extent of fibrillar PGI deposition at pH 6.0, 5.5, and 5.0 (Figs. 1, 2, 3). Cross-linking of PGI with glutaraldehyde for different times prior to
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