Acid-induced conformational changes in phosphoglucose isomerase result in its increased cell surface association and deposition on fibronectin fibrils.
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 microg/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 microg/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.
Amraei
Mohammad
M
Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
Jia
Zongjian
Z
Reboul
Pascal
P
Nabi
Ivan R
IR
D016475:3T3 Cells; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D019902:Chondrocytes / Q000378:metabolism; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005353:Fibronectins / Q000737:chemistry*; D005956:Glucose-6-Phosphate Isomerase / Q000737:chemistry*; D005976:Glutaral / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D016207:Cytokines; D005353:Fibronectins; D005956:Glucose-6-Phosphate Isomerase; D005976:Glutaral