Surface expression of a glycolytic enzyme, alfa-enolase, recognized by autoantibodies in connective tissue disorders

n systemic autoimmune diseases, autoantibodies specific for ?-enolase are detected more frequently in patients with active renal involvement. To analyze the properties of anti-?-enolase antibodies and the distribution of the enzyme in the cell, mouse monoclonal and polyclonal antibodies were obtained from mice immunized with a glutathione-S-transferase-?-enolase fusion protein. Anti-?-enolase antibodies were purified from patient sera on enolase from human kidney. Using these antibodies, the distribution of ?-enolase in the cell was analyzed in subcellular fractions and in the cell membrane by flow cytometry and immunoprecipitation. Plasminogen binding was studied by an immunoenzymatic assay. We observed that ?-enolase was present in the cytosol and membrane fractions obtained from kidney and U937 cells. By flow cytometry, mouse polyclonal anti-enolase antibodies, one monoclonal and 7/9 human anti-enolase antibodies bound the membrane of U937 cells. One monoclonal antibody and mouse polyclonal anti-enolase antibodies immunoprecipitated a 48-kDa molecule from surface-labeled U937 cells and this molecule was recognized by rabbit anti-enolase antibodies. Both immunization-induced antibodies and 7/9 autoantibodies from patient sera inhibited the binding of plasminogen to ?-enolase. The results show that ?-enolase, an autoantigen in connective tissue diseases, is a cytoplasmic enzyme which is also expressed on the cell membrane, with which it is strongly associated. Anti-?-enolase autoantibodies isolated from patient sera recognize the membrane-associated form of the enzyme and/or interfere with its receptor function, thus inhibiting the binding of plasminogen. Autoantibodies specific for ?-enolase could play a pathogenic role, either by a cytopathic effect or by interfering with membrane fibrinolytic activity.