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

In systemic autoimmune diseases, autoantibodies specific for alpha -enolase are detected more frequently in patients with active renal involvement. To analyze the properties of anti-alpha -enolase antibodies and the distribut ion of the enzyme in the cell, mouse monoclonal and polyclonal antibodies w ere obtained from mice immunized with a glutathione-S-transferase-alpha -en olase fusion protein. Anti-alpha -enolase antibodies were purified from pat ient sera on enolase from human kidney. Using these antibodies, the distrib ution of alpha -enolase in the cell was analyzed in subcellular fractions a nd in the cell membrane by flow cytometry and immunoprecipitation, Plasmino gen binding was studied by an immunoenzymatic assay. We observed that alpha -enolase was present in the cytosol and membrane fractions obtained from k idney and U937 cells. By flow cytometry, mouse polyclonal anti-enolase anti bodies, one monoclonal and 7/9 human anti-enolase antibodies bound the memb rane of U937 cells. One monoclonal antibody and mouse polyclonal anti-enola se antibodies immunoprecipitated a 48-kDa molecule from surface-labeled U93 7 cells and this molecule was recognized by rabbit anti-enolase antibodies. Both immunization-induced antibodies and 7/9 autoantibodies from patient s era inhibited the binding of plasminogen to alpha -enolase. The results sho w that alpha -enolase, an autoantigen in connective tissue diseases, is a c ytoplasmic enzyme which is also expressed on the cell membrane, with which it is strongly associated. Anti-alpha -enolase autoantibodies isolated from patient sera recognize the membrane-associated form of the enzyme and/or i nterfere with its receptor function, thus inhibiting the binding of plasmin ogen. Autoantibodies specific for alpha -enolase could play a pathogenic ro le, either by a cytopathic effect or by interfering with membrane fibrinoly tic activity.