Structural basis for autoantibody recognition of phosphatidylserine-beta 2glycoprotein I and apoptotic cells

Apoptotic cells contain nuclear autoantigens that may initiate a systemic a utoimmune response. To explore the mechanism of antibody binding to apoptot ic cells, 3H9, a murine autoantibody with dual specificity for phospholipid s and DNA,was used. H chain mutants of 3H9 were constructed, expressed as s ingle-chain Fv (scFv) in Escherichia coli, and assessed for binding to phos phatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its g ermline revertant bound to dioleoyl phosphatidylserine in ELISA, and bindin g was enhanced by beta2 glycoprotein 1 (beta 2GPI), a plasma protein that s electively binds to apoptotic cells. Higher relative affinity for DOPS-beta 2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Spec ificity of the two structurally most diverse scFv for apoptotic cells was s hown by flow cytometry, and two populations of scFv-bound cells were identi fied by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potenti al to affect the clearance and processing of apoptotic cells.