Dissecting the cellular functions of annexin XI using recombinant human annexin XI-specific autoantibodies cloned by phage display

Abstract

Functional studies of cellular proteins are often complicated by the lack of well-defined monoclonal antibodies, the production of which is hampered by the highly conserved nature of these cellular proteins across species. Annexin XI, a member of the Ca2+-dependent, phospholipid-binding protein family, is an example of such a protein and was used in studies to devise a strategy using human autoimmune phage display libraries to generate reagents for biological studies of conserved cellular proteins. An IgG phage display library was generated from bone marrow of an autoimmune patient with high serum antibody titer against annexin XI, which was identified recently as an autoantigen targeted by autoantibodies in several systemic autoimmune diseases. From this phage library, a panel of human monoclonal annexin XI-specific Fabs were isolated and applied to studies of the cellular functions of annexin XI. Confocal microscopy showed a cell cycle-specific redistribution of annexin XI from the cytoplasm to the mitotic spindle. In metaphase, annexin XI was up-regulated and costained with alpha-tubulin. The subcellular distribution of annexin XI in COS-7 cells was shown to be Ca2+-dependent, and exhibited a predominantly nuclear pattern at low concentrations and a cytoplasmic pattern at high Ca2+ concentrations. Calcyclin, found previously to bind annexin XI in vitro, in vivo coated the nuclear membrane of cultured cell lines and did not colocalize with annexin XI. Ultrastructural analysis by immunoelectron microscopy revealed that annexin XI associated with specific granules in both neutrophils and eosinophils, suggesting a role in the exocytotic pathway. Our results illuminate the multifunctional nature of human annexin XI, provide the first evidence that annexin XI associates with the mitotic spindles and might play a role in cell division, and clearly illustrate the potential of phage display-derived human autoantibodies in broader analyses of the function of highly conserved cellular proteins.