SINGLE POINT MUTATIONS IN DOMAIN 1 OF ß2-GLYCOPROTEIN I DECREASE BINDING OF ANTIPHOSPHOLIPID AUTOANTIBODIES.

Eric M. Smith, G. Michael Iverson, Edward J. Victoria, David M. Marquis, Keith A. Cockerill, and Matthew D. Linnik.

La Jolla Pharmaceutical Company, San Diego, CA 92121

b2-glycoprotein I (b2GPI) is a plasma protein comprised of five complement control protein domains (CCP or "sushi") that has been shown to play an important role in the antigenic specificity of antiphospholipid autoantibodies. These autoantibodies are associated with thrombosis and recurrent fetal loss in humans. The location of the antigenic site on the b2GPI molecule has been the subject of considerable research. Previous work from this laboratory using whole domain deletion mutants of recombinant b2GPI expressed in insect cells suggested that domain 1 is important for the binding of human affinity-purified antiphospholipid antibodies.1 Antiphospholipid antibodies also bind preferentially to domain 1 expressed as a pIII fusion protein on filamentous phage (unpublished data). Here we further define the antigenic site(s) on domain 1 by using error-prone PCR to generate a library of domain 1 mutants expressed on phage. We confirm results from phage assays by expressing selected single point mutations on full-length b2GPI for testing in competitive inhibition ELISAs. The results confirm the antigenic importance of domain 1 and suggest that autoantibodies from different patients may recognize similar but not identical epitopes.

1. Iverson, G.M.; Victoria, E.J.; Marquis, D.M. Proc. Natl Acad. Sci. USA 1998, 95,15542.

Presented at the 25th Annual Conference of the La Jolla Immunologists, La Jolla, CA., Nov. 8-9, 1999.