Small nuclear ribonucleoproteins (snRNP) are prominent self antigens targeted in scleroderma and other autoimmune conditions. The overall goal of this proposal is to characterize the role of antibodies directed against snRNP in the pathogenesis of scleroderma. In recent work, we have shown that patients who recognize the oxidative modified form of 70k have scleroderma-spectrum clinical characteristics. Furthermore, we have demonstrated that the lupus-associated apoptotic form of 70k is antigenically distinct from intact 70k. We hypothesize that oxidative modified 70k exposes previously cryptic epitopes, driving the development of scleroderma-associated anti-70k immune response. This proposal will seek to test this hypothesis and define the antigenetically distinct epitopes of oxidative 70k recognized by human anti-snRNP antibodies. The four Specific Aims of this proposal are: 1) identify antibodies that specifically bind at high affinity to oxidative 70k but not to native or apoptotic 70k from scleroderma-spectrum disease patient sera; 2) define the structural modification of 70k produced by metal-catalyzed oxidation that are sufficient to induce exposure of oxidative-specific 70k epitopes; 3) map oxidative-specific 70k antibody epitopes; 4) generate monoclonal anti-oxidative 70k antibodies from phage display expression libraries. To accomplish Aim 1, we will examine at least 40 sera known to have anti-70k antibodies from our large well-characterized cohort of patients using oxidative 70k and immunoblotting. Specificity will be determined in blocking studies using molar excess of either intact, apoptotic 70k or oxidative modified 70k pre-incubated prior to immunoblotting. We anticipate that we will be able to define a panel of patients with oxidative-specific antibodies.
In Aim 2, we will use expression cloning and site-directed mutagenesis to define the products of 70k produced by metal-catalyzed oxidation. We anticipate that we will be able to define the sites of 70k that preferentially express oxidative-specific epitopes.
In Aim 3, we will use truncation and point mutation of the 70k fusion protein, along with synthetic polypeptides and oxidative-specific 70k antibodies, to define the linear B cell epitopes on oxidative-modified 70k. We predict that these experiments will serve to define the B cell epitope on oxidative-modified 70k and anticipate that this will reside in p94-194 region of 70k. Finally, in Aim 4, we will use phage Fab expression libraries to identify antibodies that specifically bind at high affinity to oxidative 70k. We anticipate that these experiments will determine whether heavy and light chain gene usage patterns are similar between libraries generated from different patients. These Fab may then serve as valuable reagents for a series of future experiments derived directly from the work proposed.
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