Principal Investigator: GORDON SHARP
Abstract: The objective is to elucidate the roles of nuclear acidic protein antigens and certain other antigens in rheumatic diseases and in basic cellular biology. Patient autoimmune antibodies directed against distinct antigens (RNP, Sm and Me) of the ubiquitous U1 small nuclear ribonucleoprotein particle (U1 snRNP) are strongly associated with connnective tissue diseases, ie. mixed connective tissue disease (MCTD), systemic lupus erythematosus (SLE), and undifferentiated connective tissue disease (UCTD), respctively. Autoantigens (RNP, Sm, Me, etc.) and their five-nine component peptides (9-70 Kdaltons), monospecific patient sera, IgG, and murine and human monoclonal antibodes will be isolated by high performance liquid chromatography ion, exchange, gel filtration, and immunoaffinity chromatography, and by gel electrophoresis. Antigens and autoantibodies will be vigorously characterized by immunological (Ouchterlony, passive hemagglutination, hemagglutination inhibition, competitive hemagglutination inhibition, immunofluorescence, counter immunoelectrophoresis, ELISA, dot, and Western blot) and biochemical (gel electrophoresis, nucleoside and amino acid compositions, sugar and lipid analysis, RNA and amino acid sequencing) methods. Autoantigenic determinants of the U1 RNP and the other anRNP peptides will be defined in terms of amino acid sequence, sugar and lipid compositions, and compared to all known peptide sequences to reveal any relationships to known viral, or "housekeeping" proteins. The 9-14K dalton Sm antigenic peptides are of particular interest because they are recognized by anti-histone monoclonal antibodies developed from autoimmune MRL mouse hybridomas. The question of the immunogenicity of the defined antigenic determinants is central to understanding these autoimmune responses and will be addressed. Cellular localization and co-localizations of the antigens with regard to function in RNA splicing will be investigated by sophisticated high resolution digitized image immunofluorescence microscopy. Alterations in the antigen locations throughout the cel cycle and during transcriptional inhibition will also be evaluated. Co-localization studies will be complemented by biochemical reconstitution studies of the ineractions of snRNPs with hnRNPs for recognition of intron sequences. The heterogeneity of the human autoimmune response will be investigated by various methods including description of individual, specific peptide recognition by patient sera and the production of a human monoclonal antibody library. Human monoclonals will be produced through either EBV transformation or myeloma/lymphoblastoid cell fusions. The product of autoimmune response, the immune complex, will be biochemically characterized and its distribution studied for specific autoantibody-antigen reactivities.
Funding Period: 1977-08-01 - 1992-07-31
more information: NIH RePORT