AUTOIMMUNITY TO NUCLEAR ANTIGENS

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In autoimmune diseases such as lupus, scleroderma, Sjogren's syndrome and dermato/polymyositis there are distinctive sets of autoantibodies to nuclear and nucleolar antigens which can be used as immunological markers to separate one disease from the other. Many of the autoantigens have been identified and are evolutionarily conserved molecules which have important cellular functions including DNA synthesis, transcription, RNA processing and translation. The newest hypothesis which will be examined is that immune responses in these diseaseS are antigen-driven with the antigen(s) being a component of a larger immunogenic particle. The second hypothesis related to the conservation of the epitope and function of the particle is that the autoimmunogenic region (AIR) may be an active site of the particle and therefore contributing to function. A combination of immunoelectron microscopy (immuno EM) and fluorescence light microscopy (LM) will be used to detect nuclear antigens in different windows of cell metabolism to determine if a set of autoantigens such as that in scleroderma can be co- localized in common structural regions at some point in cell metabolism. Double-label colloidal gold immuno EM will be used with different sized gold particles and the targets will be cells in which interference with function induced by chemical and other manipulations is associated with structural changes. cDNA clones encoding SS-B/La, DNA topoisomerase I and 34 KD fibrillarin protein will be generated by antibody screening of cDNA expression libraries or by synthetic oligonucleotide hybridization. Restriction fragments of these clones will be inserted into expression plasmids and subclones generated. The fusion proteins of these subclones will be analyzed for their reactivity with human autoantibodies to identify the sequence(s) containing the AIRs. From the sequence data, short length polypeptides will be synthesized and again examined for reactivity with autoantibodies to more closely define the boundaries of the AIRs. This information in conjunction with the hydrophilicity profile of the antigen deduced from the cDNA sequence will be used to determine if there are special features of the protein surface related to auto-antigenicity. Finally, antibodies to AIR and to non-AIR peptides as controls will be tested to determine the importance of the AIR in the function of their respective native proteins.