AUTOANTIBODY EPITOPE MAPPING OF THE 21-HYDROXYLASE ANTIGEN IN AUTOIMMUNE ADDISONS-DISEASE

Patients with idiopathic Addison's disease are characterized by cytopl asmic adrenal autoantibodies, detectable by indirect immunofluorescenc e of cryocut sections of human adrenal cortex. Recently, autoantibodie s that bind a 55-kilodalton protein in the microsomal fraction of adre nal gland extracts identified to be the cytochrome P450 enzyme 21-hydr oxylase have been found in Addisonian patient sera. We confirm the fin ding and report here the autoantigenic epitopes involved in the autoan tibody reactivity using recombinant DNA technology. Six cDNA fragments spanning different regions of the 21-hydroxylase gene were expressed as fusion proteins with glutathione S-transferase in Escherichia coli. Immunoblot analyses were used to evaluate the reactivity of the recom binant proteins with patients' sera to determine the autoepitopes invo lved. We found that a conserved region (amino acids 164-356) reacted w ith 25 of 30 adrenal autoantibody-positive sera tested. One serum samp le reacted only with the amino portion of the al-hydroxylase (amino ac ids 1-162). In addition, 4 other enzymes important to steroid hormone biosynthesis, 11 beta-hydroxylase, 17 alpha-hydroxylase, side-chain cl eavage enzyme P450, and 3 beta-hydroxysteroid dehydrogenase, were expr essed in E. coli, but none of them gave positive autoantibody reaction s by Western blot assays, even using sera from 5 patients with type I autoimmune polyglandular syndrome. The availability of recombinant ant igens has permitted structural analysis of the autoepitopes involved i n the autoimmune response to 21-hydroxylase in Addison's disease. Our findings should lend to the development of a simple and specific tool for immunodiagnosis of the disease.