A conformation-dependent epitope in Addison's disease and other endocrinological autoimmune diseases maps to a carboxyl-terminal functional domain ofhuman steroid 21-hydroxylase

Idiopathic Addison's disease develops as a consequence of autoimmune destru ction of steroid-producing cells in the adrenal gland. A major autoantigen is 21-hydroxylase (210H; P450c21), which is involved in the biosynthesis of cortisol and aldosterone in the adrenal cortex. We selected a number of fu nctionally important 210H amino acid substitutions, found in patients with congenital adrenal hyperplasia, to study their effects on the binding of 21 0H autoantibodies (21OHAb) to 21OH. The ability of 21OHAb to bind in vitro transcribed and translated wild-type 210H and five different 210H mutant pr oteins was quantified by liquid-phase assays. Sera from 21OHAb-positive pat ients with idiopathic Addison's disease (n = 24), Graves' disease (n = 3), and insulin-dependent diabetes mellitus (n = 1) were used. While the P105L, delE196, and G291S mutations had no effect on autoantibody binding, the P4 53S mutation had a considerable effect, and the R483P mutation almost compl etely abolished binding. Synthetic peptides corresponding to linear epitope s defined by amino acids 447-461 and 477-491 were unable to compete with wi ld-type 210H for binding to autoantibodies, Direct 210H DNA sequencing coul d not reveal any specific genetic variation in alleles found in 21OHAb-posi tive patients. We conclude that the region involving R483 plays a key role in the formation of a three-dimensional epitope in a functionally important C-terminal domain of the enzyme.