Autoantibody Binding to Steroid 21-hydroxylase – Effect of Five Mutations

Steroid 21-hydroxylase (21-OH) is a key haem containing steroidogenic enzyme and a major adrenal specific autoantigen. Cys 428 in 21-OH is thought to have an important role in haem binding and we now describe the effects of mutations at Cys 428 (to Ser, Arg and Phe) on 21-OH autoantibody binding. Expression of wild type and mutated 21-OH was carried out using an in vitro transcription/translation (TnT) system and reactivity of 21-OH autoantibodies with mutated 21-OH analysed by western blotting (in the case of unlabelled proteins) or immunoprecipitation assay (IPA) (in the case of 35S-labelled proteins). All 3 substitutions at Cys 428 had similar effects on 21-OH autoantibody binding and each one caused a reduction in autoantibody binding to about 50 of wild type in the case of IPA and to about 70 of wild type in the case of western blotting analysis. In addition to mutations at Cys 428, we studied 2 naturally occurring mutations at Pro 30 to Leu and He 172 to Asn which are associated with diminished 21-OH enzyme activity. The Pro 30 mutation had no effect, but the He 172 mutation caused a reduction in 21-OH autoantibody binding in the IPA to about 80 of wild type.Overall, our studies emphasise the close relationship between the 21-OH aminoacid sequences important for 21-OH enzyme activity and 21-OH autoantibody binding.