title: Autoantibody binding to steroid 21-hydroxylase--effect of five mutations.
abstract: 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 Ile 172 to Asn which are associated with diminished 21-OH enzyme activity. The Pro 30 mutation had no effect, but the Ile 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.
authors.affiliation: FIRS Laboratories, Parc Ty-Glas, Llanishen, Cardiff, UK.
authors.forename: H
authors.initials: H
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Asawa
authors.forename: M
authors.initials: M
authors.lastname: Powell
authors.forename: S
authors.initials: S
authors.lastname: Chen
authors.forename: B R
authors.initials: BR
authors.lastname: Smith
authors.forename: J
authors.initials: J
authors.lastname: Furmaniak
mesh_terms: D000224:Addison Disease / Q000276:immunology; D000818:Animals; D001323:Autoantibodies / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D003545:Cysteine / Q000235:genetics / Q000276:immunology; D015870:Gene Expression; D006801:Humans; D007532:Isoleucine / Q000235:genetics / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011233:Precipitin Tests; D011392:Proline / Q000235:genetics / Q000276:immunology; D013255:Steroid 21-Hydroxylase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D001323:Autoantibodies; D007532:Isoleucine; D011392:Proline; D013255:Steroid 21-Hydroxylase; D003545:Cysteine