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 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.
Tanaka
H
H
FIRS Laboratories, Parc Ty-Glas, Llanishen, Cardiff, UK.
Asawa
T
T
Powell
M
M
Chen
S
S
Smith
B R
BR
Furmaniak
J
J
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
D001323:Autoantibodies; D007532:Isoleucine; D011392:Proline; D013255:Steroid 21-Hydroxylase; D003545:Cysteine