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Analysis of the interaction between human steroid 21-hydroxylase and various monoclonal antibodies using comparative structural modelling

¹ FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK, ² Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK, ³ Faculty of Integrated Arts and Sciences, Hiroshima University, Higashihiroshima, 739-8521 Japan, ⁴ Department of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK and ⁵ Department of Medical and Surgical Sciences, University of Padova, Via Ospedale Civile 105, 35128 Padova, Italy

(Correspondence should be addressed to J Furmaniak at FIRS Laboratories address; Email: firs{at}rsrltd.eclipse.co.uk)

Objective: To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison’s disease.

Design: Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH–21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity.

Methods: Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [³H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed.

Results: M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8% ± 24% (n = 6), 86.7% ± 7.6% (n = 6) and 86.5% ± 10.6% (n = 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5 ± 8.3% activity remaining; P < 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model.

Conclusions: A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding.