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

• Published in: European journal of endocrinology Vol. 153; no. 6; pp. 949 - 961
• Main Authors: Miguel, Ricardo Núñez, Chen, Shu, Nikfarjam, Laleh, Kominami, Shiro, Carpenter, Byron, Dal Pra, Chiara, Betterle, Corrado, Zanchetta, Renato, Nakamatsu, Takashi, Powell, Michael, Hewer, Rachel, Blundell, Tom L, Smith, Bernard Rees, Furmaniak, Jadwiga
• Format: Journal Article
• Language: English
• Published: Colchester European Society of Endocrinology 01.12.2005; Portland Press
• Subjects: Animals; Antibodies, Monoclonal - immunology; Antigen-Antibody Reactions; Autoantibodies - immunology; Binding Sites; Binding Sites, Antibody; Biological and medical sciences; Endocrinopathies; Epitopes; Experimental Studies; Fundamental and applied biological sciences. Psychology; Humans; Medical sciences; Mice; Models, Immunological; Models, Molecular; Sequence Homology, Nucleic Acid; Steroid 21-Hydroxylase - genetics; Steroid 21-Hydroxylase - immunology; Steroid 21-Hydroxylase - metabolism; Vertebrates: endocrinology; Human; Enzyme; Oxidoreductases; Monoclonal antibody; Endocrinology; Comparative study; Steroid 21-monooxygenase
• ISSN: 0804-4643; 1479-683X
• DOI: 10.1530/eje.1.02044

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.