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


METHODS
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.
Miguel
Ricardo Núñez
RN
FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK.
Chen
Shu
S
Nikfarjam
Laleh
L
Kominami
Shiro
S
Carpenter
Byron
B
Dal Pra
Chiara
C
Betterle
Corrado
C
Zanchetta
Renato
R
Nakamatsu
Takashi
T
Powell
Michael
M
Hewer
Rachel
R
Blundell
Tom L
TL
Smith
Bernard Rees
BR
Furmaniak
Jadwiga
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000276:immunology*; D001665:Binding Sites; D001666:Binding Sites, Antibody; D000939:Epitopes; D006801:Humans; D051379:Mice; D018448:Models, Immunological; D008958:Models, Molecular; D012689:Sequence Homology, Nucleic Acid; D013255:Steroid 21-Hydroxylase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D000939:Epitopes; D013255:Steroid 21-Hydroxylase