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

Abstract

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 [ 3 H]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 ¼ 6), 86.7%^7.6% (n ¼ 6) and 86.5%^10.6% (n ¼ 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. European Journal of Endocrinology 153 949-961

Το Φάσμα Των Μεταλλάξεων Του Γονιδίου CYP21A2 Σε Ασθενείς Με Συγγενή Υπερπλασία Επινεφριδίων... Ες Γην Εναλίαν Κυπρον

“…ανάπτυξη του εμβρυικού επινεφριδίου ρυθμίζεται πρωταρχικά από την εμβρυική ACTH, η οποία δρα με αυτοκρινικό/παρακρινικό τρόπο. Η δράση αυτή Το ένζυμο CYP21 όπως φαίνεται στην εικόνα 9 στον άνθρωπο έχει φυσιολογικά 494 αμινοξέα και έχει μοριακή μάζα περίπου 52 kDa(95)(96)(97). Πριν από την κλωνοποίηση του γονιδίου CYP21Α2, η γενετική αποτύπωση του HLA αποτελούσε την κύρια μεθοδο για προγεννητική διάγνωση(114).…”