Functional characterization of 102-amino acid-deleted form of human aromatase (Δ102-aromatase) 1

Abstract

A truncate form of human aromatase cDNA that corresponds to the recently identified rat cortical type aromatase mRNA variant (Yamada-Mouri et al., J. Steroid Biochem. Molec. Biol., 60: 325–329, 1997) has been generated, and the amino-terminus deleted form of the enzyme has been expressed in CHO cells. The resulting product lacking 102 residues from the N-terminus of aromatase (i.e. 102-aromatase) showed an extremely low enzyme activity using an ‘In-cell’ assay. A strong aromatase activity, however, was observed for the Δ102-aromatase using an in vitro method on the solublized preparations. The in vitro activity was dependent on both incubation time and NADPH concentration as well as inclusion of NADPH-cytochrome P450 reductase in the assay mixture. The average turnover rate of aromatization of the reconstituted Δ102-aromatase was 6.8 min −1. The results of the immunosuppression assay suggested that Δ102-aromatase still holds the epitope interactive to MAb3-2C2, a monoclonal antibody raised agaist human placental aromatase P450. Furthermore, the IC 50 values of MAb3-2C2 were determined to be 24 and 23 μg/ml for the whole homogenate and the 105 000× g precipitate fractions prepared from the truncated aromatase expressing cells, respectively, whereas an IC 50 of 1.3 μg/ml was shown for the full-length human aromatase. These results indicate that the Δ102-aromatase P450 can be expressed and is catalytically competent as the full-length enzyme, but the epitope structure for the monoclonal antibody MAb3-2C2 is altered from that of the native enzyme. In addition, the intracellular distribution of Δ102-aromatase may be different from that of the wild-type enzyme, explaining why very low activity was measured using an ‘In-cell’ assay.