Abstract
Purpose: :
To analyze the enzymatic activity and protein stability of novel CYP1B1 mutations previously identified in Spanish patients with primary open-angle glaucoma (POAG).
Methods: :
The CYP1B1 mutations Arg145Trp, Ala189Pro, Ala330Ser, and Val409Phe were generated in vitro by site-directed mutagenesis using as template a cDNA encoding wild type CYP1B1 cloned into the mammalian expression vector pcDNA 3.1, and taged with the myc-epitope at the C-terminal end. To determine the enzymatic activity and protein stability of these mutations HEK-293T cells were transiently transfected with the corresponding cDNAs. The time course of the enzymatic activity was analyzed by determining the ethoxyresorufin O-deethylation (EROD) activityin transfected cells using a fluorimetric assay. Protein stability was studied by western blot of transfected cells exposed to cycloheximide for various time points. Recombinant CYP1B1 proteins were detected using an anti-myc antibody. Wild type CYP1B1 was used as a control in all the assays.
Results: :
The enzymatic activity (at 2 h) and protein stability (at 8 h) of the studied CYP1B1 mutations were 3.1 to 6.5 and 1.3 to 5.5 times lower, respectively, than that of the wild type protein.
Conclusions: :
Overall, the results show that the CYP1B1 mutations studied are associated with a reduced EROD enzymatic activity and protein stability, supporting their potential involvement in the development of POAG.