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J. Escribano, M.-P. López-Garrido, M. Coca-Prados, F. Sánchez-Sánchez; Functional Analysis of CYP1B1 Mutations Found in Spanish Patients With Primary Open-Angle Glaucoma. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3120.
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To analyze the enzymatic activity and protein stability of novel CYP1B1 mutations previously identified in Spanish patients with primary open-angle glaucoma (POAG).
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.
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.
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.
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