Abstract
Purpose: :
To investigate the functional consequences of five missense mutations in PDE6C, the gene encoding the alpha’- subunit of the cone-specific phosphodiesterase. Mutations in PDE6C are a rare cause of autosomal recessive achromatopsia.
Methods: :
Expression constructs applying the pFastBac HTb vector (Invitrogen, Carlsbad, USA) for human PDE6C/PDE5-chimeras were cloned and achromatopsia associated mutations introduced by an in vitro mutagenesis strategy. Proteins were expressed in the baculovirus-Sf9-expression system and monitored by SDS-PAGE, Western blotting and Coomassie staining. The proteins were purified using affinity chromatography on a His-bind resin (Novagen, Darmstadt, Germany). The functionality of wild type and mutant recombinant proteins was analyzed by a series of assays including gel filtration, enzymatic activity, and inhibition by Zaprinast, IBMX and the inhibitory pγ-subunit.
Results: :
All mutations result in considerable to total loss of PDE6C-specific enzymatic activity and/or altered substrate binding, as well as altered binding and inhibition by Zaprinast, IBMX and the inhibitory pγ-subunit.
Keywords: retinal degenerations: hereditary • proteins encoded by disease genes • mutations