In vitro enzymology sepiapterin PAH variants
Description
The aim of this study was to assess if sepiapterin increases PAH activity and has chaperone effects on PAH variants.
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Two separate studies were conducted; the first study involved a single transfection of 62 variants and the second study involved a co-transfection of 15 variant couplets. COS-7 cells transiently transfected with plasmid vectors carrying different PAH mutations (5 pg DNA/cell) were cultured in the presence of 0, 5, or 20 µM of sepiapterin for 48 hours. The cells were then harvested, lysed, and used for enzymatic activity determination. The assay mixture that included 22.35 mM HEPES sodium salt (Na-HEPES) with pH 7.0, 2 U/µL mg/mL catalase, 10 µM ferrous ammonium sulphate, and 1 mM L-Phe, was pre-incubated with the cell lysate for 5 minutes and the reaction was then initiated by the addition of 75 μM BH4 in 100 mM dithiothreitol (DTT). The reaction mixture was incubated for 60 minutes at 25°C and stopped with the addition of 2 µL of acetic acid. The amount of L-tyrosine production was measured and quantified by high-performance liquid chromatography (HPLC). Each sample was run in triplicate. The effect of sepiapterin in enzymatic activity was determined in comparison to non-treated cells, after subtraction of the cellular background signal. Background activity signal was determined using non-transfected cells treated with equivalent concentrations of sepiapterin. The coding DNA (cDNA) of human PAH (expressed sequence tag [EST] clone obtained from imaGenes) was cloned into the pcDNA3.1/myc-His C vector (Life Technologies). PAH sequence variations were introduced using the polymerase chain reaction (PCR)-based QuikChange site-directed mutagenesis kit (Stratagene). Authenticity of the mutagenesis was verified by DNA sequencing. Wild-type (WT) and variant PAH cDNAs were then subcloned into the pMAL-c2X DEST Gateway vector encoding an N-terminal maltose binding protein and the Factor Xa cleavage site. Gateway technology (Invitrogen) was used to subclone the WT and variant vectors into the eukaryotic expression vector pEF-DEST51 (Invitrogen), and further validate by sequencing.
Institutions
- PTC Therapeutics (United States)New Jersey, South Plainfield
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Funders
- PTC Therapeutics (United States)New Jersey, South Plainfield