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A. Aherne, A. Kennan, S.J. Bowne, S.P. Daiger, P.C. Engel, G.J. Farrar, P.F. Kenna, P. Humphries; Functional Analysis of Mutations in IMPDH1 Gene Implicated in RP10 Form of Retinitis Pigmentosa . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3564.
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Purpose: Mutations in the IMPDH1 gene have recently been identified in the RP10 form of retinitis pigmentosa. IMPDH1 is widely expressed in all tissues of the body, yet mutations only cause disease in the retina. IMPDH1 catalyses the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP), which is the rate limiting step in guanine nucleotide biosynthesis. The aim of this work was to evaluate the effects of two established RP mutations, Arg224Pro and Asp226Asn, on the properties and activity of the IMPDH1 enzyme, with a view to establishing the disease mechanism that causes degeneration of photoreceptors in this form of RP. Methods: The Arg224Pro and Asp226Asn substitutions were introduced into the native IMPDH1 cDNA sequence using site-directed mutagenesis. The sequences were then cloned into bacterial and mammalian expression vectors that incorporate a His-tag at the N-terminus of the protein to aid detection and purification. IMPDH1 proteins were over-expressed in bacterial and mammalian cells, and soluble and insoluble fractions were isolated to determine protein solubility. Protein expression was confirmed by SDS-PAGE with Coomassie staining and Western analysis using anti-His antibodies. Pure enzyme was quantified and the specific enzyme activity was calculated by measuring the absorbance increase at 340nm as NADH was produced. Protein computer modelling studies were undertaken to predict the effect of amino acid alterations on protein folding. Results: A high level of protein expression was achieved and pure IMPDH1 protein was successfully isolated using His-tag purification techniques. Preliminary results suggested a difference in solubility is detectable between the normal and mutant IMPDH1 proteins when expressed in E.coli. The specific enzyme activity of the normal and mutant proteins was calculated, and all proteins were found to exhibit similar levels of activity. Conclusions: The mutations under investigation do not lie in the active site of the IMPDH1 protein but rather in the CBS2 domain. These mutations do not appear to have an effect on the activity of the enzyme but may have an effect on its folding, solubility or stability.
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