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Astra Dinculescu, Susan Bolch, Frank M Dyka, Manoj Kulkarni, Chiab Simpson, W Clay Smith, William W Hauswirth; Phenotypic consequences of amino acid substitutions at the S163 position in C1QTNF5 protein following expression in murine RPE. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3052. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The S163R mutation in C1QTNF5 protein causes an autosomal dominant disorder known as late-onset retinal degeneration (L-ORD), characterized by the presence of thick deposits between the RPE and Bruch's membrane. We have previously shown that the S163R C1QTNF5 mutant in mice exhibits a reversely polarized distribution in-vivo, being routed towards the basal rather than apical RPE. Here, we study the behavior of other mutants with amino-acid substitutions at the same S163 position in the globular domain of C1QTNF5.
We generated scAAV vectors (AAV2 quad YF) expressing the following HA-tagged mutant C1QTNF5 proteins under the control of an RPE-specific BEST1 promoter (1012 vector genomes/mL): S163A, S163T, S163E, S163Y, and S163F. Effects of the mutant C1QTNF5 proteins were analyzed in-vitro following transient transfections in HEK cells, and in-vivo in mouse RPE cells. The vectors were delivered subretinally into one eye of 2 month-old C57BL/6 mice. The eyes were examined by non-invasive imaging methods, including color digital fundus imaging and spectral-domain optical coherence tomography (SD-OCT). Fundus autofluorescence (AF) images were also acquired and correlated with SD-OCT. Mutant protein expression was detected by immunohistochemistry and Western Blotting.
All examined substitutions at the S163 positions, except S163E, led to secreted proteins capable of forming dimers, trimers, and multimeric species under non-reducing and non-denaturing conditions, similar to wild-type C1QTNF5 behavior. These mutants were present in cell culture media following plasmid transfections in HEK cells. The S163E mutant was retained in the RPE as globular aggregates. Other mutants accumulated intracellularly and their apical RPE secretion was impaired compared to wild-type C1QTNF5. Punctate areas of regularly spaced, intense autofluorescence dots were detected in the fundus AF images as early as 2 months following expression of S163T and S163A mutants in the RPE.
These results suggest that all mutant proteins examined have a higher propensity to aggregate compared to wild-type protein. Uniquely, the S163E mutation abolishes the ability of the C1QTNF5 protein to be secreted. None of the mutants examined led to thick basal RPE deposits associated with the S163R mutation.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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