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Fabian Garces, Robert S Molday; Biochemical analysis of mutations in transmembrane domain one of ABCA4 responsible for Stargardt Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2018;59(9):367.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the ABCA4 gene cause Stargardt macular degeneration (STGD1). The purpose of this study is to examine how STGD1-causing mutations in the transmembrane domain one (TMD1) of ABCA4 affect the folding, trafficking and function of ABCA4 and lead to STGD1.
The topology and amino acid sequences of the TMDs of ABCA4 were predicted in silico by hidden Markov algorithms and refined using the ABCA1 structure to model ABCA4. Missense mutations within the TMD1 region were produced by site-directed-mutagenesis. WT and mutants of ABCA4 were immunopurified using a 1D4-column against a 1D4-tag placed at the C-terminus of ABCA4. ABCA4 expression levels in transected HEK293T cells was determined by western blotting. Cellular localization of ABCA4 in transfected COS7 cells was measured by confocal microscopy. ATPase activity was determined by a luminescence assay and retinal binding activity was determined using radio-labeled retinal.
Of the 17 missense mutations analyzed, 70% had significantly lower expression levels than wildtype ABCA4 (WT), whereas the remaining 30% expressed near WT levels. Mutants with low expression levels were localized to the endoplasmic reticulum (ER) in transfected cells suggesting that some misfolding may be occurring. Mutants expressing near WT levels displayed a vesicular expression pattern similar to WT ABCA4. At the functional level, mutants with low expression levels had drastically reduced levels of retinal-binding and ATPase activity, 25% or below WT levels. Mutants expressing near WT levels had retinal-binding activity between 60-70% of WT levels, with the exception of R653C, which bound retinal at 13% of WT levels. The basal ATPase activity was similar to WT for all mutants with normal expression levels. The retinal stimulated ATPase activity increased between 1.9-2.5 fold for these mutants with the exception of R653C, whose ATPase activity was not stimulated by retinal.
Our results suggest that protein misfolding is correlated with low protein expression levels and a severely decreased functional activity of ABCA4. The residue R653 is important for substrate binding and likely forms part of the substrate binding site. These findings provide direct biochemical evidence of the effects that STGD1-causing mutations in TMD1 have on ABCA4.
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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