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Yaroslav Tsybovsky, Krzysztof Palczewski; Structural and Functional Changes in the ABCA4 Transporter Associated with Mutations that Cause Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2685.
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© ARVO (1962-2015); The Authors (2016-present)
Recessive retinal degeneration caused by mutations in the ABCA4 gene, commonly diagnosed as Stargardt disease, is characterized by high genetic heterogeneity and complex molecular pathogenesis. It has been hypothesized that both reduced functional activity of mutated ABCA4 and its compromised folding contribute to the severity of the disease. The purpose of this work was to investigate changes in structure and activity of ABCA4 resulting from a relatively common complex L541P/A1038V mutation and to develop an approach that allows characterization of ABCA4 mutants at the level of protein structure.
The wild-type (WT) human ABCA4 protein, L541P and A1038V mutants, and the L541P/A1038V double mutant were transiently expressed in mammalian cells and purified to homogeneity. Basal and all-trans-retinal-stimulated ATPase activities were measured after reconstitution in lipid vesicles. Three-dimensional protein structures were obtained using transmission electron microscopy (EM) of negatively stained samples and single particle analysis.
We developed an approach that allows EM analysis of ABCA4 utilizing limited amounts of recombinant protein expressed in mammalian cells. This analysis revealed that, at a resolution of 19 Å, WT human ABCA4 is structurally equivalent to the native protein that we earlier obtained from bovine photoreceptors. We found that mutations affected structure and function of ABCA4 to various extents. In the case of the A1038V mutant, only local conformational changes were detected in the cytoplasmic moiety of the protein, combined with a reduced ATPase activity that could be stimulated with all-trans-retinal. The structure of the L541P protein was substantially different from that of WT ABCA4, suggesting a more dramatic effect of this mutation. The complex L541P/A1038V mutation induced strong structural heterogeneity, indicative of destabilization and misfolding, and a tendency to form aggregates. This was accompanied by a severely reduced ATPase activity.
The L541P/A1038V mutant of ABCA4 demonstrated a diminished activity and severe misfolding, exemplifying the complexity of molecular pathogenesis of Stargardt disease. Our studies provide a framework for structural characterization of disease-causing mutations of the ABCA4 transporter.
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