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D. A. Mans, K. Boldt, J. van Reeuwijk, S. J. Letteboer, R. Roepman, M. Ueffing; Quantitative Protein Complex Analysis of Lebercilin Mutations Reveals Loss of Interaction With Intraflagellar Transport Proteins in Leber Congenital Amaurosis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2582.
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We have previously shown that the LCA5 gene encoding lebercilin is mutated in patients with Leber congenital amaurosis (LCA). Lebercilin localizes to primary cilia and interacts with proteins involved in microtubule dynamics, as determined by tandem affinity purification followed by mass spectrometry. We here present a more sensitive affinity proteomics approach to i) expand the lebercilin-interactome and ii) to identify the molecular disease pathogenesis resulting from nonsense mutations in LCA5.
We have used affinity purification in combination with Stable Isotope Labeling by Amino acids in Cell culture (SILAC) in HEK293T cells using SF-TAP tagged full-length lebercilin, and variants identified in LCA5 (p.P493TfsX1 and p.Q279X). Protein complexes were identified via mass spectrometry and MaxQuant-based quantitative analysis. Presence or absence of proteins in the eluates was confirmed by western blot analysis. Immunofluorescent stainings were performed of ciliated retinal pigment epithelial (hTERT-RPE1) cells and mouse retinal sections to examine endogenous (co-)localization of the associated proteins.
Using SILAC, we were able to identify the association of lebercilin with a novel subset of mostly centrosomal and ciliary proteins. Amongst these proteins were most of the known intraflagellar transport (IFT) complex A and complex B members. Interestingly, we found that nonsense lebercilin mutations in LCA5 patients disrupt specifically the interaction with the IFT proteins, while others were retained. The results were validated by immunohistochemistry and western blotting.
We have identified the association of lebercilin with the IFT transport machinery, which is lost in LCA patients with mutations in LCA5. These data suggest that the association of lebercilin with the IFT machinery is important for the development and/or maintenance of normal photoreceptor function in the retina, rendering LCA5 a retinal ciliopathy.
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