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Nageswara Rao Kollu, Linjing Li, Manisha Anand, Hemant Khanna; Dissecting the Role of X-Linked Retinitis Pigmentosa-Causing Protein RPGR (RP GTPase regulator) in Photoreceptor Protein Trafficking. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4642.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in RPGR (retinitis pigmentosa GTPase regulator) are associated with more than 80% of X-linked RP. However, the function of RPGR and mode of associated disease pathogenesis are unclear. Based on its localization to sensory cilia and association with transport assemblies, we postulated that RPGR is involved in regulating protein trafficking to photoreceptor sensory cilia (PSC) or outer segment (OS). The purpose of these studies is to assess the effect of loss of RPGR on the composition of OS of mouse photoreceptors.
PSC was isolated from 4 month-old wild type (WT) or Rpgr-ko mouse retinas using density gradient centrifugation. This stage was selected because no obvious degeneration can be observed by this age. Immunoblotting and immunofluorescence were carried to validate the purity of PSC using specific markers. To evaluate protein composition of the PSC, samples were subjected to in-gel digestion and analyzed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). The raw data files were processed and then searched against the SwissProt database using Mascot Search engine. Search results were loaded into Scaffold software for comparison between samples. Data were categorized using Database for Annotation, Visualization, and Integrated Discovery (DAVID) and Panther software. Exponentially Modified Protein Abundance Index (emPAI) was used for analysis of abundance of proteins. We used immunoblotting and immunofluorescence to validate differential expression of specific proteins.
Immunoblot analysis of the PSC showed enrichment of OS proteins and absence inner segment and mitochondrial proteins. LC-MS/MS analysis showed a total of 1595 proteins in the PSC. Prior to additional analysis, obvious contaminants such as keratins, mitochondrial, and ribosomal proteins were removed. We found alterations in the levels of proteins belonging primarily to proteasomal cascade and ciliary transport pathways in Rpgr-ko mice. No change was detected in the levels of proteins involved in phototransduction cascade.
Our results suggest that RPGR depletion results in a subtle but significant deficit in the composition of the PSC. As these alterations are detected prior to onset of degeneration, we reckon that such changes contribute to the pathogenesis of RPGR-associated XLRP.
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