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SM Hauck, S Suppmann, T Meitinger, M Ueffing; Secreted proteins from Müller Glia and Photoreceptors: candidates for cell to cell communication . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3623.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Degeneration of retinal photoreceptor cells (RPR) is a major cause of blindness. Within the retina, Müller glia cells (MG) support RPR survival by yet unknown means. In addition, experimental evidence suggests that rod photoreceptors support cone cell survival. Thus, secreted factors from MG as well as from RPR may be released to protect RPR including cones from cell death. Our efforts have been focused on the detection of potentially protective factors for RPR secreted by MG and RPR. We propose here a very sensitive method to search for secreted proteins, including potential neuroprotective factors produced by retinal cells endogenously. Methods: Müller glia cells (MG) and retinal photoreceptors (RPR) were isolated from pig retina and cultured with 35-trans-S-label. The conditioned media was collected after 16 hours of labeling. The proteins were precipitated, dissolved in urea/thiourea 2D-Lysisbuffer and resolved by 2D-electrophoresis. The gels were then silver stained and autoradiographed. We discriminated between contamination from intracellular proteins within the pattern of secreted proteins by differential 2D gel display. From the silver stained gels, protein spots were excised, digested with trypsin and peptide mass fingerprint spectra or sequence spectra were recorded in a Bruker Reflex III mass spectrometer or a Micromass Q-TOF. Proteins were identified by comparison with publicly accessible databases. Results: We could detect 20 proteins clearly secreted by RPR and more than 100 distinct proteins from MG of which the majority has been identified. We will present the secretome of both retinal cell types as a basis for further functional analysis of the detected proteins. Conclusion: 2D gel analysis and mass spectrometry are a very sensitive method to identify specifically defined subsets of proteins synthesized by retinal cells. Because MG and RPR as well as rods and cones are interdependent in survival and function, identification of those molecules involved in the cell to cell communication offers the opportunity to functionally analyse discrete signaling molecules involved in this interaction.
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