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A.F. Goldberg, L.M. Ritter, N. Khattree, L. Dang; Structure and Activity of Non–Fusogenic Peripherin/rds in Transgenic Rod Photoreceptors . Invest. Ophthalmol. Vis. Sci. 2006;47(13):826.
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© ARVO (1962-2015); The Authors (2016-present)
Peripherin/rds (P/rds) is required for photoreceptor outer segment (OS) architecture in a manner that is not currently understood. Defects in this protein’s cytoplasmic C–terminus cause retinal degenerations in humans and mice. A C–terminal helical region (CHR) has been proposed to catalyze membrane fusion events required for OS renewal. This investigation is designed to examine whether a non–fusogenic variant of P/rds can support photoreceptor viability and normal OS structure.
Non–fusogenic P/rds was generated by using site–directed mutagenesis to neutralize key charged residues in the CHR (E321L, K324A). A transgene construct consisting of the opsin promoter region, the non–fusogenic P/rds coding region and a protamine 1 polyadenylation sequence was used to generate transgenic mice that were backcrossed onto C57BL6/J and rds (+/–) backgrounds. Western blot, immunoprecipitation, and sedimentation analyses were used to assess protein structure. Light, fluorescence, and electron microscopy were used to characterize retinal structure, transgenic protein localization, and photoreceptor/RPE ultrastructure and viability.
Non–fusogenic P/rds is detected in retinas of transgenic mice by Western blot analysis; the polypeptide displays normal electrophoretic mobility and post–translational modifications, including N–linked glycosylation and intermolecular disulfide formation. It assembles normally into a tetrameric form that is properly targeted and localized to photoreceptor outer segments. Expression of this transgene effects a rescue of OS structure in rds (+/–) retinas. The presence of non–fusogenic P/rds in photoreceptors with a normal complement of endogenous WT P/rds does not measurably affect OS morphology or photoreceptor viability in 6–12 week old animals, but results in an increased frequency of phagosomes in the RPE.
Loss of fusogenic activity does not compromise P/rds biosynthesis, tetrameric subunit assembly, or localization to OSs in transgenic murine photoreceptors. Incorporation of non–fusogenic P/rds into WT–containing tetramers does not prevent disk renewal or compromise photoreceptor viability in young mice, but may affect regulation of disk shedding and/or phagocytosis.
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