May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
Structure and Activity of Non–Fusogenic Peripherin/rds in Transgenic Rod Photoreceptors
Author Affiliations & Notes
  • A.F. Goldberg
    Eye Research Institute, Oakland University, Rochester, MI
  • L.M. Ritter
    Eye Research Institute, Oakland University, Rochester, MI
  • N. Khattree
    Eye Research Institute, Oakland University, Rochester, MI
  • L. Dang
    Eye Research Institute, Oakland University, Rochester, MI
  • Footnotes
    Commercial Relationships  A.F. Goldberg, None; L.M. Ritter, None; N. Khattree, None; L. Dang, None.
  • Footnotes
    Support  NIH Grants EY013246 and EY014803
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 826. doi:
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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)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: photoreceptors • protein structure/function • retinal degenerations: cell biology 
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