April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Cell Specific Processing Of Pikachurin, A Protein Involved In Retinal Synaptogenesis
Author Affiliations & Notes
  • Jianzhong Han
    Neuroscience, UMDNJ-New Jersey Med Sch, Newark, New Jersey
  • Jianfeng Wang
    Neuroscience, UMDNJ-New Jersey Med Sch, Newark, New Jersey
  • Ellen Townes-Anderson
    Neuroscience, UMDNJ-New Jersey Med Sch, Newark, New Jersey
  • Footnotes
    Commercial Relationships  Jianzhong Han, None; Jianfeng Wang, None; Ellen Townes-Anderson, None
  • Footnotes
    Support  NIH Grant R01EY012031, R21EY018175, and FM Kirby Foundation
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 5427. doi:
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      Jianzhong Han, Jianfeng Wang, Ellen Townes-Anderson; Cell Specific Processing Of Pikachurin, A Protein Involved In Retinal Synaptogenesis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5427.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Pikachurin (PK) is a newly identified protein with exceptionally high mRNA abundance in the retina (Sato et al. Nature Neurosci. 2008). The murine PK is expected to have 1,017 amino acids (~110 Kd), bind to α-dystroglycan (DG), and has been localized mainly in the synaptic cleft of ribbon synapses. Its knockout selectively affected synaptogenesis between photoreceptor and bipolar cells. However, many issues are still not clear, e.g. a) In addition to photoreceptors, do other retinal cells, in particular Müller cells, also synthesize PK? b) What is its functional role in synapse formation? To begin to address these issues and gain better understanding of this new protein, this study examined endogenous and exogenous expression profiles of PK in a variety of cell types.

Methods: : PK expression was investigated by Western blot (WB) using a polyclonal antibody (gift of Dr. T. Furukawa, Osaka Bioscience Institute) raised against an N-terminal fragment of murine PK (28-350 aa). To express exogenous PK or DG, a bicistronic expression vector was used to simultaneously produce target protein and a separate nuclear form of GFP. Immunofluorescence was performed to examine the localization of PK in cultured cells.

Results: : Unexpectedly, a 60 Kd band was the predominant endogenous signal for PK in WB of human, pig, and salamander retinas. A similar protein was also abundant in Y79 cells, a human retinoblastoma cell line expressing photoreceptor markers. Double staining for PK and β-DG in untreated Y79 cells showed punctate immunofluorescence of PK on the cell surface. PK localization and expression were not affected by DG overexpression. Furthermore, an anti-His antibody bound a single band of ~60 Kd in WB of Y79 cells expressing a PK variant with a His tag at the C-terminus. In contrast, overexpressed exogenous PK in primary porcine Müller cells and HEK293 cells, both with negligible levels of endogenous PK, was found to be mainly ~260 Kd and ~125 Kd and primarily intracellular in localization.

Conclusions: : Our data suggest the existence of cell-specific posttranslational processing of the PK protein. In retina-derived cells, PK, either endogenous or exogenous, is cleaved into two major parts, the N-terminal and the C-terminal domains (designated α and β fragments), which are close in size in WB. The α fragment is an extracellular protein and binds to plasma membrane through an unknown mechanism that is likely to be DG-independent. In cells lacking this processing mechanism, exogenously expressed PK is not cleaved and exists as monomers and dimers. The differential processing suggests there may be multiple functions for PK in the retina.

Keywords: retina • protein modifications-post translational • cell membrane/membrane specializations 

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