July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Wild Type and Mutant Retinoschisin Subunits Co-assemble When Expressed in the Same Cells
Author Affiliations & Notes
  • Duo Sun
    Ophthalmology, Regeneron Pharmaceuticals Inc, Ossining, New York, United States
  • Yang Liu
    Ophthalmology, Regeneron Pharmaceuticals Inc, Ossining, New York, United States
  • Nicole Zazzarino
    Therapeutic Proteins, Regeneron, Tarrytown, New York, United States
  • Jifang Tao
    Therapeutic Proteins, Regeneron, Tarrytown, New York, United States
  • Joel Martin
    Therapeutic Proteins, Regeneron, Tarrytown, New York, United States
  • William Olson
    Therapeutic Proteins, Regeneron, Tarrytown, New York, United States
  • Jingtai Cao
    Ophthalmology, Regeneron Pharmaceuticals Inc, Ossining, New York, United States
  • Carmelo Romano
    Ophthalmology, Regeneron Pharmaceuticals Inc, Ossining, New York, United States
  • Footnotes
    Commercial Relationships   Duo Sun, Regeneron (E); Yang Liu, Regeneron (E); Nicole Zazzarino, Regeneron (E); Jifang Tao, Regeneron (E); Joel Martin, Regeneron (E); William Olson, Regeneron (E); Jingtai Cao, Regeneron (E); Carmelo Romano, Regeneron (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3050. doi:
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      Duo Sun, Yang Liu, Nicole Zazzarino, Jifang Tao, Joel Martin, William Olson, Jingtai Cao, Carmelo Romano; Wild Type and Mutant Retinoschisin Subunits Co-assemble When Expressed in the Same Cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3050.

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

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Abstract

Purpose : X-linked retinoschisis (XLRS) is caused by mutations in the gene encoding the secreted protein retinoschisin (RS1). Of these, nonsense or frame shift mutations are predicted to result in absence of a full-length protein. However, many disease-causing mutations are missense ones allowing for production of full length misfolded protein that is retained intracellularly. RS1 gene delivery via viral vectors to RS1 KO mice has resulted in significant preservation of retinal structure and function. However, there is no information available concerning subunit assembly by individual cells expressing a misfolded mutant and wild-type proteins. We used in vitro RS1 expression and secretion assays to investigate the co-assembly of mutant and wild type RS1 to better understand the potential of gene supplementation therapy.

Methods : Study 1. HEK293 cells were transiently transfected with plasmids containing differentially tagged WT mouse RS1 (6xHis) and mutant RS1 (C59S and R141C RS1) (myc) singly and in combination. Protein expression, secretion and subunit assembly of his-RS1 and myc-RS1 were analyzed by western blot (WB) and ELISA. Study 2. Co-expression and interactions were also studied by transfecting WT RS1 plasmid into CHO cells stably expressing mutant RS1. The co-assembly of WT and mutant RS1 was further investigated by immuno-precipitating mutant RS1 using anti–myc antibody-Protein G beads, and the bound fraction was analyzed by WB.

Results : WT RS1 from both transient and stable transfections showed a single octamer band on non-reducing WB, whereas C59S myc-RS1 only exhibited dimer, and no secreted product was observed from R141C myc-RS1 (intracellular protein was present). Co-expression of WT RS1 with both mutants RS1 after either transient or stable transfections demonstrated RS1 octamer detected by all antibodies, anti-RS1, anti-His, and anti-myc, indicating the presence of hetero-octamer. Co-assembly and secretion were confirmed by co-immunoprecipitation. ELISA showed co-expression of WT RS1 with either mutant types resulted in decreased amounts of RS1.

Conclusions : Mutant RS1 interferes with the expression/secretion of WT RS1 when co-expressed, and co-assembly of mutant and WT RS1 subunits into hetero-octamers may have deleterious effects on RS1 function. The molecular physiology of WT RS1 and the consequences of hetero-octamer formation with disease causing mutants remain to be elucidated.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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