July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
The impact of CHED- and FECD-associated SLC4A11 mutations on protein localization in human corneal endothelial cells.
Author Affiliations & Notes
  • Doug D Chung
    Ophthalmology, Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Anthony J Aldave
    Ophthalmology, Stein Eye Institute, UCLA, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Doug Chung, None; Anthony Aldave, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2919. doi:
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      Doug D Chung, Anthony J Aldave; The impact of CHED- and FECD-associated SLC4A11 mutations on protein localization in human corneal endothelial cells.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2919.

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

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Abstract

Purpose : Mutations identified in the solute carrier family 4 member 11 (SLC4A11) gene, encoding a cell surface protein, are associated with two distinct corneal endothelial dystrophies, congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy (FECD). To gain insight into how homozygous or compound heterozygous SLC4A11 missense mutations lead to CHED while heterozygous SLC4A11 missense mutations lead to FECD, we performed immunocytochemistry experiments to determine the effect of CHED- and FECD-associated SLC4A11 mutations on protein localization in human corneal endothelial cells.

Methods : Hemagglutinin (HA)-tagged SLC4A11 constructs encoding either the wild-type SLC4A11 protein (control), CHED-associated SLC4A11 mutant proteins (p.Arg125His, p.Glu143Lys, p.Ser213Leu, p.Ala269Val, p.Cys386Arg, p.Thr401Lys, p.Leu843Pro), or a FECD-associated SLC4A11 mutant protein (p.Gly709Glu) were transiently transfected into a human corneal endothelial cell line, HCEnC-21T. Approximately 48 hours after transfection, cells were fixed with 4% paraformaldehyde solution, permeabilized with 0.25% Triton X-100, and sequentially incubated with mouse anti-HA primary antibodies and donkey anti-mouse Alexa Fluor 594 secondary antibodies. DAPI was used to stain nuclei. Immunocytochemistry images were captured using fluorescence microscopy.

Results : The wild-type SLC4A11 protein primarily localized to the plasma membrane of the corneal endothelial cells, as expected. In contrast, although three CHED-associated SLC4A11 mutant proteins (p.Arg125His, p.Ala269Val, p.Cys386Arg) demonstrated primarily cell surface localization, the remaining four CHED-associated SLC4A11 mutants and the FECD-associated p.Gly709Glu mutant exhibited perinuclear targeting with partial or complete loss of cell surface localization.

Conclusions : CHED- and FECD-associated SLC4A11 mutations may lead to corneal endothelial cell dysfunction by interfering with cell surface localization of the SLC4A11 protein in human corneal endothelial cells. The demonstration of this phenomenon in human corneal endothelial cells corroborates previous findings by other groups who have performed either cell surface processing assays and/or localization studies in kidney-derived cells (HEK293 and MDCK) that have been transfected with SLC4A11 mutant constructs.

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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