June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
The effect of CHED- and FECD4-associated SLC4A11 mutations on the subcellular localization of SLC4A11 isoforms 2 and 3
Author Affiliations & Notes
  • Doug Chung
    Jules Stein Eye Institute, Los Angeles, California, United States
  • Wenlin Zhang
    Jules Stein Eye Institute, Los Angeles, California, United States
  • Angela Chen
    Jules Stein Eye Institute, Los Angeles, California, United States
  • Charlene Choo
    Jules Stein Eye Institute, Los Angeles, California, United States
  • Dominic Williams
    Jules Stein Eye Institute, Los Angeles, California, United States
  • Anthony Aldave
    Jules Stein Eye Institute, Los Angeles, California, United States
  • Footnotes
    Commercial Relationships   Doug Chung None; Wenlin Zhang None; Angela Chen None; Charlene Choo None; Dominic Williams None; Anthony Aldave None
  • Footnotes
    Support  Support was provided by Knights Templar Eye Foundation Career Starter Grant (D.D.C), Walton Li Chair in Cornea and Uveitis (A.J.A), National Eye Institute Grant P30EY000331 (core grant), the Stotter Revocable Trust (SEI Cornea Division), and unrestricted grant from Research to Prevent Blindness to Stein Eye Institute
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 659. doi:
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    • Get Citation

      Doug Chung, Wenlin Zhang, Angela Chen, Charlene Choo, Dominic Williams, Anthony Aldave; The effect of CHED- and FECD4-associated SLC4A11 mutations on the subcellular localization of SLC4A11 isoforms 2 and 3. Invest. Ophthalmol. Vis. Sci. 2023;64(8):659.

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

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Abstract

Purpose : To characterize the subcellular localization of SLC4A11 isoforms 2 and 3 in human corneal endothelial cells (hCEnC) and determine the impact of SLC4A11 mutations associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy type 4 (FECD4) on SLC4A11 protein localization in hCEnC.

Methods : Variant-specific SLC4A11WT hCEnC lines were generated by stable transduction of SLC4A11-/- hCEnC with lentiviruses containing either SLC4A11 variant 2 (V2) or variant 3 (V3), the two major variants expressed in ex vivo hCEnC and encoding isoforms 2 and 3, respectively. SLC4A11MU hCEnC lines were generated by stable transduction with lentiviruses containing SLC4A11MU expression vectors harboring CHED-/FECD4-associated SLC4A11 mutations. Subcellular localization of SLC4A11 was performed in primary hCEnC and generated SLC4A11WT and SLC4A11MU hCEnC lines by immunostaining using various anti-SLC4A11 antibodies (both pan and isoform-specific) and organelle markers.

Results : Immunostaining in primary hCEnC and SLC4A11WT CEnC lines demonstrated SLC4A11 predominately localizing at the cell surface while partially colocalizing with mitochondrial marker COX4 within punctate structures but absent within the mitochondrial filamentous structures independently stained with COX4. Minimal to weak colocalization of SLC4A11 with Golgi marker GM130 and endoplasmic reticulum marker PDI was observed in primary hCEnC. Stably transduced CHED-associated SLC4A11 V2MU c.374G>A and SLC4A11 V3MU c.326G>A hCEnC, and FECD-associated SLC4A11 V2MU c.2224G>A and SLC4A11 V3MU c.2176G>A hCEnC displayed mainly cell surface localization of SLC4A11, similar to their wild type counterparts. CHED-associated SLC4A11 V2MU c.2263C>T and SLC4A11 V3MU c.2215C>T hCEnC exhibited mostly perinuclear staining of SLC4A11, while CHED-associated SLC4A11 V2MU c.1813C>T and SLC4A11 V3MU c.1765C>T hCEnC demonstrated the loss of SLC4A11 protein expression.

Conclusions : SLC4A11 isoforms 2 and 3 predominantly localize to the cell surface in hCEnC and partially co-stain with COX4 in punctate mitochondrial structures. While previous studies have investigated the impact of SLC4A11 mutations on protein localization and abundance in transiently transfected HEK293 cells, we demonstrate selected CHED-associated SLC4A11 mutations lead to the loss of SLC4A11 cell surface localization in stably transduced hCEnC.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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