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SLC4A11 in Blinding Endothelial Corneal Dystrophies: Characterization, Molecular Defect and Potential Therapeutic Strategy

  • Author / Creator
    Malhotra, Darpan
  • Endothelial Corneal Dystrophies (ECD), genetic blinding diseases with a heterogeneous pathophysiology, are the leading cause of endothelial keratopathy (corneal transplants). Patients manifest symptoms, including corneal edema with increased corneal thickness and loss of corneal endothelial cells (CEnC), which form the corneal endothelium and rest on their basement membrane, the Descemet’s membrane (DM). Patients suffer blurred vision with progressive deterioration until partial or complete blindness arises. We lack a clear understanding of ECD and no effective therapeutic options are available for patients, leaving them to undergo corneal transplants. Global shortage of donor corneas is a growing concern that makes it essential to understand the defects underlying these dystrophies, to create new therapeutic strategies.
    Two prominent blinding corneal dystrophies, rare pediatric-onset congenital hereditary endothelial dystrophy (CHED) and some cases of late-onset and prevalent (4-12% incidence) Fuchs endothelial corneal dystrophy (FECD), are caused by SLC4A11 mutations. SLC4A11, an integral membrane solute transport protein, is expressed in the endothelial layer of the cornea. More than 90 ECD mutations have been reported in SLC4A11, but the underlying mechanisms of the disease are incompletely understood. Three N-terminal variants of SLC4A11 (v1, v2, v3) are expressed in humans but recent controversies in the corneal expression of these variants demands studies to conclusively identify the relevant SLC4A11 isoform for corneal studies. Using a combination of bioinformatic analysis, RNA quantification and protein analysis with variant-specific custom antibodies, we identified SLC4A11 v2 as predominant in human corneal endothelium. We also found the transcription start site in v2 is Methionine at position 36 in human cornea, as opposed to the originally reported Methionine 1.
    We further attempted to understand ECD in light of SLC4A11 and the mechanisms by which its mutations cause ECD. SLC4A11 functions as a solute transporter in removing excessive fluid from the cornea which is essential to maintain corneal transparency and clear vision. Mutations compromising SLC4A11-mediated solute transport lead to fluid accumulation in the cornea, which explains corneal edema and increased corneal thickness. However, loss of solute transport does not explain the progressive decline in CEnC density in patients. We found that SLC4A11 is a cell adhesion molecule (CAM) adhering CEnC to their basement membrane, the DM, in human cornea. We designed a fluorescent in vitro cell adhesion assay using bovine DM and HEK293 cell expression system that enabled testing of multiple samples in 96-well format.
    Results in this thesis revealed that SLC4A11 interacts with DM to promote cell adhesion. SLC4A11’s CAM function is conserved across mammalian species. SLC4A11’s third extracellular loop (SLC4A11-EL3) anchors human CEnC to DM as revealed by inhibition of adhesion by anti-SLC4A11-EL3 antibody. ECD-causing mutations in SLC4A11-EL3 ablate its cell adhesion function, without affecting cell surface trafficking, or transport activity. In an energy-minimized three-dimensional model of SLC4A11-EL3, these mutations cluster and are buried within EL3 structure, suggesting they do not directly form the binding site. SLC4A11 interacts with principal DM protein, COL8A2, as revealed by GST pull-down and mass-spectrometry. Experiments in primary CEnC isolated from donor human corneas confirmed the physiological role of SLC4A11 as a CAM in promoting adhesion to DM. Primary human CEnC lose SLC4A11 when cultured. Growing primary cultured human CEnC on bovine DM restored SLC4A11 expression along with its interaction partner, COL8A2. Engineered SLC4A11-EL3-containing chimeric protein, STIC (SLC4A11-EL3 Transmembrane-GPA Integrated Chimera), restores cell adhesion to DM, confirming the role of EL3 in cell adhesion and its potential applications in ECD therapeutics. SLC4A11 is the first solute transport protein to be discovered as a CAM. SLC4A11-mediated cell adhesion to DM is defective in ECDs, providing an explanation for pathological CEnC loss in patients and a potential therapeutic strategy for ECD treatment.

  • Subjects / Keywords
  • Graduation date
    Fall 2019
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-j7zy-0p29
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.