June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Hereditary Corneal Endothelial Dystrophy Protein, SLC4A11, has a role in Endothelial Cell Adhesion
Author Affiliations & Notes
  • Darpan Malhotra
    Biochemistry, University of Alberta, Edmonton, Alberta, Canada
  • Martin Jung
    Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, Homburg, Saarland, Germany
  • Richard Zimmermann
    Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, Homburg, Saarland, Germany
  • Joseph R Casey
    Biochemistry, University of Alberta, Edmonton, Alberta, Canada
  • Footnotes
    Commercial Relationships   Darpan Malhotra, None; Martin Jung, None; Richard Zimmermann, None; Joseph Casey, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1472. doi:
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      Darpan Malhotra, Martin Jung, Richard Zimmermann, Joseph R Casey; Hereditary Corneal Endothelial Dystrophy Protein, SLC4A11, has a role in Endothelial Cell Adhesion. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1472.

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

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Abstract

Purpose : Mutations of corneal endothelial cell (CEC) basolateral membrane transport protein, SLC4A11, give rise to some cases of Fuchs endothelial corneal dystrophy (FECD) and congenital hereditary endothelial dystrophy (CHED). These diseases are marked by loss of CEC from their attachment site on the underlying Descemet’s membrane (DM). The presence of FECD mutations in SLC4A11 third extracellular loop (EL3), a region with no apparent role in transport function, led us to wonder whether SLC4A11 has essential roles beyond membrane transport. We examined whether SLC4A11 promotes CEC adhesion to the DM.

Methods : Bovine DM was proteolyzed and coated onto 96-well dishes to perform cell adhesion assays. HEK293 cells, transfected with cDNA encoding GFP and SLC4A11 (or empty vector), were plated on the coated wells. Mild mechanical disruption was used to remove loosely bound cells. Adhering cells were quantified by measuring GFP fluorescence as a surrogate for cell number. The effect of three FECD-causing mutations of EL3 on cell adhesion was also examined. To assess potential DM binding sites in EL3, four synthetic peptides corresponding to regions of EL3 were tested for their ability to disrupt cell adhesion. To identify the interaction partner of SLC4A11 in DM, glutathione-S-transferase (GST) pull down assays were performed with GST-EL3 fusion protein and proteolyzed DM peptides. Bound peptides were identified by mass spectrometry. Two way ANOVA was used for statistical analysis.

Results : SLC4A11 expressing cells adhered to DM-coated dishes to a significantly higher degree (p=0.0001) than vector transfected cells. FECD-causing point mutations in SLC4A11 EL3 reduced cell adhesion compared to WT-SLC4A11 (p=0.0001). Synthetic peptides corresponding to first half of EL3 significantly reduced adhesion of SLC4A11 expressing cells, but not vector-transfected controls (p=0.01). Eluate from GST-EL3 pull down assays revealed the collagens COL8A1 and COL8A2 as predominant in SLC4A11-binding fraction.

Conclusions : These data suggest that SLC4A11 promotes the attachment of CECs to DM via the large EL3, with the binding site located in first half of the loop. COL8A1 and/or COL8A2 may be the interaction partner of SLC4A11 in DM. Interestingly, FECD-causing EL3 mutants decreased cell adhesion to DM. Together these data suggest that disruption of SLC4A11 cell adhesion function contributes to corneal dystrophy pathology.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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