Role of SLC4A11 in Corneal Dystrophies

• Author / Creator

  Loganathan, Sampath Kumar

• Congenital hereditary endothelial corneal dystrophy type 2 (CHED2), Harboyan Syndrome (HS) and Fuchs endothelial corneal dystrophy (FECD) are caused by SLC4A11 mutations. The majority of SLC4A11 point mutations cause SLC4A11 misfolding and retention in the endoplasmic reticulum (ER). This impairs the ability of SLC4A11 to facilitate water flux across membranes. To develop treatments for the patients with corneal dystrophies caused by SLC4A11 mutations, we tested the feasibility to rescue misfolded SLC4A11 protein to the plasma membrane as a therapeutic approach. Functional activity assays were carried out in transfected HEK293 cells expressing SLC4A11 variants in combinations representing the state found in CHED2 carriers, CHED2 affected individuals, FECD individuals and unaffected individuals. These cells manifest respectively about 60%, 5%, and 25% of water flux activity, relative to the unaffected (WT alone). ER-retained CHED2 mutant SLC4A11 protein could be rescued to the plasma membrane, where it conferred 25-30% of WT water flux level. Further, cells expressing some ER-retained CHED2 mutants cultured at 30 °C supported increased water flux compared to 37 °C cultures. Caspase activation and cell vitality assays revealed that expression of SLC4A11 mutants in HEK293 cells does not induce cell death. Hence it can be concluded that therapeutics able to increase cell surface localization of ER-retained SLC4A11 mutants hold promise to treat CHED2 and FECD patients. About one third of all reported SLC4A11 mutants are found in the cytoplasmic domain (residues 1-370), indicating an important role in function, yet the role of the cytoplasmic domain is unknown. Interestingly, a catalytically inactive CHED2 mutant R125H resides in the cytoplasmic domain, indicating a role of this domain in SLC4A11 membrane transport activity. While testing the presence of a possible transport pore that transverses through the membrane domain and extends into cytoplasmic domain of SLC4A11, we found that the cytoplasmic domain is essential for the stability of the membrane domain. Fusion of soluble proteins, including green fluorescent protein or mNectarine failed to rescue the SLC4A11 membrane domain to the cell surface. Homology modeling studies show that the SLC4A11 cytoplasmic domain has a similar structural fold as AE1 (SLC4A1) cytoplasmic domain. Fusion of AE1 cytoplasmic domain to SLC4A11 membrane domain assisted the protein in maturing to the cell surface, yet with no functional activity, indicating a role of the cytoplasmic domain in the transport function of SLC4A11. Further, we found that the SLC4A11 cytoplasmic domain and membrane domain specifically associate when co-expressed separately in HEK293 cells. The monomers of SLC4A11 cytoplasmic domain dimerize independently of the membrane domain. Overall, we conclude that the cytoplasmic domain is essential for the function, stabilization and dimerization of SLC4A11 protein.

• Subjects / Keywords

  • Corneal dystrophies CHED FECD
  • SLC4A11 eye diseases
  • ER retention and protein misfolding
  • Water transport
  • Membrane protein

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R31G0J30Z

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.