Structure-led Studies of SLC4 Transporters in Physiology and Pathophysiology

• Author / Creator

  Badior, Katherine

• AE1 mediates electroneutral Cl-/HCO3- exchange in red blood cells and kidney. The X-ray crystal structure of AE1 at 3.5 Å resolution provided the first human SLC4 protein structure at resolution sufficient to gain mechanistic insights, to AE1 specifically and to SLC4 family members by extension. Here, the structure of the AE1 membrane domain was used to guide studies of SLC4 proteins AE1 (SLC4A1) and SLC4A11.
  SLC4A11 mutants cause the blinding corneal dystrophy Congenital Hereditary Endothelial Dystrophy (CHED) and some cases of Fuchs Endothelial Corneal Dystrophy (FECD). A three-dimensional homology model of the SLC4A11 membrane domain was created, using the AE1 structure as a template. To assess the validity of the homology model we predicted molecular phenotypes of de novo membrane domain mutations, on the basis of the model. The biochemical properties of these mutants were consistent with predictions, revealing the reliability of the homology model. The model was used to map forty-five identified corneal dystrophy-causing mutations, and for each mutation the disease-causing effect was rationalized and categorized. Amongst membrane domain mutants 73% were predicted to cause disease by affecting protein folding and 27% by directly affecting protein function.
  AE1 is the predominant integral membrane protein in red blood cells (RBCs). Senescent RBCs are cleared from circulation by macrophages, which recognize RBCs coated by AE1-directed auto-antibodies. Senescence auto-antibodies react with AE1 residues 812-830, a region which has characteristics of both extracellular and intracellular localization. To investigate the discrepancy in localization of the AE1 812-830 region, we tested the accessibility of AE1 residues to extracellular, membrane-impermeant probes. In live HEK293 cells, residues within the AE1 812-830 region demonstrated extracellular accessibility inconsistent with the intracellular localization seen in the X-ray crystal structure. Conversely, in fixed HEK293 cells, 84% of AE1 molecules displayed intracellular localization of the 812-830 region. Taken together, these data indicate that the AE1 812-830 senescent antigen undergoes transient intracellular to extracellular reorientation. This reorientation was not abolished by inhibiting transporter movements required for anion exchange or restricted by modifications used to crystallize AE1. Extracellular antibody directed against the AE1 812-830 region bound AE1 expressed in HEK293 cells but was unable to bind AE1 in RBCs, suggesting a role of the glycocalyx in limiting RBC opsonization.
  IgG eluted from RBCs could immunoprecipitate AE1 but did not bind to AE1 on immunoblots. This indicates that the antibody requires a conformational epitope. Thus, senescent cell IgG recognizes a conformational epitope of AE1, comprised of a reorienting region with limited surface exposure. This collectively indicates that the red blood cell senescence signalling is mediated by extracellular exposure of the AE1 senescent cell epitope.
  Together, these structure-led studies provide new insight into established (AE1 senescence) and emerging (SLC4A11) fields of SLC4 protein physiology and pathophysiology

• Subjects / Keywords

  • SLC4
  • Red blood cell senescence
  • Homology modelling

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-st0k-j018

• 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.