Molecular phenotype of SLC4A11 missense mutants: Setting the stage for personalized medicine in corneal dystrophies

Abstract

SLC4A11 mutations cause cases of congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (HS), and Fuchs endothelial corneal dystrophy (FECD). Defective water reabsorption from corneal stroma by corneal endothelial cells (CECs) leads to these corneal dystrophies. SLC4A11, in the CEC basolateral membrane, facilitates transmembrane movement of H 2 O, NH 3 , and H + -equivalents. Some SLC4A11 disease mutants have impaired folding, leading to a failure to move to the cell surface, which in some cases can be corrected by the drug, glafenine. To identify SLC4A11 mutants that are targets for folding-correction therapy, we examined 54 SLC4A11 missense mutants. Cell-surface trafficking was assessed on immunoblots, by the level of mature, high molecular weight, cell surface-associated form, and using a bioluminescence resonance energy transfer assay. Low level of cell surface trafficking was found in four out of 18 (20%) of FECD mutants, 19/ out of 31 (61%) of CHED mutants, and three out of five (60%) of HS mutants.Amongst ER-retained mutants, 16 showed increased plasma membrane trafficking when grown at 30 • C, suggesting that their defect has potential for rescue. CHED-causing point mutations mostly resulted in folding defects, whereas the majority of FECD missense mutations did not affect trafficking, implying functional impairment. We identified mutations that make patients candidates for folding correction of their corneal dystrophy.

Ophthalmic Nonsteroidal Anti-Inflammatory Drugs as a Therapy for Corneal Dystrophies Caused by SLC4A11 Mutation

“…A BRET-based assay measured total cell surface abundance of SLC4A11 mutants with or without the drug. 12 In these assays, luciferase fused to the SLC4A11 N-terminus, releases a photon of luminescent light. The lipid-anchored plasma membranelocalized protein, K-Ras, has yellow fluorescent protein (YFP) fused to its C-terminus.…”

“…pAMC1 encoding N-terminally HA-tagged human SLC4A11 in pcDNA3.1 20 or wild-type (WT) Luc-SLC4A11 with N-terminal luciferase tag 12 were used as template for mutagenesis experiments to create point mutants.…”

“…12,22,23 HEK293 cells were transiently cotransfected in 100-mm dishes with 0.7 lg cDNA encoding Nterminal luciferase-tagged human SLC4A11 (splicing variant 2 of human SLC4A11 (891 amino acid, NCBI reference: NG_017072.1) and 4.9 lg cDNA encoding K-Ras-YFP (acceptor labeled protein). HEK293 cells transfected with 0.7 lg cDNA encoding Luc-SLC4A11 and 4.9 lg of pcDNA3.1 were included for each variant tested in this study (WT SLC4A11 and mutants W240S, R282P, E399K, T431I, G583D, G709E, E143K, R209W, S213L, S232N, R233C, C386R, G394R, T4501K, G417R, G418D, G464D, L473R, S489L, T584K, C611R, R755Q, R755W, P773L, T833M, R869C, L873P, S213P, R488K, and L843P).…”

“…11 When studied in model cells, the most common molecular phenotype of mutant membrane proteins is mild misfolding. 12 This biosynthetic defect renders the protein incapable of transit from the endoplasmic reticulum (ER) site of synthesis to its normal cellular location. Note that SLC4A11 localization in corneas of endothelial dystrophy patients has not been examined; the conclusion of ER retention presently rests on data from cell culture models.…”

“…7,10,[14][15][16][17][18][19] Most SLC4A11 mutations lead to biosynthetic defects marked by accumulation in the ER, and protein degradation before reaching the plasma membrane. 12 A small-scale drug screen showed that Glafenine, a disused NSAID, was able to move some SLC4A11 mutants to the cell surface, 20 suggesting that other NSAIDs might also have therapeutic potential. We thus set out to screen NSAIDs available as eye drops for their ability to correct the cell-surfacing trafficking of SLC4A11 proteins, using a bioluminescence resonance energy transfer (BRET) assay that enabled rapid analysis of SLC4A11 cell surface abundance.…”