Characterization of Novel SLC4A11 Mutations Identified in Recessive Congenital Hereditary Endothelial Dystrophy (CHED2)

Abstract

Purpose:: Congenital Hereditary Endothelial Dystrophy (CHED) is an uncommon bilateral disorder characterized by altered corneal endothelium and Descemetâ€^TMs membrane that lead to a variable reduction of vision. SLC4A11 is a transmembrane protein, classified as a member of the SLC4 bicarbonate transporter family. Here, we identify and describe three novelnew SLC4A11 mutations, C386R, E143K and R755W associated with CHED2 disease. P reviously reported CHED2 mutations R869C, S489L, G464D and R755Q were also further characterized.

Methods:: SLC4A11 protein was characterized following expression in transiently transfected HEK293 cells with the cDNA of HA-tagged SLC4A11 mutants and wild type (WT).

Results:: Immunoblots of the cell lysates showed that E143K and R755W are expressed as doublet of ~80 kDa but C386R also expressed a faint ~120 kDa band. Surface processing analysis revealed that the 120 kDa band is predominantly expressed on the plasma membrane both for SLC4A11 WT and C386R mutant but, the 80 kDa protein is retained intracellularly. Consistently, confocal microscopy images reveal that the mutants localized intracellularly. To study glycosylation of the WT and mutant proteins, lysates of HEK293 transfected cells were treated with N-Glycosidase F. Immunoblot analysis of the samples revealed that the 120 kDa band corresponds to the fully glycosylated form of the protein while the 80 kDa band showed a slight shift in molecular weight consistent with a core glycosylated form of the protein. Reduction of incubation temperature of cells improved the processing of aberrant folded proteins to the cell surface. 16 hs after transfection HEK293 were incubated at 30°C for 24 h and lysates collected. Immunoblot experiments showed that mutants C386R, R869C and S489L expressed in cells grown at 30°C had an increase in expression of the fully glycosylated 120 kDa band.

Conclusions:: Therefore we conclude that SLC4A11 mutants have a reduced expression probably due to an increase degradation of improperly folded protein. mutants mostly They show little or no mature glycosylation and expression at the plasma membrane. However, reduction of incubation temperature improves processing to the plasma membrane.

Keywords: cornea: basic science • proteins encoded by disease genes • ion transporters