Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies

Darpan Malhotra,Sampath K Loganathan,Anthony M Chiu,Chris M Lukowski,Joseph R Casey

doi:10.1038/s41598-019-46094-y

Abstract

Two blinding corneal dystrophies, pediatric-onset congenital hereditary endothelial dystrophy (CHED) and some cases of late-onset Fuchs endothelial corneal dystrophy (FECD), are caused by SLC4A11 mutations. Three N-terminal SLC4A11 variants: v1, v2 and v3 are expressed in humans. We set out to determine which of these transcripts and what translated products, are present in corneal endothelium as these would be most relevant for CHED and FECD studies. Reverse transcription PCR (RT-PCR) and quantitative RT-PCR revealed only v2 and v3 mRNA in human cornea, but v2 was most abundant. Immunoblots probed with variant-specific antibodies revealed that v2 protein is about four times more abundant than v3 in human corneal endothelium. Bioinformatics and protein analysis using variant-specific antibodies revealed that second methionine in the open reading frame (M36) acts as translation initiation site on SLC4A11 v2 in human cornea. The v2 variants starting at M1 (v2-M1) and M36 (v2-M36) were indistinguishable in their cell surface trafficking and transport function (water flux). Structural homology models of v2-M36 and v3 suggest structural differences but their significance remains unclear. A combination of bioinformatics, RNA quantification and isoform-specific antibodies allows us to conclude that SLC4A11 variant 2 with start site M36 is predominant in corneal endothelium.

Highlights

Defects in the endothelial “pump” give rise to stromal fluid accumulation, significantly thickening the layer
Reverse transcription PCR (RT-PCR) and qRT-PCR further revealed the presence of only v2 and v3 transcripts in human cornea and the endothelium, where variant 2 is the predominant transcript in the endothelium
Antibodies targeting variant 2 at M1 (v2-M1) and v2-M36 SLC4A11 confirmed the presence of SLC4A11 v2-M36 in the human cornea

Summary

Introduction

Defects in the endothelial “pump” give rise to stromal fluid accumulation, significantly thickening the layer. The genome database reveals three N-terminal variants of human SLC4A11: 918 amino acid splice form 1 (NCBI Reference Sequence: NP_001167561.1), 891 amino acid splice form 2 (NP_114423) and 875 amino acid splice form 3 (NP_001167560). These variants have been respectively called SLC4A11-A, -B and – C23. The three N-terminal variants may affect the structure and/or function of the N-terminal cytoplasmic domain, with potential impact on transport activity. This underscores the need to identify the SLC4A11 isoform(s) present in human cornea. We further characterized the cell surface trafficking, transport functions and structural differences of SLC4A11 variants

Methods

Results

Conclusion