Abstract
Purpose: :
Gelatinous drop-like corneal dystrophy (GDLD) is a rare hereditary disease characterized by bilateral corneal amyloidosis. The responsible gene for GDLD is the tumor associated calcium transducer 2 (TACSTD2) gene, which encodes a single-pass transmembrane protein. The purpose of this study was to elucidate the molecular mechanism by which the TACSTD2 protein regulates the subcellular localization and the stability of claudin proteins.
Methods: :
We constructed lentiviral vectors harboring the expression cassettes for the claudin (CLDN) 1, 4, and 7 genes. Plasmid vectors harboring the expression cassettes for the V5 epitope-fused TACSTD2 gene, with or without several deletion mutations, were also constructed. Plasmid vectors with missense mutations for the AxxxG motif within the transmembrane domain of the TACSTD2 protein by replacing the alanine and/or glycine residue with valine were also constructed. HeLa cells, stably expressing one of the 3 CLDN genes, were lysed and immunoprecipitated using an anti-ubiquitin antibody and subjected to western blotting. To identify the region responsible for the binding of the TACSTD2 protein to CLDN 1 and 7 proteins, the stably-expressing HeLa cells were further transfected with the wild or above-stated TACSTD2 gene mutation. The transfected cells were lysed and immunoprecipitated using an anti-V5 antibody and subjected to western blotting.
Results: :
CLDN 1 and 7 proteins were co-immunoprecipitated by anti-ubiquitin antibody, yet the CLDN 4 protein was not. The mutated TACSTD2 protein lacking any of the partial amino acid sequences of its extracellular domain or transmembrane domain almost completely lost the binding activity to the CLDN 1 and 7 proteins, while that lacking its intracellular domain was found to retain the binding activity to the CLDN 1 and 7 proteins almost at the same level as the wild TACSTD2 protein.
Conclusions: :
Our findings indicate that the extracellular and transmembrane regions of the TACSTD2 protein are essential for the binding of this protein to CLDN 1 or 7 proteins and that alanine and glycine residues of the AxxxG motif are also important. The protein degradation of CLDN 1 and 7 proteins was at least in part attributable to the ubiquitin-proteasome system, while that of CLDN 4 was not.
Keywords: cornea: basic science • cornea: epithelium • pathobiology