Cellular Localization and Processing of USH3A Protein Clarin–1 in Transiently Transfected Cell Lines

Abstract

Abstract: : Purpose: To investigate the cellular localization, stability and processing of mutant and wild–type human clarin–1 in transiently transfected neural and non–neural cell lines. Methods: Human retinal cDNA library was used to amplify the main transcript of clarin–1 (accession number NM_174878). The cDNA was cloned into a hemagglutinin (HA)–tagged expression vector. The mutant cDNA constructs were generated by the QuickChange site–directed in vitro mutagenesis kit. The constructs were used to transfect cultured BHK–21, HeLa and mouse primary neuron cells. Subcellular localization and processing of produced polypeptides were examined by confocal immunofluorescent microscopy and Western blot analysis. Results: Transfection experiments show that wild–type (WT) clarin–1 is targeted to the plasma membrane. Staining of mutant clarin–1 proteins representing human USH3A mutations resembled that of the endoplasmic reticulum. To determine the stability of clarin–1 polypeptides, protein synthesis in transfected BHK–21 cells was interrupted by cycloheximide treatment. After a four–hour treatment, WT clarin–1, and the mutants M120K and I153_L154delinsM, were still detectable, whereas the mutants N48K and L150P were almost completely vanished. In neurons, WT clarin–1 is present in the cell soma and along the neuronal extensions in "beads on a string" fashion. This may indicate expression of clarin–1 in the regions of synaptic contacts. The studies with the mutant clarin–1 constructs in mouse retinal primary cultures are ongoing. Western blot analysis reveals that WT Clarin–1 exists as a monomer and a dimer on SDS PAGE gels. PNGase F treatment of WT Clarin–1 results in an electrophoretic mobility shift consistent with the removal of sugar residues. PNGase F–treated N48K mutant protein did not show an electrophoretic shift suggesting that the mutation abolishes the glycosylation site. These results confirm that clarin–1 is a glycoprotein. Conclusions: Our data suggest that the wild–type clarin–1 is a glycoprotein, which is associated with plasma membranes and partially with internal membrane structures in BHK–21 cells. Distribution and stability of the mutant proteins were altered. This may imply that the pathogenesis of USH3A is associated with defective intracellular trafficking and/or instability of the mutant proteins.

Keywords: proteins encoded by disease genes • retinal degenerations: hereditary • retinal degenerations: cell biology