Disease-causing mutations in the CLRN1 gene alter normal CLRN1 protein trafficking to the plasma membrane

Abstract

Purpose: Mutations of clarin 1 (CLRN1) cause Usher syndrome type 3 (USH3). To determine the effects of USH3 mutations on CLRN1 function, we examined the cellular distribution and stability of both normal and mutant CLRN1 in vitro. We also searched for novel disease-causing mutations in a cohort of 59 unrelated Canadian and Finnish USH patients.

Methods: Mutation screening was performed by DNA sequencing. For the functional studies, wild-type (WT) and mutant CLRN1 genes were expressed as hemagglutinin (HA) tagged fusion proteins by transient transfection of BHK-21 cells. Subcellular localization of CLRN1-HA was examined by confocal microscopy. The N-glycosylation status of CLRN1 was studied by using the N-glycosidase F (PNGase F) enzyme and western blotting. Cycloheximide treatment was used to assess the stability of CLRN1 protein.

Results: We found three previously reported pathogenic mutations, p.A123D, p.N48K, and p.Y176X, and a novel sequence variant, p.L54P, from the studied USH patients. The WT HA-tagged CLRN1 was correctly trafficked to the plasma membrane, whereas mutant CLRN1-HA proteins were mislocalized and retained in the endoplasmic reticulum. PNGase F treatment of CLRN1-HA resulted in an electrophoretic mobility shift consistent with sugar residue cleavage in WT and in all CLRN1 mutants except in p.N48K mutated CLRN1, in which the mutation abolishes the glycosylation site. Inhibition of protein expression with cycloheximide indicated that WT CLRN1-HA remained stable. In contrast, the CLRN1 mutants showed reduced stability.

Conclusions: WT CLRN1 is a glycoprotein localized to the plasma membrane in transfected BHK-21 cells. Mutant CLRN1 proteins are mislocalized. We suggest that part of the pathogenesis of USH3 may be associated with defective intracellular trafficking as well as decreased stability of mutant CLRN1 proteins.

Figure 1

Sequence conservation around the p.L54P and p.A123D changes. Amino acids conserved in evolution are marked in bold, and mutations are marked with red. Mutations are also marked with an asterisk. Abbreviations: Homo sapiens (HS), Pan troglodytes (Pt), Bos taurus (Bt), Mus musculus (Mm), Gallus gallus (Gg), Danio rerio (Dr).

Figure 2

Predicted membrane topology of CLRN1. Disease-associated mutations are marked; the frameshift and nonsense mutations are marked with red amino acids, missense mutations are marked with blue amino acids, and the deleted amino acids replaced by an insertion are marked with green. Transmembrane regions (1–4) were predicted using a TMHMM2.0 program [36]. The mutations studied in this article are marked with red.

Figure 3

Optical coherence tomographs of a normal control and three USH3 patients. A: 32-year-old healthy control with visual acuity (VA) of 20/20. B: 8-year-old USH3 patient with heterozygous p.Y176X and p.N48K mutations and VA of 20/20. The arrow points to the region of retinal thinning in patient’s macula. C: 35-year-old USH3 patient with homozygous p.A123D mutation and VA of 20/25. The arrow points to the schisis-like change in patient’s macula. D: 52-year-old USH3 patient with homozygous p.N48K mutation and VA of 20/30. The arrow points to the intraretinal cysts. Scale bar represents 1 mm.

Figure 4

Cellular localization of WT CLRN1-HA protein in transfected BHK-21 cells. In panels B and E the cells were immunostained with HA antibody (red). In panel A the cells were immunostained with a plasma membrane specific antibody (green) and in panel D with ER specific antibody (green). The right-most panels (C and F) show the overlay of both CLRN1-HA and the organelle-specific double staining. Yellow-orange staining indicates an overlap of the CLRN1-HA protein (red) and subcellular markers (green). Cells were viewed with a confocal immunofluorescence microscope, magnification 63×. Scale bar represents 10 μm.

Figure 5

Cellular localization of the disease-causing mutant CLRN1-HA polypeptides. The transfected BHK-21 cells were double immunostained with HA antibody (red) in panels B, E, H, K, and N showing the localization of mutant CLRN1-HA. In panels A, D, G, J, and M the cells were stained with the ER marker (green). The right-most panels C, F, I, L, and O show the overlay of the mutant CLRN1- HA staining (red) and ER-specific staining (green). Yellow-orange staining indicates colocalization of these stainings. Cells were viewed with a confocal immunofluorescence microscope. Scale bar represents 10 µm.

Figure 6

Cellular localization of the p.L54P, p.N48K and WT CLRN1-HA polypeptides. The transfected BHK-21 cells were immunostained with HA antibody (red) showing the localization of WT CLRN1-HA (E), the novel sequence alteration p.L54P mutated CLRN1-HA (B) and the known disease-causing p.N48K mutated CLRN1-HA (H). The same cells were immunostained with plasma membrane –specific antibody (green) in panels A, D, and G. Double-staining shows that WT CLRN1-HA (F) and the p.L54P mutated CLRN1-HA (C) colocalize (yellow) with the plasma membrane marker whereas the known mutation p.N48K (I) does not colocalize with the plasma membrane marker. Cells were viewed with a confocal immunofluorescence microscope. Scale bar represents 10 µm.

Figure 7

Stability of WT and mutant CLRN1-HA polypeptides in transfected BHK-21 cells. Cells were transiently transfected either with WT or mutant CLRN1-HA cDNAs. At 48 h posttransfection protein synthesis was stopped by incubating the cells for 4 h in the presence of 50 µg/ml of cycloheximide. Cells were viewed with a Zeiss Axioplan 2 fluorescence microscope. The scale bar represents 50 µm.

Figure 8

Western blot analysis of the wild-type and mutant CLRN1-HA polypeptides. BHK-21 cells were transfected with the indicated HA-tagged CLRN1 plasmids. Nontransfected cells (0-BHK) were used as controls. Polypeptides were resolved on 12% SDS–PAGE, and anti-HA antibodies were used to probe the blots. Samples were untreated (-) or treated (+) with deglycosylating enzyme (PNGase F). The molecular weights of the protein bands are indicated on the left and right sides of the figure.