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

Isosomppi J, Västinsalo H, Geller SF, Heon E, Flannery JG, Sankila EM - Mol. Vis. (2009)

Bottom Line: 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.In contrast, the CLRN1 mutants showed reduced stability.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.

View Article: PubMed Central - PubMed

Affiliation: Folkhälsan Institute of Genetics, Department of Molecular Genetics, Helsinki, Finland.

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.

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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.
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f4: 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.

Mentions: To determine the cellular localization of the CLRN1 protein, we cloned the transcript of CLRN1 (NM_174878) into a HA-tagged phCMV mammalian expression vector. We generated mutant cDNA constructs by the QuikChange site-directed mutagenesis kit. The mutant and WT proteins were transiently expressed in BHK-21 cells. Their refined cellular localizations were visualized by anti-HA, anti-ER (protein disulfide isomerase), and anti-plasma membrane (sodium potassium ATPase) antibodies, using confocal immunofluorescence microscopy. WT CLRN1 was observed throughout the plasma membrane of transfected cells (Figure 4A-C), but it also showed partial colocalization with the ER marker (Figure 4D-F), indicating its normal processing through the ER to the plasma membrane. In contrast, all known mutant proteins analyzed colocalized almost exclusively within the ER, suggesting that they are retained therein and prevented from targeting to the plasma membrane (Figure 5). The novel sequence alteration p.L54P localized to the plasma membrane (Figure 6A-C) similar to WT CLRN1 (Figure 6D-F), and unlike the known mutation p.N48K (Figure 6G-I). The biologic significance of the p.L54P alteration found in a patient in heterozygous form remains thus unclear.


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

Isosomppi J, Västinsalo H, Geller SF, Heon E, Flannery JG, Sankila EM - Mol. Vis. (2009)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742642&req=5

f4: 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.
Mentions: To determine the cellular localization of the CLRN1 protein, we cloned the transcript of CLRN1 (NM_174878) into a HA-tagged phCMV mammalian expression vector. We generated mutant cDNA constructs by the QuikChange site-directed mutagenesis kit. The mutant and WT proteins were transiently expressed in BHK-21 cells. Their refined cellular localizations were visualized by anti-HA, anti-ER (protein disulfide isomerase), and anti-plasma membrane (sodium potassium ATPase) antibodies, using confocal immunofluorescence microscopy. WT CLRN1 was observed throughout the plasma membrane of transfected cells (Figure 4A-C), but it also showed partial colocalization with the ER marker (Figure 4D-F), indicating its normal processing through the ER to the plasma membrane. In contrast, all known mutant proteins analyzed colocalized almost exclusively within the ER, suggesting that they are retained therein and prevented from targeting to the plasma membrane (Figure 5). The novel sequence alteration p.L54P localized to the plasma membrane (Figure 6A-C) similar to WT CLRN1 (Figure 6D-F), and unlike the known mutation p.N48K (Figure 6G-I). The biologic significance of the p.L54P alteration found in a patient in heterozygous form remains thus unclear.

Bottom Line: 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.In contrast, the CLRN1 mutants showed reduced stability.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.

View Article: PubMed Central - PubMed

Affiliation: Folkhälsan Institute of Genetics, Department of Molecular Genetics, Helsinki, Finland.

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.

Show MeSH
Related in: MedlinePlus