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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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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.
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f8: 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.

Mentions: Based on computational predictions, the molecular weight of WT CLRN1-HA with no modifications is 26.81 kDa [35]. WT CLRN1-HA has a single putative N-glycosylation site at amino acid position 48 [23,28]. To study CLRN1 glycosylation in vitro, we treated HA-tagged WT CLRN1 and p.N48K mutated CLRN1 polypeptides produced in transiently transfected BHK-21 cells with PNGase F, an enzyme that removes all N-linked oligosaccharide side chains from glycoproteins. Western blot analysis of WT CLRN1-HA showed several bands ranging from 23 up to 98 kDa (Figure 8, lane 1). After PNGase F treatment, two major bands at 27 kDa and 48 kDa were observed (Figure 8, lane 2). In contrast, PNGase F treatment had no effect on p.N48K samples: the same 23, 27, and 48 kDa bands were observed in both PNGase F treated and untreated samples (Figure 8, lanes 3 and 4). These results confirm that CLRN1 is a glycoprotein like previously reported and that the p.N48K mutation disrupts the protein’s single glycosylation site [28]. The presence of a 48 kDa band suggests that CLRN1 has a tendency to form dimers. The other studied mutations, p.M120K, p.A123D, p.L150P, and p.I153_L154delinsM, were glycosylated (Figure 8, lanes 7–16), and clearly showed decreased molecular weights following PNGase F treatment. Their glycoform patterns were, however, different from that of the WT, probably reflecting their retention in the ER.


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)

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

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

f8: 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.
Mentions: Based on computational predictions, the molecular weight of WT CLRN1-HA with no modifications is 26.81 kDa [35]. WT CLRN1-HA has a single putative N-glycosylation site at amino acid position 48 [23,28]. To study CLRN1 glycosylation in vitro, we treated HA-tagged WT CLRN1 and p.N48K mutated CLRN1 polypeptides produced in transiently transfected BHK-21 cells with PNGase F, an enzyme that removes all N-linked oligosaccharide side chains from glycoproteins. Western blot analysis of WT CLRN1-HA showed several bands ranging from 23 up to 98 kDa (Figure 8, lane 1). After PNGase F treatment, two major bands at 27 kDa and 48 kDa were observed (Figure 8, lane 2). In contrast, PNGase F treatment had no effect on p.N48K samples: the same 23, 27, and 48 kDa bands were observed in both PNGase F treated and untreated samples (Figure 8, lanes 3 and 4). These results confirm that CLRN1 is a glycoprotein like previously reported and that the p.N48K mutation disrupts the protein’s single glycosylation site [28]. The presence of a 48 kDa band suggests that CLRN1 has a tendency to form dimers. The other studied mutations, p.M120K, p.A123D, p.L150P, and p.I153_L154delinsM, were glycosylated (Figure 8, lanes 7–16), and clearly showed decreased molecular weights following PNGase F treatment. Their glycoform patterns were, however, different from that of the WT, probably reflecting their retention in the ER.

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