Disease-causing mutations in the CLRN1 gene alter normal CLRN1 protein trafficking to the plasma membrane
Disease-causing mutations in the CLRN1 gene alter normal CLRN1 protein trafficking to the plasma membrane
复制标题
CLRN1 基因中的致病突变会改变正常的 CLRN1 蛋白向质膜的运输
作者：
J. Isosomppi;Hanna Västinsalo;S. Geller;E. Héon;J. Flannery;E. Sankila
目的 Clarin 1 (CLRN1) 突变会导致 3 型亚瑟综合征 (USH3)。为了确定 USH3 突变对 CLRN1 功能的影响，我们在体外检查了正常和突变 CLRN1 的细胞分布和稳定性。我们还在 59 名不相关的加拿大和芬兰 USH 患者队列中寻找新的致病突变。方法通过DNA测序进行突变筛查。对于功能研究，通过瞬时转染 BHK-21 细胞将野生型 (WT) 和突变型 CLRN1 基因表达为血凝素 (HA) 标记的融合蛋白。通过共聚焦显微镜检查 CLRN1-HA 的亚细胞定位。通过使用 N-糖苷酶 F (PNGase F) 酶和蛋白质印迹研究了 CLRN1 的 N-糖基化状态。使用放线菌酮处理来评估 CLRN1 蛋白的稳定性。结果 我们从所研究的 USH 患者中发现了三种先前报告的致病性突变：p.A123D、p.N48K 和 p.Y176X，以及一种新的序列变异体：p.L54P。 WT HA 标记的 CLRN1 被正确运输到质膜，而突变的 CLRN1-HA 蛋白被错误定位并保留在内质网中。 PNGase F 处理 CLRN1-HA 导致电泳迁移率变化，与 WT 和所有 CLRN1 突变体中的糖残基裂解一致，但 p.N48K 突变的 CLRN1 除外，其中突变废除了糖基化位点。用放线菌酮抑制蛋白质表达表明 WT CLRN1-HA 保持稳定。相反，CLRN1 突变体表现出稳定性降低。结论 WT CLRN1 是一种定位于转染的 BHK-21 细胞质膜上的糖蛋白。突变的 CLRN1 蛋白定位错误。我们认为 USH3 的部分发病机制可能与细胞内运输缺陷以及突变 CLRN1 蛋白稳定性降低有关。
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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影响因子：
9.8
作者：
Ahmed, ZM;Riazuddin, S;Wilcox, ER
通讯作者：
Wilcox, ER
影响因子：
3.5
作者：
Alagramam, KN;Yuan, HJ;Smith, RJH
通讯作者：
Smith, RJH
影响因子：
9.8
作者：
Weston, MD;Luijendijk, MWJ;Kimberling, WJ
通讯作者：
Kimberling, WJ
视网膜色素变性转基因大鼠模型中视紫红质错误分选和组成型激活的表征。
DOI：
--
发表时间：
2000
期刊：
Investigative ophthalmology & visual science.
影响因子：
--
作者：
Green,ES;Menz,MD;LaVail,MM;Flannery,JG
通讯作者：
Flannery,JG
影响因子：
4.4
作者：
Eudy, JD;Yao, SF;Sumegi, J
通讯作者：
Sumegi, J