Correction of the Disease Phenotype of Myocilin-Causing Glaucoma by a Natural Osmolyte
Correction of the Disease Phenotype of Myocilin-Causing Glaucoma by a Natural Osmolyte
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DOI：
10.1167/iovs.08-3151
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发表时间：
2009-08-01
影响因子：
4.4
通讯作者：
Yam, Gary Hin-Fai
中科院分区：
文献类型：
--
作者：
Jia, Li-Yun;Gong, Bo;Yam, Gary Hin-Fai
目的。表征导致青少年发病的开角型青光眼 (JOAG) 的新型 Asp384Asn (D384N) 突变体肌纤蛋白 (MYOC)，并研究天然渗透剂三甲胺 N-氧化物 (TMAO) 对突变表型的校正。方法。招募了一个中国JOAG家庭，并从44名家庭成员的外周血中提取了基因组DNA。对 MYOC 的编码区进行了测序。 200 名没有高眼压或青光眼的个体（>60 岁）作为对照对象。将全长人野生型 MYOC cDNA 克隆到 p3xFLAG-myc-CMV-25 中，并通过定点诱变引入错义突变。用小分子化学伴侣处理转染的人小梁网细胞。通过联合免疫沉淀-Western印迹分析来分析分泌的MYOC。将细胞内肌纤蛋白分为 Triton X-100 可溶性和不溶性级分，并通过蛋白质印迹分析进行分析。通过免疫荧光测定细胞内聚集物和细胞凋亡。通过密度梯度分级和蛋白质印迹分析来分析TMAO对亚细胞肌纤蛋白分布的影响。结果。一本小说 C. 1150G>鉴定出 MYOC 发生变化。 optineurin、WDR36 和 CYP1B1 的筛选显示不存在致病多态性。突变的 D384N 肌纤蛋白溶解度降低，易于聚集且不分泌。用TMAO处理转染细胞提高了D384N突变体的溶解度，并以剂量反应方式校正了分泌。 TMAO 减少了 D384N 突变体在内质网 (ER) 中的分布，减轻了 ER 应激，并挽救了细胞免于凋亡。结论。结果表明TMAO具有分子伴侣活性，促进突变型MYOC的折叠和分泌。这种由化学伴侣辅助的治疗方法可用于治疗青光眼。 （投资眼科可见科学。2009 年；50：3743-3749）DOI：10.1167/iovs.08-3151
PURPOSE. To characterize a novel Asp384Asn (D384N) mutant myocilin (MYOC) that causes juvenile-onset open-angle glaucoma (JOAG) and investigate the correction of mutant phenotype by a natural osmolyte, trimethylamine N-oxide (TMAO).METHODS. A Chinese JOAG family was recruited and genomic DNA was extracted from peripheral blood obtained from 44 family members. Coding regions of the MYOC were sequenced. Two hundred individuals (>60 years old) without ocular hypertension or glaucoma were the control subjects. Full-length human wild-type MYOC cDNA was cloned in p3xFLAG-myc-CMV-25 and missense mutation was introduced by site-directed mutagenesis. Transfected human trabecular meshwork cells were treated with small-molecule chemical chaperones. Secreted MYOC was analyzed by combined immunoprecipitation-Western blot analysis. Intracellular myocilin was fractionated into Triton X-100-soluble and insoluble fractions, and analyzed by Western blot analysis. Intracellular aggregate and apoptosis were assayed by immunofluorescence. The effect of TMAO on subcellular myocilin distribution was analyzed by density gradient fractionation, followed by Western blot analysis.RESULTS. A novel c. 1150G>A change of MYOC was identified. Screening of optineurin, WDR36, and CYP1B1 showed an absence of disease-causing polymorphisms. Mutated D384N myocilin had reduced solubility and was aggregation-prone and nonsecreted. Treatment of transfected cells with TMAO improved the solubility of the D384N mutant, which was corrected for secretion in a dose-response manner. TMAO reduced the distribution of the D384N mutant in the endoplasmic reticulum (ER), alleviated ER stress, and rescued cells from apoptosis.CONCLUSIONS. The results indicate that TMAO, with chaperoning activity, facilitated the folding and secretion of mutant MYOC. This therapeutic approach assisted by a chemical chaperone can be developed for treating glaucoma. (Invest Ophthalmol Vis Sci. 2009; 50: 3743-3749) DOI: 10.1167/iovs.08-3151