Intracellular sequestration of hetero-oligomers formed by wild-type and glaucoma-causing myocilin mutants
Intracellular sequestration of hetero-oligomers formed by wild-type and glaucoma-causing myocilin mutants
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DOI：
10.1167/iovs.04-0300
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发表时间：
2004-10-01
影响因子：
4.4
通讯作者：
Raymond, V
中科院分区：
文献类型：
--
作者：
Gobeil, S;Rodrigue, MA;Raymond, V
目的。为了研究心肌蛋白（MYOC）的olfactomedin结构域（也称为小梁网诱导的糖皮质激素反应（TIGR）基因）突变导致常染色体显性开角型青光眼的机制，研究了野生型（WT） MYOC蛋白单独表达或与Q368X或K423E疾病相关多肽同时表达时的结构和特性。分析了人房水组织和人小梁网（HTM）组织中心肌蛋白的含量。用编码WT MYOC、突变和/或表位标记cdna的表达载体转染COS-7和永生化人小梁网（iHTM）细胞系。通过双表位标记和/或Western blot分析对MYOC蛋白进行了表征。MYOC多肽在房水、热媒组织、转染的COS-7和iHTM细胞系中形成高度相似的低聚物。这些复合物的大小从116 kDa到200 kDa以上不等。最小的复合物，大约116 kDa，是由两个MYOC单体之间的二聚化产生的。150-kDa复合物在房水中的表达最强。用Q368X或K423E cDNA共转染WT构建体可产生心肌WT/心肌突变体异源二聚体和更高分子量的异源寡聚物复合物。两种细胞系的细胞外培养基中均分泌WT同聚物复合物，而Q368X和K423E突变/突变体的同聚物和WT/突变体的异聚物仍被隔离在细胞内。异质WT/突变体复合物的形成可能是突变型心肌蛋白多肽产生常染色体显性开角型青光眼的关键机制。异常WT/突变复合物的细胞内隔离可能导致表达心肌细胞的功能障碍，并可能导致POAG的显性负作用。
PURPOSE. To investigate mechanism(s) by which mutations in the olfactomedin domain of myocilin ( MYOC), also known as the trabecular meshwork - induced glucocorticoid response ( TIGR) gene, cause autosomal dominant open-angle glaucoma, the structure and properties of wild-type (WT) MYOC protein were examined, when expressed alone or simultaneously with the Q368X or K423E disease-associated polypeptides.METHODS. Myocilin was analyzed in human aqueous humor and human trabecular meshwork (HTM) tissues. COS-7 and immortalized human trabecular meshwork (iHTM) cell lines were transfected with expression vectors encoding WT MYOC, mutated, and/or epitope-tagged cDNAs. MYOC proteins were characterized by double-epitope tagging procedures and/or Western blot analysis.RESULTS. MYOC polypeptides formed highly similar oligomers in aqueous humor, HTM tissues, transfected COS-7, and iHTM cell lines. These complexes ranged in size from 116 kDa to more than 200 kDa. The smallest complex, approximately 116 kDa, resulted from dimerization between two MYOC monomers. Expression of a 150-kDa complex was strongest in aqueous humor. Cotransfections of the WT construct with either the Q368X or K423E cDNA produced MYOCWT/MYOCmutant heterodimers and higher molecular weight hetero-oligomeric complexes. WT homo-oligomeric complexes were secreted in the extracellular media of both cell lines whereas the Q368X and K423E mutant/mutant homomultimers and heteromeric WT/mutant oligomers remained sequestered intracellularly.CONCLUSIONS. Formation of heteromeric WT/mutant complexes may provide a critical mechanism by which mutant myocilin polypeptides produce autosomal dominant open-angle glaucoma. The intracellular sequestration of abnormal WT/mutant complexes could lead to the malfunction of MYOC-expressing cells and to POAG potentially involving a dominant negative effect.