Kanagavalli Janakaraj, Pandaranayaka P J Eswari, Krishnadas Subbaiah Ramasamy, Krishnaswamy Sankaran, Sundaresan Periasamy
Department of Genetics, Aravind Medical Research Foundation, Tamilnadu, India.
Mol Vis. 2007 Jul 13;13:1161-8.
Mutations in the myocilin gene (MYOC) leading to a perturbed outflow of aqueous in the trabecular meshwork (TM) has been associated with the pathophysiology of glaucoma. This study examines the expression of normal and mutant myocilin (Gly367Arg) in cultured TM cells.
Normal and mutant MYOC cDNA constructs were used to transfect the TM cells. In order to confirm the method of transfection, reverse transcriptase polymerase chain reaction (RT-PCR) was carried out. Further, confocal microscopic analysis was used to determine the cellular localization of myocilin protein. The extracellular nature of myocilin in the culture supernatant and cell lysates of the transfected cells was analyzed by western blot. Molecular modeling was done earlier using a knowledge based consensus method which involved threading the protein into the identified pentein fold for the COOH-terminal part. Molecular dynamics was carried out using GROMACS for the mutant model which was built using the native myocilin model.
The Gly367Arg mutation causes accumulation of myocilin protein within TM cells with extensively reduced secretion contrary to wild type myocilin being characterized by intracellular localization and extracellular secretion. Further, Gly367Arg mutation occurs in a hydrophobic region which leads to burial of a charged residue. The dynamics suggests large conformational change is required to accommodate the mutation favoring aggregation of the protein.
Our results suggest that Gly367Arg is a potential mutation that causes malfunction of TM cells either by dominant negative effect or gain of function of mutant myocilin. The structural model suggests that the mutated myocilin could aggregate, implying the possible role of Gly367Arg in causing Primary open angle glaucoma (POAG).
肌纤凝蛋白基因（MYOC）突变导致小梁网（TM）房水流出受阻，这与青光眼的病理生理学相关。本研究检测了正常和突变型肌纤凝蛋白（Gly367Arg）在培养的TM细胞中的表达。
使用正常和突变型MYOC cDNA构建体转染TM细胞。为了确认转染方法，进行了逆转录聚合酶链反应（RT-PCR）。此外，使用共聚焦显微镜分析来确定肌纤凝蛋白的细胞定位。通过蛋白质印迹分析转染细胞培养上清液和细胞裂解物中肌纤凝蛋白的细胞外性质。早期使用基于知识的共识方法进行分子建模，该方法涉及将蛋白质穿入已鉴定的COOH末端部分的五聚体折叠中。使用GROMACS对基于天然肌纤凝蛋白模型构建的突变体模型进行分子动力学研究。
与野生型肌纤凝蛋白的细胞内定位和细胞外分泌特征相反，Gly367Arg突变导致肌纤凝蛋白在TM细胞内积累，分泌大量减少。此外，Gly367Arg突变发生在一个疏水区，导致一个带电荷的残基被掩埋。动力学表明需要大的构象变化来适应突变，这有利于蛋白质聚集。
我们的结果表明，Gly367Arg是一个潜在的突变，它通过显性负效应或突变型肌纤凝蛋白的功能获得导致TM细胞功能障碍。结构模型表明，突变的肌纤凝蛋白可能聚集，这意味着Gly367Arg在导致原发性开角型青光眼（POAG）中可能起作用。
