Zimmerman C C, Lingappa V R, Richards J E, Rozsa F W, Lichter P R, Polansky J R
Cellular Pharmacology Laboratory, Department of Ophthalmology, University of California San Francisco, San Francisco, CA 94143-0730, USA.
Mol Vis. 1999 Aug 23;5:19.
To examine possible effects of the E323K mutation in the trabecular meshwork glucocorticoid response (TIGR) gene (also known as myocilin [MYOC]), using assays of translocational processing through the endoplasmic reticulum (ER). The E323K mutation was of particular interest, since the mutation shows a strong association with early onset open-angle glaucoma, but has a minimal predicted effect on protein structure.
Normal and mutant TIGR cDNA constructs were used to generate protein products in the presence of endoplasmic reticulum (ER) membranes, using an assay previously developed to detect alterations in the ER translocation function. "Paused" regions for potential protein modifications were defined by proteinase K (PK) sensitivity in the presence of ER membranes, with the ability to restart translocation when treated with EDTA. The effects of the E323K mutation were evaluated, as well as mutations located on either side of E323K (G246R, G364V, P370L) as the other mutations had substantial predicted structural changes in addition to clear disease associations.
The native TIGR molecule was observed to have a paused region that corresponds to the region of highest olfactomedin (OLF) homology. The E323K mutation, located near the beginning of this region, dramatically altered the normal pattern of nascent proteins observed in the translocational pausing assay. A prominent band appeared with the E323K mutation, which could represent a new product or a marked enhancement of a faint band normally seen, approximately 3 kDa higher than the major paused band. The other TIGR mutants examined did not show this effect.
The major translocational pause that starts near the beginning of the region of high OLF homology may help to explain the high frequency of glaucoma-associated mutations in this area. The observed effect of the E323K mutation on the products of translocational processing suggests a delay in the normal pausing process of TIGR biogenesis. This delay points to a potentially distinct pathogenic mechanism for E323K as compared with the other TIGR mutations so far evaluated.
通过内质网（ER）转位加工分析，研究小梁网糖皮质激素反应（TIGR）基因（也称为肌纤蛋白[MYOC]）中E323K突变的可能影响。E323K突变特别引人关注，因为该突变与早发性开角型青光眼密切相关，但对蛋白质结构的预测影响极小。
使用正常和突变型TIGR cDNA构建体，在存在内质网膜的情况下生成蛋白质产物，采用先前开发的检测内质网转位功能改变的分析方法。通过在存在内质网膜的情况下蛋白酶K（PK）敏感性来定义潜在蛋白质修饰的“暂停”区域，在用EDTA处理时能够重新开始转位。评估了E323K突变的影响，以及位于E323K两侧的突变（G246R、G364V、P370L），因为除了明确的疾病关联外，其他突变还具有显著的预测结构变化。
观察到天然TIGR分子有一个与最高嗅觉介质（OLF）同源性区域相对应的暂停区域。位于该区域起始附近的E323K突变显著改变了转位暂停分析中观察到的新生蛋白质的正常模式。E323K突变出现了一条明显的条带，这可能代表一种新产物或通常可见的微弱条带的显著增强，比主要暂停条带高约3 kDa。所检测的其他TIGR突变体未显示此效应。
在高OLF同源性区域起始附近开始的主要转位暂停可能有助于解释该区域青光眼相关突变的高频率。观察到的E323K突变对转位加工产物的影响表明TIGR生物合成的正常暂停过程出现延迟。与迄今为止评估的其他TIGR突变相比，这种延迟指出了E323K潜在的独特致病机制。
