Aroca-Aguilar José-Daniel, Sánchez-Sánchez Francisco, Martínez-Redondo Francisco, Coca-Prados Miguel, Escribano Julio
Area de Genética, Facultad de Medicina/Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete, Spain.
Mol Vis. 2008;14:2097-108. Epub 2008 Nov 21.
Heterozygous mutations in the myocilin gene (MYOC) cause glaucoma by an unknown mechanism. MYOC encodes an extracellular protein of unidentified function that undergoes intracellular endoproteolytic processing in the secretory pathway. It has been described that co-expression of wild-type/mutant myocilin reduces the secretion of the wild-type protein and that single expression of glaucoma myocilin mutants reduces its proteolytic processing. However, the effect of wild-type myocilin on mutant myocilin secretion and how mutant myocilin affects the proteolytic processing of wild-type myocilin have not been investigated. We herein analyze these two issues.
We modeled the heterozygous state for 4 missense (E323K, R346T, P370L, D380A) and 1 nonsense (Q368X) myocilin mutants by transiently co-expressing each mutant with the wild-type protein in HEK-293T cells. Recombinant mutant and wild-type myocilin in both culture media and cellular fractions were quantified by western immunoblot and densitometry.
A 24 h transient co-expression of each myocilin mutant with the wild-type protein elicited an augmented secretion of the mutant forms from 1.5 fold (D380A) to 5.4 fold (E323K). Under such conditions, extracellular mutant myocilin represented up to 20% of the total mutant protein. Other than this effect, secreted wild-type myocilin significantly decreased from 2.6 fold (E323K) to 36 fold (Q368X). When myocilin proteolytic processing was enhanced (96 hour co-expression) the extracellular amount of wild-type processed myocilin diminished from approximately 2.1 fold (E323K) to 6.3 fold (P370L). Nonreducing SDS-PAGE indicated that extracellular myocilin resulting from 24 h co-expression of wild-type myocilin and each of the 4 missense mutants forms hetero-oligomers and that glaucoma mutations do not increase the size of myocilin aggregates.
Increased extracellular levels of mutant myocilin expressed in heterozygosis may play a relevant role in glaucoma pathogenesis. This effect is likely the result of intracellular mutant/wild-type myocilin hetero-oligomerization.
肌纤蛋白基因（MYOC）的杂合突变通过未知机制导致青光眼。MYOC编码一种功能不明的细胞外蛋白，该蛋白在分泌途径中经历细胞内蛋白水解加工。已有研究表明，野生型/突变型肌纤蛋白的共表达会降低野生型蛋白的分泌，且青光眼肌纤蛋白突变体的单一表达会减少其蛋白水解加工。然而，野生型肌纤蛋白对突变型肌纤蛋白分泌的影响以及突变型肌纤蛋白如何影响野生型肌纤蛋白的蛋白水解加工尚未得到研究。我们在此分析这两个问题。
我们通过在HEK-293T细胞中瞬时共表达每个突变体与野生型蛋白，对4个错义突变（E323K、R346T、P370L、D380A）和1个无义突变（Q368X）的肌纤蛋白突变体的杂合状态进行建模。通过蛋白质免疫印迹和密度测定法对培养基和细胞组分中的重组突变型和野生型肌纤蛋白进行定量。
每个肌纤蛋白突变体与野生型蛋白共表达24小时，会使突变体形式的分泌增加，从1.5倍（D380A）到5.4倍（E323K）。在这种情况下，细胞外突变型肌纤蛋白占总突变蛋白的比例高达20%。除此之外，分泌的野生型肌纤蛋白显著减少，从2.6倍（E323K）到36倍（Q368X）。当肌纤蛋白的蛋白水解加工增强（共表达96小时）时，野生型加工后的肌纤蛋白的细胞外量从约2.1倍（E323K）减少到6.3倍（P370L）。非还原SDS-PAGE表明，野生型肌纤蛋白与4个错义突变体中的每一个共表达24小时产生的细胞外肌纤蛋白形成异源寡聚体，且青光眼突变不会增加肌纤蛋白聚集体的大小。
杂合状态下表达的突变型肌纤蛋白细胞外水平升高可能在青光眼发病机制中起相关作用。这种效应可能是细胞内突变型/野生型肌纤蛋白异源寡聚化的结果。
