Chen Tong, Li XiaLian, Yang Yu, Church Robert L
Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA, USA.
Mol Vis. 2002 Oct 11;8:372-88.
[corrected] MP19 is the second most abundant major intrinsic protein of the lens fiber cell membrane. A specific heritable mutation at amino acid 15 in the MP19 protein, termed MP19To3, results in total cataract and microphthalmia in the mouse. The goals of this study were to determine the specific localization of MP19 in the cell membrane and to determine whether the mutant MP19To3 protein migrates to the cell membrane in a similar fashion to normal MP19.
MP19 and MP19To3 cDNAs were cloned into two different sets of expression vectors. The first set was composed of two vectors, pEGFP-N1 and pDsRed2-N1. The first vector expressed green fluorescent protein and the second expressed a red fluorescent protein when transfected into mammalian cells. The two lens membrane protein cDNAs were separately cloned into the vectors so that the cDNA was at the 5'-end of the fluorescent protein coding DNA. These vectors expressed each of the lens proteins fused to the fluorescent protein upon transfection into mammalian cell cultures. The second vector set was a single vector, pcDNA4/TO which must be induced in the transfected cells by tetracycline in order to express the cloned cDNAs. Each of the membrane cDNAs coupled to the fluorescent protein coding region was cut out of the first vector set and cloned into pcDNA4/TO and stable clones were isolated. Each of the prepared plasmids was transfected into human and chick embryo lens epithelial cells and human T-RexTM-293 cells. The fluorescent cells were viewed using confocal and episcopic-fluorescence microscopy.
Each of the transfected plasmids expressed fluorescent protein in all three cell lines. MP19 was observed to transport to the cell membrane. When compared to the distribution of another, separate fusion protein consisting of a signal peptide that targets to cell membranes fused to EGFP, MP19 did not distribute uniformly on the membrane, but appeared to localize into "spots" or pools of fluorescent material around the cell membrane. In contrast, MP19To3 protein appeared to not distribute to the cell membrane; it instead appeared to collect in a particular subcellular compartment within the cell.
The distribution of MP19 and MP19To3 in the cell appeared to be quite distinct. MP19 was observed to distribute to the cell membrane while MP19To3 did not. The fact that the MP19To3 did not traffic to the membrane, instead appearing to be trapped within a subcellular compartment within the cell sheds further light on the cause of the cataract and microphthalmia observed in the MP19To3 mutation, and further sheds information on the pathway of MP19 transport to the cell membrane.
[已校正]MP19是晶状体纤维细胞膜中含量第二丰富的主要内在蛋白。MP19蛋白第15位氨基酸处的一种特定遗传突变，称为MP19To3，会导致小鼠出现完全性白内障和小眼症。本研究的目的是确定MP19在细胞膜中的具体定位，并确定突变型MP19To3蛋白是否以与正常MP19相似的方式迁移到细胞膜。
将MP19和MP19To3的cDNA克隆到两组不同的表达载体中。第一组由两个载体组成，即pEGFP-N1和pDsRed2-N1。当转染到哺乳动物细胞中时，第一个载体表达绿色荧光蛋白，第二个载体表达红色荧光蛋白。将这两种晶状体膜蛋白cDNA分别克隆到载体中，使cDNA位于荧光蛋白编码DNA的5'端。将这些载体转染到哺乳动物细胞培养物中后，它们会表达与荧光蛋白融合的每种晶状体蛋白。第二组载体是单个载体pcDNA4/TO，必须在转染细胞中用四环素诱导才能表达克隆的cDNA。将与荧光蛋白编码区偶联的每个膜cDNA从第一组载体中切出，克隆到pcDNA4/TO中，并分离出稳定的克隆。将制备的每种质粒转染到人及鸡胚晶状体上皮细胞和人T-RexTM-293细胞中。使用共聚焦显微镜和落射荧光显微镜观察荧光细胞。
每种转染的质粒在所有三种细胞系中均表达荧光蛋白。观察到MP19转运到细胞膜。与另一种由靶向细胞膜的信号肽与EGFP融合组成的单独融合蛋白的分布相比，MP19在膜上分布不均匀，而是似乎定位在细胞膜周围的“斑点”或荧光物质池中。相比之下，MP19To3蛋白似乎没有分布到细胞膜；相反，它似乎聚集在细胞内的一个特定亚细胞区室中。
MP19和MP19To3在细胞中的分布似乎非常不同。观察到MP19分布到细胞膜，而MP19To3则没有。MP19To3没有运输到细胞膜，而是似乎被困在细胞内的一个亚细胞区室中，这一事实进一步揭示了在MP19To3突变中观察到的白内障和小眼症的原因，并进一步提供了有关MP19运输到细胞膜途径的信息。
