Lindsey Rose Kristie M, Gourdie Robert G, Prescott Alan R, Quinlan Roy A, Crouch Rosalie K, Schey Kevin L
Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston 29403, USA.
Invest Ophthalmol Vis Sci. 2006 Apr;47(4):1562-70. doi: 10.1167/iovs.05-1313.
Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, is a water-permeable channel, has a role in fiber cell adhesion, and is essential for fiber cell structure and organization. The purpose of this study was to identify proteins that interact with the C terminus of AQP0, by using a proteomics approach, and thus further elucidate the role of AQP0 in the human lens.
AQP0 C-terminal peptides and AQP0 antibody affinity chromatography were used for affinity purification of interacting human lens proteins. Purified proteins were digested with trypsin, analyzed by liquid chromatography (LC)-tandem mass spectrometry and identified after database searching and manual examination of the mass spectral data. Colocalization of AQP0 with filensin and CP49, two proteins identified after mass spectrometric analysis, were examined by immunoconfocal and immunoelectron microscopy of lens sections.
The proteomics approach used to identify affinity-purified proteins revealed the lens-specific intermediate filament proteins filensin and CP49. With immunoconfocal microscopy, regions of colocalization of AQP0 with filensin and CP49 at the fiber cell plasma membrane in the lens cortex were defined. Immunoelectron microscopy confirmed that filensin and AQP0 were present in the same membrane compartments.
These studies suggest a novel interaction between an aquaporin water channel and intermediate filaments, an interaction through which AQP0 may maintain lens fiber cell shape and organization.
水通道蛋白0（AQP0）是晶状体中最丰富的膜蛋白，是一种水通透通道，在纤维细胞黏附中起作用，对纤维细胞的结构和组织至关重要。本研究的目的是通过蛋白质组学方法鉴定与AQP0 C末端相互作用的蛋白质，从而进一步阐明AQP0在人晶状体中的作用。
使用AQP0 C末端肽和AQP0抗体亲和色谱法对相互作用的人晶状体蛋白进行亲和纯化。用胰蛋白酶消化纯化的蛋白质，通过液相色谱（LC）-串联质谱分析，并在数据库搜索和对质谱数据进行人工检查后进行鉴定。通过晶状体切片的免疫共聚焦和免疫电子显微镜检查AQP0与丝状肌动蛋白和CP49（质谱分析后鉴定出的两种蛋白质）的共定位。
用于鉴定亲和纯化蛋白的蛋白质组学方法揭示了晶状体特异性中间丝蛋白丝状肌动蛋白和CP49。通过免疫共聚焦显微镜，确定了晶状体皮质中纤维细胞质膜上AQP0与丝状肌动蛋白和CP49的共定位区域。免疫电子显微镜证实丝状肌动蛋白和AQP0存在于同一膜区室中。
这些研究表明水通道蛋白水通道与中间丝之间存在一种新的相互作用，AQP0可能通过这种相互作用维持晶状体纤维细胞的形状和组织。
