Oka Mikako, Kudo Hiroaki, Sugama Norio, Asami Yuko, Takehana Makoto
Department of Molecular Function and Physiology, Kyoritsu University of Pharmacy, Tokyo, Japan.
Mol Vis. 2008 Apr 25;14:815-22.
Beaded filaments are lens cell-specific intermediate filaments composed of two proteins: filensin and phakinin (CP49). Filensin and phakinin are believed to function in the maintenance of lens transparency. To elucidate the function of filensin and phakinin at the molecular level, we examined the degradation of these two proteins in normal and cataractous rat lenses.
A hereditary cataract model, the Shumiya cataract rat (SCR), was used for these studies. Anti-filensin antibodies were raised against three different regions of the protein, the rod domain, the inner region of the tail domain, and the outer region of the tail domain. Anti-filensin and anti-phakinin antibodies were used to examine the conformation of degradation of filensin and phakinin by western blot analysis and fluorescent immunocytochemistry of cryosectioned lenses.
In the normal lens, filensin was processed from a 94 kDa protein to proteins of 50 kDa and 38 kDa. Similarly, phakinin was processed from a 49 kDa protein to one of 40 kDa. The concentrations of filensin and phakinin in the rat lens cortex fluctuated with age and decreased during cataractogenesis. The 50 kDa form of filensin decreased significantly before opacification. In the normal lens, phakinin, the filensin rod domain, and the filensin inner tail domain localized to membrane lining regions in the shallow cortex and to the central region of the cytoplasm in the deep cortex. The COOH-terminal domain of filensin localized to the membrane lining region in the deep cortex. In pre-cataractous lenses, phakinin and the filensin rod domain localized primarily to the membranes lining the shallow cortex region and were distributed throughout the cytoplasm of lens fiber cells in the deep cortex.
The 50 kDa form of filensin is important for the localization of beaded filaments in lens fiber cells and for lens transparency.
串珠状细丝是晶状体细胞特异性中间丝，由丝状晶状体蛋白和晶状体蛋白（CP49）两种蛋白质组成。丝状晶状体蛋白和晶状体蛋白被认为在维持晶状体透明度方面发挥作用。为了在分子水平上阐明丝状晶状体蛋白和晶状体蛋白的功能，我们研究了这两种蛋白质在正常和白内障大鼠晶状体中的降解情况。
使用遗传性白内障模型舒米亚白内障大鼠（SCR）进行这些研究。针对该蛋白质的三个不同区域，即杆状结构域、尾部结构域内部区域和尾部结构域外部区域，制备了抗丝状晶状体蛋白抗体。使用抗丝状晶状体蛋白和抗晶状体蛋白抗体，通过蛋白质印迹分析和冷冻切片晶状体的荧光免疫细胞化学，来检测丝状晶状体蛋白和晶状体蛋白降解的构象。
在正常晶状体中，丝状晶状体蛋白从94 kDa的蛋白质加工成50 kDa和38 kDa的蛋白质。同样，晶状体蛋白从49 kDa的蛋白质加工成40 kDa的蛋白质。大鼠晶状体皮质中丝状晶状体蛋白和晶状体蛋白的浓度随年龄波动，并在白内障形成过程中降低。丝状晶状体蛋白的50 kDa形式在浑浊化之前显著降低。在正常晶状体中，晶状体蛋白、丝状晶状体蛋白杆状结构域和丝状晶状体蛋白内部尾部结构域定位于浅皮质的膜内衬区域和深皮质细胞质的中央区域。丝状晶状体蛋白的COOH末端结构域定位于深皮质的膜内衬区域。在白内障前期晶状体中，晶状体蛋白和丝状晶状体蛋白杆状结构域主要定位于浅皮质区域的膜内衬，并且分布在深皮质晶状体纤维细胞的整个细胞质中。
丝状晶状体蛋白的50 kDa形式对于串珠状细丝在晶状体纤维细胞中的定位以及晶状体透明度很重要。
