Fukagawa Takashi, Takafuji Kazuaki, Tachibanaki Shuji, Kawamura Satoru
Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan.
Center of Medical Innovation and Translational Research, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
PLoS One. 2017 Mar 14;12(3):e0173908. doi: 10.1371/journal.pone.0173908. eCollection 2017.
Rods and cones are both photoreceptors in the retina, but they are different in many aspects including the light response characteristics and, for example, cell morphology and metabolism. These differences would be caused by differences in proteins expressed in rods and cones. To understand the molecular bases of these differences between rods and cones, one of the ways is to compare proteins expressed in rods and cones, and to find those expressed specifically or dominantly. In the present study, we are interested in proteins in the outer segment (OS), the site responsible for generation of rod- or cone-characteristic light responses and also the site showing different morphology between rods and cones. For this, we established a method to purify the OS and the inner segment (IS) of rods and also of cones from purified carp rods and cones, respectively, using sucrose density gradient. In particular, we were interested in proteins tightly bound to the membranes of cone OS. To identify these proteins, we analyzed proteins in some selected regions of an SDS-gel of washed membranes of the OS and the IS obtained from both rods and cones, with Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) using a protein database constructed from carp retina. By comparing the lists of the proteins found in the OS and the IS of both rods and cones, we found some proteins present in cone OS membranes specifically or dominantly, in addition to the proteins already known to be present specifically in cone OS.
视杆细胞和视锥细胞都是视网膜中的光感受器，但它们在许多方面存在差异，包括光反应特性，例如细胞形态和新陈代谢。这些差异是由视杆细胞和视锥细胞中表达的蛋白质差异所导致的。为了理解视杆细胞和视锥细胞之间这些差异的分子基础，一种方法是比较视杆细胞和视锥细胞中表达的蛋白质，并找出那些特异性或优势表达的蛋白质。在本研究中，我们关注的是外段（OS）中的蛋白质，外段是产生视杆细胞或视锥细胞特征性光反应的部位，也是视杆细胞和视锥细胞之间形态不同的部位。为此，我们建立了一种方法，分别从纯化的鲤鱼视杆细胞和视锥细胞中，利用蔗糖密度梯度纯化视杆细胞和视锥细胞的外段及内段（IS）。特别是，我们对紧密结合在视锥细胞外段膜上的蛋白质感兴趣。为了鉴定这些蛋白质，我们使用从鲤鱼视网膜构建的蛋白质数据库，通过液相色谱-串联质谱（LC-MS/MS）分析了从视杆细胞和视锥细胞获得的外段和内段洗涤膜的SDS凝胶中一些选定区域的蛋白质。通过比较视杆细胞和视锥细胞的外段和内段中发现的蛋白质列表，我们发现了一些特异性或优势存在于视锥细胞外段膜上的蛋白质，此外还有已知特异性存在于视锥细胞外段的蛋白质。
