Proteomic Analysis of the Loss of Function induced by Changes in Molecular Structure of ClC-5 Chloride Channel

ClC-5 氯离子通道分子结构变化引起的功能丧失的蛋白质组学分析

• 批准号: 14571035
• 负责人: SAKAMOTO Hisato
• 金额: $ 2.24万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571035/
• 关键词: ClC-5 channel; sorting; mutation; Dent's disease; proteome; lectin family protein; siRNA; C1C-5チャネル; ClC-5チャネル

To elucidate how the mutation in CLCN5 affect on the trafficking of each gene product, four Dent's disease related mutations of human ClC-5, two-missense type (S270R and R280P) and two frameshift in C-terminus type (658delC and 2079/2080insA), were comparatively examined in detail. As a result, we demonstrated that the synthesized proteins of the missense mutants were correctly trafficked to the early endosome in the same manner as the wild-type (WT) ClC-5, and in contrast that the two truncated frameshift mutations resulted in the retainment of the sorting in the endoplasmic reticulum (ER). This is the first to demonstrate the diversity of the sorting disorder in the molecular mechanism of Dent's disease. In the next step, we extensively examined to identify specific protein(s) that is crucial for the ClC-5 sorting using proteomic analysis. The proteomic approach was used to generate differential protein expression maps between the mock-transfected cells and the transfected cells expressing WT or-2079/2080insA mutation. Finally, the analysis using two-dimensional electrophoresis identified 3 proteins out of approximately 1,000 spot visualized on each gel, which were expressed at relatively higher levels (300-fold) in the WT-than the mock-or mutant-transfected cells. Furthermore, a peptide mass of one of 3 proteins was identified by MALDI-TOF mass spectrometry as a molecule (GA) belongs to the lectin family. Subsequently the suppression of this identified molecule using the siRNA resulted in the sorting disorder of ClC-5 channel and significant inhibition of the endocytosis of albumin in the OK cell expressing native ClC-5 channel. These findings provide novel and important insights that a protein belongs to learn family might play an important roles in the sorting regulation of ClC-5 channel.

为了阐明CLCN 5基因突变对各基因产物转运的影响，我们对4种与Dent病相关的人ClC-5基因突变进行了详细的比较研究，包括两种错义突变（S270 R和R280 P）和两种C端移码突变（658 delC和2079/2080 insA）。因此，我们证明了错义突变体的合成蛋白以与野生型（WT）ClC-5相同的方式被正确地运输到早期内体，相反，两个截短的移码突变导致内质网（ER）中的分选保留。这是第一次证明Dent病分子机制中分选障碍的多样性。在下一步中，我们使用蛋白质组学分析广泛检查以鉴定对ClC-5分选至关重要的特定蛋白质。蛋白质组学方法用于产生模拟转染细胞和表达WT或-2079/2080insA突变的转染细胞之间的差异蛋白质表达图谱。最后，使用二维电泳的分析在每个凝胶上可见的约1，000个斑点中鉴定出3种蛋白质，其在WT中的表达水平比模拟转染或突变体转染的细胞相对更高（300倍）。此外，通过MALDI-TOF质谱法鉴定了3种蛋白质之一的肽质量，作为属于凝集素家族的分子（GA）。随后，使用siRNA抑制该鉴定的分子导致ClC-5通道的分选紊乱，并显著抑制表达天然ClC-5通道的OK细胞中白蛋白的内吞作用。这些发现为clC-5通道的分选调控提供了新的重要的见解。

项目成果

Matsuo T., Sakamoto H.et al.: "Diversity in the Sorting Disorder Induced by the Mutant C1C-5 Chloride Channels Responsible for Dent's disease"Kitasato Medicine. 33(in press). (2004)

Matsuo T.、Sakamoto H.等人：“导致登特氏病的突变 C1C-5 氯离子通道诱导的分选紊乱的多样性”Kitasato Medicine。

松尾孝俊, 坂本尚登, 他: "ClC-5クロライドチャネルの分子構造・機能解析による遺伝性腎疾患の病態解明"日腎会誌. 45. 204 (2003)

Takatoshi Matsuo、Naoto Sakamoto 等人：“通过 ClC-5 氯离子通道的分子结构和功能分析阐明遗传性​​肾病的病理生理学”，日本肾脏病学会杂志 45. 204 (2003)。

Matsuo, T., H.Sakamoto, S.Inoue, I.Naito, Y.Kobayashi, M.Higashihara: "Diversity in the Sorting Disorder Induced by the Mutant ClC-5 Chloride Channel Responsible for Dent's Disease"KITASATO MEDICINE. (in press). (2004)

Matsuo, T.、H.Sakamoto、S.Inoue、I.Naito、Y.Kobayashi、M.Higashihara：“导致 Dent 病的突变 ClC-5 氯化物通道诱导的分选紊乱的多样性”KITASATO MEDICINE。

松尾孝俊, 坂本尚登 他: "レクチンファミリー蛋白によるC1C-5クロライドチャネルのソーティング制御"日腎会誌. 46・3. (2004)

Takatoshi Matsuo、Naoto Sakamoto 等：“凝集素家族蛋白对 C1C-5 氯离子通道的排序”，日本肾病学会杂志 46・3。

松尾孝俊, 坂本尚登 他: "ClC-5遺伝子変異による細胞内小胞輸送障害分子機構の遺伝子導入解析"日腎会誌. 44・3. 312 (2002)

Takatoshi Matsuo、Naoto Sakamoto等：“ClC-5基因突变引起的细胞内囊泡转运障碍的分子机制的基因转移分析”日本肾病学会杂志44・3.312（2002年）。