2003 Fiscal Year Final Research Report Summary
Proteomic Analysis of the Loss of Function induced by Changes in Molecular Structure of ClC-5 Chloride Channel
| Project/Area Number |
14571035
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Kidney internal medicine
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| Research Institution | Kitasato University |
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Principal Investigator |
SAKAMOTO Hisato Kitasato Univ., School of Medicine, Assistant Professor, 医学部, 講師 (80187046)
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| Co-Investigator(Kenkyū-buntansha) |
NAGABA Yasushi Kitasato Univ., School of Medicine, Assistant Professor, 医学部, 講師 (40255279)
NAITO Ichiro Shigei Medical Institute, Manager, 超病理部門, 部長
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| Project Period (FY) |
2002 – 2003
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| Keywords | ClC-5 channel / sorting / mutation / Dent's disease / proteome / lectin family protein / siRNA |
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| Research Abstract |
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.
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