| Project/Area Number |
15380072
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Kyoto Institute of Technology |
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Principal Investigator |
ODA Kohei Kyoto Institute of Technology, Department of Applied Biology, Professor, 繊維学部, 教授 (50081584)
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| Co-Investigator(Kenkyū-buntansha) |
OYAMA Hiroshi Kyoto Institute of Technology, Department of Applied Biology, Associate Professor, 繊維学部, 助教授 (50221700)
HIRAGA Kazumi Kyoto Institute of Technology, Department of Applied Biology, Assistant Professor, 繊維学部, 助手 (50252549)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥12,700,000 (Direct Cost: ¥12,700,000)
Fiscal Year 2004: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 2003: ¥9,900,000 (Direct Cost: ¥9,900,000)
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| Keywords | fatal neurodegenerative disease / serine-carboxyl pepetidases / CLN2 / novel peptidase family / sedolisin |
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| Research Abstract |
My colleagues and I determined the crystal structure of sedolisin at high resolution and defined a novel family, serine-carboxyl peptidases (sedolisins, MEROPS S53 family). Unique features of this family are as follows ; (1)subtilisin-like fold, (2)catalytic triad consisting of Ser, Glu, and Asp, (3)involvement of asparatate in the formation of an oxyanion hole. CLN2 plays a crucial role in lysosomal protein degradation and deficiency of this enzyme leads to a fatal neurodegenerative disease (Batten disease).
In this -project, structure and function relationship of CLN2 was studied for analyzing biochemical process of Batten disease.
(1)Catalytic function of h residues Ser280 and Glu77
The catalytic mechanism of this family was postulated to utilize glutamate (Glu77) as a general base abstracting a proton from the serine (Ser280) that acts as a nucleophile. The S280A mutant of CLN2 showed no enzymatic activity. In addition, CLN2 was inactivated by Ac-IAF-CHO, which bind covalently to the
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catalytic serine residue. The E77A mutant did not show any significant enzymatic activity (1/10^4 lower than that of wild-type enzyme). Coupled with the results of structure analysis, the residues Ser280 and Glu77 were identified as the catalytic residues of CLN2.
(2)Subsite structure of CLN2
CLN2 is composed of only three subsites on the non-prime side and at least three on the prime side (S_3-S_3'). CLN2 favored Phe, Tyr, or Leu at the P_1 position, suggesting that the S_1 subsite is occupied by hydrophobic amino acid residues. CLN2 preferred Ala, Arg, or Asp at the P_2 position. The result suggests that the S_2 subsite has electrostatic interactions. CLN2 prefers Ala at the P_3 position, suggesting that the S_3 subsite is small. The results described here are well consistent with the structure model of CLN2.
(3)Homology modeling of the structure of CLN2
A three-dimensional model of CLN2 was built based on the homology with sedolisin. It was clarified that the carboxyl group of Asp132 would extend out into the active site cleft and act as an anchor of the N-terminal of the substrate.
(4)Crystal structure of CLN2
Three-dimensional structure analyses of CLN2 complexes with and without new tripeptide-based inhibitors are currently under way. Less
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