| 研究課題/領域番号 |
16J09051
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| 研究機関 | 京都産業大学 |
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研究代表者 |
TONITI WARAPHAN 京都産業大学, 工学研究科, 特別研究員(DC2)
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| 研究期間 (年度) |
2016-04-22 – 2018-03-31
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| キーワード | enterotoxin / binary toxin / CPILE / x-ray crystallography |
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| 研究実績の概要 |
In the past twenty years, there were unusual outbreaks of Clostridium perfringens food poisoning in Japan. All of them lacked of the typical C. perfringens enterotoxin (CPE). On the other hand, they secreted a novel enterotoxin. The W5052 strain isolated from Tokyo outbreaks was named C. perfringens iota-like enterotoxin or CPILE (Irikura et al., 2015). This strain is consisting of two components: CPILE-a, which acts as an enzymatic ADP-ribosyltransferase and CPILE-b, a membrane binding component. This research aimed to study structure and function of the CPILE-a using x-ray crystallography and biochemical methods.
The structure of CPILE-a was solved using SHELX against the single anomalous dispersion data set of mercury-soaked crystal at 2.28 A resolution. Then the main-chain tracing was done using ARP/wARP. Furthermore, the triple Cys mutant structure was used as a template for refining the final structure of wild type CPILE-a at 2.01 A resolution. In addition, the two complex structures of NAD+-CPILE-a and NADH-CPILE-a were revealed at 1.80 A and 2.26 A, respectively. There were no obvious structure differences among three structures.
Based on the C-terminal domain structural similarity with Ia, it was expected that CPILE-a should have similar ART and NADase activities as Ia. CPILE-a showed ART activity against both α- and β/γ-actins. However, there was almost no NADase activity without actin. This finding was completely different from Ia case.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
CPILE is a binary toxin composes of two unlinked components; CPILE-a an enzymatic component and CPILE-b a binding component. Three crystal structures of CPILE-a with its cofactors have been solved and deposited in PDB including 5GTT, 5WTZ, and 5WU0. Moreover, its ADP-ribosyltransferase and NAD glycohydrolase activities also have been tested and compared to Ia. Recently, the structures and functions of CPILE-a have been studied and published (Toniti et al., 2017).
It should be noted that CPILE-a alone is non-toxic. On the other hand, high concentration of CPILE-b alone is cytotoxic and causes morphological changes of the L929 cells (Irikura et al., 2015). As a binary toxin, the cell toxicity of CPILE-a seems to enhance CPILE-b. How CPILE-a gets into the cell and reaches its target still on investigation. The crystal screening for CPILE-a-actin complex is on progression via hanging drop vapor diffusion under 4°C.
As a binding component, CPILE-b binds to the specific cell receptor and assists translocation of CPILE-a into the target cell. How CPILE-b binds to the particular cell receptor is still unclear. It probably binds as a monomer as another binary toxin does then transforms to a heptamer. Or binds as a heptamer which is already formed outside the cell. Hence, several conditions of crystal screening of CPILE-b have been screened.
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| 今後の研究の推進方策 |
According to the apo-, NAD+-, and NADH-CPILE-a crystal structures, we try to investigate the structure of Cpile-a-Actin complex by x-ray crystallography. Briefly, the complex structure of CPILE-a and actin will be crystallized at various conditions using hanging drop vapor diffusion technique. Then, the data sets of complex crystals will be collected and structure determination will perform using Molecular Replacement and so on.
The crystal structure of both monomeric and heptameric CPILE-b will be screened using standard Hampton Screening Kits. Moreover, the structures of CPILE-b including prepore and pore structures will be studied by x-ray crystallography. The biochemical functions of CPILE-b as a cell binding protein and a pore-forming protein will be explored also.
In addition, the interaction between CPILE-a and CPILE-b will be studied parallel to the CPILE-b studies. Briefly, the C-terminus His-Tag of CPILE-a will be expressed and purified for studying interaction between CPILE-a and CPILE-b including x-ray crystallography and biochemical assay.
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