Proteomics Analysis of Secretory Cysteine Protease Inhibitors and their Practical Application on Oral Health
| Project/Area Number |
15591981
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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 |
Functional basic dentistry
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| Research Institution | NIIGATA INSTITUTE OF TECHNOLOGY (2005)
The Nippon Dental University (2003-2004) |
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Principal Investigator |
SAITOH Eiichi Niigata Institute of Technology, Technology, Professor, 工学部, 教授 (40120662)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Cysteine protease inhibitors / Porphyromonas gingivalis / Proteomics analysis / Reverse zymography / Human cystatin S / Rice cystatins / Eel-CPI-1 with lectin activity / CD4-positive T cells / オリザシスタチン / ウナギ上皮レクチン / システインプロテアーゼインヒビター / 口腔細菌 / 組換え蛋白質 / 枯草菌 / リバースザイモグラフィー / ヒト唾液 / シスタチンスーパーファミリー / 魚類体表上皮粘膜 / サイトカイン誘導 / CD4陽性T細胞 / 組換えヒトシスタチン |
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| Research Abstract |
This study focused on the proteomics analysis of secretory cysteine protease inhibitors and their practical application on oral health. The results obtained are as follows ;
1.A tow dimensional gel electrophoresis system that can separate and detect cysteine protease inhibitors in biological fluids was developed.
2.A novel rice cysteine protease inhibitor, oryzacystatin III was cloned and sequenced. It was found that oryzacystain III from the calli of rice shares 56% and 89% amino acid sequence identity, respectively, with oryzacystatin I and oryzacystatin II. The sequence differs from that of oryzacystatin II in the N-terminal region (Gln^7 - Ala^<19>; in the oryzacystatin II numbering), and this region contained glycine residue (Gly^<14>), which is the evolutionarily conserved in the cystatin superfamily. Biochemical properties of oryzacystatins were characterized using recombinant protein.
3.Two cysteine protease inhibitors (Eel-CPI-1 and Eel-CPI-2) from skin mucus of the Japanese eel
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(Anguilla japonica) were isolated and characterized. Eel-CPI-1 inhibited papain (K_i=18 nM) and ficin (K_i=120 nM) competitively. Combined with the data on amino acid and sequence analysis, Eel-CPI-1 is identical to the eel lectin, AJL-2.
4.A Bacillus subtilis system that can be used to produce large quantities of recombinant human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily was developed. The DNA sequences for promoter and signal of the alkaline endoglucanase gene in Bacillus sp.KSM-S237 were employed to accomplish higher expression levels of recombinant human salivary cystatins by the first use of ΔaprE type mutant strain of B.subtilis. Our system yielded approximately 1.1 gram/liter of r-cystatin S and 0.8 g/liter of r-cystatin SA.
5.We have succeeded in producing two antibodies that discriminate the structural differences between salivary cystatins S and SN, which share more than 90% identity in amino acid sequence with cystatin SA.
6.It was found that human cystatin SA induce gamma-interferon production by CD4-positive T cells. Less
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Report
(4 results)
Research Products
(34 results)
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[Journal Article] Murine monoclonal antibody which can distinguish cystatins SA1 and SA2.2005
Author(s)
Ito, T., Komiya-Ito, A., Okuda, K., Minaguchi, K., Saitoh, E., yamada, S., Kato, T.
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Journal Title
Molecular Immunology (In press)
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[Journal Article] Molecular cloning and biochemical characterization of oryzacystatin-III, a novel member of phytocystatin from rice (Oryza sativa L.Japonica).2005
Author(s)
Ohtsubo, S., Kobayashi, H., Noro, W., Sato, K., Taniguchi, M., Saitoh, E.
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Journal Title
Abstracts of 2004 World Rice Research Conference
Pages: 175-175
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[Journal Article] Cystatin SA, a cysteine proteinase inhibitor, induces interferon-γ expression in CD4-positive cells.2004
Author(s)
Kato, T., Ito, T., Imatani, T., Minaguchi, K., Saitoh, E., Okuda, K.
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Journal Title
Biological Chemistry Vol.385,No.5
Pages: 419-422
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