| Project/Area Number |
09670249
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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 |
Experimental pathology
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| Research Institution | AICHI MEDICAL UNIVERSITY |
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Principal Investigator |
SAGA Shinsuke AICHI MEDICAL UNIVERSITY,SCHOOL OF MEDICINE,PROFESSOR, 医学部, 教授 (40144141)
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| Co-Investigator(Kenkyū-buntansha) |
SATOH Mamoru AICHI HUMAN SERVICE CENTER,INSTUITUTE FOR DEVELOPMENTAL RESEARCH,RESEARCHER, 形態学部, 研究員 (70291177)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | PDI / ERp61 / ERp72 / protein folding / immunoglobulins / molecular chaperones / deletion mutants / S-S結合 |
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| Research Abstract |
Folding and assembly of nascent polypeptides involves two kinds of proteins, molecular chaperones and the enzymes that catalyze the formation of covalent bonds. The representative of the latter is protein disulfide isomerase (PDI) that is a resident protein in the endoplasmic reticulum (ER) with thioredoxin-like domains containing active sites (CGHC) responsible for its redox activity. Similar structures are also present in ERp61 and ERp72, other ER resident proteins. These PDI family proteins are implicated in the disulfide bond formation of nascent polypeptides in the ER lumen.
In this study, the effect of PDI family proteins on reconformation of reduced and denatured immunoglobulins was examined, It was failed to detect the difference of their activity for in vitro reconformation of IgG.Unfortunately, it was very difficult to establish in vitro reconformation system for IgM and IgA due to the low solubility of denatured immunoglobulins. Western blotting of immuno-precipitates with an
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ti- alpha chain or anti- mu chain antibody from cell lysate crosslinked in situ with dithiosuccinimidyl propionate (DSP) elucidated the association of ERp61 with IgM and of ERp72 with IgA and IgM, but no association of PDI with both immunoglobulins was detected. These results indicate that different enzymes may catalyze the disulfide bond formation in different class of immunoglobulins.
In order to analyze the chaperone activity of ERp72, delution mutants of ERp72 were expressed as histidine taged proteins in E coli and purified, Analysis of their activity of binding to denatured insulin peptides and immunoglobulins is on going.
The expression of PDI family proteins in mouse F9 teratocarcinoma cells during differentiation induced with retinoic acid and dibutyryl cAMP.The differentiation clearly induced all these enzymes. In immunoprecipitation experiments combined with in situ chemical crosslinking, type IV collagen was significantly coprecipitated with PDI whereas laminin was equally coprecipitated with the three proteins. Furthermore, 210 kDa protein characteristically coprecipitated with ERp72. Less
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