Mechanism of glycoprotein sorting and transport in the cells and its application for drug design
| Project/Area Number |
16390019
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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 |
Biological pharmacy
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| Research Institution | The University of Tokyo |
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Principal Investigator |
YAMAMOTO Kazuo The University of Tokyo, Graduate School of Frontier Sciences, Professor, 大学院新領域創成科学研究科, 教授 (20174782)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUMOTO Naoki The University of Tokyo, Graduate School of Frontier Sciences, Associate Professor, 大学院新領域創成科学研究科, 助教授 (40239108)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2006: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2005: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2004: ¥6,200,000 (Direct Cost: ¥6,200,000)
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| Keywords | cargo receptor / lectin / VIP36 / ERGIC-53 / MCFD2 / pH依存性 |
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| Research Abstract |
FLAG-tagged ERGIC-53 was expressed with HA-tagged VIPL or VIP36 in 293T cells and pull down assay was performed from the cell lysates. HA-VIPL and HA-VIP36 were precipitated with ERGIC-53 indicating that these proteins interact with ERGIC-53. ERGIC-53 also interacted with MCFD2 in the presence of calcium, but neither VIP36 nor VIPL did. Surface plasmon resonance (SPR) experiment demonstrated that the interaction between ERGIC-53 and MCFD2 was susceptible to calcium ions, especially the concentration between 0.1 and 0.2 mM. MCFD2 mutants derived from F5F8D patients had three or four order of magnitude lower Ka values for ERGIC-53. Ultracentrifugation analysis and SPR experiment of soluble VIP36 indicating that sugar-binding activity of VIP36 was regulated by oligomer formation dependent on pH, which explains enhancement of avidity to sugar chains. Further FLET analysis using YGFP- and EGFP-fused VIP36 showed their interactions in the cells. In contrast, sugar-binding ability of ERGIC-53
… More
was enhanced by MCFD2 interactions. The interaction of ERGIC-53 with MCFD2 depends on calcium concentration. By pull-down assay with VIP36, novel protein constitutively interacted with VIP36 was identified as a chaperone BiP. The interaction had different characteristics from those of chaperone substrates. Immunoelectron microscopic analysis showed that the binding of VIP36 to BiP was occurred especially in the endoplasmic reticulum (ER). Interestingly, VIP36 precipitated with BiP had sugar chains resistant to endo H suggesting that VIP36 transported retrogradely could bind to BiP in the ER. The binding of PE-labeled soluble ERGIC-53, VIP36, and VIPL tetramers to HeLaS3 cells showed their preferable binding to high mannose-type sugar chains. Inhibition analysis by a panel of high mannose-type sugar derivatives demonstrated the precise specificities of them. VIP36 and VIPL recognize Man α 2Man α 2Man arm and glucosylation of its non-reducing terminal abrogated their binding. ERGIC-53 preferencially bound to M8b isoform, which is a product of ER α-mannosidase I, indicating that newly synthesized glycoproteins may transferred from calnexin to ERGIC-53 via VIPL. VIP36 fused with CD8 transmembrane domain followed by intracellular domain of CD3ζ was expressed in HeLaS3 cells. The cell surface display system enabled us to pick up clones of mutated VIP36 having distinct sugar-binding specificities from random mutated VIP36 libraries. Less
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Report
(4 results)
Research Products
(37 results)
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[Journal Article] MHC class I-like MILL mokecules are beta2-microglobulin-associated, GPI-anchored glycoproteins that do not require TAP for cell surface expression.2006
Author(s)
M.Kajikawa, B.Tomohisa, U.Tomaru, Y.Watanabe, S.Koganei, S.Kawahara, N.Matsumoto, K.Yamamoto, M.Miyazawa, k.Maenaka, A.Ishizu, M.Kasahara
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Journal Title
J.Immunol. 177
Pages: 3108-3115
Description
「研究成果報告書概要(欧文)」より
Related Report
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[Journal Article] Sugar-binding Properties of VIP36, an Intracellular Animal Lectin Operating as a Cargo Receptor.2005
Author(s)
Y.Kamiya, Y.Yamaguchi, N.Takahashi, Y.Arata, K.Kasai, Y.Ihara, I.Matsuo, Y.Ito, K.Yamamoto, Koichi Kato
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Journal Title
J.Biol.Chem. 280
Pages: 37178-37182
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Coordinated metabolism of Alcadein and amyloid protein precursor regulates FE65-dependent gene transactivation.2004
Author(s)
Y.Araki, N.Miyagi, N.Kato, T.Yoshida, S.Wada, M.Nishimura, H.Komano, T.Yamamoto, B.D.Strooper, K.Yamamoto, T.Suzuki
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Journal Title
J.Biol.Chem. 279
Pages: 24343-24354
Description
「研究成果報告書概要(欧文)」より
Related Report
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