| Project/Area Number |
13470485
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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) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥14,400,000 (Direct Cost: ¥14,400,000)
Fiscal Year 2003: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥4,900,000 (Direct Cost: ¥4,900,000)
Fiscal Year 2001: ¥6,000,000 (Direct Cost: ¥6,000,000)
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| Keywords | cargo receptor / ERGIC-53 / VIP36 / lectin / sugar specificity |
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| Research Abstract |
In secretory pathway of eucaryotic cells, newly synthesized glycoproteins are transported from the ER through the Golgi toward cell surface via transport vesicles. During this process, sorting events are performed by cargo receptors. ERGIC-53 and VIP36 having carbohydrate-binding domain homologous to leguminous lectins at lumenal region are receptors to direct specific proteins into vesicles. cDNA coding lectin domains of both receptors were constructed and expressed in E.coli. Interactions of these recombinant soluble ERGIC-53 and VIP36 with glycoproteins were analyzed using surface plasmon resonance with or without calcium ions under several pH conditions. Interaction of ERGIC-53 and IgY was not so different under the pH ranging from 6.5 to 7.2. By contrast, soluble VIP36 bound to porcine thyroglobulin stronger under the acidic condition than neutral pH. Oligomerization of soluble VIP36 occurred in relation to the enhanced association with the ligands under acidic condition. Then random oligonucleotides were introduced into cDNAs in place of the nucleotides encoding carbohydrate-binding loops of VP36 and ERGIC-53, respectively. The mutated cDNAs were expressed in CHO cells and the cells having different sugar moieties on the surfaces were enriched by flow cytometry. After cloning of the enriched cells, which have specifically changed to bind to PNA after the trasfection of mutated VIP36 cDNAs, 13 kinds of stable clones were established. These clones were classified into two groups based on the mutated cDNAs expressed in the cells and the carbohydrate-binding loops of these mutated VIP36 cDNAs were deduced to be DPDSNGGSF and DSSFNYVHA, respectively.
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