| Project/Area Number |
11480166
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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 |
Structural biochemistry
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| Research Institution | OSAKA UNIVERSITY |
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Principal Investigator |
KINOSHITA Taroh Research Institute for Microbial Diseases, Osaka Univ. Professor, 微生物病研究所, 教授 (10153165)
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| Co-Investigator(Kenkyū-buntansha) |
MURAKAMI Yoshiko Research Institute for Microbial Diseases, Osaka Univ. Research Associate, 微生物病研究所, 教務職員 (00304048)
NAGAMUNE Kisaburo Research Institute for Microbial Diseases, Osaka Univ. Research Associate, 微生物病研究所, 助手 (90314418)
OHISHI Kazuhito Research Institute for Microbial Diseases, Osaka Univ. Research Associate, 微生物病研究所, 助手 (60273702)
INOUE Norimitsu Research Institute for Microbial Diseases, Osaka Univ. Research Associate, 微生物病研究所, 助教授 (80252708)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥12,700,000 (Direct Cost: ¥12,700,000)
Fiscal Year 2000: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1999: ¥8,100,000 (Direct Cost: ¥8,100,000)
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| Keywords | GPI anchor / post translational modification / endoplasmic reticulum / GIPアンカー / 生合成経路 / 糖転移酵素 |
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| Research Abstract |
We aimed to clarify molecular mechanisms of GPI anchor biosynthesis and obtained following results.
1. We purified GPI-GlcNAc transferase that mediates the first step in GPI biosynthesis pathway. The enzyme consisted of six proteins : four previously reported and two new components. One of the new components was PIG-P that is essential for the enzyme because mutant mouse cell line defective in PIG-P is completely deficient in GPI biosynthesis. The other new component was DPM2 that is known as a regulatory component of dolichol-phosphate-mannose synthase. GPI-GlcNAc transferase activity of DPM2-deficient cells was one-third that of wild-type cells, indicating that GPI-GlcNAc transferase is also regulated by DPM2.
2. We found that human dolichol-phosphate-mannose synthase consists of DPM1, DPM2 and DPM3, that DPM2 stabilizes DPM3 and that DPM3, in turn, stabilizes catalytic DPM1.
3. We cloned PIG-M cDNA and demonstrated that PIG-M encodes a mannosyltransferase that transfers the first mannose to GPI.PIG-M had a functionally important DXD motif within a lumenal domain, suggesting that the first mannose is transferred on the lumenal side of the endoplasmic reticulum.
4. We demonstrated that PIG-O is involved in transfer of phosphoethanolamine to the third mannose. PIG-O that is most likely a catalytic component is associated with PIG-F that is also involved in the same step. Stable exprrssion of PIG-O was dependent upon PIG-F.
5. We characterized mammalian genes for GAA1, a component of GPI transamidase. Human and mouse GAA1 genes had 12 exons and spanned 4 kb. Human and mouse GAA1 genes are present in 8q24.3 and 15E, respectively. Intron 8's of those GAA1 genes were of minor class. GPI8, the other component of GPI transamidase, had a functionally essential cysteine that is conserved among GPI8 from other organisms and members of a cystein protease family, suggesting that this cystein is active site of GPI transamidase.
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