GPIアンカー合成の調節機構と合成不全症

• Project/Area Number: 14082205
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Osaka University
• Principal Investigator: 木下 タロウ Osaka University, 微生物病研究所, 教授 (10153165)
• Co-Investigator(Kenkyū-buntansha): 前田 裕輔 (前田 祐輔) 大阪大学, 微生物病研究所, 助教授 (00294124) ; 村上 良子 大阪大学, 微生物病研究所, 助手 (00304048) ; 芦田 久 京都大学, 大学院・生命科学研究科, 助手 (40379087) ; 森田 康裕 大阪大学, 微生物病研究所, 特任助手 (70397769)
• Project Period (FY): 2002 – 2006
• Project Status: Completed (Fiscal Year 2006)
• Budget Amount: ¥32,400,000 (Direct Cost: ¥32,400,000); Fiscal Year 2006: ¥4,500,000 (Direct Cost: ¥4,500,000); Fiscal Year 2005: ¥4,500,000 (Direct Cost: ¥4,500,000); Fiscal Year 2004: ¥7,200,000 (Direct Cost: ¥7,200,000); Fiscal Year 2003: ¥7,200,000 (Direct Cost: ¥7,200,000); Fiscal Year 2002: ¥9,000,000 (Direct Cost: ¥9,000,000)
• Keywords: 遺伝子 / 酵素 / 細胞・組織 / 脂質 / 糖鎖 / GPIアンカー / 小胞体 / 糖転移酵素 / 生合成 / N結合型糖質

Research Abstract

GPIアンカー生合成経路の酵素群の研究に関し、第1ステップに働くNアセチルグルコサミン転移酵素の新規構成成分PIG-Yをクローニングし、この酵素が7つのサブユニットからなる極めてユニークな単糖転移酵素であることを示した。
GPIアンカー欠損症で、後天性遺伝子病である発作性夜間血色素尿症の発症メカニズムに関し、GPIアンカー欠損クローンの拡大が良性腫瘍性に起こることを示唆する第2の遺伝子変異を証明した。
平成15年度の成果として報告したPGAP1は、タンパク質に結合直後のGPIアンカーのイノシトールから脂肪酸を除去する脱アシル化酵素である。PGAP1が機能しないとGPIアンカーは3つの脂肪鎖を持ったまま細胞表面に発現してしまう。このとき生体に何が起こるかを知るため、PGAP1ノックアウトマウスを作製した。ノックアウト個体の多くは周産期致死を示し、生き残った個体も成長が遅延した。雄は不妊であったので、不妊の機序を解析したところ、精子が卵子の透明体に付着できないことがわかった。ノックアウト個体の精子上のGPIアンカー型タンパク質のレベルを調べたところ、いくつかの蛋白質の量が正常より多かった。GPIアンカーの切断が、精子の妊性獲得に重要であるとの報告もあり、イノシトールからの脂肪酸除去不全がGPIアンカー型タンパク質の精子からの切断除去を阻害している可能性が考えられた。

Research Products

• [Journal Article] PGAP1 knockout mice show otocephaly and male infertility. (2007)
  • Author(s): Ueda, Y.
  • Journal Title: J. Biol. Chem. 282 Pages: 30373-30380
• [Journal Article] DPM1, the catalytic subunit of dolichol-)hosphate-mannose synthase, is tethered to and stabilized on. the endoplasmic reticulum membrane by DPM3. (2006)
  • Author(s): Ashida, H
  • Journal Title: J. Biol. Chem 281 Pages: 896-904
• [Journal Article] PGAP2 is essential for correct processing and stable expression of GPI-anchored proteins. (2006)
  • Author(s): Tashima, Y
  • Journal Title: Mol. Biol. Cell 17 Pages: 1410-1420
• [Journal Article] Molecular basis of clonal expansion of hematopoiesis in two patients with paroxysmal nocturnal hemoglobinuria (PNH). (2006)
  • Author(s): Inoue, N.
  • Journal Title: Blood 108 Pages: 4232-4236
• [Journal Article] DPM1, the catalytic subunit of dolichol-phosphate-mannose synthase, is tethered to and stabilized on the endoplasmic reticulum membrane by DPM3. (2006)
  • Author(s): Ashida, H.
  • Journal Title: J. Biol. Chem. 281 Pages: 896-904
• [Journal Article] A cohort study of nature of paroxysmal nocturnal hemoglobinuria (PNH) clones and PIG-A mutations in patients with aplastic anemia. (2006)
  • Author(s): Wanachiwanawin, W
  • Journal Title: Eur. J. Haematol. 76 Pages: 502-509
• [Journal Article] CHO glycosylation mutants : GPI anchor. Methods Enzymol. (2006)
  • Author(s): Maeda, Y.
  • Journal Title: Methods Enzymol. 416 Pages: 182-205
• [Journal Article] The Initial Enzyme for Glycosylphosphatidylinositol Biosynthesis Requires PIG-Y, a Seventh Component. (2006)
  • Author(s): Murakami, Y.
  • Journal Title: Mot.Biol.Cell 16 Pages: 5236-5246
• [Journal Article] DPM1, the catalytic subunit of dolichol-phosphate-mannose synthase, is tethered to and stabilized on the endoplasmic reticulum membrane by DPM3. (2006)
  • Author(s): Ashida, H.
  • Journal Title: J Biol.them 281 Pages: 896-904
• [Journal Article] PGAP2 is essential for correct processing and stable expression of GPI-anchored proteins. (2006)
  • Author(s): Tashima, Y.
  • Journal Title: Mot.Biol.Cell 17 Pages: 1410-1420
• [Journal Article] Mammalian PIG-X and yeast Pbnlp are the essential components ofglycosylphosphatidylinositol-mannosyltransferase I. (2005)
  • Author(s): Ashida, H.
  • Journal Title: Mol. Biol. Cell 16 Pages: 1439-1448
• [Journal Article] PIG-V involved in transferring the second mannose in glycosylphosphatidylinositol. (2005)
  • Author(s): Kang, J. Y.
  • Journal Title: J. Biol. Chem. 280 Pages: 9489-9497
• [Journal Article] GPI7 is the second partner of PIG-F and involved in modification of glycosylphosphatidylinositol. (2005)
  • Author(s): Shishioh, N.
  • Journal Title: J. Biol. Chem 280 Pages: 9728-9734
• [Journal Article] The initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-Y, a seventh component. (2005)
  • Author(s): Murakami, Y
  • Journal Title: Mol. Biol. Cell. 16 Pages: 5236-5246
• [Journal Article] PIG-X and yeast Pbn1p are the essential components of glycosylph osphatidylinositol-mannosyltransferase I. (2005)
  • Author(s): Ashida, H.
  • Journal Title: Mol.Biol.Cell (in press.)
• [Journal Article] GPI7 is the second partner of PIG-F and is involved in modification of glycosylphosphatidylinositol (2005)
  • Author(s): Shishioh, N.
  • Journal Title: J.Biol.Chem (in press)
• [Journal Article] GPI7PIG-V Involved in Transferring the Second Mannose in Glycosylphosphatidylinositol (2005)
  • Author(s): Kang, J.Y.
  • Journal Title: J.Biol.Chem (in press)
• [Journal Article] Surface sialic acids taken from the host allow trypanosome survivalin tsetse fly vectors. (2004)
  • Author(s): Nagamune, K.
  • Journal Title: J. Exp. Med. 199 Pages: 1445-1450
• [Journal Article] Genome-wide phenotype analysis in ES cells by regulated disruptionof the Bloom's syndrome gene. (2004)
  • Author(s): Yusa, K.
  • Journal Title: Nature 429 Pages: 896-899
• [Journal Article] Inositol-deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p. (2004)
  • Author(s): Tanaka, S
  • Journal Title: J.Biol.Chem 279 Pages: 14256-14263
• [Journal Article] Surface sialic acids taken from the host allow trypanosome survival in tsetse fly vectors. (2004)
  • Author(s): Nagamune, K.
  • Journal Title: J.Exp.Med. 199 Pages: 1445-1450
• [Journal Article] Genome-wide phenotype analysis in ES cells by regulated disruption of the Bloom's syndrome gene. (2004)
  • Author(s): Yusa, K.
  • Journal Title: Nature 429 Pages: 896-899
• [Journal Article] GPI transamidase of Trypanosoma brucei has two novel (TTAl andTTA2)and three common subunits. (2003)
  • Author(s): Nagamune, K.
  • Journal Title: Proc. Natl. Acad. Sci. USA 100 Pages: 10682-10687
• [Publications] Hong, Y.: "Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins."Mol.Biol.Cell. 14. 1780-1789 (2003)
• [Publications] Ohishi, K.: "Two subunits of glycosylphosphatidylinositol transamidase GPI8 and PIG-T form a functionally important intermolecular disulfide bridge."J.Biol.Chem. 278. 13959-13967 (2003)
• [Publications] Inoue, N.: "Molecular genetics of paroxysmal nocturnal hemoglobinuria."Int.J.Hematol.. 77. 107-112 (2003)
• [Publications] Murakami, Y.: "PIG-W is critical for inositol acylation but not for flipping of glycosylphosphatidylinositol-anchor."Mol.Biol.Cell. 14. 4285-4295 (2003)
• [Publications] Nagamune, K.: "GPI transamidase of Trypanosoma brucei has two novel (TTA1 and TTA2) and three common subunits."Proc.Natl.Acad.Sci.USA. 100. 10682-10687 (2003)
• [Publications] Tanaka, S.: "Inositol-deacylation of glycosylphosphatidylinositol-anchored proteins is mediated by mammalian PGAP1 and yeast Bst1p."J.Biol.Chem. (in press). (2004)
• [Publications] Vainauskas, S.: "Structural requirements for the recruitment of Gaa1 into a functional glycosylphosphatidylinositol transamidase complex"J.Biol.Chem.. 277. 30535-30542 (2002)
• [Publications] Hong, Y: "Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum a-toxin"EMBO J.. 21. 5047-5056 (2002)
• [Publications] Hong, Y: "Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins"Mol.Biol.Cell. (in press). (2003)
• [Publications] Ohishi, K.: "Two subunits of glycosylphosphatidylinositol transamidase GPI8 and PIG-T form a functionally important intermolecular disulfide bridge"J.Biol.Chem. (in press). (2003)