genetic and biochemical study on intracellular lipid transport and lipid functions in microdomain

细胞内脂质转运和微区脂质功能的遗传和生化研究

• 批准号: 07457545
• 负责人: NISHIJIMA Masahiro
• 金额: $ 4.93万
• 依托单位: National Institute of Infectious Diseases
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457545/
• 关键词: CHO cell; phosphatidylserine; sphingolipid; LPS; cardiolipin; phospholipase D; phosphatidylcholine; LCB1; SPT(セリンパルミトイルトランスフェラーゼ); LCB2; ホスフォリパーゼD; RhoA; ホスファチジルグリセロールリン酸; ホスファチジルセリン; CD14; マクロファージ; NF-κB; ホスァチジルセリン; NK-κB

1.We isolated CHO cell mutants defectivein PSS II from PSA-3 cells, and found that the pssB gene product, PSS II,functions as the principal enzyme in the PS formation from PE in CHO-K1 cells.2.A partial human cDNA encoding a PSS I -related peptide has been found during a homology search of DNA datavases, and then the full-length cDNA of the CHO homolog has been isolated [15]. Introduction of this CHO cDNA,designated pssB,into CHO-K1 cells results in striking increases in both serine and ethanolamine base-exchange activities. In contrast to the pssA cDNA,the pssB cDNA is incapable of elevating the choline base-exchange activity. Expression of the pssB gene in insect cells also leads to elevation of serine and ethanolamine base-exhange activities. These findings indicate that the pssB gene encodes PSS II catalyzing the serine and ethanolamine base-exchange but not the choline base-exchange.3.We showed that the inhibition of PtdSer synthase I by PtdSer is essential for the maintenance of … More a normal PtdSer levelin CHO-K1 cells and that Arg-95 of the synthase is a crucial residue for tha inhibition.We found that both sphingolipids and cholesterol were involved in insolubility of PLAP,a GPI-anchored protein, and suggested that these lipids coordinately playd a role in formation of Triton X-100-resistant complexes.5.We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and an LCB2 cDNA homolog from CHO cells, and found that the CHO LCB1 homolog encodes a component of SPT.6.Phosphatidylglycerophosphate (PGP) synthase catalyzes the committed step in cardiolipin (CL) biosyntheses, and is thought to be a key enzyme for regulating cellularphosphatidylglycerol (PG) and CL content. We purified PGP synthase from Chines hamster ovary (CHO) cells. We also isolated a homologue of yeast PGS1, which encodes PGP synthase, from CHO cells. This cDNA,designated as CPGS1, encodes a protein which exhibits 28% amino acid identity with yeast PGS1, and its calculated molecular weight is 62,329 Da.7.We found that exposure of mouse macrophage-like J774.1 cells to lipopolysaccharide (LPS) induces rapid production of the cellular dialglycerol (DAG) from phosphatidylcholine (PC). We also found that LPS stimulus activated PLD toward PC,resulting in the formation of PA which was then conerted to DAG by PAP,and that the DAG production from PC by the PLD/PAP pathway was upstream of the NF-kB activation in response to LPS in J774.1 cells. Less

1.我们从PSA-3细胞中分离了PSS II缺陷的CHO细胞突变体，发现pssB基因产物PSS II在CHO-K1细胞中由PE形成PS中起主要酶的作用。2.在DNA数据库的同源性搜索过程中发现了编码PSS I相关肽的部分人cDNA，然后分离了CHO同源物的全长cDNA [15]。将该CHO cDNA（命名为pssB）导入CHO-K1细胞中，导致丝氨酸和乙醇胺碱交换活性显著增加。与pssA cDNA相反，pssB cDNA不能提高胆碱碱交换活性。pssB基因在昆虫细胞中的表达也导致丝氨酸和乙醇胺碱交换活性的升高。这些结果表明，pssB基因编码的PSS Ⅱ催化丝氨酸和乙醇胺的碱交换，但不催化胆碱的碱交换。3. PtdSer抑制PtdSer合成酶Ⅰ是维持细胞内的 ...更多信息 PLAP是一种GPI锚定蛋白，5.从小鼠和中国仓鼠卵巢（CHO）中分离到哺乳动物LCB 1的cDNA同源物，并与正常小鼠和正常小鼠的LCB 1 cDNA同源物进行了比较。6.磷脂酰甘油磷酸（phosphatidylglycerophosphate，PGP）合成酶催化心磷脂（cardiolipin，CL）生物合成的关键步骤，被认为是调节细胞内磷脂酰甘油（phosphatidylglycerol，PG）和CL含量的关键酶。我们从中国仓鼠卵巢（CHO）细胞中纯化了PGP合酶。我们还从CHO细胞中分离了一个编码PGP合酶的酵母PGS 1同源物。该cDNA命名为CPGS 1，编码的蛋白质与酵母PGS 1的氨基酸同源性为28%，分子量为62，329 Da。7.我们发现脂多糖（lipopolysaccharide，LPS）刺激小鼠巨噬细胞样J774.1细胞后，可诱导细胞内磷脂酰胆碱（phosphatidylcholine，PC）快速合成甘油二酯（dialglycerol，DAG）。LPS刺激可激活PLD向PC转化，形成PA，PA再经PAP转化为DAG，并且PLD/PAP途径从PC转化为DAG的过程位于LPS激活NF-κ B的上游。少

项目成果

O.Kuge, K.Hasegawa, K.Saito, and M.Nishijima: "Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells" Proc.Natl.Acad.Sci.USA. (in press). (1998)

O.Kuge、K.Hasekawa、K.Saito 和 M.Nishijima：“通过磷脂酰丝氨酸介导的中国仓鼠卵巢细胞中磷脂酰丝氨酸合酶 I 的抑制来控制磷脂酰丝氨酸生物合成”Proc.Natl.Acad.Sci.USA。

M.Nishijima: "Mammalian cell mutants of membrane phospho lipid biogenesis" Trends in Cell Biol.7. 324-329 (1997)

M.Nishijima：“膜磷脂生物发生的哺乳动物细胞突变体”细胞生物学趋势7。

O.Kuge and M.Nishijima: "Phosphatidylserine synthase I and II of mammalisan cells" BBA. 1348. 151-156 (1997)

O.Kuge 和 M.Nishijima：“哺乳动物细胞的磷脂酰丝氨酸合酶 I 和 II”BBA。

H.Yamamoto: "Involvement of diacylglycerol production in activation of nuclear factor-κB by a CD14-mediated lipopolysaccharide stimulus" Biochem.J.325. 223-228 (1997)

H. Yamamoto：“通过 CD14 介导的脂多糖刺激，二酰基甘油的产生参与核因子-κB 的激活”Biochem.J.325（1997）。

O.Kuge: "Control of phosphatidylserine biosynthesis through phosphatidylserine-mediated inhibition of phosphatidylserine synthase I in Chinese hamster ovary cells" Proc.Natl.Acad.Sci.USA. (in press). (1998)

O.Kuge：“通过磷脂酰丝氨酸介导的中国仓鼠卵巢细胞中磷脂酰丝氨酸合酶 I 的抑制来控制磷脂酰丝氨酸生物合成”Proc.Natl.Acad.Sci.USA。