Molecular Biological Study on Regulation and Expression of Physiological Function of Human Lipoprotein Lipase

人脂蛋白脂肪酶生理功能调控与表达的分子生物学研究

• 批准号: 01580206
• 负责人: IKEDA Yasuyuki
• 金额: $ 1.47万
• 依托单位: National Cardiovascular Center Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01580206/
• 关键词: Lipoprotein Lipase; Enzyme Immunoassay; Macrophage; cDNA Cloning; Hyperlipidemia; Endothelial Cell; リポ蛋白リパ-ゼ(LPL); 免疫沈降

Lipoprotein lipase (LPL) is a key enzyme to catalyze triglyceride-rich lipoprotein at the first step of their metabolism in circulation. LPL enzyme takes on a functionally active form at capillary endothelium through three processes such as (1) expression of LPL gene, and systhesis and secretion of LPL (2) transport of LPL into capillary endothilium (3) binding of LPL on the surface of capillary endothelium. The study on these processes has been delayed by a lack of monospecific antibody directed to human LPL, LPL gene from a patient with LPL deficiency, and binding of LPL into bovine endothelim.(1) Biosynthesis and secretion of LPL : Macrophage-like cells (Mphi) derived from THP-1 cells were cultured with "protein labeling medium (PLM)" containing ^<35>S-methionine. The labeled LPL protein was immunoprecipitated from medium and Mphi cells by anti-human PHP-LPL antibody. The percipitated LPL protein was analyzed by 9% SDS-Page. LPL was newly synthesized in a 55 KDa protein, processed to be 60 KDa protein by N-glycosylation, secreted in a form of 61 KDa protein. The secreted LPL protein could bind on the surface of bovine endothelium.(2) Study on LPL molecule in a patient with LPL deficiency : A patient TN was judged to be LPL deficiency by a lack of LPL activity and mass in postheparin plasma. We examined LPL molecule which was newly synthesized in monocyte-derived macrophages from patient TN, and also analyzed LPL mRNA by Norther blot with a probe of THP-1 LPL cDNA. LPL protein and LPL mRNA were not detected from patient TN. Analyzing LPL gene of patient TN, we found one base deletion in LPL coding region following by a premature terminal codon by frame shift. This mutation leads no detectable LPL protein due to the absence of LPL mRNA transcript.(3) Binding of LPL in endothelium : we studied the binding of human LPL molecule on bovine endothelium. Maximum binding of LPL was estimated to be 3.4 mu mol FFA/h/cm^2 of endothelium.

脂蛋白脂酶（LPL）是脂蛋白代谢的第一步催化酶。LPL酶通过三个过程在毛细血管内皮上呈现功能活性形式：（1）LPL基因的表达，LPL的合成和分泌;（2）LPL向毛细血管内皮的转运;由于缺乏针对人LPL的单特异性抗体、LPL缺乏症患者的LPL基因以及LPL与牛内皮素的结合，对这些过程的研究一直受到阻碍。(1)LPL的生物合成和分泌：将来源于THP-1细胞的巨噬细胞样细胞（Mphi）与含有13S-甲硫氨酸的"蛋白质标记培养基（PLM）"一起培养。<35>用抗人PHP-LPL抗体从培养液和Mphi细胞中免疫沉淀标记的LPL蛋白。通过9%SDS-PAGE分析沉淀的LPL蛋白。新合成的LPL分子量为55 KDa，经N-糖基化后为60 KDa，以61 KDa的形式分泌。分泌的LPL蛋白可与牛内皮细胞表面结合。(2)1例LPL缺乏症患者LPL分子的研究：1例TN患者经肝素治疗后血浆LPL活性和质量均下降，判断为LPL缺乏症。我们检测了TN患者单核细胞源性巨噬细胞中新合成的LPL分子，并以THP-1 LPL cDNA为探针，用Norther印迹法分析了LPL mRNA。在TN患者中未检测到LPL蛋白和LPL mRNA。对TN患者LPL基因进行分析，发现LPL编码区有一个碱基缺失，并伴有一个提前的末端密码子。由于LPL mRNA转录物的缺失，该突变导致检测不到LPL蛋白。(3)LPL在内皮细胞中的结合：我们研究了人LPL分子与牛内皮细胞的结合。LPL的最大结合估计为3.4 μ mol FFA/h/cm ^2内皮细胞。

项目成果

Ikeda, Y., Takagi, A., and Yamamoto , A.: "Purification and characterization of lipoprotein lipase and hepatic triglyceride lipase from human postheparin plasma : production of monospecific antibody to the individual lipase" Biochim Biophys Acta. 1003. 25

Ikeda, Y.、Takagi, A. 和 Yamamoto, A.：“来自人肝素后血浆的脂蛋白脂肪酶和肝甘油三酯脂肪酶的纯化和表征：针对个体脂肪酶的单特异性抗体的生产”Biochim Biophys Acta。

Ikeda,Y.,Takagi,A.,Ohkaru,Y.,Nogi,K.,Iwanaga,T.,Kurooka,S.Yamamoto,A.: "A sandwichーenzyme immunoassay for the quantification of lipoーprotein lipase and hepatic triglyceride lipase in human postheparin plasma sing monoclonal antibodies to the corresponding

Ikeda, Y.、Takagi, A.、Ohkaru, Y.、Nogi, K.、Iwanaga, T.、Kurooka, S. Yamamoto, A.：“用于定量脂蛋白脂肪酶和肝细胞的夹心酶免疫测定法人肝素后血浆中的甘油三酯脂肪酶与相应的单克隆抗体

Takagi,A.,Ikeda,Y.,Yamamoto,A.: "DNA sequence of lipoprotein lipase cDNA cloned from human moncytic leukemia THPー1 cells." Nucleic Acids Research. 18. 6436 (1990)

Takagi, A.、Ikeda, Y.、Yamamoto, A.：“从人单核细胞白血病 THP-1 细胞中克隆的脂蛋白脂肪酶 cDNA 的 DNA 序列。” 18. 6436 (1990)。

高木敦子、池田康行、山村卓、山本章: "原発性I型高リポ蛋白血症患者の病因解析のシステム化" 脂質生化学研究. 31. 125-128 (1989)

Atsuko Takagi、Yasuyuki Ikeda、Takashi Yamamura、Akira Yamamoto：“原发性 I 型高脂蛋白血症患者发病机制的系统分析”脂质生物化学研究 31. 125-128 (1989)。

Takagi,A.Ikeda,Y.Yamamoto,A.: "DNA sequence of lipoprotein lipase cDNA cloned from human moncytic leukemia THPー1 cells." Nucleic Acids Research. 18. 6436 (1990)

Takagi, A. Ikeda, Y. Yamamoto, A.：“从人单核细胞白血病 THP-1 细胞中克隆的脂蛋白脂肪酶 cDNA 的 DNA 序列。核酸研究”18. 6436 (1990)。