The mutation Gly(142)->Glu in human lipoprotein lipase produces a missorted protein that is diverted to lysosomes
The mutation Gly(142)->Glu in human lipoprotein lipase produces a missorted protein that is diverted to lysosomes
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DOI：
10.1074/jbc.271.4.2139
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发表时间：
1996-01-26
影响因子：
4.8
通讯作者：
Vilaro, S
中科院分区：
文献类型：
--
作者：
Busca, R;Martinez, M;Vilaro, S
虽然脂蛋白脂酶（LPL）激活的分子表征正在取得进展，但LPL的细胞内加工、转运和分泌信号仍然知之甚少，本文的目的是研究甘氨酸142参与LPL分泌，并阐明保留在细胞内的改变的蛋白质的细胞内目的地。我们通过定点突变使人LPL cDNA突变，以产生G142 E hLPL，其中甘氨酸142被谷氨酸取代。通过瞬时转染COS 1细胞表达野生型人LPL（WT hLPL）和突变型G142 E hLPL。用Western印迹分析我们鉴定了两种蛋白具有相同分子量的单一条带，然而，培养基的Western印迹未显示突变蛋白的任何特异性条带，ELISA实验显示突变体LPL的胞外质量仅为WT蛋白的25%，表明改变的酶的分泌缺陷。肝素在WT hLPL的情况下增加LPL分泌，但当作用于G142 E hLPL转染的细胞时没有任何刺激作用。然而，肝素-琼脂糖凝胶层析显示，两种蛋白质呈现相同的肝素亲和力。代谢标记和放射免疫沉淀研究显示，WT和突变体hLPL细胞内水平随追踪时间降低。此外，亮抑酶肽对突变酶的细胞内水平具有更大的影响，从而表明其更高的细胞内降解。免疫荧光研究表明，使用共聚焦显微镜LPL标记和Lamp 1溶酶体标记在G142 E hLPL表达细胞的高度共定位。这一结果得到了免疫电子显微镜的证实，另外还显示了高尔基体中的金标记。这一发现，连同实验进行的免疫沉淀的放射性标记的LPL的糖苷内切酶H消化，表明突变酶进入高尔基室。本文报道的结果表明，G142 E hLPL不能有效地分泌到细胞外培养基中，但它被错误地分选到溶酶体中进行细胞内降解。这一发现表明，溶酶体错误分选可能是分泌型LPL的细胞质量控制机制。
While the molecular characterization of lipoprotein lipase (LPL) activation is progressing, the intracellular processing, transport, and secretion signals of LPL are still poorly known, The aim of this paper is to study the involvement of glycine 142 in LPL secretion and to elucidate the intracellular destination of the altered protein that remains inside the cell. We mutated the human LPL cDNA by site-directed mutagenesis in order to produce the G142E hLPL in which the glycine 142 was replaced by a glutamic acid. The wild type human LPL (WT hLPL) and the mutant G142E hLPL were expressed by transient transfection in COS1 cells, Using Western blot assays we identified a single band that had the same molecular weight for both proteins, However, Western blots of culture media did not reveal any specific band for the mutant protein, and ELISA experiments showed that the extracellular mass of the mutant LPL was only 25% of the WT protein, indicating defective secretion of the altered enzyme. Heparin increased LPL secretion in the case of the WT hLPL but did not have any stimulatory effect when acting on G142E hLPL-transfected cells. However, heparin-Sepharose chromatography revealed that both proteins presented the same heparin affinity, Metabolic labeling and radioimmunoprecipitation studies showed that both the WT and the mutant hLPL intracellular levels decreased upon chase time. Furthermore, leupeptin had a greater effect on the intracellular level of the mutant enzyme, thus indicating its higher intracellular degradation. Immunofluorescent studies using confocal microscopy indicated high colocalization of the LPL labeling and the Lamp1 lysosomal labeling in G142E hLPL-expressing cells. This result was confirmed using immunoelectron microscopy, which in addition showed gold labeling in Golgi stacks. This finding, together with experiments performed with endoglycosidase H digestion of immunoprecipitated radiolabeled LPL, indicated that the mutant enzyme entered the Golgi compartment. The results reported in this paper show that the G142E hLPL is not efficiently secreted to the extracellular medium, but it is missorted to lysosomes for intracellular degradation. This finding suggests that lysosomal missorting might be a mechanism of cell quality control of secreted LPL.