Fernández-Borja M, Bellido D, Vilella E, Olivecrona G, Vilaró S
Department of Biochemistry and Physiology, University of Barcelona, Spain.
J Lipid Res. 1996 Mar;37(3):464-81.
Lipoprotein lipase (LPL), a key enzyme in lipoprotein triglyceride metabolism, produces a marked increase in the retention and uptake of all classes of lipoproteins by cultured cells. It was previously shown that two different receptors are involved in mediating the LPL effects: heparan sulfate proteoglycans (HSPG) and the low density lipoprotein (LDL) receptor-related protein/alpha 2 macroglobulin receptor (LRP). By immunofluorescence we show here that cell surface-bound LPL displays a pattern that corresponds to the previously described distribution of cell surface HSPG. No evident relation to the distribution of bound activated alpha 2-macroglobulin (alpha 2M*) or to LRP was observed. By immunoelectron microscopy we found that after 30 min at 37 degrees C most of the detected alpha 2M* (70% of the total gold particles) was inside the cells and associated with endosomal vesicles. However, at the same time, 76% of the LPL remained at the cell surface, suggesting that, LPL is internalized by a slow endocytic process. Binding of triglyceride-rich lipoproteins (TRL) or LDL together with LPL led to a spectacular increase in bound lipoproteins, which completely colocalized with LPL. After incubation at 37 degrees C, LPL and 1,1'-dioctadecyl-3,3,3,'3'-tetramethylindocarbocyanine (DiI)-TRL formed large clusters on the cell surface. Immunofluorescene and quantitative immunoelectron microscopy provided evidence of co-internalization of LPL and apoE-containing TRL by a slow endocytic process. In the absence of LPL, the fibroblasts rapidly internalized DiI-LDL and showed fluorescence in central, lysosome-like vesicles. In contrast, when LPL was present, internalization of DiI-LDL involved small, widely distributed vesicles. This pattern slowly changed to one consisting of large perinuclear vesicles. LDL receptor-deficient fibroblasts internalized DiI-LDL, either with or without LPL, into small widely distributed vesicles and no central vesicles were seen. Chloroquine-treated normal fibroblasts internalized DiI-LDL in a pattern similar to that of receptor-deficient fibroblasts. Taken together our results suggest an alternative receptor-independent endocytosis pathway for LDL. This pathway is potentiated by LPL and is characterized by a slow uptake involving small vesicles that gradually reach lysosomes. We suggest that, through its interaction with HSPG, LPL provides high capacity binding sites for lipoproteins and a independent internalization pathway.
脂蛋白脂肪酶（LPL）是脂蛋白甘油三酯代谢中的关键酶，可使培养细胞对所有类型脂蛋白的保留和摄取显著增加。先前的研究表明，有两种不同的受体参与介导LPL的作用：硫酸乙酰肝素蛋白聚糖（HSPG）和低密度脂蛋白（LDL）受体相关蛋白/α2巨球蛋白受体（LRP）。通过免疫荧光，我们在此表明，细胞表面结合的LPL呈现出一种与先前描述的细胞表面HSPG分布相对应的模式。未观察到与结合的活化α2巨球蛋白（α2M*）分布或与LRP有明显关系。通过免疫电子显微镜，我们发现，在37℃下孵育30分钟后，大部分检测到的α2M*（占总金颗粒的70%）位于细胞内并与内体囊泡相关。然而，与此同时，76%的LPL仍留在细胞表面，这表明LPL是通过缓慢的内吞过程内化的。富含甘油三酯的脂蛋白（TRL）或LDL与LPL一起结合导致结合的脂蛋白显著增加，其与LPL完全共定位。在37℃孵育后，LPL和1,1'-二辛基-3,3,3,'3'-四甲基吲哚羰花青（DiI）-TRL在细胞表面形成大簇。免疫荧光和定量免疫电子显微镜提供了LPL和含载脂蛋白E的TRL通过缓慢内吞过程共同内化的证据。在没有LPL的情况下，成纤维细胞迅速内化DiI-LDL，并在中央的溶酶体样囊泡中显示荧光。相反，当存在LPL时，DiI-LDL的内化涉及小的、广泛分布的囊泡。这种模式缓慢转变为由大的核周囊泡组成的模式。缺乏LDL受体的成纤维细胞将DiI-LDL内化，无论有无LPL，均进入小的、广泛分布的囊泡，未见中央囊泡。用氯喹处理的正常成纤维细胞以与受体缺陷型成纤维细胞相似的模式内化DiI-LDL。综合我们的结果表明，LDL存在一种不依赖受体的内吞途径。该途径由LPL增强，其特征是通过逐渐到达溶酶体的小囊泡进行缓慢摄取。我们认为，通过与HSPG相互作用，LPL为脂蛋白提供了高容量结合位点和一条独立的内化途径。
