Wilsie Larissa C, Gonzales Amanda M, Orlando Robert A
Department of Biochemistry and Molecular Biology, University of New Mexico, School of Medicine, MSC08 4670 1 University of New Mexico, Albuquerque, New Mexico, 87131, USA.
Lipids Health Dis. 2006 Aug 31;5:23. doi: 10.1186/1476-511X-5-23.
Triacylglyerol-rich very low density lipoprotein (VLDL) particles are the primary carriers of fatty acids in the circulation and as such serve as a rich energy source for peripheral tissues. Receptor-mediated uptake of these particles is dependent upon prior association with apolipoprotein E (apoE-VLDL) and is brought about by cell surface heparan sulfate proteoglycans (HSPG) in some cell types and by the low density lipoprotein receptor-related protein (LRP) in others. Although LRP's role in apoE-VLDL uptake has been well studied, the identity of the HSPG family member that mediates apoE-VLDL uptake has not been established. We investigated if syndecan-1 (Syn-1), a transmembrane cell surface HSPG, is able to mediate the internalization of apoE-VLDL and examined the relationship between Syn-1 and LRP toward apoE-VLDL uptake. For this study, we used a human fibroblast cell line (GM00701) that expresses large amounts of LRP, but possesses no LDL receptor activity to eliminate its contributions toward apoE-VLDL uptake.
Although LRP in these cells is fully active as established by substantial alpha2macroglobulin binding and internalization, uptake of apoE-VLDL is absent. Expression of human Syn-1 cDNA restored apoE-VLDL binding and uptake by these cells. Competition for this uptake with an LRP ligand-binding antagonist had little or no effect, whereas co-incubation with heparin abolished apoE-VLDL internalization. Depleting Syn-1 expressing cells of K+, to block clathrin-mediated endocytosis, showed no inhibition of Syn-1 internalization of apoE-VLDL. By contrast, treatment of cells with nystatin to inhibit lipid raft function, prevented the uptake of apoE-VLDL by Syn-1.
These data demonstrate that Syn-1 is able to mediate apoE-VLDL uptake in human fibroblasts with little or no contribution from LRP and that the endocytic path taken by Syn-1 is clathrin-independent and relies upon lipid raft function. These data are consistent with previous studies demonstrating Syn-1 association with lipid raft domains.
富含三酰甘油的极低密度脂蛋白（VLDL）颗粒是循环中脂肪酸的主要载体，因此是外周组织丰富的能量来源。这些颗粒的受体介导摄取取决于先前与载脂蛋白E（apoE-VLDL）的结合，并且在某些细胞类型中由细胞表面硫酸乙酰肝素蛋白聚糖（HSPG）介导，在其他细胞类型中由低密度脂蛋白受体相关蛋白（LRP）介导。尽管LRP在apoE-VLDL摄取中的作用已得到充分研究，但介导apoE-VLDL摄取的HSPG家族成员的身份尚未确定。我们研究了跨膜细胞表面HSPG syndecan-1（Syn-1）是否能够介导apoE-VLDL的内化，并研究了Syn-1与LRP在apoE-VLDL摄取方面的关系。在本研究中，我们使用了一种人类成纤维细胞系（GM00701），该细胞系表达大量LRP，但不具有低密度脂蛋白受体活性，以消除其对apoE-VLDL摄取的影响。
尽管通过大量α2巨球蛋白结合和内化证实这些细胞中的LRP完全有活性，但apoE-VLDL的摄取却不存在。人Syn-1 cDNA的表达恢复了这些细胞对apoE-VLDL的结合和摄取。用LRP配体结合拮抗剂竞争这种摄取几乎没有影响，而与肝素共同孵育则消除了apoE-VLDL的内化。耗尽表达Syn-1的细胞中的K +以阻断网格蛋白介导的内吞作用，未显示对Syn-1介导的apoE-VLDL内化有抑制作用。相比之下，用制霉菌素处理细胞以抑制脂筏功能，可阻止Syn-1对apoE-VLDL的摄取。
这些数据表明，Syn-1能够介导人类成纤维细胞中apoE-VLDL的摄取，LRP几乎没有贡献，并且Syn-1所采用的内吞途径不依赖于网格蛋白，而是依赖于脂筏功能。这些数据与先前证明Syn-1与脂筏结构域相关的研究一致。
