A PROTEIN PARTIALLY EXPRESSED ON THE SURFACE OF HEPG2 CELLS THAT BINDS LIPOPROTEINS SPECIFICALLY IS NUCLEOLIN
A PROTEIN PARTIALLY EXPRESSED ON THE SURFACE OF HEPG2 CELLS THAT BINDS LIPOPROTEINS SPECIFICALLY IS NUCLEOLIN
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DOI：
10.1021/bi00493a028
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发表时间：
1990-10-16
期刊：
影响因子：
2.9
通讯作者：
YANG, JW
中科院分区：
文献类型：
--
作者：
SEMENKOVICH, CF;OSTLUND, RE;YANG, JW
核仁蛋白是快速生长的真核细胞的主要核仁蛋白，被认为主要（如果不是完全）位于核仁中。然而，最近的数据[Borer, R. A., Lehner, C. F., Eppenberger, H. M., and Nigg, N. A. (1989) Cell 56, 379-390]表明蛋白质在细胞核和细胞质之间不断穿梭。对 HepG2 细胞的全细胞提取物进行配体印迹研究，除了 LDL 受体外，还鉴定出 Mr 109,000 的另一种 LDL 结合蛋白。 109 kDa 蛋白通过 HPLC 部分纯化，与 LDL 受体一样，结合含有 apoB 和 apoE 的脂蛋白，但不结合 HDL。然而，与 LDL 受体不同，109-kDa 蛋白在 EDTA 和还原剂存在下结合脂蛋白，对脂蛋白的亲和力比 LDL 受体低，并且不与针对 LDL 受体产生的两种抗体发生反应。确定了源自部分纯化的 109-kDa 物种的三种独立肽的蛋白质序列，除了与已发表的核仁素序列的三个独立区域的一个残基外，它们是相同的。在免疫印迹分析中，109-kDa 蛋白与核仁素特异性抗体反应，纯化的核仁素与抗 109-kDa 抗体和 LDL 均发生反应。当完整的 HepG2 细胞在收获前用链霉蛋白酶处理时，100-kDa 蛋白质减少了 46%，而肌动蛋白（一种细胞内蛋白质）的回收不受影响。当完整的 HepG2 细胞表面被碘化并对蛋白质进行 HPLC 分级时，发现 109-kDa 蛋白质被碘化。印迹上，125I-LDL 与 109-kDa 蛋白的结合被抗 109-kDa 抗体和核仁素特异性抗体抑制。此外，抗 109-kDa 抗体显着降低了 125I-LDL 与完整 HepG2 细胞的特异性结合。这些数据表明脂蛋白结合 109-kDa 蛋白是核仁蛋白样蛋白，部分表达在细胞表面。
Nucleolin, a major nucleolar protein of rapidly growing eukaryotic cells, has been thought to be predominantly if not exclusively located in the nucleolus. Recent data however [Borer, R. A., Lehner, C. F., Eppenberger, H. M., and Nigg, N. A. (1989) Cell 56, 379-390] suggest that the protein shuttles constantly between the nucleus and cytoplasm. Ligand blotting studies of whole cell extracts of HepG2 cells identified, in addition to the LDL receptor, another LDL binding protein of Mr 109,000. The 109-kDa protein was partially purified by HPLC and, like the LDL receptor, bound apoB- and apoE-containing lipoproteins but not HDL. However, unlike the LDL receptor, the 109-kDa protein bound lipoproteins in the presence of EDTA and reducing agents, had a lower affinity for lipoproteins than the LDL receptor, and did not react with two antibodies raised against the LDL receptor. The protein sequences of three separate peptides derived from the partially purified 109-kDa species were determined and were identical except for one residue to three separate regions of the published sequence of nucleolin. On immunoblot analysis the 109-kDa protein reacted with a nucleolin-specific antibody, and purified nucleolin reacted both with anti-109-kDa antibody and with LDL. When intact HepG2 cells were treated with Pronase before harvest, there was a 46% decrease in 100-kDa protein while recovery of actin, an intracellular protein, was unaffected. When intact HepG2 cells were surface iodinated and the proteins subjected to HPLC fractionation, the 109-kDa protein was found to be iodinated. On blots, the binding of 125I-LDL to the 109-kDa protein was inhibited by anti-109-kDa antibody and by the nucleolin-specific antibody. In addition, anti-109-kDa antibody significantly decreased the specific binding of 125I-LDL to intact HepG2 cells. These data suggest that the lipoprotein-binding 109-kDa protein is nucleolin-like protein that is partially expressed on the cell surface.