La Belle M, McCall M R, Krauss R M, Forte T M
Research Medicine and Radiation Biophysics Division, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Biochim Biophys Acta. 1990 Oct 1;1046(3):288-93. doi: 10.1016/0005-2760(90)90243-q.
Previous work has shown that low-density lipoproteins (LDL) secreted by hepatoma-derived cell lines have an unusual composition compared to plasma LDL; rather than cholesteryl ester, the hepatoma cell-secreted LDL have a triacylglycerol core. We have found that they also have an increased negative charge, as judged by agarose electrophoresis. Since apolipoprotein B is a glycoprotein containing carbohydrate chains terminated with negatively charged sialic acid residues, we examined whether increased glycosylation of the apolipoprotein B from three hepatoma cell lines (Hep G2, Hep 3B and Huh 7) might account for the differences in LDL charge. The weight percent carbohydrate for Hep G2, Hep 3B and Huh 7 LDL-protein (1.1 +/- 0.2; 1.7 +/- 0.8; 0.4 +/- 0.1) was found to be extremely low compared with the 2.8-9% range we found for plasma LDL-protein, while the amount of LDL-lipid associated carbohydrate from hepatoma LDL was similar to that we found in plasma LDL. Furthermore, desialation of hepatoma cell-secreted LDL with neuraminidase did not normalize the negative charge to that of neuraminidase-treated plasma LDL. Western blots of thrombin proteolytic fragments indicated that, in addition to the T1-T4 fragments seen in plasma apolipoprotein B, apolipoprotein B of hepatoma-derived LDL produced four to five new fragments (T5-T9), suggesting increased exposure of proteolytic sites. Western blotting of the new fragments with antibodies specific for known apolipoprotein B sequences suggests that many of the new cleavage sites cluster in or near the putative LDL receptor recognition site.
先前的研究表明，肝癌衍生细胞系分泌的低密度脂蛋白（LDL）与血浆LDL相比，其组成不同寻常；肝癌细胞分泌的LDL不是以胆固醇酯为核心，而是以三酰甘油为核心。我们发现，通过琼脂糖电泳判断，它们的负电荷也有所增加。由于载脂蛋白B是一种糖蛋白，其碳水化合物链末端带有带负电荷的唾液酸残基，我们研究了来自三种肝癌细胞系（Hep G2、Hep 3B和Huh 7）的载脂蛋白B糖基化增加是否可能导致LDL电荷的差异。与我们在血浆LDL-蛋白中发现的2.8%-9%的范围相比，Hep G2、Hep 3B和Huh 7的LDL-蛋白的碳水化合物重量百分比（分别为1.1±0.2；1.7±0.8；0.4±0.1）极低，而肝癌LDL中与脂质相关的碳水化合物量与我们在血浆LDL中发现的相似。此外，用神经氨酸酶对肝癌细胞分泌的LDL进行去唾液酸化处理后，其负电荷并未恢复到经神经氨酸酶处理的血浆LDL的水平。凝血酶蛋白水解片段的蛋白质印迹分析表明，除了在血浆载脂蛋白B中看到的T1-T4片段外，肝癌衍生LDL的载脂蛋白B产生了四到五个新片段（T5-T9），这表明蛋白水解位点的暴露增加。用针对已知载脂蛋白B序列的特异性抗体对新片段进行蛋白质印迹分析表明，许多新的切割位点聚集在假定的LDL受体识别位点或其附近。
