Ji Z S, Brecht W J, Miranda R D, Hussain M M, Innerarity T L, Mahley R W
Gladstone Institute of Cardiovascular Disease, San Francisco, California 94141-9100.
J Biol Chem. 1993 May 15;268(14):10160-7.
Addition of apolipoprotein (apo) E to rabbit beta-very low density lipoproteins (beta-VLDL) has been shown to result in a marked enhancement of their binding and uptake by various cell types. Apolipoprotein E binds to lipoprotein receptors and proteoglycans. To distinguish between apoE binding to these sites, cells were treated with heparinase. Heparinase treatment of receptor-negative familial hypercholesterolemic (FH) fibroblasts and human hepatoma cells (HepG2) released 30-40% of newly synthesized cell surface 35S-labeled proteoglycans and decreased the binding of beta-VLDL+apoE to FH and normal fibroblasts and HepG2 cells by more than 80%. Furthermore, heparinase treatment significantly decreased the uptake of fluorescently labeled beta-VLDL+apoE by HepG2 cells and decreased cholesteryl ester synthesis in FH fibroblasts by 75%. Likewise, canine chylomicron remnants enriched in apoE demonstrated enhanced binding that was 80% inhibited by heparinase treatment of HepG2 cells. Heparinase treatment did not affect beta-VLDL (without added apoE) or low density lipoprotein (LDL) binding to these cells or the binding activity of beta-VLDL+apoE to the LDL receptor-related protein (LRP) or to the LDL receptor on ligand blots. Chinese hamster ovary (CHO) mutant cells lacking the synthesis of either heparan sulfate (pgsD-677) or all proteoglycans (pgsA-745) did not display any enhanced binding of the beta-VLDL+apoE. By comparison, wild-type CHO cells demonstrated enhanced binding of beta-VLDL+apoE that could be abolished by treatment with heparinase. These mutant cells and wild-type CHO cells possessed a similar amount of LRP, as determined by ligand blot analyses and by alpha 2-macroglobulin binding, and possessed a similar amount of LDL receptor activity, as determined by LDL binding. Therefore, we would interpret these data as showing that heparan sulfate proteoglycan may be involved in the initial binding of the apoE-enriched remnants with the subsequent involvement of the LRP in the uptake of these lipoproteins. It remains to be determined whether the heparan sulfate proteoglycan can function by itself in both the binding and internalization of the apoE-enriched remnants or whether the proteoglycan is part of a complex with LRP that mediates a two-step process, i.e. binding and subsequent internalization by the receptor.
已证明向兔β-极低密度脂蛋白（β-VLDL）中添加载脂蛋白（apo）E会导致其与多种细胞类型的结合及摄取显著增强。载脂蛋白E与脂蛋白受体和蛋白聚糖结合。为区分apoE与这些位点的结合情况，用肝素酶处理细胞。用肝素酶处理受体阴性的家族性高胆固醇血症（FH）成纤维细胞和人肝癌细胞（HepG2），可释放出30%-40%新合成的细胞表面35S标记的蛋白聚糖，并使β-VLDL+apoE与FH和正常成纤维细胞以及HepG2细胞的结合减少80%以上。此外，肝素酶处理显著降低了HepG2细胞对荧光标记的β-VLDL+apoE的摄取，并使FH成纤维细胞中的胆固醇酯合成减少75%。同样，富含apoE的犬乳糜微粒残粒表现出增强的结合，用肝素酶处理HepG2细胞可抑制80%。肝素酶处理不影响β-VLDL（未添加apoE）或低密度脂蛋白（LDL）与这些细胞的结合，也不影响β-VLDL+apoE与配体印迹上的低密度脂蛋白受体相关蛋白（LRP）或低密度脂蛋白受体的结合活性。缺乏硫酸乙酰肝素（pgsD-677）或所有蛋白聚糖（pgsA-745）合成的中国仓鼠卵巢（CHO）突变细胞未表现出β-VLDL+apoE的任何增强结合。相比之下，野生型CHO细胞表现出β-VLDL+apoE的增强结合，用肝素酶处理可消除这种结合。通过配体印迹分析和α2-巨球蛋白结合测定，这些突变细胞和野生型CHO细胞具有相似数量的LRP，通过LDL结合测定，它们具有相似数量的LDL受体活性。因此，我们认为这些数据表明硫酸乙酰肝素蛋白聚糖可能参与富含apoE的残粒的初始结合，随后LRP参与这些脂蛋白的摄取。硫酸乙酰肝素蛋白聚糖是否能单独在富含apoE的残粒的结合和内化中发挥作用，或者该蛋白聚糖是否是与LRP形成的复合物的一部分，介导一个两步过程，即结合和随后被受体内化，仍有待确定。
