Nicodeme E, Benoist F, McLeod R, Yao Z, Scott J, Shoulders C C, Grand-Perret T
Laboratoire GlaxoWellcome, Centre de Recherche, 25 avenue du Quebec, ZA de Courtaboeuf, 91951 Les Ulis cedex, France.
J Biol Chem. 1999 Jan 22;274(4):1986-93. doi: 10.1074/jbc.274.4.1986.
The microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apoB-containing lipoproteins. To investigate the role of MTP in lipoprotein assembly, we determined the ability of carboxyl-terminally truncated forms of apoB to be secreted from cells treated with the MTP inhibitor 4'-bromo-3'-methylmetaqualone (Benoist, F., Nicodeme, E., and Grand-Perret, T. (1996) Eur. J. Biochem. 240, 713-720). In Caco-2 and mhAT3F cells that produce apoB100 and apoB48, the inhibitor preferentially blocked apoB100 secretion. When the inhibitor was tested on McA-RH7777 cells stably transfected with cDNAs encoding human apoB100, apoB72, apoB53, apoB29, and apoB18, the secretion of apoB100, apoB72, and apoB53 was preferentially impaired relative to apoB48 and shorter forms. To delineate the region between apoB48 and apoB53 that has a high requirement for MTP, we used puromycin to generate a range of truncated forms of apoB in HepG2 cells. The secretion of apoB53 and longer forms of apoB was markedly affected by low concentrations of the MTP inhibitor (approximately 1 microM), whereas apoB51 and smaller forms of apoB were only affected at higher concentrations (> 10 microM). The size-related sensitivity to MTP inhibitor was not due to late processing or retention, since the same result was observed when nascent lipoproteins were isolated from the endoplasmic reticulum. The MTP inhibitor did not alter the density of the secreted lipoproteins, indicating that each apoB polypeptide requires a minimally defined amount of lipid to attain a secretable conformation. Our results suggest that the folding of the domain between apoB51 and apoB53 has a high requirement for lipid. This domain is predicted to form amphipathic alpha-helices and to bind lipid reversibly. It proceeds and is followed by rigid amphipathic beta-sheets that are predicted to associate with lipid irreversibly. We speculate that these domains enable apoB to switch from a stable lipid-poor conformation in apoB48 to another lipid-rich conformation in apoB100 during lipoprotein assembly.
微粒体甘油三酯转运蛋白（MTP）是含载脂蛋白B（apoB）的脂蛋白组装和分泌所必需的。为了研究MTP在脂蛋白组装中的作用，我们测定了apoB羧基末端截短形式从用MTP抑制剂4'-溴-3'-甲基异喹啉（Benoist，F.，Nicodeme，E.，和Grand-Perret，T.（1996）Eur. J. Biochem. 240，713 - 720）处理的细胞中分泌的能力。在产生apoB100和apoB48的Caco - 2和mhAT3F细胞中，该抑制剂优先阻断apoB100的分泌。当在稳定转染了编码人apoB100、apoB72、apoB53、apoB29和apoB18的cDNA的McA - RH7777细胞上测试该抑制剂时，相对于apoB48和较短形式，apoB100、apoB72和apoB53的分泌优先受损。为了确定apoB48和apoB53之间对MTP有高需求的区域，我们在HepG2细胞中使用嘌呤霉素产生一系列apoB的截短形式。低浓度（约1 microM）的MTP抑制剂显著影响apoB53和较长形式的apoB的分泌，而apoB51和较小形式的apoB仅在较高浓度（>10 microM）时受到影响。对MTP抑制剂的大小相关敏感性不是由于后期加工或滞留，因为从内质网分离新生脂蛋白时观察到相同的结果。MTP抑制剂没有改变分泌的脂蛋白的密度，表明每个apoB多肽需要最小限定数量的脂质以获得可分泌的构象。我们的结果表明，apoB51和apoB53之间区域的折叠对脂质有高需求。该区域预计形成两亲性α - 螺旋并可逆地结合脂质。它之前和之后是预计与脂质不可逆结合的刚性两亲性β - 折叠。我们推测这些区域使apoB在脂蛋白组装过程中能够从apoB48中稳定的贫脂构象转变为apoB100中另一种富脂构象。
