Lu Song, Yao Ying, Cheng Xiangying, Mitchell Sonya, Leng Shuangying, Meng Songmei, Gallagher James W, Shelness Gregory S, Morris Gabriel S, Mahan James, Frase Sharon, Mansbach Charles M, Weinberg Richard B, Black Dennis D
Children's Foundation Research Center at Le Bonheur Children's Medical Center and Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee 38103, USA.
J Biol Chem. 2006 Feb 10;281(6):3473-83. doi: 10.1074/jbc.M502501200. Epub 2005 Dec 7.
Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.
新生猪肠道载脂蛋白A-IV的表达受膳食脂质的高度调控，提示其在脂质吸收中发挥作用。新生猪肠细胞中载脂蛋白A-IV的组成型过表达使富含三酰甘油（TG）的脂蛋白的基底外侧三酰甘油分泌增强4.9倍（Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929 - 31937）。为研究这种增强作用的机制，用四环素调控表达系统（Tet-On）转染IPEC-1细胞。在用油酸孵育的细胞中，观察到培养基中强力霉素浓度与基底外侧载脂蛋白A-IV和三酰甘油分泌之间存在剂量反应关系。载脂蛋白A-I的类似调控表达并未增强脂质分泌。经强力霉素处理的细胞分泌的富含三酰甘油的脂蛋白的平均直径大于未处理细胞（87.0 nm对53.4 nm）。基底外侧载脂蛋白B分泌减少。使用相同的表达系统，全长人载脂蛋白A-IV（376个氨基酸）；一种缺乏C末端EQQQ重复序列的“猪样”人载脂蛋白A-IV（361个氨基酸）；以及进一步截短至343个氨基酸的“鸡样”载脂蛋白A-IV在IPEC-1细胞中表达。随着蛋白质分泌增加，表达全长人载脂蛋白A-IV的细胞三酰甘油分泌增加2倍；与之形成鲜明对比的是，表达“猪样”人载脂蛋白A-IV的细胞三酰甘油分泌增加25倍，脂蛋白直径增加27倍。当人载脂蛋白A-IV进一步截短产生“鸡样”蛋白时，三酰甘油分泌受到抑制。我们得出结论，猪载脂蛋白A-IV的过表达通过增加分泌脂蛋白的大小以剂量依赖方式增强基底外侧三酰甘油分泌。这些数据表明，人载脂蛋白A-IV蛋白中344至354位残基的区域对其增强脂质分泌的能力至关重要，可能是通过使额外的核心三酰甘油包装到乳糜微粒颗粒中实现的。富含EQQQ的区域可能在人类乳糜微粒包装中起抑制或调节作用。
