脂蛋白B 之重摺疊/重組裝與其應用

Abstract

脂蛋白B (Apolipoprotein B, apo B) 為極低密度脂蛋白(very-low-density lipoprotein ,VLDL) 與低密度脂蛋白 (low-density lipoprotein, LDL) 的主要組成蛋白。因為脂蛋白B具有高度疏水性與巨大分子量，直到本研究之前，尚未有完整脂蛋白B的摺疊研究。在本研究中，我們使用準靜態過臨界點摺疊法將脂蛋白B摺疊，並且於單分子的層次觀測其摺疊過程。藉由與脂蛋白B的C端截短突變蛋白(脂蛋白B-48與脂蛋白B-29)比較，可證實脂蛋白B序列的N端可協助脂質開始吸附，而C端的長短則可決定所攜帶脂質量的多寡。藉由脂蛋白重摺疊技術，我們用於製作包含有超順磁性氧化鐵奈米粒子(super-paramagnetic iron oxide nanoparticles, SPIONPs) 與抗癌藥物M4N (tetra-O-methyl nordihydroguaiaretic acid)的脂蛋白B重組脂質球 (reconstituting apoB lipoparticle, rABL)。含有超順磁性氧化鐵奈米粒子的rABL可以增強老鼠肝臟的T2核磁共振影像對比 (Magnetic resonance imaging, MRI)，表示其有成為包裹核磁共振顯影劑載體之潛力。另外，包覆M4N的rABL也被證實可以降低癌細胞的存活率。總而言之，在本研究中，我們證明了脂蛋白B的重新摺疊可於單分子層次觀測，並且此一脂蛋白B重組脂質球系統可以用來當作自然且效果佳之的疏水性與兼親性藥物載體。

Apolipoprotein (apo) B is the major structural component of very-low-density lipoprotein (VLDL) and low-density lipoprotein (LDL). The large size and extreme hydrophobicity of apoB render examination of its lipidation process an experimental challenge. In the present study, we showed that apoB lipidation and its folding reaction intermediates could be followed at single molecule level by a five-step (on-path) folding process. When carboxyl-terminal-truncated mutants apoB-29 and apoB-48, representing the amino-terminal 29% and 48%, respectively, of the full-length apoB, were used for comparison, we observed that the refolded apoB resembled the native LDL. Thus the process of lipidation in vitro recapitulates that of biosynthesis of apoB. Furthermore, hydrophobic super-paramagnetic iron oxide nanoparticles (SPIONPs) and the anti-cancer drug M4N (tetra-O-methyl nordihydroguaiaretic acid), used as an imaging enhancer and lipophilic drug model, respectively, were incorporated into the reconstituting apoB lipoparticle (rABL), leading to the formation of SPIONPs- and M4N- containing rABL (SPIO@rABL and M4N@rABL, respectively). SPIO@rABL presented significant hepatic contrast enhancement in T2-weighted magnetic resonance imaging in BALB/c mice, suggesting its potential application as a medical imaging contrast agent. M4N@rABL could reduce the viability of the cancer cell line A549. The current study also demonstrates that apoB can be used as hydrophobic/amphiphilic molecules carrier and be monitored at single molecule level, too.