基因重組Apolipoprotein C3擇殖與表現及與不同曲度脂質球結合能力之探討

Abstract

Apolipoprotein C3是一個小分子蛋白質，只有79個胺基酸，是脂蛋白中極低密度脂蛋白和高密度脂蛋白的組成成員之一，近期的研究中指出它會和特定的磷脂質結合，且在肝臟VLDL的生合成中可能扮演了攜帶脂質和VLDL precursor融合、幫助VLDL precursor成熟角色，但其分子機制尚未完全瞭解。先前有研究指出，有些和脂質結合的蛋白質，例如golgins，對於脂質膜的曲度很敏感，從高度彎曲到平坦的liposome有不同的結合喜好，且會影響其脂質的運送。基於apo C3也具有運送脂質的功能，我們想要探究apo C3和不同脂質膜度結合的差異及apo C3在VLDL成熟機制中扮演的角色。我們利用基因重組的方式將apo C3擇殖到E. coli和yeast系統，表現出重組的apo C3蛋白質，利用fat western lipid protein overlay assay確認了重組的apo C3蛋白會和磷脂質結合，具有正常的生理功能。接著利用合成出不同大小的仿生脂肪球 (Au@lipid) 來探討apo C3對脂肪球膜曲度的喜好。本研究發現apo C3傾向於和較小的仿生脂肪球結合，因此推測和過大的脂肪球結合可能會使apo C3釋出，這可能暗示了生理中apo C3參與VLDL成熟的角色，未來我們將會探討體外重組的LDL與apo C3之結合作用。

Apolipoprotein C3 is a small (79 amino acid) protein that is one of the component of very low density lipoprotein (VLDL) and high density lipoprotein (HDL). Previous study suggested that apo C3 showed robust binding to phospholipids and played an intracellular role in promoting bulk lipid incorporation during VLDL assembly, but the mechanism remained unknown. Previous golgins study also indicated the size (surface curvature) of lipid droplets might affect the binding ability of lipoprotein. We surmise that , apo C3 may follow the similar mechanism. It is intriguing to reveal the binding and dissociation mechanism between apo C3 and lipid droplets, and to define the possible role of apo C3 in VLDL maturation mechanism. In this study, we cloned and expressed human apo C3 protein in both E. coli and yeast expression systems. Through fat western lipid protein overlay assay, we confirmed that the recombinant apo C3 protein bound with specific phospholipids. The result also indicates the recombinant apo C3 possesses normal physical function. Different sizes lipo-particle were synthesized to investigate the binding preference of apo C3. Our results showed that apo C3 tight bound with smaller lipo-particles than those larger ones. We speculate large particles may promote apo C3 release. These results may reveal the possible role of apoC3 in VLDL maturation process.