變異脂蛋白元AV功能之研究

Abstract

高三酸甘油脂血症(HTG)是冠心病的獨立危險因子。根據先前的研究，三酸甘油脂代謝不良與脂蛋白元A5基因(APOA5)的多型性(polymorphism)相關。APOAV可經由幫助lipoprotein lipase (LPL)進行水解反應，進而降低血液中的三酸甘油脂含量。不過APOAV完整的功能與作用機制到目前都不甚清楚。 本實驗室於2003年發現一東方族群特有的single nucleotide polymorphism (SNP) APOA5 c.553G>T，造成第185胺基酸由glycine變為cysteine。具有此SNP的病人罹患高脂血症的機率較正常人高，此一現象啟發本實驗室的研究方向：是否此SNP會破壞或減低APOAV的功能。 本實驗室過去曾以HEK293細胞為實驗工具，探討三種APOAV變異型蛋白幫助LPL水解功能上的差異，然而結果並不顯著。推測原因可能由於細胞表現的蛋白量不足，以及細胞中有無法排除的內源性APOAV蛋白。為了改善上述問題，本論文改用大腸桿菌表現APOAV蛋白。本論文中利用site directed mutagenesis製造十九種變異型蛋白，分別以DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine)模擬人類VLDL構造，及apoa5 基因剔除小鼠的VLDL進行實驗，希望得知第185胺基酸影響AV功能的原因。實驗結果在DMPC系統與基因剔除小鼠VLDL系統有相同趨勢，即野生型APOAV蛋白促進LPL的能力明顯高於其他十九種變異型(DMPC部分P<0.00014，基因剔除小鼠VLDL部分P<0.00012)。此結果證實185G在影響LPL水解三酸甘油脂的重要性，也呼應了臨床上SNP病人較易產生高脂血症的現象。另外，根據實驗結果，APOAV功能上的差異，與185位置胺基酸的親水性，酸鹼度，大小等特質並無明顯關聯，提示185G除了原先認知的與脂蛋白結合之外，或許還有與LPL活化相關的功能。

Hypertriglyceridemia (HTG) is an independent risk factor for coronary heart disease. According to the previous studies, triglyceride (TG) metabolic disorder was associated with the apolipoprotein A5 (APOA5) gene polymorphisms. APOAV can modulate the triacylglycerol hydrolase activity of lipoprotein lipase (LPL) through direct activation or indirect effects to reduce the plasma TG. However, the mechanism of APOAV modulation is still unclear. We have identified a c.553G>T polymorphism, which was found in oriental populations only, cause a G185C substitution effect. Moreover, patients with c.553G>T polymorphism tend to suffer from hypertriglyceridemia. These phenomena inspired us that substitution of G185C might results in losing, or at least lowering the function of APOAV. In our previous studies, we had expressed the recombinant APOAV protein by HEK293 cell model. However, the activation of LPL revealed no significant. Explanations of this result might be the unavoidable intrinsic APOAV, and the inadequate protein concentration expressed by cell. Overcoming the problems mentioned above, we shifted the expression system to bacteria (E.coli BL21). To study the function of 185G, we generate 19 kinds of mutant-type APOAV proteins by site-direct mutagenesis. Before applying to the animal model, we constructed a synthetic system of DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine), which is similar to VLDL. We found that in the DMPC system, mutant-type APOAV did decrease its function on the activation of LPL comparing to that of wild-type (P<0.00014). Besides, mutant-type APOAV proteins show lower abilities to hydrolyze TG in VLDL from apoa5 knockout mice (P<0.00012). It may somehow suggest the importance of 185G. We repeated the experiment with apoa5 knockout mice model, similar to DMPC system, 185G showed its importance in hydrolysing TG by LPL. It could probably bring up a clue why patients with c.553G>T polymorphism come across the problem with hypertriglyceridemia more often. Meanwhile it seems the characteristic of amino acid (hydrophilic, hydrophobic, or size) dose not affect so much.