| Summary: | 碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 98 === 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.
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