Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taiwan.
Clin Chim Acta. 2012 Jan 18;413(1-2):246-50. doi: 10.1016/j.cca.2011.09.045. Epub 2011 Oct 8.
Apolipoprotein A5 (APOA5) over-expression enhances lipolysis of triglyceride (TG) through stimulation of lipoprotein lipase (LPL) activity; however, an APOA5 G185C variant was found associated with hypertriglyceridemia. The aim of this study was, therefore, to explore the importance of APOA5 185GG in the activation of LPL.
A fragment containing mature human APOA5 cDNA was obtained by RT-PCR and subcloned into pET-15b vector. Site-directed mutagenesis was performed to generate 19 variants. Recombinant human APOA5 wild type and variants were produced in Escherichia coli, and then activation of LPL was measured.
Activity of APOA5 variants on LPL-mediated 1,2-dimyristoyl-sn-glycero-3-phosphocholine hydrolysis was reduced by 17 to 74% in comparison to wild type APOA5 (P<0.0001). All variants also showed reduced activation (P<0.0001) of LPL-mediated hydrolysis of very low-density lipoprotein (VLDL); activation abilities of APOA5 variants ranged from 31 to 81% of wild-type APOA5.
APOA5 residue 185G is very important in LPL-mediated VLDL hydrolysis, and any mutation at this residue will decrease LPL activation and concomitant TG modulation.
载脂蛋白 A5（APOA5）的过表达通过刺激脂蛋白脂肪酶（LPL）的活性增强甘油三酯（TG）的脂肪分解；然而，已经发现 APOA5 G185C 变体与高甘油三酯血症相关。因此，本研究的目的是探讨 APOA5 185GG 在 LPL 激活中的重要性。
通过 RT-PCR 获得包含成熟人 APOA5 cDNA 的片段，并将其亚克隆到 pET-15b 载体中。通过定点诱变生成 19 种变体。在大肠杆菌中产生重组人 APOA5 野生型和变体，并测量 LPL 的激活。
与野生型 APOA5 相比（P<0.0001），APOA5 变体对 LPL 介导的 1,2-二肉豆蔻酰基-sn-甘油-3-磷酸胆碱水解的活性降低了 17%至 74%。所有变体也显示出 LPL 介导的极低密度脂蛋白（VLDL）水解的激活降低（P<0.0001）；APOA5 变体的激活能力范围为野生型 APOA5 的 31%至 81%。
APOA5 残基 185G 在 LPL 介导的 VLDL 水解中非常重要，该残基的任何突变都会降低 LPL 的激活和伴随的 TG 调节。
