Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA.
Hum Mol Genet. 2013 Apr 1;22(7):1424-31. doi: 10.1093/hmg/dds559. Epub 2013 Jan 7.
A common synonymous single nucleotide polymorphism in exon 12 of the low-density lipoprotein receptor (LDLR) gene, rs688, has been associated with increased plasma total and LDL cholesterol in several populations. Using immortalized lymphoblastoid cell lines from a healthy study population, we confirmed an earlier report that the minor allele of rs688 is associated with increased exon 12 alternative splicing (P < 0.05) and showed that this triggered nonsense-mediated decay (NMD) of the alternatively spliced LDLR mRNA. However, since synonymous single nucleotide polymorphisms may influence structure and function of the encoded proteins by co-translational effects, we sought to test whether rs688 was also functional in the full-length mRNA. In HepG2 cells expressing LDLR cDNA constructs engineered to contain the major or minor allele of rs688, the latter was associated with a smaller amount of LDLR protein at the cell surface (-21.8 ± 0.6%, P = 0.012), a higher amount in the lysosome fraction (+25.7 ± 0.3%, P = 0.037) and reduced uptake of fluorescently labeled LDL (-24.3 ± 0.7%, P < 0.01). Moreover, in the presence of exogenous proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein that reduces cellular LDL uptake by promoting lysosomal degradation of LDLR, the minor allele resulted in reduced capacity of a PCSK9 monoclonal antibody to increase LDL uptake. These findings are consistent with the hypothesis that rs688, which is located in the β-propeller region of LDLR, has effects on LDLR activity beyond its role in alternative splicing due to impairment of LDLR endosomal recycling and/or PCSK9 binding, processes in which the β-propeller is critically involved.
低密度脂蛋白受体 (LDLR) 基因第 12 外显子中的一个常见同义单核苷酸多态性(rs688) 与多个群体的血浆总胆固醇和 LDL 胆固醇升高有关。我们使用来自健康研究人群的永生化淋巴母细胞系，证实了先前的一项报告，即 rs688 的次要等位基因与第 12 外显子的选择性剪接增加有关(P < 0.05)，并表明这触发了异常剪接的 LDLR mRNA 的无意义介导的衰变 (NMD)。然而，由于同义单核苷酸多态性可能通过共翻译效应影响编码蛋白的结构和功能，我们试图测试 rs688 是否在全长 mRNA 中也具有功能。在表达 LDLR cDNA 构建体的 HepG2 细胞中，该构建体被设计为包含 rs688 的主要或次要等位基因，后者与细胞表面的 LDLR 蛋白量减少(-21.8 ± 0.6%，P = 0.012)，溶酶体分数增加(+25.7 ± 0.3%，P = 0.037)，荧光标记的 LDL 摄取减少(-24.3 ± 0.7%，P < 0.01)。此外，在存在外源性蛋白水解酶原激活物枯草杆菌蛋白酶/凝血酶 9 (PCSK9)时，该蛋白通过促进 LDLR 的溶酶体降解来降低细胞摄取 LDL 的能力，次要等位基因导致促进 LDL 摄取的 PCSK9 单克隆抗体的能力降低。这些发现与假设一致，即位于 LDLR 的β-三叶螺旋区的 rs688 除了在选择性剪接中的作用外，对 LDLR 活性有影响，这是由于 LDLR 内体再循环和/或 PCSK9 结合受损，而β-三叶螺旋区在这些过程中起着关键作用。
