Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
Department of Medicine and the Canadian Centre for Health and Safety in Agriculture, University of Saskatchewan, Saskatoon, Saskatchewan S7N 2Z4, Canada.
Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Sep;1866(9):158991. doi: 10.1016/j.bbalip.2021.158991. Epub 2021 Jun 9.
In eukaryotic organisms, two unrelated acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2, catalyze the final step of the triacylglycerol biosynthetic pathway. Both enzymes are highly expressed in lipogenic tissues, such as adipose tissue, small intestine and the liver. DGAT2 has a prominent role in hepatocyte lipid metabolism synthesizing triacylglycerols that are utilized for very low-density lipoprotein assembly. However, due to the lack of useful antibodies to detect endogenous DGAT2 protein, it has been difficult to determine how this enzyme functions at the cellular level. We have unsuccessfully tested many commercial antibodies as well as our own "in-house" antibodies. There is currently no evidence that DGAT2 undergoes processing such that antigenic epitopes to these antibodies are removed. As an alternative, many studies have utilized epitope tagged versions of DGAT2 overexpressed in cells. These approaches can provide valuable information about a protein, but can be subject to artifacts, such as mislocalization, misregulation, protein aggregation and abnormal protein-protein interactions. In this study, we used gene editing with CRISPR/Cas9 to add three consecutive FLAG epitopes to the C-terminus of endogenous DGAT2 in HepG2 cells. HepG2 cells, derived from a human hepatocellular carcinoma, have been routinely used as a cell model to study human hepatocyte lipid and lipoprotein metabolism. Using this system allowed us to successfully detect DGAT2 expressed from its endogenous locus in HepG2 cells by immunoblotting with anti-FLAG antibodies.
在真核生物中，两种不相关的酰基辅酶 A：二酰基甘油酰基转移酶（DGAT）酶，DGAT1 和 DGAT2，催化三酰基甘油生物合成途径的最后一步。这两种酶在脂肪组织（如脂肪组织、小肠和肝脏）中高度表达。DGAT2 在肝细胞脂质代谢中起着重要作用，合成用于极低密度脂蛋白组装的三酰基甘油。然而，由于缺乏用于检测内源性 DGAT2 蛋白的有用抗体，因此很难确定该酶在细胞水平上的功能。我们已经测试了许多商业抗体和我们自己的“内部”抗体，但都没有成功。目前没有证据表明 DGAT2 经历了加工，从而去除了这些抗体的抗原表位。作为替代方案，许多研究已经利用细胞中过表达的 DGAT2 的表位标记版本。这些方法可以提供有关蛋白质的有价值信息，但可能会出现伪影，例如定位错误、调节异常、蛋白质聚集和异常的蛋白质-蛋白质相互作用。在这项研究中，我们使用 CRISPR/Cas9 基因编辑在 HepG2 细胞内源性 DGAT2 的 C 末端添加三个连续的 FLAG 表位。HepG2 细胞源自人类肝癌，已常规用于研究人类肝细胞脂质和脂蛋白代谢的细胞模型。使用该系统，我们能够成功地通过用抗 FLAG 抗体进行免疫印迹检测到 HepG2 细胞中从其内源基因座表达的 DGAT2。
