Department of Gastroenterology and Hepatology, Xiamen University Zhongshan Hospital, Xiamen, Fujian Province, 361001, China; Department of Digestive Diseases, School of Medicine, Xiamen University, Xiamen, Fujian Province, 361001, China.
Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, 55902, USA.
Int J Biochem Cell Biol. 2021 Feb;131:105906. doi: 10.1016/j.biocel.2020.105906. Epub 2020 Dec 26.
Apolipoprotein H (APOH), also known as beta2-glycoprotein I (beta2-GPI), is an acute phase protein in hepatitis B virus (HBV) infection and binds to hepatitis B surface antigen (HBsAg) with high-affinity. APOH expression is upregulated by HBV and the large surface protein (LHBs), but also elevated in HBV-related hepatoma cells. Previous studies show that intracellular retention of HBsAg induces endoplasmic reticulum (ER) stress, a key driver of hepatocyte damage during chronic liver injury, but the mechanisms are unclear. We hypothesize that APOH mediates HBV-induced ER stress through increased retention of HBsAg.
VR-APOH-myc and VR-LHBs-flag plasmids were constructed by PCR using pcDNA3.1(-)-APOH or an HBV expression vector, respectively. APOH and ER stress markers were examined at protein and mRNA levels by Western Blot or RT-qPCR. HBsAg titer was assayed by ELISA. RNA-seq was performed to elucidate the transcriptional impact of APOH manipulation in HBV-producing cells (HepG2.2.15 cells).
We found that HBV upregulates APOH expression in 293 T cells, and APOH overexpression subsequently inhibits secretion of HBsAg. Next, we show that LHBs overexpression in conjunction with APOH leads to ER stress in 293 T cells, as evidenced by production of the binding immunoglobulin protein (BiP) and C/EBP homologous protein (CHOP), as well as increased splicing of X-box binding protein 1 (XBP1). We further observed that loss of beta2-GPI reduced CHOP expression in HepG2.2.15 cells, while beta2-GPI overexpression enhanced CHOP production.
The interaction of beta2-GPI and HBV initiates ER stress through driving intracellular retention of HBsAg and activates the UPR.
载脂蛋白 H（APOH），也称为β2-糖蛋白 I（β2-GPI），是乙型肝炎病毒（HBV）感染中的一种急性期蛋白，与乙型肝炎表面抗原（HBsAg）具有高亲和力。APOH 的表达受 HBV 和大表面蛋白（LHBs）上调，但在 HBV 相关肝癌细胞中也升高。先前的研究表明，HBsAg 的细胞内滞留会引起内质网（ER）应激，这是慢性肝损伤过程中肝细胞损伤的关键驱动因素，但机制尚不清楚。我们假设 APOH 通过增加 HBsAg 的滞留来介导 HBV 诱导的 ER 应激。
通过 PCR 使用 pcDNA3.1（-）-APOH 或 HBV 表达载体分别构建 VR-APOH-myc 和 VR-LHBs-flag 质粒。通过 Western Blot 或 RT-qPCR 检测 APOH 和 ER 应激标志物在蛋白质和 mRNA 水平上的表达。通过 ELISA 测定 HBsAg 滴度。在产生 HBV 的细胞（HepG2.2.15 细胞）中进行 RNA-seq 以阐明 APOH 操作的转录影响。
我们发现 HBV 在 293T 细胞中上调 APOH 的表达，并且 APOH 过表达随后抑制 HBsAg 的分泌。接下来，我们表明 LHBs 的过表达与 APOH 一起导致 293T 细胞中的 ER 应激，这表现为结合免疫球蛋白蛋白（BiP）和 C/EBP 同源蛋白（CHOP）的产生，以及 X 盒结合蛋白 1（XBP1）的剪接增加。我们进一步观察到在 HepG2.2.15 细胞中丢失β2-GPI 会降低 CHOP 的表达，而β2-GPI 过表达会增强 CHOP 的产生。
β2-GPI 和 HBV 的相互作用通过驱动 HBsAg 的细胞内滞留引发 ER 应激，并激活 UPR。
