State Key Laboratory of Virology. Hubei province Key Laboratory of Allergy and Immune-related diseases, Medical Research Institute, Department of Immunology of Wuhan University School of Basic Medical Sciences, Wuhan 430071, China.
The Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Biochim Biophys Acta Gen Subj. 2017 May;1861(5 Pt A):1036-1045. doi: 10.1016/j.bbagen.2017.02.014. Epub 2017 Feb 14.
Hepatitis C virus (HCV) infection causes chronic liver diseases, liver fibrosis and even hepatocellular carcinoma (HCC). However little is known about any information of N-glycan pattern in human liver cell after HCV infection.
The altered profiles of N-glycans in HCV-infected Huh7.5.1 cell were analyzed by using mass spectrometry. Then, lectin microarray, lectin pull-down assay, reverse transcription-quantitative real time PCR (RT-qPCR) and western-blotting were used to identify the altered N-glycosylated proteins and glycosyltransferases.
Compared to uninfected cells, significantly elevated levels of fucosylated, sialylated and complex N-glycans were found in HCV infected cells. Furthermore, Lens culinaris agglutinin (LCA)-binding glycoconjugates were increased most. Then, the LCA-agarose was used to precipitate the specific glycosylated proteins and identify that fucosylated modified annexin A2 (ANXA2) and heat shock protein 90 beta family member 1 (HSP90B1) was greatly increased in HCV-infected cells. However, the total ANXA2 and HSP90B1 protein levels remained unchanged. Additionally, we screened the mRNA expressions of 47 types of different glycosyltransferases and found that α1,6-fucosyltransferase 8 (FUT8) was the most up-regulated and contributed to strengthen the LCA binding capability to fucosylated modified ANXA2 and HSP90B1 after HCV infection.
HCV infection caused the altered N-glycans profiles, increased expressions of FUT8, fucosylated ANXA2 and HSP90B1 as well as enhanced LCA binding to Huh7.5.1.
Our results may lay the foundation for clarifying the role of N-glycans and facilitate the development of novel diagnostic biomarkers and therapeutic targets based on the increased FUT8, fucosylated ANXA2 and HSP90B1 after HCV infection.
丙型肝炎病毒（HCV）感染可导致慢性肝病、肝纤维化甚至肝细胞癌（HCC）。然而，人们对 HCV 感染后人类肝细胞中 N-糖链模式的任何信息知之甚少。
采用质谱法分析 HCV 感染 Huh7.5.1 细胞后 N-糖链谱的改变。然后，使用凝集素微阵列、凝集素下拉实验、逆转录-定量实时 PCR（RT-qPCR）和 Western blot 鉴定改变的 N-糖基化蛋白和糖基转移酶。
与未感染细胞相比，HCV 感染细胞中发现岩藻糖基化、唾液酸化和复杂 N-聚糖水平显著升高。此外，扁豆凝集素（LCA）结合糖缀合物增加最多。然后，使用 LCA-琼脂糖沉淀特异性糖基化蛋白，并鉴定出 HCV 感染细胞中 fucosylated 修饰的 annexin A2（ANXA2）和热休克蛋白 90β家族成员 1（HSP90B1）显著增加。然而，总 ANXA2 和 HSP90B1 蛋白水平保持不变。此外，我们筛选了 47 种不同糖基转移酶的 mRNA 表达，发现α1,6-岩藻糖基转移酶 8（FUT8）上调最为显著，有助于增强 HCV 感染后 LCA 与 fucosylated 修饰的 ANXA2 和 HSP90B1 的结合能力。
HCV 感染导致 N-糖链谱改变、FUT8、fucosylated ANXA2 和 HSP90B1 表达增加以及 LCA 与 Huh7.5.1 的结合增强。
我们的研究结果可能为阐明 N-糖链的作用奠定基础，并为基于 HCV 感染后 FUT8、fucosylated ANXA2 和 HSP90B1 的增加开发新型诊断生物标志物和治疗靶点提供依据。
