Identification And Characterization Of Abnormal Glycoconjugates In Human Hepatic Cells During Hepatitis C Virus Infection

Background:Hepatitis C virus(HCV)infection causes chronic liver diseases,liver fibrosis and even hepatocellular carcinoma(HCC).HCV infects nearly 71 million people worldwide,approximately 399,000 people die each year from hepatitis C-related liver diseases,according to the 2017 WHO report.The total numbers of HCV infection and morbidity rates in China ranks first in the world,and keep rising in recent years.The current standard of care(peginterferon-?/ribavirin),and recently developed direct antiviral agents(DAAs)have constituted significant contribution to the progress in HCV treatment.However,these drugs are expensive and because HCV has a high degree of variability,in the course of treatment may induce resistance mutant strains.Glycosylation is one of the most essential post-translational modifications in proteins.About 80%of proteins in cells were glycosylated.It is primarily catalyzed by various glycosyltransferase and glycosidase which are mostly located in the Golgi apparatus in cells.With the development of glycoprotein identification techniques,more and more studies show that abnormal glycosylation of glycoproteins,especially N-glycosylation may serve as biomarkers for various diseases(including cancer)in the diagnosis and prognosis evaluation.Detection of the glycopattern of host cells and the profiling of N-linked glycans from isolated glycoproteins can reflect the progression of viral infection,which may be a promising new target for the diagnosis and treatment of viral infection.Objective:In recent years,it has become the focus of research to find key host factors and post-translational modification regulatory factors involved in the infection and immunity of human hepatitis virus.In this study,we investigated the abnormal glycosylation and glycoconjugates in human hepatic cells during HCV infection.Method:In this study,the alteration of N-linked glycans in Huh7.5.1 cells after HCVcc infection was examined by MALDI-TOF-MS.Lectin microarray which is composed of 40 lectins was prepared to analyze the changes of glycan pattern of glycoproteins during HCVcc infection.Meanwhile,the differences in the expression of fucosyltransferases between Huh7.5.1 control cells and HCVcc-infected cells were investigated using qRT-qPCR.Additionally,the mRNA expression profiling microarray was used to detect the mRNA change after HCVcc infection.Results:In MALDI-TOF-MS,23 N-glycans structures were proposed from the HCVcc-infected Huh7.5.1 cells and Huh7.5.1 cells,of which 21 N-glycans and 14 N-glycans were derived from HCVcc-infected Huh7.5.1 cells and Huh7.5.1 cells,respectively.There were 12 common N-glycan structures in both uninfected Huh7.5.1 cells and HCVcc-infected cells,but they displayed in different intensities.Changes in the relative variation of N-glycans in HCV infection are summarized and the proportions of fucosylated,sialylated and complex glycan type were higher in HCVcc-infected cells than in the Huh7.5.1 control.Using the Lectin microarray found that LCA,EC A and AC A lectins in HCVcc-infected cells showed a statistically significant binding glycan than control.The results were then verified by flow cytometry and confocal microscopy analysis.The differential glycoproteins from HCVcc-infected cells bound by LCA were identified by LC/MS/MS,and HSP90B1(heat shock protein 90 beta family member 1)and ANXA2(annexin A2)were selected as the two candidate molecules.We observed that the total levels of these two proteins were not increased in HCVcc-infected cells compared to Huh7.5.1 cells,but the ?1,6 fucosylation levels of HSP90B1and ANXA2 were enhanced by HCV infection.Meanwhile,the fucosylation-related Fut8 expression and catalytic activity were increased in HCVcc-infected cells comapred to that of control cells.Further studies have shown that FUT8 enhances fucosylation of HSP90B1 and ANXA2 after HCV infection.On the other hand,FUT8 which was up-regulated in HCV infection,promotes the proliferation of HCV-infected cells by activating the PI3K-AKT signaling pathway;induces resistance to HCV against antiviral drugs;reduces the inhibitory effect of antiviral drugs on HCV virus and results in an increase of HCV replication.In the mRNA microarray,TNNT1,one of the most up-regulated gene,was selected as the candidate taget.TNNT1 expression were verified by qRT-PCR analysis.TNNT1 can up-regulate the expression of cell-cycle related genes,promote cell proliferation and increase the HCV replication.Further study showed that the protein MTA2 could interact with TNNT1.Conclusions:This study focused on abnormally expressed glycoconjugates and other molecules in HCV infection.Our results will lay the foundation for clarifying the role of N-glycans in HCV related liver diseases and may provide valuable information to unveil the molecular mechanisms in HCV infection and facilitate the discovery of new diagnostic biomarkers and new anti-HCV drugs by targeting N-glycans synthesis.