Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a processing enzyme of antigenic peptides presented to major histocompatibility complex (MHC) class I molecules. ERAP1-dependent trimming of epitope repertoire determines an efficacy of adoptive CD8+ T-cell responses in several viral diseases; however, its role in hepatitis B virus (HBV) infection remains unknown. Here, we show that the serum level of ERAP1 in patients with chronic hepatitis B (CHB) (n = 128) was significantly higher than that of healthy controls (n = 44) (8.78 ± 1.82 vs. 3.52 ± 1.61, p< 0.001). Furthermore, peripheral ERAP1 level is moderately correlated with HBV DNA level in patients with CHB (r = 0.731, p< 0.001). HBV-transfected HepG2.2.15 cells had substantially increased ERAP1 expression and secretion than the germline HepG2 cells (p< 0.001). The co-culture of ERAP1-specific inhibitor ERAP1-IN-1 pretreated HepG2.2.15 cells or ERAP1 knockdown HepG2.2.15 cells with CD8+ T cells led to 14–24% inhibition of the proliferation of CD8+ T cells. Finally, liquid chromatography tandem mass spectrometry (LC-MS/MS) test demonstrated that ERAP1-IN-1 blocks completely the production of a 9-mers peptide (30–38, LLDTASALY) derived from Hepatitis B core antigen (HBcAg). The predictive analysis by NetMHCpan-4.1 server showed that human leukocyte antigen (HLA)-C*04:01 is a strong binder for the 9-mers peptide in HepG2.2.15 cells. Taken together, our results demonstrated that ERAP1 trims HBcAg to produce 9-mers LLDTASALY peptides for binding onto HLA-C*04:01 in HepG2.2.15 cells, facilitating the potential activation of CD8+ T cells.
World Health Organization (WHO) Global hepatitis report, 2017 has shown that in 2015, 257 million people were estimated who have been chronically infected with hepatitis B virus (HBV) and more than 887,000 patients die annually due to chronic HBV infection (CHB)-related diseases worldwide. The risk of CHB after acute infection is reduced to < 5% for immunocompetent adults. A vigorous CD8+ T-cell response that exhibits antiviral activity by producing interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) or by directly killing the infected hepatocytes is generated to control and clear HBV.
A fundamental component of host immunity against viral infection relies on the presentation of endogenously derived peptides by major histocompatibility complex class I (MHC-I) molecules. These peptide MHC-I (pMHC-I) complexes are presented on the cell surface and recognized by CD8+ cytotoxic T lymphocytes (CTLs). Antigenic peptides are generated by the degradation of proteasome, translocation into the endoplasmic reticulum (ER) lumen by the transporter associated with antigen processing (TAP), and trimming of excess N-terminal amino acids by aminopeptidases. The endoplasmic reticulum aminopeptidase 1 (ERAP1) belongs to the oxytocinase subfamily of M1 zinc metallopeptidases and trims the N-terminus of peptides to the optimal size of 8–9 amino acids for loading onto MHC-I. pMHC-I complexes are then shuttled to the cell surface for antigen presentation, which triggers antigen-specific cytolytic and effector T-cell responses. The regulation of ERAP1 expression has been shown to substantially affect the presentation of immunodominant epitopes, thereby altering the MHC I immunopeptidome profiling. However, whether ERAP1 is involved in HBV-specific immune response, namely, the antigenic peptide presentation remains unknown. In this study, we analyzed ERAP1 expression in HBV-transfected HepG2.2.15 cells and peripheral ERAP1 level in patients with CHB. We further explored the enzymatic activity of ERAP1 in HepG2.2.15 cells by the specific ERAP1 inhibitor ERAP1-IN-1, especially its effects on HBV-derived peptides and CD8+ T-cell stimulation. Finally, the binding of ERAP1-trimmed HBV-derived peptides with MHC class I molecules was predicted.
Serum samples were obtained from patients with CHB (n = 128) and healthy controls (HC) (n = 44) in the Second Hospital of Anhui Medical University from June 2018 to July 2021. The baseline and laboratory data of patients with CHB and health controls are shown in Table 1. CHB was defined as the persistent presence of serum hepatitis B surface antigen (HBsAg) for > 6 months, and serum tests were performed before receiving any anti-HBV treatment. The exclusion criteria were as follows: (1) coinfection with other hepatotropic viruses (hepatitis A/C/D/E virus) or human immunodeficiency virus (HIV); (2) the coexistence of autoimmune hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, drug hepatitis, primary biliary cirrhosis, or hepatocellular carcinoma. The study was conducted in the principles of the Declaration of Helsinki and was approved by the Ethics Committee for the Second Hospital of Anhui Medical University, and all patients have signed informed consent.
Baseline and laboratory data of patients with CHB and health controls (HC).
Fasting serum level of alanine aminotransferase (ALT, IU/L), aspartate aminotransferase (AST, IU/L), total bilirubin (TBIL, μmol/L), albumin (ALB, g/L), and gamma-glutamyl transpeptidase (GGT, IU/L) was detected by AU5800 biochemistry analyzer (Beckman Coulter, CA, United States). HBV genotyping was based on line probe assays and genotype-specific PCR (Fosun Long March Medical Science, Shanghai, China) according to the manufacturer’s instructions. The supernatants from HepG2 and HepG2.2.15 cells treated or left untreated with ERAP1-IN-1 (50 μm) (HY-133125, MedChem Express, New Jersey, United States) were collected 72 h later. HBsAg and HBeAg in the sera and supernatants were measured using commercially available kits with chemiluminescence apparatus (ARCHITECT i2000SR; Abbott diagnostics, IL, United States). HBV viral loads in the sera and supernatants were measured using the real-time polymerase chain reaction (Mx3000p, Agilent Technologies, CA, United States) according to the manufacturer’s instructions.
The inhibitory potency of ERAP1-IN-1 for ERAP1 was determined using an established fluorogenic assay as previously described.
HepG2. 2.15 cells that are characterized by having stable HBV expression and replication and the germline HepG2 cells were purchased from Shanghai Fuheng Biotechnology. Both cell lines were cultured in Dulbecco’s modified eagle medium (DMEM) containing stable glutamine, supplemented with 10% heat-inactivated fatal bovine serum (FBS) (Gibco) and penicillin (100 IU/ml)/streptomycin (100 μg/ml), and incubated at 37°C, 5% CO 2. Both cell lines were authenticated using short tandem repeat (STR) analysis in combination with sex-typing gene amelogenin detection and compared with DSMZ STR cell line profiles before the use.
HepG2 and HepG2.2.15 cells were lysed in a radio immunoprecipitation assay (RIPA) buffer supplemented with proteinase inhibitors. About 40 μg of proteins was loaded and separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The protein was then transferred onto a polyvinylidene fluoride (PVDF) membrane, blocked in 5% (w/v) non-fat milk, and incubated with the primary antibodies. The source of the primary antibody was anti-ARTS1/ERAP1 antibody (Abcam, ab124669) and secondary antibody was the Goat Anti-rabbit antibody (Abcam, ab6721). Finally, the protein blots were visualized using enhanced chemiluminescent (ECL) system (Thermo Fisher Scientific, United States). The expression levels of ERAP1 were assessed using ImageJ software (v1.8.0, National Institutes of Health) and were normalized against β-actin.
The peripheral ERAP1 was detected using enzyme-linked immunosorbent assay (ELISA) kits (OM534481, Omni mAbs, United States) according to the manufacturer’s instructions.
The small interfering RNA target ERAP1 (Si-ERAP1) and the negative control siRNA (SiControl) oligos were synthesized by Jima Biotechnology (Shanghai, China). The sequences of the siRNA-ERAP1 were as follows: siERAP1 #1, 5′-GGG CGA GUC UCA UUA ACA ATT-3′ and siERAP1 #2, 5′-UUG UUA AUG AGA CUC GCC CTT-3′. The siControl oligos were as follows: siControl #1, 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and siControl #2, 5′-ACG UGA CAC GUU CGG AGA ATT-3′. Lipofectamine 3,000 reagent (L3000015, Invitrogen, United States) was used to perform transfection according to the manufacturer’s protocol. The siRNA-transfected and mock HepG2.2.15 cells were incubated at 37°C in 5% CO 2, and ERAP1 knockdown was verified at 48 h post-transfection via western blotting analysis.
Peripheral blood mononuclear cells (PBMCs) were prepared from the heparinized venous blood of healthy volunteers by Ficoll-paque plus (GE, United States) density gradient centrifugation. Human naïve CD8+ T cells were sorted by positive magnetic-activated cell sorting (MACS, Miltenyi Biotec, United States) according to the manufacturer’s instructions. The cell purity was confirmed by flow cytometry using an anti-mouse CD8 antibody (eBioscience, CA, United States).
HepG2.2.15 cells were treated or left untreated by ERAP1-IN-1 (50 μm) for 72 h followed by the treatment with mitomycin C (10 μg/ml) for 2.5 h. Cells were washed 5 times with PBS to remove residual ERAP1-IN-1 and mitomycin C and resuspended in DMEM at a final concentration of 1 × 10 4/ml. The isolated CD8+ T lymphocytes and HepG2.2.15 cells (10:1) were seeded in the 96-well plate, supplemented with recombinant human IL-2 (200 U/ml), and cultured for 14 days. The proliferation of CD8+ T cells was determined by WST-8/CCK-8 at 0, 3, 5, 7, 10, and 14 days. Briefly, 10 μl of the WST-8/CCK-8 solution (Biosharp, Shanghai, China) was added to each well and incubated for 4 h. Finally, the absorbance was measured at 450 nm.
To evaluate ERAP1-IN-1 cytotoxicity against HepG2.2.15 cells, 100 μl (10 4/ml) cell suspension was dispensed into 96-well plate, and varying concentrations (0, 20, 40, 60, 80, and 100 μm) of ERAP1-IN-1 was added and cultured for 72 h. About 10 μl of the WST-8/CCK-8 solution (Biosharp, Shanghai, China) was added to each well and incubated for 4 h, and the absorbance was measured at 450 nm. All experiments were performed at least three times and the relative cell viability (%) was expressed as a percentage relative to untreated control cells. Cell viability (%) = OD value of ERAP1-IN-1 group/OD value of control group × 100%.
After 72 h of treatment with ERAP1-IN-1, HepG2.2.15 cells were harvested for peptidome analysis. SDS with DTT (SDT) buffer (4% SDS, 100 mM Tris-HCL, 1 mM DTT, pH7.6) was used for cell lysis and protein extraction. The extracted protein was quantified with the BCA (bicinchoninic acid) Protein Assay Kit (Bio-Rad, United States). The protein suspensions were digested with 4 μg trypsin (Promega, Madison, United States) in 40 μl 25 mM NH 4 HCO 3 buffer overnight at 37°C. The digested peptides of each sample were desalted on C18 Cartridges (Empore™ SPE Cartridges C18, Sigma), concentrated by vacuum centrifugation, and reconstituted in 40 μl of 0.1% (v/v) formic acid.
Liquid chromatography tandem mass spectrometry analysis was performed on a timsTOF Pro mass spectrometer (Bruker) that was coupled to Nanoelute (Bruker Daltonics). Peptides were separated using a linear gradient of buffer (84% acetonitrile and 0.1% formic acid) for 120 min at a flow rate of 300 nl/min and a Thermo Scientific reverse-phase nano Easy-spray column (Thermo Scientific PepMap C18, 2 μm particle size, 100 A pore size, 75 μm i.d). The mass spectrometer was opera
