title: Role of asparagine at position 562 in dimerization and immunogenicity of the hepatitis E virus capsid protein.
abstract: The hepatitis E virus (HEV) capsid protein, pORF2, contains 2 potential N-glycosylation sites, N137 and N310, located in the S domain, and one site, N562, in the P domain. The last domain located at positions 454-606 aa forms a protruding spike from the shell, with N562 being located in the apical center of the spike, which is also a cell-attachment region and neutralizing antigenic site. Here, we expressed in Pichia pastoris a recombinant polypeptide p179 comprising the region of 439-617 aa of the HEV pORF2 as well as a set of 4 mutant proteins containing substitutions of Q, D, P and Y instead of N at position 562. All proteins were shown to be secreted from yeast. Using SDS-PAGE, Western blot analysis and tunicamycin treatment assay, we showed that the wild-type (wt) protein, p179N562, and 2 mutant variants, p179N562Q and p179N562D, formed homodimers but only the wt protein was shown to be glycosylated. As homodimers, all 3 proteins were immunoreactive with a neutralizing monoclonal antibody (5G5); however, they did not immunoreact with 5G5 after denaturation into monomers. Two other mutant variants, p179N562P and p179N562Y, did not form homodimers but were immunoreactive with the 5G5 antibody. The wt protein was shown to be less immunoreactive with 5G5 than the mutant variants in a double-antibody sandwich ELISA, suggesting a role of glycosylation at N562 in reducing antibody binding. In vitro neutralization experiments showed a more efficient neutralization with mouse antibody against p179N562P and p179N562Y than against the other 3 proteins. These findings indicate that specific substitutions at position 562 have a more measurable effect on the activity of the HEV neutralizing epitope than dimerization or glycosylation of the structural protein. Furthermore, the secretion of monomers fully immunoreactive may call into question the importance of dimerization for an effective presentation of HEV neutralization epitopes.
authors.affiliation: Department of Microbiology and Immunology, Southeast University, School of Medicine, 87 Dingjiaqiao Road, Nanjing, Jiangsu 210009, China. Electronic address: ruobing_008@163.com.
authors.forename: Mingjie
authors.initials: M
authors.lastname: Xu
authors.affiliation: Department of Microbiology and Immunology, Southeast University, School of Medicine, 87 Dingjiaqiao Road, Nanjing, Jiangsu 210009, China. Electronic address: nouredine_behloul@hotmail.com.
authors.forename: Nouredine
authors.initials: N
authors.lastname: Behloul
authors.affiliation: Department of Microbiology and Immunology, Southeast University, School of Medicine, 87 Dingjiaqiao Road, Nanjing, Jiangsu 210009, China. Electronic address: wenjiyue139@aliyun.com.
authors.forename: Jiyue
authors.initials: J
authors.lastname: Wen
authors.affiliation: Department of Microbiology and Immunology, Southeast University, School of Medicine, 87 Dingjiaqiao Road, Nanjing, Jiangsu 210009, China. Electronic address: sxzjh1018@126.com.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Microbiology and Immunology, Southeast University, School of Medicine, 87 Dingjiaqiao Road, Nanjing, Jiangsu 210009, China. Electronic address: jihongmeng@163.com.
authors.forename: Jihong
authors.initials: J
authors.lastname: Meng
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D057134:Antibodies, Neutralizing / Q000378:metabolism; D001216:Asparagine / Q000378:metabolism*; D002460:Cell Line; D005260:Female; D006031:Glycosylation; D016752:Hepatitis E virus / Q000276:immunology*; D006801:Humans; D051379:Mice; D062105:Molecular Docking Simulation; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D057134:Antibodies, Neutralizing; C400971:ORF2 protein, Hepatitis E virus; D014764:Viral Proteins; D001216:Asparagine
keywords: Capsid protein; Dimerization; Glycosylation; Hepatitis E virus; Mutation; Neutralization antibodies