title: Evidences for the existence of intermolecular disulfide-bonded oligomers in the H3 hemagglutinins expressed in insect cells.
abstract: The hemagglutinin (HA) protein as the predominant antigen, executes receptor binding and membrane fusion, which critically influence the virological characteristics of influenza viruses. The literature contained scattered data showing reduction-sensitive HA oligomers when HA proteins were analyzed under non-reducing conditions. However, whether the reduction-sensitive HA oligomers are inter-monomer disulfide-bonded has not been studied. Here, we showed: (1) the detection of β-mercaptoethanol-sensitive H3 HA oligomers was not affected by the treatment of cells with iodoacetamide prior to cell solubilization; (2) H3 HA oligomers were present on cell surfaces; (3) H3 HA oligomers had higher density than monomers; and (4) mutation of all the five C-terminal cysteines completely abolished the formation of H3 HA oligomers. Furthermore, mutant HAs with mutations of TM cysteines, CT cysteines or all five cysteines had decreased thermal stability but increased fusion activity in comparison with wildtype HA. In conclusion, this study has presented enough evidence for the existence of inter-monomer S-S H3 HA oligomers formed by five C-terminal cysteines, and suggested that all five C-terminal cysteines exerted opposite effects on HA thermal stability and fusion activity.
authors.affiliation: State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China.
authors.forename: Shun
authors.initials: S
authors.lastname: Xu
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Zhou
authors.forename: Qiliang
authors.initials: Q
authors.lastname: Liu
authors.forename: Kang
authors.initials: K
authors.lastname: Liu
authors.forename: Chunyi
authors.initials: C
authors.lastname: Xue
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: Zheng
authors.forename: Dongyu
authors.initials: D
authors.lastname: Luo
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Cao
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004220:Disulfides / Q000378:metabolism*; D006388:Hemagglutinins / Q000378:metabolism*; D006801:Humans; D007313:Insecta / Q000166:cytology*; D008623:Mercaptoethanol / Q000008:administration & dosage / Q000737:chemistry
chemical_list: D004220:Disulfides; D006388:Hemagglutinins; D008623:Mercaptoethanol