N-glycosylation modification of heat shock protein gp96 affects its immunological function.

Peng GUO; Changfei LI; Ying JU; Erlong LIU; Han ZHANG; Jun HU; Songdong MENG

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N-glycosylation modification of heat shock protein gp96 affects its immunological function.

Author: Peng GUO ¹ ; Changfei LI ¹ ; Ying JU ¹ ; Erlong LIU ² ; Han ZHANG ¹ ; Jun HU ³ ; Songdong MENG ¹
Author Information

1. CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
2. Heat Shock Biotechnology Co., Ltd., Beijing 100123, China.
3. Cominghealth Biotechnology Co., Ltd., Beijing 100101, China.
Publication Type:Journal Article
Keywords: ATPase; N-glycosylation modification; adjuvant; antigen presentation; heat shock protein gp96
MeSH: Adjuvants, Immunologic; Animals; CD8-Positive T-Lymphocytes/metabolism*; Glycosylation; Heat-Shock Proteins; Influenza Vaccines; Mice
From: Chinese Journal of Biotechnology 2021;37(11):4036-4046
CountryChina
Language:Chinese
Abstract: N-glycosylation modification, one of the most common protein post-translational modifications, occurs in heat shock protein gp96. The purpose of this study is to investigate the effect of N-glycosylation modification on immunologic function of the recombinant gp96 using the mutant gp96 in N-glycosylation sites. Firstly, wild-type and mutant gp96 proteins were expressed by insect expression system and their glycosylation levels were detected. To determine the effect of N-glycosylation on gp96 antigen presentation function, the IFN-γ+ CD8+ T cells in gp96-immunized mice and secretion level of IFN-γ were examined by flow cytometry and ELISA. The ATPase activity of gp96 was further detected by the ATPase kit. Finally, the effect of N-glycosylation on adjuvant function of gp96 for influenza vaccine was investigated in immunized mice. It was found that total sugar content of mutant recombinant gp96 was reduced by 27.8%. Compared to the wild type recombinant gp96, mutations in N-glycosylation sites resulted in decreased antigen presentation ability and ATPase activity of gp96. Furthermore, influenza vaccine-specific T cell levels induced by mutant gp96 as adjuvant were dramatically reduced compared to those by wild type recombinant gp96. These results demonstrate that N-glycosylation modification is involved in regulation of ATPase activity and antigen presentation function of gp96, thereby affecting its adjuvant function. The results provide the technical bases for development of gp96- adjuvanted vaccines.