N-糖基化修饰对热休克蛋白gp96免疫学功能的影响

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

摘要:

文中旨在以N-糖基化位点突变的重组热休克蛋白gp96为对象，研究N-糖基化修饰对其免疫功能的影响。首先利用昆虫表达系统表达野生型和突变型gp96蛋白，并检测其糖基化水平。进一步通过体外和体内实验，利用流式细胞术和酶联免疫吸附试验 (Enzyme linked immunosorbent assay，ELISA) 检测小鼠CD8+IFN-γ+ T细胞亚群和IFN-γ的分泌，查明糖基化对gp96抗原呈递功能的影响，进一步用ATPase试剂盒检测gp96的ATPase活性。最后通过小鼠免疫实验探究糖基化对gp96疫苗佐剂功能和活化流感疫苗特异性T细胞的影响。结果显示，N-糖基化修饰位点突变后，重组gp96蛋白总含糖量下降了27.8%。与野生型重组蛋白相比，突变gp96的抗原呈递能力减弱，同时ATPase活性明显降低。同时与野生型重组gp96相比，突变gp96佐剂活化流感疫苗特异性T细胞水平也明显减少。这些结果表明，N-糖基化修饰参与调节gp96的ATPase活性和抗原呈递功能，进而影响其疫苗佐剂功能，为开发基于gp96的佐剂疫苗提供了依据。

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.