Yin Xiaoxue, Li Xiaoyu, Mu Liangliang, Han Kailiang, Wu Hairong, Wu Liting, Wang Anli, Ying Wantao, Ye Jianmin
School of Life Sciences, South China Normal University, Institute of Modern Aquaculture Science and Engineering, Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture, Guangzhou, 510631, PR China.
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, 102206, PR China; College of Life Science and Bioengineering, Beijing University of Technology, Beijing, 100124, PR China.
Talanta. 2020 Nov 1;219:121346. doi: 10.1016/j.talanta.2020.121346. Epub 2020 Jul 5.
N-glycan modification is reported to be important in regulating the structure and function of immunoglobulins in mammals. While, the study on teleost immunoglobulin glycosylation is still limitted. In this study, we constructed a TNP-antigen driven model, and detected the site-specific N-glycans of PBS-immunized and TNP-specific Oreochromis niloticus serum IgM through O-labeling and nanoLC-MS/MS. These methods are widely used for peptide enrichment and protein modification identification, but rarely used in detecting the level of N-glycosylation in teleost Igs that driven by specific antigen. The results revealed that there are four N-glycosylation sites in O.niloticus IgM heavy chain, namely, the Asn-315 site in the CH2 domain, the Asn-338 site in the CH3 domain, and the Asn-509 and Asn-551 sites in the CH4 domain, All of the four residues were efficiently N-glycosylated. After immunized with TNP-antigen, the signal strength of oligomannose in the TNP-specific IgM in primary mass spectrometry was significantly higher than that in the PBS-immunized IgM. Notably, the TNP-specific IgM had an Asn-509 site fully occupied with oligomannose, while only a small amount of oligomannose was found in the PBS-immunized IgM of this site. N-glycans in other sites were mainly complex-type with a low content of fucosylation and sialylated. The oligomannose in TNP-specific IgM was further verified to be essential for the binding of IgM and MBL. These results demonstrated that the TNP-antigen induced the site-specific oligomannose modification of O.niloticus IgM heavy chain, and played an important role in the interaction of IgM and MBL, which provided insights into the evolutionary understanding of the IgM oligomannose modification and function.
据报道，N-聚糖修饰在调节哺乳动物免疫球蛋白的结构和功能方面很重要。然而，硬骨鱼免疫球蛋白糖基化的研究仍然有限。在本研究中，我们构建了一个TNP抗原驱动模型，并通过O标记和纳升液相色谱-串联质谱法检测了经PBS免疫和TNP特异性尼罗罗非鱼血清IgM的位点特异性N-聚糖。这些方法广泛用于肽富集和蛋白质修饰鉴定，但很少用于检测由特定抗原驱动的硬骨鱼免疫球蛋白中的N-糖基化水平。结果显示，尼罗罗非鱼IgM重链中有四个N-糖基化位点，即CH2结构域中的Asn-315位点、CH3结构域中的Asn-338位点以及CH4结构域中的Asn-509和Asn-551位点，这四个残基均被有效地进行了N-糖基化。用TNP抗原免疫后，一级质谱中TNP特异性IgM中低聚甘露糖的信号强度明显高于经PBS免疫的IgM。值得注意的是，TNP特异性IgM的Asn-509位点被低聚甘露糖完全占据，而在经PBS免疫的IgM中该位点仅发现少量低聚甘露糖。其他位点的N-聚糖主要是复合型，岩藻糖基化和唾液酸化含量较低。进一步证实，TNP特异性IgM中的低聚甘露糖对于IgM与MBL的结合至关重要。这些结果表明，TNP抗原诱导了尼罗罗非鱼IgM重链的位点特异性低聚甘露糖修饰，并在IgM与MBL的相互作用中发挥了重要作用，这为深入了解IgM低聚甘露糖修饰及其功能的进化提供了思路。
