Relative content detection of oligomannose modification of IgM heavy chain induced by TNP-antigen in an early vertebrate through nanoLC-MS/MS
Relative content detection of oligomannose modification of IgM heavy chain induced by TNP-antigen in an early vertebrate through nanoLC-MS/MS
复制标题
通过 nanoLC-MS/MS 检测早期脊椎动物 TNP 抗原诱导的 IgM 重链寡甘露糖修饰的相对含量
DOI：
10.1016/j.talanta.2020.121346
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发表时间：
2020-11-01
期刊：
影响因子：
6.1
通讯作者：
Ye，Jianmin
中科院分区：
文献类型：
--
作者：
Yin，Xiaoxue;Li，Xiaoyu;Ye，Jianmin
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中是重要的。然而，硬骨鱼类免疫球蛋白糖基化的研究还很有限。本研究构建了TNP-抗原驱动模型，通过18 O-标记和nanoLC-MS/MS检测了PBS免疫和TNP特异性尼罗罗非鱼血清IgM中的位点特异性N-糖基化。这些方法广泛用于肽段富集和蛋白质修饰鉴定，但很少用于检测由特异性抗原驱动的硬骨鱼Ig中的N-糖基化水平。结果表明，尼罗罗非鱼IgM重链上存在4个N-糖基化位点，即CH 2区的Asn-315位点、CH 3区的Asn-338位点、CH 4区的Asn-509和Asn-551位点，这4个残基均被有效地N-糖基化。经TNP抗原免疫后，TNP特异性IgM中寡聚甘露糖的信号强度显著高于PBS免疫的IgM。值得注意的是，TNP特异性IgM具有完全被寡聚甘露糖占据的Asn-509位点，而在PBS免疫的IgM的该位点中仅发现少量的寡聚甘露糖。其他位点的N-聚糖主要为复合型，岩藻糖基化和唾液酸化含量较低。进一步证实了TNP特异性IgM中的寡聚甘露糖对于IgM与MBL的结合是必需的。这些结果表明，TNP抗原诱导尼罗罗非鱼IgM重链发生位点特异性寡聚甘露糖修饰，并在IgM与MBL相互作用中发挥重要作用，为进一步了解IgM寡聚甘露糖修饰及其功能的进化机制提供了线索。
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 through18O-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 inO.niloticusIgM 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 ofO.niloticusIgM 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.