Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan, China.
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China.
Front Immunol. 2018 Nov 14;9:2645. doi: 10.3389/fimmu.2018.02645. eCollection 2018.
Immunoglobulin M (IgM) is the major antibody in teleost fish and plays an important role in humoral adaptive immunity. The N-linked carbohydrates presenting on IgM have been well documented in higher vertebrates, but little is known regarding site-specific N-glycan characteristics in teleost IgM. In order to characterize these site-specific N-glycans, we conducted the first study of the N-glycans of each glycosylation site of the grass carp serum IgM. Among the four glycosylation sites, the Asn-262, Asn-303, and Asn-426 residues were efficiently glycosylated, while Asn-565 at the C-terminal tailpiece was incompletely occupied. A striking decrease in the level of occupancy at the Asn-565 glycosite was observed in dimeric IgM compared to that in monomeric IgM, and no glycan occupancy of Asn-565 was observed in tetrameric IgM. Glycopeptide analysis with liquid chromatography-electrospray ionization tandem mass spectrometry revealed mainly complex-type glycans with substantial heterogeneity, with neutral; monosialyl-, disialyl- and trisialylated; and fucosyl-and non-fucosyl-oligosaccharides conjugated to grass carp serum IgM. Glycan variation at a single site was greatest at the Asn-262 glycosite. Unlike IgMs in other species, only traces of complex-type and no high-mannose glycans were found at the Asn-565 glycosite. Matrix-assisted laser desorption ionization analysis of released glycans confirmed the overwhelming majority of carbohydrates were of the complex-type. These results indicate that grass carp serum IgM exhibits unique N-glycan features and highly processed oligosaccharides attached to individual glycosites.
免疫球蛋白 M（IgM）是硬骨鱼类中的主要抗体，在体液适应性免疫中发挥重要作用。在高等脊椎动物中，IgM 上的 N-连接糖基化已得到充分证实，但关于硬骨鱼类 IgM 中特定糖基化位点的 N-糖链特征知之甚少。为了研究这些特定的 N-糖链，我们首次对草鱼血清 IgM 每个糖基化位点的 N-糖链进行了研究。在四个糖基化位点中，Asn-262、Asn-303 和 Asn-426 残基被有效糖基化，而 C 末端尾部的 Asn-565 残基则未完全被占据。与单体 IgM 相比，二聚体 IgM 中 Asn-565 糖基化位点的占据程度显著降低，而四聚体 IgM 中则未观察到 Asn-565 的糖基化。通过液相色谱-电喷雾串联质谱的糖肽分析，发现草鱼血清 IgM 主要结合有复杂型聚糖，且具有显著的异质性，包括中性、单涎酰基、二涎酰基和三涎酰基以及岩藻糖基和非岩藻糖基寡糖。在单个位点的聚糖变化中，Asn-262 糖基化位点的变化最大。与其他物种的 IgM 不同，在 Asn-565 糖基化位点仅发现痕量的复杂型聚糖，而没有高甘露糖型聚糖。释放聚糖的基质辅助激光解吸电离分析证实，绝大多数碳水化合物均为复杂型。这些结果表明，草鱼血清 IgM 具有独特的 N-糖链特征和高度加工的寡糖连接到各个糖基化位点。
