Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.
Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.
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DOI：
10.1074/mcp.m114.039537
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发表时间：
2014-11
期刊：
影响因子：
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通讯作者：
Wuhrer M
中科院分区：
文献类型：
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作者：
Bondt A;Rombouts Y;Selman MH;Hensbergen PJ;Reiding KR;Hazes JM;Dolhain RJ;Wuhrer M
免疫球蛋白G恒定片段（Fc）的n -链糖基化已被证明在病理和生理事件中发生变化，并强烈影响抗体炎症特性。相比之下，对Fab-linked n -糖基化知之甚少，约20%的IgG携带这种糖基化。本研究提出了一种高通量的工作流程来分析从5 μl血清中纯化的多克隆IgG的Fab和Fc糖基化。我们能够检测和量化37种不同的n -聚糖，通过MALDI-TOF-MS分析，在反射阳性模式下，使用一种新的链接特异性衍生唾液酸。该方法应用于174例妊娠队列样本，揭示Fab糖基化特征及其随妊娠的变化。数据分析显示Fab和Fc之间的糖基化存在显著差异，特别是在半乳糖基化和唾液基化水平、分割GlcNAc的发生率以及高甘露糖结构的存在方面，Fab部分的糖基化水平都高于Fc，而Fc则表现出更高的聚焦化水平。此外，我们观察到怀孕期间和分娩后的一些变化。相对于产后时间点，Fab n -聚糖唾液化增加，二分化减少，并且在整个过程中观察到Fab n -聚糖几乎完全半乳糖化。Fc糖基化变化与之前描述的结果相似，妊娠期间半乳糖基化和唾液基化增加，二切减少。我们期望在本文中建立的IgG Fab和Fc的平行分析将对揭示这些聚糖在（自身）免疫中的作用很重要，这些聚糖可能通过人凝集素的识别或抗原结合的调节来介导。
The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.