Multi-isotype Glycoproteomic Characterization of Serum Antibody Heavy Chains Reveals Isotype- and Subclass-Specific N-Glycosylation Profiles
Multi-isotype Glycoproteomic Characterization of Serum Antibody Heavy Chains Reveals Isotype- and Subclass-Specific N-Glycosylation Profiles
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DOI：
10.1074/mcp.ra118.001185
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发表时间：
2019-04-01
影响因子：
7
通讯作者：
Costello, Catherine E.
中科院分区：
文献类型：
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作者：
Chandler, Kevin Brown;Mehta, Nickita;Costello, Catherine E.
抗体是连接先天性和适应性免疫系统以提供抗感染保护的关键糖蛋白。抗体的同种型/亚类、CH 2结构域上的共翻译N-糖基化以及通过ER和Golgi期间N-连接聚糖的重塑是Fc结构域内编程抗体效应子功能的已知变量。通过单克隆治疗的研究，已经观察到从抗体N-聚糖中添加或去除特定单糖残基可以影响抗体的效力，突出了彻底表征抗体N-糖基化的重要性。尽管IgG通常具有单个N-糖基化位点并且被充分研究，但是在某些疾病中作为第一反应者的其他抗体同种型（例如伊加和IgM）具有2至5个位点/抗体单体，并且关于它们的N-糖基化知之甚少。在这里，我们采用nLC-MS/MS方法，使用步进能量较高的能量碰撞解离来表征循环血清抗体的N-聚糖库和位点占有率。我们同时测定了个体健康供体中IgG 1、IgG 4、IgA 1、IgA 2和IgM的位点特异性N-连接聚糖库。与IgG 1相比，IgG 4中G1 S1 F的相对丰度较高，而G1 FB的相对丰度较低。IgA 1和IgA 2主要显示双触角N-聚糖。检测到具有丝氨酸（S93）或脯氨酸（P93）的IgA 2变体。在来自供体亚组的血清样本中，我们检测到在位置93处含有脯氨酸残基的未修饰肽;这种取代强烈不利于N92处的N-糖基化。IgM位点N46、N209和N272主要显示复杂聚糖，而位点N279和N439显示高甘露糖糖型的相对丰度较高。这种多同种型方法是开发一个平台的关键一步，该平台用于定义不同同种型的疾病特异性N-聚糖特征，以帮助调整抗体以诱导保护。数据可通过ProteomeXchange获得，标识符为PXD 010911。
Antibodies are critical glycoproteins that bridge the innate and adaptive immune systems to provide protection against infection. The isotype/subclass of the antibody, the co-translational N-glycosylation on the CH2 domain, and the remodeling of the N-linked glycans during passage through the ER and Golgi are the known variables within the Fc domain that program antibody effector function. Through investigations of monoclonal therapeutics, it has been observed that addition or removal of specific monosaccharide residues from antibody N-glycans can influence the potency of antibodies, highlighting the importance of thoroughly characterizing antibody N-glycosylation. Although IgGs usually have a single N-glycosylation site and are well studied, other antibody isotypes, e.g. IgA and IgM, that are the first responders in certain diseases, have two to five sites/monomer of antibody, and little is known about their N-glycosylation. Here we employ a nLC-MS/MS method using stepped-energy higher energy collisional dissociation to characterize the N-glycan repertoire and site occupancy of circulating serum antibodies. We simultaneously determined the site-specific N-linked glycan repertoire for IgG1, IgG4, IgA1, IgA2, and IgM in individual healthy donors. Compared with IgG1, IgG4 displayed a higher relative abundance of G1S1F and a lower relative abundance of G1FB. IgA1 and IgA2 displayed mostly biantennary N-glycans. IgA2 variants with the either serine (S93) or proline (P93) were detected. In digests of the sera from a subset of donors, we detected an unmodified peptide containing a proline residue at position 93; this substitution would strongly disfavor N-glycosylation at N92. IgM sites N46, N209, and N272 displayed mostly complex glycans, whereas sites N279 and N439 displayed higher relative abundances of high-mannose glycoforms. This multi-isotype approach is a crucial step toward developing a platform to define disease-specific N-glycan signatures for different isotypes to help tune antibodies to induce protection. Data are available via ProteomeXchange with identifier PXD010911.