From the ‡Center for Biomedical Mass Spectrometry, Boston University School of Medicine, Boston, Massachusetts.
Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts.
Mol Cell Proteomics. 2019 Apr;18(4):686-703. doi: 10.1074/mcp.RA118.001185. Epub 2019 Jan 18.
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 -glycosylation on the CH2 domain, and the remodeling of the -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 -glycans can influence the potency of antibodies, highlighting the importance of thoroughly characterizing antibody -glycosylation. Although IgGs usually have a single -glycosylation site and are well studied, other antibody isotypes, 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 -glycosylation. Here we employ a nLC-MS/MS method using stepped-energy higher energy collisional dissociation to characterize the -glycan repertoire and site occupancy of circulating serum antibodies. We simultaneously determined the site-specific -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 -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 -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 -glycan signatures for different isotypes to help tune antibodies to induce protection. Data are available via ProteomeXchange with identifier PXD010911.
抗体是至关重要的糖蛋白，它们连接先天免疫系统和适应性免疫系统，为抗感染提供保护。抗体的同型/亚型、CH2 结构域上的共翻译糖基化以及在通过内质网和高尔基体时的 -连接糖链重塑，是 Fc 结构域中影响抗体效应功能的已知变量。通过对单克隆治疗药物的研究发现，抗体糖基化中添加或去除特定单糖残基可以影响抗体的效力，这凸显了彻底表征抗体糖基化的重要性。虽然 IgG 通常只有一个糖基化位点，并且研究得比较透彻，但其他抗体同型，如 IgA 和 IgM，它们是某些疾病的早期反应者，每个抗体分子有 2 到 5 个糖基化位点，而它们的糖基化情况知之甚少。在这里，我们采用 nLC-MS/MS 方法，使用逐步能量高能碰撞解离来表征循环血清抗体的 -聚糖库和位点占有率。我们同时确定了单个健康供体中 IgG1、IgG4、IgA1、IgA2 和 IgM 的位点特异性 -连接糖链库。与 IgG1 相比，IgG4 显示出更高相对丰度的 G1S1F 和更低相对丰度的 G1FB。IgA1 和 IgA2 主要显示双天线 -聚糖。检测到 IgA2 变体中的丝氨酸（S93）或脯氨酸（P93）。在一组供体血清的消化物中，我们检测到一个含有脯氨酸残基的未修饰肽，该残基位于位置 93；这种取代会强烈不利于 N92 的糖基化。IgM 位点 N46、N209 和 N272 主要显示复杂糖型，而位点 N279 和 N439 显示出高甘露糖糖型的相对丰度更高。这种多同型方法是开发用于定义不同同型的疾病特异性 -聚糖特征的平台的关键步骤，有助于调整抗体以诱导保护。数据可通过 ProteomeXchange 获得，标识符为 PXD010911。
