In Planta Glycan Engineering and Functional Activities of IgE Antibodies
In Planta Glycan Engineering and Functional Activities of IgE Antibodies
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DOI：
10.3389/fbioe.2019.00242
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发表时间：
2019-09-25
影响因子：
5.7
通讯作者：
Steinkellner, Herta
中科院分区：
文献类型：
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作者：
Montero-Morales, Laura;Maresch, Daniel;Steinkellner, Herta
人免疫球蛋白E （IgE）是最广泛糖基化的抗体同型，因此在其Fab和Fc结构域的7个n -糖苷位（NGS）上附着的聚糖可以调节其功能。然而，靶向修饰多糖基化蛋白中的聚糖仍然是一个挑战。在这里，我们应用了一种体内方法，允许操纵IgE n -聚糖，使用曲妥珠单抗等效IgE （HER2-IgE）作为模型。利用植物的固有特征，即大部分同质的n -聚糖复合物的合成和对聚糖工程的敏感性，我们产生了HER2-IgE的靶向糖型，与血清IgE中的糖型非常相似。植物源性HER2-IgE表现出n -聚糖末端为GlcNAc、半乳糖或唾液酸，缺乏或携带核心焦点和木糖。我们不仅能够调节自然修饰有复杂n -聚糖的5种NGSs，而且还能够诱导通常未被占用的NGS6靶向糖基化，从而增加HER2-IgE的总体糖基化含量。重组人细胞源性HER2-IgE表现出较大的n -聚糖异质性。所有HER2-IgE变体均表现出与靶抗原和高亲和受体Fc epsilon RI的糖基化非依赖性结合，并随后具有类似的触发肥大细胞脱颗粒的能力。相比之下，与低亲和力受体CD23 （Fc epsilon RII）的结合受到聚糖谱的调节，随着聚糖以GlcNAc残基终止，与IgE变体的结合增加。在这里，我们提供了一种高效的植物方法，在多重糖基化IgE上生成定义的糖型，从而可以精确地探索糖基化依赖性活性。
Human immunoglobulin E (IgE) is the most extensively glycosylated antibody isotype so glycans attached to the seven N-glycosites (NGS) in its Fab and Fc domains may modulate its functions. However, targeted modification of glycans in multiply glycosylated proteins remains a challenge. Here, we applied an in vivo approach that allows the manipulation of IgE N-glycans, using a trastuzumab equivalent IgE (HER2-IgE) as a model. Taking advantage of plant inherent features, i.e., synthesis of largely homogeneous complex N-glycans and susceptibility to glycan engineering, we generated targeted glycoforms of HER2-IgE largely resembling those found in serum IgE. Plant-derived HER2-IgE exhibited N-glycans terminating with GlcNAc, galactose or sialic acid, lacking, or carrying core fucose and xylose. We were able to not only modulate the five NGSs naturally decorated with complex N-glycans, but to also induce targeted glycosylation at the usually unoccupied NGS6, thus increasing the overall glycosylation content of HER2-IgE. Recombinant human cell-derived HER2-IgE exhibited large N-glycan heterogeneity. All HER2-IgE variants demonstrated glycosylation-independent binding to the target antigen and the high affinity receptor Fc epsilon RI, and subsequent similar capacity to trigger mast cell degranulation. In contrast, binding to the low affinity receptor CD23 (Fc epsilon RII) was modulated by the glycan profile, with increased binding to IgE variants with glycans terminating with GlcNAc residues. Here we offer an efficient in planta approach to generate defined glycoforms on multiply glycosylated IgE, allowing the precise exploration of glycosylation-dependent activities.