Generation and Characterization of Native and Sialic Acid-Deficient IgE.
Generation and Characterization of Native and Sialic Acid-Deficient IgE.
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DOI：
10.3390/ijms232113455
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发表时间：
2022-11-03
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
--
作者：
IgE抗体及其聚糖结构的有效表征对于理解其在过敏中的功能和在抗体免疫治疗的新兴变态反应肿瘤学领域中是必需的。我们报告了天然和唾液酸缺陷的糖工程化人IgE的产生、糖谱分析和功能分析。通过人IgE类特异性亲和基质纯化由人胚肾细胞产生的抗体，并通过SDS-PAGE和尺寸排阻色谱法（SEC）确认结构完整性。对肿瘤相关抗原硫酸软骨素蛋白聚糖4（CSPG 4-IgE）和人表皮生长因子受体2（HER 2-IgE）具有特异性的纯化IgE不含游离轻链等副产物。使用神经氨酸酶-A，我们产生唾液酸缺陷型CSPG 4-IgE作为示例糖工程抗体。通过亲水相互作用液相色谱法（HILIC）-高效液相色谱法（HPLC）对天然和糖工程化IgE进行的比较聚糖分析表明唾液酸末端残基和差异聚糖谱的丢失。天然和糖工程化CSPG 4-IgE识别表达人Fcε RI的大鼠嗜碱性白血病RBL-SX 38细胞和表达CD 23/Fcε RII的人RPMI-8866 B淋巴细胞表面上的Fc受体，并与表达CSPG 4的A2058人黑色素瘤细胞结合，证实Fab介导的识别。当在细胞表面上交联时，两种IgE均触发RBL-SX 38脱粒。我们证明了重组天然和唾液酸缺陷型IgE的有效生成和功能能力。
Efficient characterization of IgE antibodies and their glycan structures is required for understanding their function in allergy and in the emerging AllergoOncology field for antibody immunotherapy. We report the generation, glyco-profiling and functional analysis of native and sialic acid-deficient glyco-engineered human IgE. The antibodies produced from human embryonic kidney cells were purified via a human IgE class-specific affinity matrix and structural integrity was confirmed by SDS-PAGE and size-exclusion chromatography (SEC). Purified IgEs specific for the tumor-associated antigens Chondroitin Sulfate Proteoglycan 4 (CSPG4-IgE) and Human Epidermal Growth Factor Receptor 2 (HER2-IgE) were devoid of by-products such as free light chains. Using neuraminidase-A, we generated sialic acid-deficient CSPG4-IgE as example glyco-engineered antibody. Comparative glycan analyses of native and glyco-engineered IgEs by Hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC) indicated loss of sialic acid terminal residues and differential glycan profiles. Native and glyco-engineered CSPG4-IgEs recognized Fc receptors on the surface of human FcεRI-expressing rat basophilic leukemia RBL-SX38 cells, and of CD23/FcεRII-expressing human RPMI-8866 B-lymphocytes and bound to CSPG4-expressing A2058 human melanoma cells, confirming Fab-mediated recognition. When cross-linked on the cell surface, both IgEs triggered RBL-SX38 degranulation. We demonstrate efficient generation and functional competence of recombinant native and sialic acid-deficient IgEs.
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影响因子：
5.8
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通讯作者：
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