Ligand binding and antigenic properties of a human neonatal Fc receptor with mutation of two unpaired cysteine residues
Ligand binding and antigenic properties of a human neonatal Fc receptor with mutation of two unpaired cysteine residues
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DOI：
10.1111/j.1742-4658.2008.06551.x
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发表时间：
2008-08-01
期刊：
影响因子：
5.4
通讯作者：
Sandlie, Inger
中科院分区：
文献类型：
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作者：
Andersen, Jan T.;Justesen, Sune;Sandlie, Inger
新生儿Fc受体（FcRn）是一种主要组织相容性复合物I类相关分子，可调节IgG和白蛋白的半衰期。此外，FcRn指导IgG跨粘膜上皮和胎盘的转运，还增强中性粒细胞的吞噬作用。这一新知识为基于IgG和白蛋白的诊断和治疗的设计提供了激励。为了在体外研究FcRn并选择和表征FcRn结合剂，需要大量可溶性人FcRn。在本报告中，我们探讨了FcRn重链的两个游离半胱氨酸残基（C48和C251）对可溶性人FcRn的整体结构和功能的影响，并描述了一种基于去除这些残基的改进的细菌生产策略，产生类似于70 mg.L-1的复性可溶性人FcRn发酵。经圆二色谱、表面等离子体共振和MALDI-TOF肽图分析证实了其结构和功能的完整性。该策略通常可以被翻译为大规模生产的其他主要组织相容性复合体I类相关的分子与非功能性未配对的半胱氨酸残基。此外，在重链偶联的琼脂糖凝胶上进行亲和纯化后，分析了用单独的FcRn重链免疫的山羊中的抗FcRn应答。重要的是，纯化的抗体阻断了两种配体与可溶性人FcRn的结合，因此被导向两个结合位点。这意味着FcRn重链（事先未与人β 2-微球蛋白组装）含有可溶性人FcRn中发现的相关表位，因此足以获得任一配体结合位点的结合剂。这一发现将极大地促进此类粘合剂的选择和表征。
The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule that regulates the half-life of IgG and albumin. In addition, FcRn directs the transport of IgG across both mucosal epithelium and placenta and also enhances phagocytosis in neutrophils. This new knowledge gives incentives for the design of IgG and albumin-based diagnostics and therapeutics. To study FcRn in vitro and to select and characterize FcRn binders, large quantities of soluble human FcRn are needed. In this report, we explored the impact of two free cysteine residues (C48 and C251) of the FcRn heavy chain on the overall structure and function of soluble human FcRn and described an improved bacterial production strategy based on removal of these residues, yielding similar to 70 mg.L-1 of fermentation of refolded soluble human FcRn. The structural and functional integrity was proved by CD, surface plasmon resonance and MALDI-TOF peptide mapping analyses. The strategy may generally be translated to the large-scale production of other major histocompatibility complex class I-related molecules with nonfunctional unpaired cysteine residues. Furthermore, the anti-FcRn response in goats immunized with the FcRn heavy chain alone was analyzed following affinity purification on heavy chain-coupled Sepharose. Importantly, purified antibodies blocked the binding of both ligands to soluble human FcRn and were thus directed to both binding sites. This implies that the FcRn heavy chain, without prior assembly with human beta 2-microglobulin, contains the relevant epitopes found in soluble human FcRn, and is therefore sufficient to obtain binders to either ligand-binding site. This finding will greatly facilitate the selection and characterization of such binders.