FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.
FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.
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DOI：
10.1016/j.jbc.2023.104743
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发表时间：
2023-06
影响因子：
4.8
通讯作者：
Buchholz, Christian J
中科院分区：
文献类型：
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作者：
Riechert, Vanessa;Hein, Sascha;Visser, Mayken;Zimmermann, Mathias;Wesche, Jan;Adams, Philipp A;Theuerkauf, Samuel A;Jamali, Arezoo;Wangorsch, Andrea;Reuter, Andreas;Pasternak, Alexander O;Hartmann, Jessica;Greinacher, Andreas;Herrera-Carrillo, Elena;Berkhout, Ben;Cichutek, Klaus;Buchholz, Christian J
Fc受体参与多种生理和疾病相关反应。其中，FcγRIIA(CD32a)在病原体识别和血小板生物学中具有激活功能，是潜伏感染HIV-1的T淋巴细胞的潜在标志物。后者并非没有争议，原因是T-B细胞结合物和巨噬细胞增多症带来的技术挑战，以及缺乏区分密切相关的Fc、γ、RII亚型的抗体。为了获得FcγRIIA的高亲和力结合蛋白，通过核糖体展示筛选设计的Ankyrin重复蛋白(DARPins)文库，以与其胞外区结合。对FcγRIIB的反选消除了与两种异构体交叉反应的结合子。已鉴定的DARPins与FCγRIIA结合，未检测到与FcγRIIB的结合。它们与FcγRIIA的亲和力在低纳摩尔范围内，可以通过裂解His-Tag和二聚化来增强。有趣的是，DARPin和FcγRIIA之间的络合物形成遵循两态反应模型，而与FcγRIIB的区分是基于单个氨基酸残基。在流式细胞术中，DARPin F11检测到Fcγriia+细胞，即使它们在细胞总数中所占比例不到1%。对原代人类血细胞的图像流分析证实，F11导致了一小部分T淋巴细胞表面模糊但可靠的染色。当与血小板孵育时，F11抑制其聚集的效果与无法区分两种FcγRII亚型的抗体相同。所选的DARPins是研究血小板聚集的独特的新工具，以及FcγRIIA在潜伏的HIV-1储存库中的作用。
Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.