化学固定剂对细胞膜上生物分子相互作用动力学测量的影响

IF 2.3 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Journal of Membrane Biology Pub Date : 2024-04-01 Epub Date: 2024-01-11 DOI:10.1007/s00232-024-00305-4
Tianbao Dong, Shengyang Wan, Yanhui Wang, Yaru Fu, Pengcheng Wang
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引用次数: 0

摘要

了解配体与膜蛋白之间的相互作用对于药物设计和优化非常重要。虽然使用活细胞进行研究是可取的,但在某些情况下并不可行,因此需要进行细胞固定以减少细胞运动和降解。本研究比较了五种固定剂(即甲醛蒸汽(FV)、多聚甲醛(PFA)、丙酮、甲醇和乙醇)对配体示踪法动力学测量的影响。我们发现这五种固定剂对凝集素-糖相互作用的影响都不大。然而,抗体与受体之间的相互作用却受到凝固剂固定剂的明显干扰。丙酮固定法使抗人表皮生长因子受体 2(HER2)抗体与细胞膜上的 HER2 的结合模式从 1:2 变为 1:1,而甲醇和乙醇可能通过去除细胞膜上的 HER2 受体而取消了抗体结合。在较低温度下进行甲醇固定时,尽管结合模式为 1:1,但抗体仍能与细胞膜结合。此外,虽然细胞形态对凝集素与糖的相互作用没有实质性影响,但它确实可以改变抗体与受体相互作用的结合模式。我们的研究结果为基于细胞的动力学研究中固定剂的选择提供了启示。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Effects of Chemical Fixatives on Kinetic Measurements of Biomolecular Interaction on Cell Membrane.

Effects of Chemical Fixatives on Kinetic Measurements of Biomolecular Interaction on Cell Membrane.

Understanding the interaction between ligands and membrane proteins is important for drug design and optimization. Although investigation using live cells is desirable, it is not feasible in some circumstances and cell fixation is performed to reduce cell motion and degradation. This study compared the effects of five fixatives, i.e., formaldehyde vapor (FV), paraformaldehyde (PFA), acetone, methanol, and ethanol, on kinetic measurements via the LigandTracer method. We found that all five fixatives exerted insignificant effects on lectin-glycan interaction. However, antibody-receptor interaction is markedly perturbed by coagulant fixatives. The acetone fixation changed the binding of the anti-human epidermal growth factor receptor 2 (HER2) antibody to HER2 on the cell membrane from a 1:2 to a 1:1 binding model, while methanol and ethanol abolished the antibody binding possibly by removal of the HER2 receptors on the cell membrane. The capability of binding was retained when methanol fixation was performed at lower temperatures, albeit with a binding model of 1:1 instead. Moreover, whereas cell morphology does not exert a substantial impact on lectin-glycan interaction, it can indeed modify the binding model of antibody-receptor interaction. Our results provided insights into the selection of fixatives for cell-based kinetic studies.

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来源期刊
Journal of Membrane Biology
Journal of Membrane Biology 生物-生化与分子生物学
CiteScore
4.80
自引率
4.20%
发文量
63
审稿时长
6-12 weeks
期刊介绍: The Journal of Membrane Biology is dedicated to publishing high-quality science related to membrane biology, biochemistry and biophysics. In particular, we welcome work that uses modern experimental or computational methods including but not limited to those with microscopy, diffraction, NMR, computer simulations, or biochemistry aimed at membrane associated or membrane embedded proteins or model membrane systems. These methods might be applied to study topics like membrane protein structure and function, membrane mediated or controlled signaling mechanisms, cell-cell communication via gap junctions, the behavior of proteins and lipids based on monolayer or bilayer systems, or genetic and regulatory mechanisms controlling membrane function. Research articles, short communications and reviews are all welcome. We also encourage authors to consider publishing ''negative'' results where experiments or simulations were well performed, but resulted in unusual or unexpected outcomes without obvious explanations. While we welcome connections to clinical studies, submissions that are primarily clinical in nature or that fail to make connections to the basic science issues of membrane structure, chemistry and function, are not appropriate for the journal. In a similar way, studies that are primarily descriptive and narratives of assays in a clinical or population study are best published in other journals. If you are not certain, it is entirely appropriate to write to us to inquire if your study is a good fit for the journal.
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