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Biochemistry

基于免疫染色的红细胞蛋白动态改变检测

Published: March 17, 2023
doi:
10.3791/64843
,
1Department of Molecular and Cellular Sports Medicine,German Sport University Cologne, 2Biorheology Research Laboratory, Menzies Health Institute,Griffith University Gold Coast

Summary

捕获去核红细胞蛋白质活化的动态变化带来了方法学挑战，例如保留急性刺激的动态变化以供以后评估。所提出的方案描述了样品制备和染色技术，这些技术能够保存和分析相关的蛋白质变化和随后的检测。

Abstract

红细胞（RBC）蛋白的抗体标记是一种常用的半定量方法，用于检测总蛋白质含量的变化或蛋白质活化状态的急性改变。它有助于评估红细胞治疗，表征某些疾病状态的差异以及描述细胞一致性。检测急性改变的蛋白质活化（例如，通过机械转导）需要足够的样品制备，以保留其他暂时的蛋白质修饰。基本原理包括固定所需红细胞蛋白的靶结合位点，以实现特异性一抗的初始结合。对样品进行进一步处理，以保证二抗与相应一抗结合的最佳条件。非荧光二抗的选择需要额外的处理，包括生物素-亲和素偶联和应用3,3-二氨基联苯胺-四盐酸盐（DAB）进行染色，需要在显微镜下实时控制染色，以便及时停止氧化，从而停止染色强度。对于染色强度检测，使用标准光学显微镜拍摄图像。在该方案的修改中，可以应用荧光素偶联的二抗，其优点是不需要进一步的开发步骤。然而，该过程需要将荧光物镜连接到显微镜上进行染色检测。鉴于这些方法的半定量性质，必须提供几种对照染色剂来解释非特异性抗体反应和背景信号。在这里，我们介绍了染色方案和相应的分析过程，以比较和讨论不同染色技术各自的结果和优势。

Introduction

红细胞 （RBC） 在心血管系统中穿行 70 至 140 天，平均红细胞年龄约为 115 天 1,2。衰老或受损的红细胞通过噬红细胞作用从循环中去除，这是一种由巨噬细胞驱动的高效清除过程3。这些细胞的预定寿命是在分化和成熟过程中放弃细胞器（包括细胞核、线粒体和核糖体）的结果之一4。因此，循环红细胞缺乏翻译机制，排除了新蛋白质的合成3。因此，对现有蛋白质的动态翻译后修饰代表了响应作用于红细胞的细胞外和细胞内应激源的急性生化调节的唯一可行机制5。

机械力似乎是导致红细胞内生化途径激活或调节的主要细胞外线索。在红细胞膜6中发现机械敏感蛋白Piezo1激发了研究这些细胞中机械激活信号传导的几条研究线7。例如，最近的进展表明，红细胞的物理性质受到蛋白质8的急性和动态变化的积极调节，其中包括翻译后磷酸化和泛素化9。由于这些正常修饰在某些疾病中有所不同9,10,11，因此确定RBC蛋白的激活状态似乎具有科学和临床意义，特别是与机械生物学过程有关。

红细胞蛋白活化状态急性变化的测定带来了一些方法学挑战。例如，储存红细胞样品以供以后分析需要保存修饰的红细胞蛋白，因为翻译后修饰是不持久的。此外，众所周知，经典的蛋白质检测方法（例如蛋白质印迹）很难在红细胞中标准化，因为相对于血红蛋白的蛋白质丰度较低，血红蛋白占这些细胞中蛋白质含量的~98%12。因此，在研究重要红细胞蛋白（例如一氧化氮合酶的红细胞特异性亚型（RBC-NOS））的急性修饰时，基于抗体的化学保存红细胞染色一直是首选方法13,14。RBC-NOS已被证明可以酶促产生一氧化氮（NO），这对于基本的RBC特性（包括RBC变形性）似乎是必不可少的15,16,17。红细胞-NOS的翻译后修饰调节催化酶活性，丝氨酸1177残基的磷酸化被描述为增加酶活性，而丝氨酸114或苏氨酸495残基的磷酸化与红细胞-NOS活性降低有关18,19。

总的来说，红细胞蛋白的临时修饰有助于重要的细胞功能，并且能够检测这些修饰蛋白的标准化方案具有很高的价值。在这里，我们提出了两种不同的方案，它们利用特异性抗体来促进RBC-NOS蛋白活化的检测，并讨论了数据分析和解释的建议。

通过测量丝氨酸1177残基处RBC-NOS磷酸化的增加来评估所描述方案的性能，以响应于反映人体脉管系统内发生的机械力（5 Pa）的机械力。

Protocol

此处描述的协议符合《赫尔辛基宣言》，并已获得德国科隆体育大学（2013 年 9 月 16 日）和格里菲斯大学（2019/808）伦理委员会的批准。对志愿者进行筛选以确保没有相关病理，并提供书面知情同意书。 1. 使用免疫组织化学方案对红细胞蛋白进行染色 注意： 材料表中提供了所需化学品和材料的详细清单。以下部分描述了所需溶液?…

Representative Results

所提出的方案描述了促进检测红细胞蛋白急性改变的方法，并在一种众所周知的机械敏感蛋白质改变上进行了测试：丝氨酸1177残基处红细胞-NOS的磷酸化。从健康志愿者那里获得全血，随后分成两个单独的等分试样。将给定的血液样品暴露于生理量级（5 Pa）的机械剪切应力下300秒，先前显示其在丝氨酸117714处引起RBC-NOS磷酸化。在停止机械剪切暴露后，立即将血液样品固定在多聚…

Discussion

最近的文献高度表明，RBC-NOS蛋白对于调节RBC变形性至关重要15,22,23，这反过来又促进了它们通过狭窄的毛细血管24。蛋白质活性高度依赖于翻译后蛋白质修饰，特别是某些残基的磷酸化18。感兴趣的焦点在于磷酸化位点1177，其与RBC-NOS蛋白23的活化有关。这种蛋白质的改变已?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

LK感谢澳大利亚政府研究培训计划奖学金的支持。

Materials

3,3′-Diaminobenzidin -tetrahydrochloride Hydrate Sigma/Merck D5637 DAB
Ammoniumchloride  Merck /Millipore 101145 NH4Cl
Centrifuge 5427 R  Eppendorf 5409000010
Coverslips VWR 631-0147 
di-sodium Hydrogen Phosphate Dihydrate  Merck /Millipore 106580 Na2HPO4. 2 H2O
Disposable transfer pipettes VWR 612-6803
Entellan Merck /Millipore 107961 rapid mounting medium for microscopy
Ethanol denaturated using 1 % methyl ethyl ketone (MEK) Hofmann 642
Glucose-Oxidase Sigma/Merck G2133
Grease pencil  Dako S 2002
Horse-radish peroxidase/ExtrAvidin−Peroxidase Sigma/Merck E-2886 HRP
Hydrochloric acid  Merck /Millipore 109057 HCl
Hydrogen peroxide, 30% Merck /Millipore 107203 H2O2
ImageJ Software Freeware
Laser-assisted optical rotational cell analyser (LORCA) RR Mechatronics Ektacytometer instrument used for shearing
Methanol Merck /Millipore 106009
Microscope slides VWR 630-1985
Nickel(II)-sulfate Hexahydrate  Sigma/Merck N4882 NiSO4.6H2O
Normal Goat serum Agilent/DAKO X0907 NGS
Paraformaldehyde Merck /Millipore 818715 PFA
Pipettes Eppendorf Reference 2 VWR 613-5836/ 613-5839
Rabbit Anti-phospho eNOS Antibody (Ser1177) Merck/Millipore 07-428-I Primary Antibody
Reaction tubes, 2ml Eppendorf 30120094
Secondary Antibody goat anti rabbit Agilent/DAKO E0432 Secondary Antibody
Skim milk powder Bio-Rad 170-6404
Sodium chloride  Merck /Millipore 106404 NaCl
Sodium Dihydrogen Phosphate Monohydrate Merck /Millipore 106346 NaH2PO4.H2O
Sodium hydroxide, 1 M Merck /Millipore 150706 NaOH
Tris(hydroxymethyl)-aminomethane Merck /Millipore 108382 Tris
Trypsin Sigma/Merck T7409
Tween20  Merck /Millipore 822184
Whatman Glas microfiber filter, quality GF/F Merck /Millipore WHA1825047
Xylol VWR Chemicals 2,89,73,465
ß-D-Glucose monohydrate Merck /Millipore 14431-43-7
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Tags

Keywords: ImmunostainingRed Blood Cell ProteinsMechanobiologyPost-translational ModificationsRed Cell MechanosignalingProtein FixationImmunohistochemistryAntibody Staining
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Grau, M., Kuck, L. Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins. J. Vis. Exp. (193), e64843, doi:10.3791/64843 (2023).
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