Analysis of leukocyte membrane protein interactions using protein microarrays
Analysis of leukocyte membrane protein interactions using protein microarrays
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DOI：
10.1186/1471-2091-6-2
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发表时间：
2005-03-01
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影响因子：
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通讯作者：
Barclay, A. Neil
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文献类型：
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作者：
Letarte, Michelle;Voulgaraki, Despina;Barclay, A. Neil
背景：蛋白质微阵列代表了一种新兴的蛋白质组学工具，用于并行研究多种蛋白质-蛋白质相互作用。足够比例的固定蛋白必须保持活性构象和取向，以便对抗体和配体结合进行敏感和特异性检测。为了建立表征白细胞膜蛋白之间弱相互作用的蛋白阵列技术，我们选择了人白细胞膜蛋白CD200 （OX2）及其细胞表面受体（hCD200R）作为模型系统。由于抗体-抗原反应通常比受体-配体结合具有更高的亲和力，我们首先分析了单克隆抗体（mAb）对固定CD200R的正常和突变形式的反应性。结果：荧光标记的单抗DX147、DX136和OX108对固定化重组hCD200R细胞外区域具有特异性反应，反应范围为0.1-40 μ g ml(-1)，对应的灵敏度限为0.01-0.05飞莫/点。利用捕获抗体定向hCD200R，发现DX147与一个与更密切相关的DX136和OX108表位在空间上不同的表位发生反应。瞬时转染的细胞产生了一组hCD200R结构域1突变的可溶性重组蛋白，直接排列而不纯化，并筛选与三个单抗的结合。一些显示与阻断单抗DX136和OX108的结合减少，表明这些表位与CD200结合位点非常接近。hCD200与直接固定的大鼠、小鼠和hCD200R通过多聚配体结合，以生物素化的hCD200偶联到fitc标记的亲和素包被珠的形式。结论：我们利用蛋白芯片技术实现了不同抗体对固定化CD200R的敏感、特异性和可重复性检测，并在配体结合位点附近定位了两个单抗的抗原表位。我们还检测到CD200与其受体的结合，这是一种低亲和力的相互作用，使用珠子呈现多价配体。我们的结果证明了蛋白质阵列的定量方面及其在同时检测多种蛋白质相互作用，特别是白细胞膜蛋白之间发现的弱相互作用方面的潜在用途。
Background: Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R.Results: Fluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1-40 mu g ml(-1) corresponding to a limit of sensitivity of 0.01-0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads.Conclusion: We have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays. We also detected CD200 binding to its receptor, a low affinity interaction, using beads presenting multivalent ligands. Our results demonstrate the quantitative aspects of protein arrays and their potential use in detecting simultaneously multiple protein-protein interactions and in particular the weak interactions found between leukocyte membrane proteins.