The Interaction Affinity between Vascular Cell Adhesion Molecule-1 (VCAM-1) and Very Late Antigen-4 (VLA-4) Analyzed by Quantitative FRET
The Interaction Affinity between Vascular Cell Adhesion Molecule-1 (VCAM-1) and Very Late Antigen-4 (VLA-4) Analyzed by Quantitative FRET
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DOI：
10.1371/journal.pone.0121399
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发表时间：
2015-03-20
期刊：
影响因子：
3.7
通讯作者：
Chiou, Arthur
中科院分区：
文献类型：
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作者：
Chakraborty, Sandeep;Hu, Shih-Yang;Chiou, Arthur
非常迟抗原-4 （Very late antigen-4, vca -4）是整合素超家族的一员，它与其主要对抗配体血管细胞粘附分子-1 （vascular cell adhesion molecule-1, VCAM-1）相互作用，在白细胞粘附血管内皮和免疫突触形成中起重要作用。然而，这些蛋白的不规则表达也可能导致一些自身免疫性疾病和转移性癌症。因此，量化VCAM-1/VLA-4相互作用的相互作用亲和力对于进一步了解这种相互作用的性质和药物发现具有重要意义。在这项研究中，我们报告了一种基于“溶液中”稳态有机荧光团的定量荧光共振能量转移（FRET）测定，以解离常数（K-d）来量化这种相互作用。在FRET实验中，我们使用Alexa Fluor 488- vla4偶联物作为供体，Alexa Fluor 546-VCAM-1作为受体。通过FRET信号分析，确定了这种相互作用的K-d为41.82 +/- 2.36 nM。为了进一步证实我们的估计，我们利用表面等离子体共振（SPR）技术得到了K-d = 39.60 +/- 1.78 nM，这与FRET得到的结果吻合得很好。这是首次报道的应用基于有机荧光团的“溶液中”简单定量FRET法获得VCAM-1/ vla4相互作用解离常数的工作，也是第一次对这种相互作用进行量化。此外，K-d的值可以作为异常蛋白-蛋白相互作用的指标；因此，该试验有可能进一步发展成为VLA-4/VCAM-1以及其他蛋白质-配体相互作用的药物筛选平台。
Very late antigen-4 (VLA-4), a member of integrin superfamily, interacts with its major counter ligand vascular cell adhesion molecule-1 (VCAM-1) and plays an important role in leukocyte adhesion to vascular endothelium and immunological synapse formation. However, irregular expressions of these proteins may also lead to several autoimmune diseases and metastasis cancer. Thus, quantifying the interaction affinity of the VCAM-1/VLA-4 interaction is of fundamental importance in further understanding the nature of this interaction and drug discovery. In this study, we report an 'in solution' steady state organic fluorophore based quantitative fluorescence resonance energy transfer (FRET) assay to quantify this interaction in terms of the dissociation constant (K-d). We have used, in our FRET assay, the Alexa Fluor 488-VLA-4 conjugate as the donor, and Alexa Fluor 546-VCAM-1 as the acceptor. From the FRET signal analysis, K-d of this interaction was determined to be 41.82 +/- 2.36 nM. To further confirm our estimation, we have employed surface plasmon resonance (SPR) technique to obtain K-d = 39.60 +/- 1.78 nM, which is in good agreement with the result obtained by FRET. This is the first reported work which applies organic fluorophore based 'in solution' simple quantitative FRET assay to obtain the dissociation constant of the VCAM-1/VLA-4 interaction, and is also the first quantification of this interaction. Moreover, the value of K-d can serve as an indicator of abnormal protein-protein interactions; hence, this assay can potentially be further developed into a drug screening platform of VLA-4/VCAM-1 as well as other protein-ligand interactions.