Antibody Epitopes on G Protein-Coupled Receptors Mapped with Genetically Encoded Photoactivatable Cross-Linkers
Antibody Epitopes on G Protein-Coupled Receptors Mapped with Genetically Encoded Photoactivatable Cross-Linkers
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DOI：
10.1021/bi401289p
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发表时间：
2014-03-04
期刊：
影响因子：
2.9
通讯作者：
Sakmar, Thomas P.
中科院分区：
文献类型：
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作者：
Ray-Saha, Sarmistha;Huber, Thomas;Sakmar, Thomas P.
我们开发了一种策略，用于创建与含有光交联剂的G蛋白偶联受体（gpcr）结合的单克隆抗体（mab）的表位图。以人CXC趋化因子受体4 （CXCR4）为模型系统，我们在细胞外环2 （EC2）的不同位置遗传地加入了对叠氮多- l -苯丙氨酸（azF）。然后，我们利用靶向功能丧失研究和基于微孔板的全细胞光交联，绘制了azF-CXCR4变体与mAb 12G5的相互作用图谱。我们使用了一种新的全细胞酶联免疫吸附测定法来定量交联效率。12G5主要与CXCR4的EC2中的184、178和189残基交联。将数据映射到CXCR4的晶体结构显示出独特的单抗表位足迹，光交联残基聚集在功能缺失位点周围。我们还使用靶向光交联方法研究了人CC趋化因子受体5 （CCR5）与PRO 140（一种抑制人类免疫缺陷病毒-1细胞进入的人源化单抗）和2D7的相互作用。单抗在CCR5的EC2上产生明显的交联模式。pro140主要与EC2的氨基末端残基174和175交联，而2D7主要与残基170、176和184交联。这些结果被映射到CCR5与maraviroc复合物的最新晶体结构中，显示出由EC2形成的maraviroc结合缝隙尖端的交联残基。作为在gpcr上定位单克隆抗体表位的策略，我们的靶向光交联方法是对功能缺失突变结果的补充，对于研究具有不连续表位的单克隆抗体尤其有用。
We developed a strategy for creating epitope maps of monoclonal antibodies (mAbs) that bind to G protein-coupled receptors (GPCRs) containing photo-cross-linkers. Using human CXC chemokine receptor 4 (CXCR4) as a model system, we genetically incorporated the photolabile unnatural amino acid p-azido-L-phenylalanine (azF) at various positions within extracellular loop 2 (EC2). We then mapped the interactions of the azF-CXCR4 variants with mAb 12G5 using targeted loss-of-function studies and photo-cross-linking in whole cells in a microplate-based format. We used a novel variation of a whole cell enzyme-linked immunosorbent assay to quantitate cross-linking efficiency. 12G5 cross-linked primarily to residues 184, 178, and 189 in EC2 of CXCR4. Mapping of the data to the crystal structure of CXCR4 showed a distinct mAb epitope footprint with the photo-cross-linked residues clustered around the loss-of-function sites. We also used the targeted photo-cross-linking approach to study the interaction of human CC chemokine receptor 5 (CCR5) with PRO 140, a humanized mAb that inhibits human immunodeficiency virus-1 cellular entry, and 2D7. The mAbs produced distinct cross-linking patterns on EC2 of CCR5. PRO 140 cross-linked primarily to residues 174 and 175 at the aminoterminal end of EC2, and 2D7 cross-linked mainly to residues 170, 176, and 184. These results were mapped to the recent crystal structure of CCR5 in complex with maraviroc, showing cross-linked residues at the tip of the maraviroc binding crevice formed by EC2. As a strategy for mapping mAb epitopes on GPCRs, our targeted photo-cross-linking method is complementary to loss-of-function mutagenesis results and should be especially useful for studying mAbs with discontinuous epitopes.