Unknown

Dataset Information

0

Antibody epitopes on g protein-coupled receptors mapped with genetically encoded photoactivatable cross-linkers.


ABSTRACT: 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 amino-terminal 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.

SUBMITTER: Ray-Saha S 

PROVIDER: S-EPMC3985944 | BioStudies | 2014-01-01

REPOSITORIES: biostudies

Similar Datasets

2007-01-01 | S-EPMC1855447 | BioStudies
1000-01-01 | S-EPMC3039059 | BioStudies
2004-01-01 | S-EPMC524989 | BioStudies
1000-01-01 | S-EPMC3239297 | BioStudies
1000-01-01 | S-EPMC5695146 | BioStudies
2002-01-01 | S-EPMC136018 | BioStudies
1000-01-01 | S-EPMC5807542 | BioStudies
2020-01-01 | S-EPMC7525492 | BioStudies
2009-01-01 | S-EPMC2853233 | BioStudies
2011-01-01 | S-EPMC3019634 | BioStudies