1. Identification and characterization of Nanobodies targeting the EphA4 receptor
- Author
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Philip Van Damme, Bart De Strooper, Susan J. Little, Peter Weber, Lies Schoonaert, Robin Lemmens, Wim Robberecht, Peter Joyce, Ludo Van Den Bosch, Mieke Timmers, Gholamreza Hassanzadeh-Ghassabeh, Maarten Dewilde, Bart Roucourt, Laura Rué, Jurgen Haustraete, Adam P. Curnock, and Lucía Chávez-Gutiérrez
- Subjects
0301 basic medicine ,EPHRIN-BINDING ,FUSION PROTEIN ,single-domain antibody (sdAb,Nanobody) ,Cell ,Antibody Affinity ,Biochemistry ,Receptor tyrosine kinase ,CORTICOSPINAL TRACT ,protein-protein interaction ,Mice ,0302 clinical medicine ,CYCLIC PEPTIDE ANTAGONIST ,Medicine and Health Sciences ,SPINAL-CORD-INJURY ,SINGLE-DOMAIN ANTIBODIES ,Receptor ,biology ,phosphorylation ,Methods and Resources ,Receptor, EphA4 ,inhibition ,3. Good health ,Cell biology ,medicine.anatomical_structure ,Phosphorylation ,receptor-tyrosine kinase ,ligand-binding domain ,EPHA7 ,EphA4 ,VHH ,SMALL MOLECULES ,Protein–protein interaction ,Cell Line ,03 medical and health sciences ,Protein Domains ,REGENERATION ,medicine ,Ephrin ,Animals ,Humans ,Molecular Biology ,BLOOD-BRAIN-BARRIER ,Biology and Life Sciences ,Cell Biology ,Single-Domain Antibodies ,Fusion protein ,Molecular biology ,NERVOUS-SYSTEM ,030104 developmental biology ,regeneration ,biology.protein ,030217 neurology & neurosurgery ,AXONS - Abstract
The ephrin receptor A4 (EphA4) is one of the receptors in the ephrin system that plays a pivotal role in a variety of cell-cell interactions, mostly studied during development. In addition, EphA4 has been found to play a role in cancer biology as well as in the pathogenesis of several neurological disorders such as stroke, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (ALS), and Alzheimer's disease. Pharmacological blocking of EphA4 has been suggested to be a therapeutic strategy for these disorders. Therefore, the aim of our study was to generate potent and selective Nanobodies against the ligand-binding domain of the human EphA4 receptor. We identified two Nanobodies, Nb 39 and Nb 53, that bind EphA4 with affinities in the nanomolar range. These Nanobodies were most selective for EphA4, with residual binding to EphA7 only. Using Alphascreen technology, we found that both Nanobodies displaced all known EphA4-binding ephrins from the receptor. Furthermore, Nb 39 and Nb 53 inhibited ephrin-induced phosphorylation of the EphA4 protein in a cell-based assay. Finally, in a cortical neuron primary culture, both Nanobodies were able to inhibit endogenous EphA4-mediated growth-cone collapse induced by ephrin-B3. Our results demonstrate the potential of Nanobodies to target the ligand-binding domain of EphA4. These Nanobodies may deserve further evaluation as potential therapeutics in disorders in which EphA4-mediated signaling plays a role. ispartof: JOURNAL OF BIOLOGICAL CHEMISTRY vol:292 issue:27 pages:11452-11465 ispartof: location:United States status: published
- Published
- 2017