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J Biol Chem. 2017 Jul 7;292(27):11452-11465. doi: 10.1074/jbc.M116.774141. Epub 2017 May 19.

Identification and characterization of Nanobodies targeting the EphA4 receptor.

Author information

1
From the KU Leuven-University of Leuven, Department of Neurosciences, Experimental Neurology and Leuven Research Institute for Neuroscience and Disease (LIND), 3000 Leuven, Belgium.
2
VIB, Center for Brain and Disease Research, Laboratory of Neurobiology, 3000 Leuven, Belgium.
3
KU Leuven-University of Leuven, Laboratory for Signal Integration in Cell Fate Decision, 3000 Leuven, Belgium.
4
Vertex Pharmaceuticals (Europe) Ltd., Biology Department, OX14 4RW Abingdon, United Kingdom.
5
KU Leuven, Department of Neurosciences and Leuven Research Institute for Neuroscience and Disease (LIND), 3000 Leuven, Belgium.
6
Protein Service Facility, Inflammation Research Center, VIB, Ghent University, 9052 Ghent, Belgium.
7
Nanobody Service Facility, VIB, Vrije Universiteit Brussel, 1050 Brussels, Belgium.
8
Institute of Neurology, University College London, WC1E 6BT London, United Kingdom, and.
9
University Hospitals Leuven, Department of Neurology, 3000 Leuven, Belgium.
10
From the KU Leuven-University of Leuven, Department of Neurosciences, Experimental Neurology and Leuven Research Institute for Neuroscience and Disease (LIND), 3000 Leuven, Belgium, Wim.Robberecht@kuleuven.be.

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.

KEYWORDS:

EphA4; VHH; inhibition; ligand-binding domain; phosphorylation; protein-protein interaction; receptor-tyrosine kinase; regeneration; single-domain antibody (sdAb,Nanobody)

PMID:
28526745
PMCID:
PMC5500810
DOI:
10.1074/jbc.M116.774141
[Indexed for MEDLINE]
Free PMC Article

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