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Biochemistry. 2017 Aug 1;56(30):3962-3971. doi: 10.1021/acs.biochem.7b00436. Epub 2017 Jul 21.

Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport.

Author information

1
Department of Biochemistry, University of Zurich , Winterthurerstrasse 190, 8057 Zurich, Switzerland.
2
Department of Fundamental Neurosciences, University of Lausanne , Rue du Bugnon 9, 1005 Lausanne, Switzerland.
3
VIB Center for Structural Biology, VIB , 1050 Brussels, Belgium.
4
Structural Biology Brussels, Vrije Universiteit Brussel , 1050 Brussels, Belgium.
5
Institute of Biochemistry, Biocenter, Goethe-University Frankfurt , Max-von-Laue-Straβe 9, 60438 Frankfurt am Main, Germany.
6
Department of Applied Chemistry, School of Engineering, The University of Tokyo , Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
7
Institute of Parasitology, University of Zurich , Winterthurerstrasse 266a, 8057 Zurich, Switzerland.

Abstract

The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.

PMID:
28731329
DOI:
10.1021/acs.biochem.7b00436
[Indexed for MEDLINE]

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