Generation and Characterization of Anti-VGLUT Nanobodies Acting as

Jul 21, 2017 - The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transp...
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Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport Stephan Schenck,*,†,● Laura Kunz,† Daniela Sahlender,‡,@ Els Pardon,§,∥ Eric R. Geertsma,†,⊥ Iaroslav Savtchouk,‡ Toshiharu Suzuki,# Yvonne Neldner,† Saša Štefanić,○ Jan Steyaert,§,∥ Andrea Volterra,‡ and Raimund Dutzler*,† †

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

S Supporting Information *

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. esicular glutamate transporters (VGLUTs) fill synaptic vesicles (SVs) at the presynaptic terminal with Lglutamate, the major excitatory neurotransmitter of the vertebrate brain.1,2 Besides this obvious role, VGLUTs potentially also contribute to the regulation of quantal size,3−5 vesicle release probability,6 and vesicular synergy.7−9 They might therefore be involved in numerous pathologies such as schizophrenia, neuropathic pain, and epilepsy.10−13 In SVs, the secondary active transport of glutamate by VGLUTs is driven by the activity of the vacuolar-type ATPase (V-ATPase) that generates a proton electrochemical gradient (ΔμH+) upon hydrolysis of ATP. A large body of evidence supports the idea that the membrane potential component (ΔΨ) of ΔμH+ is the major driving force for the transport of glutamate into synaptic vesicles.1,2,14−17 Research during the past decade has revealed a transport mechanism that is surprisingly intricate. In particular, the role of protons,16,18−20 chloride,15−17,20 potassium and sodium ions17,21 in the transport cycle and the influence of other SV membrane transporters on glutamate loading16,22,23 remains controversial. Our understanding of the transport cycle would undoubtedly benefit from molecular tools that facilitate the structural and functional characterization of the transporter.

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© XXXX American Chemical Society

A number of small molecule inhibitors are known to inhibit VGLUTs.24 Some of these are substrate analogues, such as amino-cyclopentane-1,3-dicarboxylate (trans-ACPD) or 4methyl-glutamate that block VGLUTs by competition with low potencies (Ki values of 0.7 and 0.4 mM, respectively) and might potentially be transported. They also interfere with plasma membrane glutamate transporters (EAATs) and metabotropic glutamate receptors. Several other potent effectors inhibit VGLUTs by less clear mechanisms such as the dyes Chicago Sky Blue, Trypan Blue, Evans Blue, Rose Bengal, and Brilliant Yellow.25−28 Despite their potency, specificity is not guaranteed in this class of inhibitors. For example, Evans Blue is known to affect ionotropic glutamate receptors, and Rose Bengal, although very potent with a Ki of 19 nM, also interferes with the V-ATPase. This has limited the application of small molecule inhibitors to isolated synaptic vesicles, and even in this comparably defined environment, side effects have to be taken into account. Thus, in the cellular Received: May 7, 2017 Revised: July 6, 2017

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DOI: 10.1021/acs.biochem.7b00436 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

obtained from Abcam (catalog no. ab9106). For immunocytochemistry, a monoclonal mouse anti-myc antibody (Millipore, catalog no. 05-724, clone 4A6) was used. Secondary HRP-conjugated antibodies were obtained from Bio-Rad and secondary antibodies for immunocytochemistry from Molecular Probes. For Western blots, proteins were transferred to polyvinylidene fluoride (PVDF) membranes prior to detection with ECL substrate (Millipore) on a Fuji LAS3000 imaging system. Chemicals. Detergents were obtained from Anatrace and other chemicals from Sigma unless stated otherwise. Nanobody Expression and Purification. Nanobodies were expressed in the periplasm of Escherichia coli MC1061 cells in Terrific Broth (TB) supplemented with 100 μg/mL ampicillin. Expression was induced with either 1 mM isopropyl β-D-1-thiogalactopyranoside for expression from a pMESy4 plasmid or 0.02% arabinose for expression from a pBXNPHM3 plasmid at an OD600 of 0.6−0.8. Expression continued for 3 h at 37 °C. After being harvested, cells were suspended in 50 mM Tris (pH 8) and 0.5 mM EDTA. After addition of 0.5 mg/mL lysozyme, the suspension was gently agitated for 30 min at room temperature (RT) and adjusted to 150 mM NaCl, 15 mM imidazole, 5 mM MgSO4, and 0.1 mg/mL DNase I. Cells were subsequently broken by sonication on ice, and debris was spun down for 30 min at 20000g. The supernatant was incubated in batch with 2 mL of nickel-nitrilotriacetic acid (NiNTA) resin for 4 L of bacterial culture for 2 h at 4 °C. The resin was washed with 20−30 column volumes (CV) of 150 mM NaCl and 25 mM imidazole (pH 7.5) and eluted in 10 mL of 150 mM NaCl and 250 mM imidazole (pH 7.5). After elution, PreScission Protease was added at a 1:20 molar ratio to cleave off the His10-MBP fusion protein during dialysis against 150 mM NaCl, 15 mM imidazole, and 20 mM Hepes-NaOH (pH 7.4) for 3 h at 4 °C. Subsequently, the sample was incubated with 0.5−1 mL of Ni-NTA resin to rebind the cleaved His10-MBP. The flow-through containing Nbs was concentrated to 0.5 mL using Amicon spin concentrators and subjected to size exclusion chromatography (SEC) on a Superdex 75 10/300 column (GE Healthcare) to separate the remaining traces of the His 10 -MBP protein. Fractions containing Nbs were pooled and used at concentrations of 0.2−1 mg/mL for functional investigations or further concentrated to 5−10 mg/mL for large scale complex formation with purified rVGLUT1. Nbs were stored at 4 °C or flash-frozen in liquid N2 for long-term storage at −80 °C. Venus-YFP− or mCherry−Nb fusion proteins were expressed and purified in tsA201 cells as described below for VGLUTs except for wash and elution buffers, which did not contain detergent. Expression and Purification of a Thermophilic ATP Synthase. The thermophilic His6-tagged ATP synthase from Bacillus sp. PS3 (TF0F1) encoded on plasmid pTR19ASDS was expressed in TB supplemented with 100 μg/mL ampicillin in E. coli DK8 cells (with the native unc operon deleted) under a constitutively active promoter as described previously.16 For expression, cells were grown in TB at 37 °C to an OD600 of 1− 3. After being harvested by centrifugation, the pellet was resuspended in 50 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, and 1 mg/mL lysozyme under gentle agitation at 25 °C for 30 min. After addition of MgSO4 (5 mM), the suspension was sonicated on ice. DNase I, Na2SO4, and sodium cholate were added to final concentrations of 1 mg/mL, 250 mM, and 0.7% (w/v), respectively. The suspension was stirred for 20 min at 25 °C

context of a neuron, the impact on other relevant proteins cannot be excluded. Recently, peptides29 and endogenous protein factors30 have been identified as inhibitors of vesicular glutamate transport, but they yet require detailed characterization and also lack VGLUT specificity.30,31 Besides their use in transporter pharmacology, molecules that target VGLUT may also be promising tools for structural studies. In crystallography, the resistance of a membrane protein to crystallization has frequently been overcome by specific binding proteins termed crystallization chaperones. Most crystallization chaperones that have been successfully used to date are derived from immunoglobulins.32 Among these, nanobodies (Nbs), the variable binding domains of heavy-chain camelid antibodies, are a particularly promising scaffold.33−36 Different from classical immunoglobulins, their protruding antigen binding site can effectively access buried epitopes, such as clefts or substrate entry sites of membrane transporters. Their simple architecture and superior biochemical properties together with wellestablished selection strategies make Nbs very attractive for membrane protein research.37,38 Besides their general suitability as crystallization chaperones, they were also shown to be useful as conformation-specific biosensors or to trap distinct conformations of a membrane protein thus interfering with its function.39,40 Here we describe the characterization of Nbs targeting rat VGLUT1 (rVGLUT1). Following the immunization of llamas, we identified four Nbs that bind the cytosolic face of the transporter, with two acting as potent inhibitors. We show that, by fusion to fluorescent proteins, the same binders can be used to label and inhibit VGLUT inside cells. The inhibitory anti-VGLUT Nbs thus provide useful pharmacological tools for characterizing the transport properties of VGLUTs in their cellular environment.

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MATERIALS AND METHODS DNA Constructs. Initial screening was performed with Nbs containing a C-terminal His6 tag from the vector pMESy4.38 For large scale purifications and functional characterizations, Nbs were expressed from the FX plasmid pBXNPHM335,36 as a C-terminal fusion to maltose binding protein (MBP) with an intercalary PreScission protease recognition sequence (LEVLFQ/GP). MBP was preceded by a pelB leader sequence followed by a His10 tag. Except for crystallization, all Nbs expressed as MBP fusion proteins contained a C-terminal myc tag. For expression in HEK cells, polymerase chain reaction (PCR)-amplified inserts were cloned into pcDNA3.1 derivatives for fragment exchange cloning.41 C-Terminally SBPtagged proteins were cloned into vector pcDXC3MS (Addgene #49030). For N- and C-terminal Venus-YFP-encoding plasmids, pcDXNSMG3 (Addgene #49029) and pcDXC3GMS (Addgene #49031) were used, respectively. For fusion proteins with mCherry, Venus-YFP was exchanged for mCherry using the Venus-YFP-flanking KpnI sites in the respective expression plasmids. Rat VGLUT1 cDNA (UniProt entry Q62634) and rat VGAT cDNA (UniProt entry O35458) were kindly provided by S. Takamori (Doshisha University, Kyoto, Japan). All constructs were verified by Sanger sequencing. Antibodies. Rabbit antibodies against VGLUT1 (catalog no. 135302) and VGLUT2 (catalog no. 135403), a guinea pig anti-VGLUT1 antibody (catalog no. 135304), and monoclonal antibody Cl7.2 against synaptophysin 1 (catalog no. 101011) were obtained from Synaptic Systems (Göttingen, Germany). The biotinylated goat anti-rabbit antibody was obtained from Sigma (catalog no. B8895). A rabbit anti-Myc antibody was B

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Biochemistry and centrifuged at 20000g and 4 °C. The pellet was resuspended in 100 mM KCl, 20 mM imidazole (pH 7.6), 5 mM MgSO4, and 1% DDM, stirred for 45 min at 25 °C, and centrifuged for 30 min at 20000g. The supernatant was incubated for 2 h at 25 °C in batch with ∼1.5 mL of Talon resin (Clontech) per liter of culture and washed with 20 CV of 100 mM KCl, 20 mM imidazole (pH 7.6), 5 mM MgSO4, and 0.05% DDM. TF0F1 was eluted with 250 mM imidazole (pH 7.6), 50 mM KCl, 5 mM MgSO4, and 0.05% DDM and dialyzed for 2−3 h at 25 °C against 20 mM NaCl, 20 mM Hepes-NaOH (pH 7.5), and 5 mM MgSO4. The sample was injected onto a MonoQ column (GE Healthcare), eluted with a NaCl gradient, and subsequently subjected to SEC on a Superdex 200 10/300 column (GE Healthcare) equilibrated in 100 mM KCl, 10 mM Hepes-KOH (pH 7.5), and 5 mM MgSO4. Fractions were analyzed for the presence of all subunits in the holoenzyme by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). TF0F1 was stored at 4 °C for several weeks without loss of activity. VGLUT Expression and Purification. VGLUT constructs were expressed in adherent tsA201 cells by transient transfection with a calcium phosphate precipitate as described previously.16 Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 units/mL penicillin, and 50 μg/mL streptomycin until confluency was reached and then split in a 1:5 ratio for transfection. For transfection of a single dish, 20 μg of plasmid DNA at 1 mg/mL in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA was mixed with 50 μL of 2.5 M CaCl2., 500 μL of H2O, and 500 μL of 50 mM BES (pH 7.0), 280 mM NaCl, and 2 mM Na2HPO4. The transfection mix was added to the cells after the mixture became slightly opaque. For large transfections, 50 mL of the transfection mix was prepared and added to 550 mL of a cell suspension, which was then seeded on dishes. The cells were placed at 2.2% CO2 in a humidified incubator for 36 h, harvested, and frozen in liquid N2 for storage at −80 °C. For purification, cells were lysed in 40 mL of lysis buffer/L of culture containing 300 mM KCl, 40 mM TrisHCl (pH 7.6), 2 mM EDTA, 2% DDM, and 1 mM phenylmethanesulfonyl fluoride. After incubation for 1 h at 4 °C, insoluble material was pelleted for 20 min at 8000g and the supernatant was incubated in batch with 0.5 mL of streptavidin resin (UltraLink, Pierce) and rotated at 4 °C for 2 h. The beads were washed with 20 CV of 300 mM KCl, 40 mM Tris-HCl (pH 7.6), 2 mM EDTA, and 0.05% DDM, and protein was eluted with 3 CV of 100 mM KCl, 13 mM Tris-HCl (pH 7.6), 0.6 mM EDTA, 0.03% DDM, and 3 mM D-(+)-biotin. Protein was concentrated to 1 mg/mL for reconstitution with TF0F1, or to 10 mg/mL for subsequent preparative SEC on a Superdex 200 10/300 column. For fluorescence-detection size-exclusion chromatography detecting the fluorescence of either tryptophan or Venus-YFP, a sample of the protein at a concentration of 0.1−0.5 mg/mL was used directly after elution from the affinity matrix without a further concentration step. Excess protein was flash-frozen in liquid N2 in 40 μL aliquots for storage at −80 °C. Anti-VGLUT Nanobody Generation in a Llama. VGLUT-specific nanobodies were generated as previously described.35,36,38 In brief, a llama (Lama glama) was immunized six times with a total of ∼1 mg of rVGLUT1AQmin in 150 mM NaCl, 20 mM Hepes (pH 7.4), and 0.03% DDM. Four days after the final boost, blood was taken to isolate peripheral blood

lymphocytes. RNA was purified from these lymphocytes and reverse transcribed by PCR to obtain cDNA. The resulting library was cloned into phage display vector pMESy438 bearing a C-terminal His6 and a CaptureSelect sequence tag (Glu-ProGlu-Ala). Eleven different VGLUT-specific nanobodies belonging to 10 different families were selected by biopanning. For this, rVGLUTAQmin devoid of the purification tags was incorporated at a VGLUT concentration of 100 μg/mL into liposomes containing 0.5% (w/w of total lipids) biotinylphosphatidylethanolamine (Avanti, catalog no. 870282) as described previously.16 The VGLUT liposomes were either coated directly on a solid phase or captured on Neutravidincoated plates. Phages bound to the antigen were recovered by limited trypsinization. After two rounds of selection, periplasmic extracts were prepared and subjected to enzymelinked immunosorbent assay (ELISA) screens.38 Generation of Nanobodies in Alpacas. Immunization of alpacas was performed four times in 14 day intervals using each time 100 μg of the purified rVGLUT1AQmin−Nb9 complex [in 150 mM NaCl, 10 mM Hepes (pH 7.4), and 0.03% DDM]. The antigen was mixed in a 1:1 (v/v) ratio with GERBU Fama adjuvant (GERBU Biotechnik GmbH, Heidelberg, Germany) and injected subcutaneously in 100 μL aliquots into the shoulder and neck region. At the end of immunization, 60 mL of blood was collected for preparation of the lymphocyte RNA, which was subsequently used to create cDNA by RT-PCR to amplify the Nb repertoire. The Nb sequences were cloned by fragment exchange cloning41 into a PmlI-linearized pDX phagemid vector using 336 ng of the Nb and 1 μg of the plasmid DNA. The resulting phage display library was screened by biopanning against the immobilized target. For that purpose, rVGLUT or the rVGLUT−Nb9 complex was either directly coated or immobilized as proteoliposomes containing biotinylphosphatidylethanolamine on Neutravidin-coated plates. Immunocytochemistry. Primary cortical neuronal cultures were prepared from embryonic day 18 (E18) B6 mouse brains as described previously.42 For that purpose, cells were plated on coverslips coated with poly-D-lysine (50 μg/mL) and laminin (2 μg/mL). Neuronal cultures were grown in Neurobasal medium supplemented with B27, glutamax, and penicillin/ streptomycin (Gibco). For immunofluorescence microscopy, neurons were fixed with 4% paraformaldehyde in phosphatebuffered saline (1×PBS) for 15 min. Subsequently, cells were quenched with 10 mM glycine in 1×PBS for 10 min. After permeabilization with 0.1% Triton X-100 for 5 min, cells were blocked in 1×PBS with 1% bovine serum albumin (BSA) for 15 min. Coverslips were subsequently incubated with Nbs for 45 min. After being washed with 1×PBS, cells were incubated with primary antibodies, including mouse monoclonal anti-Myc (Millipore) and guinea pig anti-VGLUT1 antibodies (Synaptic Systems), in 1×PBS and 0.5% BSA for an additional 45 min. After washes with 1×PBS, coverslips were incubated with secondary antibodies (Molecular Probes) and mounted in Prolong antifade (Molecular Probes). Coverslips were analyzed on a Leica confocal microscope at 63× magnification. Preparation of Crude SVs from Rat Brains. Ten adult Wistar rats were euthanized according to Swiss animal protection regulations using CO2. Crude synaptic vesicles (LP2 fraction) were isolated from rat brains by subcellular fractionation as described previously.43 LP2 was suspended in 320 mM sucrose and 10 mM Hepes (pH 7.4), adjusted to 2.5 mg/mL, and stored at −80 °C. C

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Biochemistry Liposome Preparation. For reconstitution of VGLUT and TF0F1, 40 mg/mL soybean phospholipids (type II-S from Sigma, catalog no. P5638) in 5% n-octyl β-D-glucopyranoside (OG) were mixed with cholesterol (5 mg/mL in 7% OG) to a cholesterol content of ∼25% (molar ratio), and dialysis buffer and proteins were subsequently added. Lipids and proteins were mixed at a ratio (w/w) of ∼50 for TF0F1 or ∼25 for VGLUT1. The final lipid concentration was 2.5 mg/mL after adjustment with dialysis buffer. The solution was dialyzed against 100 mM KCl, 2 mM Mg2SO4, and 5 mM MOPS-KOH (pH 7.3) for 12 h at 4 °C. After dialysis, excess DDM was complexed by addition of a 2-fold molar excess of 2,6-di-Omethyl-β-cyclodextrin (Sigma, catalog no. H0513) for at least 30 min. Prior to uptake measurements, 1−2 mL of liposomes was subjected to gel filtration on 15 mL Sephadex G25 columns to exchange the extraluminal solution to 100 mM potassium gluconate, 2 mM MgSO4, and 5 mM MOPS (pH 7.3). The turbid fractions eluting from the column were pooled and kept on ice until glutamate uptake was measured. For measurement of inhibition, Nbs were added either before dialysis (in a 3-fold molar excess relative to VGLUT1) to the premix containing VGLUT1, TF0F1, and lipids or after liposome preparation. Immunoprecipitation of Synaptic Vesicles with Magnetic Beads. For immunoprecipitation of SVs with streptavidin-coated magnetic beads (Dynabeads, M280, Thermo Fisher), 1.5 mL reaction tubes were blocked overnight in 150 mM NaCl, 10 mM MOPS (pH 7.3), and 2% (w/v) BSA. Beads were coated with saturating amounts of biotinylated goat antirabbit antibody. After being washed with 150 mM NaCl, 10 mM MOPS (pH 7.3), and 0.1% BSA, beads were additionally incubated with rabbit anti-VGLUT1 or anti-VGLUT2 antibody, and excess antibodies were washed out with the same buffer. As a control, no further antibody was added. As isoform-specific VGLUT antibodies were raised against the C-terminal cytoplasmic tails, overlap with the Nb epitopes could be excluded. Before immunoprecipitation, 100 μg of LP2 was suspended in 150 mM NaCl, 10 mM MOPS (pH 7.3), and 0.1% BSA in the presence or absence of additional 2% DDM to solubilize the membranes. Ten micrograms of either Nb3 or Nb9 (each with a C-terminal myc tag) was added to the LP2 suspension and incubated for 30 min. The LP2/Nb suspension was transferred to blocked 1.5 mL reaction tubes together with the antibody-coated magnetic beads and incubated for 45 min at room temperature while being slowly rotated. Beads were subsequently washed four times with 150 mM NaCl, 10 mM MOPS (pH 7.3), and 0.1% BSA with or without 0.05% DDM. With the last washing step, beads were transferred to a fresh, nonblocked 1.5 mL reaction tube, and captured organelles or proteins were eluted from the beads with 24 μL of 5× Laemmli buffer. Twelve microliters of the eluate was subjected to SDS− PAGE and transferred to PVDF membranes. Nbs were detected using a rabbit anti-myc antibody and synaptophysin using a monoclonal mouse antibody. See Figure S4 for a layout of the experiment. Co-Expression of Fluorescent Nbs and VGLUT1 in HEK Cells. The Nbs were cloned into plasmids encoding either Nterminal or C-terminal Venus-YFP in frame. For mCherry versions, the plasmids were modified by cloning mCherry at the KpnI sites flanking both ends of Venus-YFP. Wild-type (WT) rVGLUT1 was cloned into a plasmid lacking Venus-YFP. For co-expression with mCherry-tagged Nbs, VGLUT1 was expressed with a C-terminal Venus-YFP fusion. All constructs bore a myc-tag and an SBP-tag either preceding or following

Venus-YFP (see DNA Constructs for details of the plasmid). tsA201 HEK cells were transfected with calcium phosphate as described for production of VGLUT1. The precipitate was removed after 15 h, and the cells were supplemented with fresh medium and transferred to 5% CO2. For co-expression of WT rVGLUT1 and Nbs, plasmid DNA was transfected at a ratio of 5:1 (VGLUT:Nb). Cells were imaged at 20× magnification using epi-fluorescence. Glutamate Uptake. Uptake of glutamate was measured at 32 °C in 100 mM potassium gluconate, 2 mM MgSO4, and 5 mM MOPS (pH 7.3) (uptake buffer) and final concentrations of 4 mM ATP, 40 μM potassium glutamate, 10 mM potassium aspartate, and 2 μCi of [3H]glutamic acid (GE Healthcare) (reaction mix) by addition of liposomes to a 10× reaction mix. The reaction was stopped by the transfer of 100−500 μL of a liposome suspension into 4 mL of ice-cold uptake buffer. The liposomes were subsequently filtered through nitrocellulose filters and washed three times with 3 mL of ice-cold uptake buffer, and trapped radioactivity was measured by liquid scintillation with a PerkinElmer β-counter. Proteoliposomes containing a total of 10−20 μg of VGLUT1 were assayed per data point. For uptake of glutamate into the LP2 fraction, 100 μg was assayed per data point under the same conditions. For inhibition, Nbs were added in 5-fold molar excess to the estimated VGLUT content in this fraction based on previously published literature data44 (∼10 μg of Nb/100 μg of LP2) 20 min before the initiation of uptake. Crystallization and X-ray Structure Determination. For crystallization, equimolar amounts of Nb9 and aNb were mixed and purified by SEC on a Superdex 200 10/300 column. The fractions corresponding to the Nb−Nb heterodimer were pooled and concentrated to 25 mg/mL. Crystals were grown by vapor diffusion in sitting drops by mixing 1 μL of protein with 1 μL of a reservoir solution containing 3.5 M sodium formate, and drops were equilibrated against 500 μL of a reservoir solution. Crystals were harvested, cryoprotected by addition of mother liquor containing additional 30% ethylene glycol, and flash-frozen in liquid propane. Data were collected on frozen crystals on the X06DA beamline at the Swiss Synchrotron Light Source of the Paul Scherrer Institute on a PILATUS 2M-F Detector (Dectris). Data were processed with XDS45 (Table S1), and the structure was determined by Molecular replacement with Phaser46 using the structure of a modified nanobody as a search model. The model was rebuilt with COOT47 and refined with PHENIX48 (Table S1). The final model contains 7373 protein atoms and 479 waters. Atomic coordinates and structure factors are available in the Protein Data Bank (PDB entry 5OCL).

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RESULTS Immunization and Nanobody Selection. To generate Nbs that target the membrane portion of rVGLUT1, we used a truncated and nonglycosylated mutant of the transporter. The truncated protein, which encompasses residues 58−515, lacks the entire N-terminus preceding transmembrane α-helix 1 and a large part of the highly flexible C-terminus. Furthermore, the sites for N-linked glycosylation, Asn92 and Asn93, were mutated to alanine and glutamine, respectively. This construct (rVGLUT1AQmin) was overexpressed and purified from human embryonic kidney tsA201 cells. The homogeneity and lack of glycosylation of the purified protein were verified by SEC, SDS−PAGE, and mass spectrometry (Figure S1). Following the immunization of a llama (L. glama) with DDM-solubilized D

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Biochemistry rVGLUT1AQmin, 11 ELISA-positive Nbs could be retrieved by phage display from a cDNA library generated from isolated lymphocytes using rVGLUT1AQmin proteoliposomes as bait (Figure S2). These Nbs were successfully overexpressed as Histag fusion proteins in the periplasm of E. coli and purified via Ni-NTA chromatography. We validated the binding of the purified Nbs to rVGLUT1AQmin by monitoring the co-elution of both proteins via SEC. From the initial selection of 11 Nbs, we could confirm complex formation for four of them (i.e., Nb3, -7, -9, and -10). As an example, a large scale preparation of the rVGLUT1AQmin−Nb3 complex is shown in Figure 1.

Figure 2. Immunocytochemical labeling of VGLUT-positive boutons with purified Nbs. Mouse primary cortical neurons were labeled with a VGLUT1-specific antibody and myc-tagged (A) Nb9 or (B) Nb3. Nuclei were stained with DAPI. Cells were imaged with a confocal microscope at 63× magnification. Left panels show the signal of a fluorescently labeled anti-guinea pig antibody binding to the antiVGLUT1 antibody. Middle panels show the signal of the myc-tagged Nbs. Right panels show a merged image of both pictures. Rectangles indicate magnified regions shown in the bottom row. In case of Nbs, part of the background might originate from binding to VGLUT2.

Figure 1. Large scale preparation of the rVGLUT1−Nb3 complex. The chromatogram shows a size exclusion chromatography profile of purified rVGLUT1AQmin after removal of the purification tag that was incubated with an excess of purified Nb3. The peak at 11.9 mL corresponds to the rVGLUT1AQmin−Nb3 complex, and the peak at 17.8 mL is dominated by excess Nb3. Symbols: rVGLUT1AQmin (●), Nb3 (▼), 3C-protease (▲), and the cleaved affinity tag (*). The molecular weights of marker proteins (left lane) are indicated in kilodaltons.

VGLUT2 antibodies that recognize the C-terminal tail of the transporters were then used to pull down either intact SVs or the solubilized transporters. The co-immunoprecipitated Nbs were subsequently detected by Western blotting with an antimyc antibody. Detection of the SV-resident protein synaptophysin served as a control for the completeness of SV solubilization. In case of a luminal epitope, Nbs would be detectable only in the presence of detergent. As seen in Figure 3A, both Nbs co-immunoprecipitated with intact SVs, which is compatible with a cytosolic epitope in VGLUTs. Because the Nbs were also detected in the VGLUT2 pull down with solubilized SVs (although with a weaker signal because of the lower abundance of VGLUT2 in total brain preparations), they likely do not discriminate between the two major VGLUT isoforms. This is expected, given the high degree of identical residues (87.5%) between the two paralogs in the core region of both transporters. Anti-VGLUT Nbs as Inhibitors of Transport. To clarify whether the binding of the anti-VGLUT Nbs would be conformation-specific, thereby locking a specific state of the transporter, we tested if the addition of Nbs would block the transport of glutamate into VGLUT-containing proteoliposomes and native SVs. For this purpose, WT rVGLUT1, expressed and purified from tsA201 cells, was co-reconstituted with a thermophilic ATP synthase (to generate VGLUT1TF0F1 liposomes)16 in the absence of Nbs or after incubation

Anti-VGLUT1 Nbs as Tools for Immunocytochemistry. Because the Nbs were selected with a truncated and nonglycosylated transport protein, we tested if native VGLUT1 would be recognized in neurons. For that purpose, we purified C-terminally myc-tagged Nb3 and Nb9 and analyzed the staining pattern by immunocytochemistry in primary cortical neuronal cultures. Both Nbs stained synaptic boutons and faithfully reproduced the staining pattern of a guinea pig anti-VGLUT1 antibody (Figure 2), whereas denaturation of the Nbs largely abolished specific labeling (Figure S3). Thus, Nb3 and Nb9, which were generated in response to the injection of an engineered VGLUT1 construct, also bind native VGLUT1 in SVs, which makes them suitable tools for immunocytochemistry. However, because the labeling requires permeabilization of membranes, these experiments do not allow us to draw any conclusion about the localization of the epitope. Localization of the Epitopes. To address the question of whether binding occurs on the cytosolic or luminal side of rVGLUT1, we isolated a crude preparation of SVs (LP2 fraction) from rat brains. We added purified myc-tagged Nb3 or Nb9 in the presence or absence of detergent to suspended LP2 vesicles (see Figure S4 for the experimental layout). Magnetic beads coated with isoform-specific anti-VGLUT1 or antiE

DOI: 10.1021/acs.biochem.7b00436 Biochemistry XXXX, XXX, XXX−XXX

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decreased, thus indicating that the inhibition is due to a true blockage of the transporter. Because Nb3 and Nb9 bound to intact SVs (Figure 3A), we also quantified the uptake of glutamate into isolated crude SVs (LP2 fraction) from rat brains after addition of either Nb (Figure S5A). Also with SVs, we could confirm the inhibition of glutamate uptake in the presence of Nb3 or Nb9 (Figure 3C), further supporting their inhibitory activity upon binding to a cytosolic epitope of the transporter. Intracellular Expression of Nanobodies Fused to Fluorescent Proteins. Previously, a Nb recognizing a certain conformation of the β2-adrenoceptor fused to green fluorescent protein (GFP) could be used to track the state of the receptor by being recruited to membranes from a cytosolic pool.39 Because we could show that Nb3 and Nb9 bind the cytosolic face of VGLUT1, we tested whether they could be expressed as fusions of fluorescent proteins in the cytosol of tsA201 cells and label co-expressed VGLUT1 inside cells. Upon expression of the Nbs in tsA201 cells as Venus-YFP or mCherry fusion proteins, a cytosolic localization of the fusion proteins was apparent (Figure S6A,B). We expressed the Nbs with an Nterminal or a C-terminal Venus-YFP (NV or CV) or mCherry (NC or CC). The expressed proteins were amenable to purification and eluted as monodisperse species via SEC (Figure 4A). SDS−PAGE under reducing and nonreducing conditions further confirmed the absence of artificial covalent oligomers as a result of aberrant disulfide bridge formation (Figure S6C). When the Nb−GFP fusion proteins were coexpressed with rVGLUT1 (devoid of a GFP tag), their cytosolic distribution changed to a more punctuate staining (Figure S6A,B), resembling the localization of an rVGLUT1−VenusYFP fusion protein (Figure S6D). The recruitment to intracellular membranes in the presence of VGLUT1 indicates that the Nb−GFP fusion proteins are functionally expressed in the cytosol of tsA201 cells and also confirms a cytosolic epitope for Nb7 and Nb10 (Figure S6A,B). Upon co-expression of rVGLUT1−Venus-YFP (rVGLUT1CV) with mCherry-tagged Nb9 (NC-Nb9), near-perfect colocalization was observed, which was absent when the Nb was co-expressed with the N-terminally Venus-YFP-tagged rat vesicular GABA transporter (NV-rVGAT) (Figure S6E). After scale-up of expression to a liter of adherently cultivated tsA201 cells (corresponding to 100 10 cm dishes), we isolated ∼2 mg of purified NV-Nb9. Because only modest levels of protein are required for functional experiments, these mammalian cells provide an attractive expression platform for the generation of fluorescent binders in numerous applications without the requirement of extensive optimizations. Purified NV-Nb9 was fully functional as shown by complex formation with solubilized rVGLUT1 (Figure 4A) and by labeling of synaptic vesicles in neuronal cultures without the use of a dye-labeled secondary antibody (Figure 4B). Structure of an Anti-VGLUT Nb in Complex with an Alpaca Nanobody. As all anti-VGLUT Nbs selected from llama recognize a cytoplasmic epitope of the transporter, we attempted to generate a distinct set of binders by immunization of alpacas (Vicugna pacos) with a VGLUT−Nb9 complex with the hopes of identifying Nbs interacting from the luminal side. Following the preparation of a phage display library, we succeeded in isolating an alpaca nanobody [aNb (Figure S7)] that recognizes Nb9 and the VGLUT−Nb9 complex, but not the transporter itself (Figure S8). Whereas the screening with binary complexes of various VGLUT1 constructs with Nb3 and

Figure 3. Sidedness of Nb binding and inhibition of transport. (A) Coimmunoprecipitation experiments of Nbs with synaptic vesicles (SVs) confirm a cytoplasmic epitope of the transporter. A crude preparation of SVs incubated with myc-tagged Nb3 (3) and Nb9 (9) was pulled down with VGLUT1 or VGLUT2 antibodies immobilized on magnetic beads (no detergent). The same procedure was repeated with solubilized SVs (detergent). Nbs were detected on immunoblots with an anti-myc antibody (α-myc, top). An antibody recognizing the protein synaptophysin present in synaptic vesicles was used as a control (α-Syp, bottom). Immunoblots of the respective solutions prior to pull down are shown at the left. Control lanes show pull-down experiments with an anti-rabbit antibody. The molecular weight is indicated on the right. (B) Inhibition of uptake of glutamate by proteoliposomes. Uptake of [3H]glutamate by proteoliposomes containing purified WT rVGLUT1 mixed in a 3-fold stoichiometric excess with Nb3, -7, -9, and -10 prior to co-reconstitution of the transporter with a proton pump. (C) Inhibition of uptake of glutamate by synaptic vesicles. Uptake of [3H]glutamate by crude rat SVs (100 μg of LP2) incubated with excess Nb3 and Nb9. Uptake in the absence of ATP was subtracted, and data were normalized to control uptake. Data in panels B and C were measured in triplicate. Error bars represent the standard deviation of the mean.

with Nb3, -7, -9, or -10. In the respective samples, Nbs were added in 3-fold molar excess to rVGLUT1 prior to the formation of the proteoliposomes to ensure that all transporters would be bound, independent of their orientation in the membrane (Figure S5A). As shown in Figure 3B, all four Nbs decreased the rate of uptake of glutamate by proteoliposomes with a nearly complete inhibition by Nb3 and Nb9. To exclude the possibility that this effect is due to interference with the reconstitution of VGLUT1, we also tested the inhibition by addition of Nb3 and Nb9 to the outside of preformed VGLUT1-TF0F1 liposomes prior to the uptake measurements (Figure S5B). Even in this case, the rate of uptake was strongly F

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Figure 4. Labeling of VGLUT inside cells with tagged Nbs. (A) Formation of a complex with labeled Nbs. Fluorescent Nbs purified from tsA201 cells were incubated with nonfluorescent purified rVGLUT1 and subjected to SEC. N-Terminally Venus-YFP-tagged Nb9 (Nb9) co-elutes with rVGLUT1 and shifts its elution volume toward higher molecular weights. The elution volume of an N-terminally Venus-YFP-tagged control Nb (NbX) was not affected by the presence of rVGLUT1. The elution of rVGLUT1 (VGLUT) in the absence of Nbs was monitored via tryptophan fluorescence; for all other samples, elution was monitored by Venus-YFP fluorescence. (B) VGLUT in synaptic termini labeled by a guinea pig antiVGLUT1 antibody (left) and purified NV-Nb9 (center). The merged images (right) show nearly perfect overlap.

ternary complex (Figure 5 and Figure S8B). The structure of the aNB−NB9 complex thus has revealed the architecture of a specific VGLUT binder, and it displays the interactions leading to Nb−Nb recognition.

Nb9 or a ternary complex consisting of aNb bound to the VGLUT−Nb9 assembly did not allow us to obtain any crystals, we succeeded with crystallization and structure determination of a complex consisting of aNb bound to Nb9 at a resolution of 2.2 Å (Table S1 and Figure S9A). The asymmetric units of these crystals contain four copies of the Nb heterodimer all exhibiting the same binding mode (Figure 5 and Figure S9B). The complex buries 1342 Å2 of the

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DISCUSSION As the responsible transporters for the loading of synaptic vesicles with the neurotransmitter glutamate, VGLUTs play a central role in neurotransmission. Specific inhibitors of transport would thus provide valuable tools for improving our understanding of this process, and they may also be pharmacologically relevant in a number of VGLUT-related pathologies. However, the intracellular localization of VGLUTs makes it very demanding to identify specific small molecule effectors in live cells in a high-throughput manner and to rule out adverse effects on other steps in the synaptic vesicle cycle or postsynaptic effects, such as activation or inhibition of glutamate receptors. In this study, we have generated Nbs against rat VGLUT1. Because of their high specificity, these proteinaceous binders provide ideal tools for a specific targeting of VGLUT function. We found that two of the selected Nbs act as efficient VGLUT inhibitors by recognizing a cytoplasmic epitope (Figure 3A and Figure S6A,B,E), which permitted us to interfere with the transport of glutamate into reconstituted proteoliposomes and SVs (Figure 3B,C). These Nbs likely do not discriminate between VGLUT1 and -2, and although this has not been tested, they might potentially also bind to VGLUT3 and orthologues from related species. Given the high degree of conservation, it is to be expected that they also recognize human VGLUT1 (which shares 99% of identical residues with rVGLUT1). Although Nbs originate from secreted proteins and contain a disulfide bridge, we succeeded in expressing both inhibitory Nbs as functional binders in the cytosol of HEK cells (Figure S6A,B,E), which makes them promising tools for targeting VGLUTs inside cells. For that purpose, the Nbs could be delivered with viruses or by transfection to a number of cell types, including neurons. By using cell-type-specific promoters, the targeted expression of Nbs could be used to block VGLUT activity in the cells of interest, for example in dopaminergic or

Figure 5. Structure of a heterodimeric Nb9−aNb complex. Both proteins are shown as ribbons with the constant and variable regions of the aNb colored beige and blue and those of Nb9 colored green and red, respectively. Termini, the locations of the variable complementary regions (CDRs), and secondary structure elelments are indicated.

combined molecular surface and involves residues of all three variable regions (CDR1−CDR3) of the aNb that predominantly bind to the constant region of Nb9 (Figure 5 and Figure S9B). The specificity of aNb toward Nb9 could be explained by a small number of interactions with residues at the turn of the variable CDR2 region connecting β4 and β5 of Nb9, which are not common to all llama nanobodies. The bulk of the variable part, including the long CDR3 region of Nb9 (connecting β8 and β9), is remote from the interaction surface and thus remains accessible for interactions with VGLUT in a G

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determined the structure. S.S. and R.D. wrote the paper with input from all other authors.

cholinergic neurons, where glutamate has been found to act as a co-transmitter.7,8 Apart from their application in neurons, these Nbs could also help to settle the controversy about the release of vesicular glutamate from astrocytes, which are thought to play an important role in synaptic plasticity using glutamate as a gliotransmitter.49−52 Such experiments would complement tissue- and isoform-specific VGLUT knockout experiments. Because the Nbs can be expressed in large quantities, they could also be introduced into host cells by microinjection. Their fusion to fluorescent proteins would allow for monitoring of the success of a microinjection and dispersal of the Nb inside the cell. Recently, VGLUTs have been successfully targeted to the plasma membrane of HEK cells and Xenopus laevis oocytes for the study of their transport mechanism by electrophysiology and cellular uptake assays.20 In these model systems, inhibitory Nbs should readily block VGLUTs by co-expression and thus help to further dissect the transport mechanisms. VGLUTmediated transport has also been investigated in reconstituted liposomes and by generating hybrid SVs that are fused with liposomes to modulate their internal contents.15−17,20 Also in such experiments, and in experiments that are based on purified SVs,21 highly specific inhibitors of VGLUTs could prove to be useful. One controversial question concerns the glutamateindependent conduction of ions by VGLUTs,16,17,20 as it is still not clear whether the flux is channel-like (resembling a leak) or linked to movements in the transporter. Here, the blockage of movements in VGLUT by Nbs could provide important insights. Finally, our characterization of anti-VGLUT Nbs in a cellular environment reinforced the general usefulness of these protein binders as intracellular tools that should be widely applicable for both membrane-bound and cytosolic targets.

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Funding

This work was supported by the Swiss National Science Foundation through the NCCR Transcure. Nanobody selection was supported by Instruct, part of the European Strategy Forum on Research Infrastructures (ESFRI), and the Hercules Foundation Flanders. E.R.G. acknowledges support from the Human Frontier Science Program (long-term fellowship LT00899/2008) and the German Research Foundation through the Cluster of Excellence Frankfurt “Macromolecular Complexes”. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Eva Beke for technical assistance during nanobody selection in llamas, Ines A. Ehrnstorfer for providing a control nanobody, Peter Deplazes (University of Zurich) for supporting alpaca immunization, and Dagmar Schaefer and Gregor Fischer (Laboratory Animals Service Center, University of Zurich) for providing and euthanizing rats. We acknowledge the Functional Genomics Center of the ETHZ/UZH for mass spectrometry and thank Prof. Shigeo Takamori (Doshisha University) for the cDNAs of rVGLUT1 and rVGAT.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00436. Supplementary Figures S1−S9 and supplementary Table S1 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Raimund Dutzler: 0000-0002-2193-6129 Present Addresses ●

S.S.: Laboratory of Biomolecular Research, Paul Scherrer Institut, CH-5232 Villigen, Switzerland. @ D.S.: Bioelectron Microscopy Core Facility, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland. Author Contributions

S.S. designed research, purified proteins, and performed biochemical experiments. L.K. characterized alpaca Nbs and crystallized proteins. D.S., I.S., and A.V. performed immunocytochemistry. E.P., E.R.G., and J.S. generated and selected Nbs with VGLUT1. T.S. designed pTR19ASDS carrying His-tagged ATP synthase. Y.N. and S.Š. generated and selected alpaca Nbs. S.S., L.K., D.S., E.P., A.V., and R.D. analyzed data. R.D. H

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