Tracing of neurons plays an essential role in elucidating neural networks in the brain and spinal cord. Cholera toxin B subunit (CTB) is already widely used as a tracer although its use is limited by the need for immunohistochemical detection. A new construct incorporating non-canonical azido amino acids (azido-CTB) offers a novel way to expand the range and flexibility of this neuronal tracer. Azido-CTB can be detected rapidly in vivo following intramuscular tongue injection by ‘click’ chemistry, eliminating the need for antibodies. Cadmium selenide/zinc sulfide (CdSe/ZnS) core/shell nanoparticles were attached to azido-CTB by strain-promoted alkyne–azide cycloaddition to make a nano-conjugate. Following tongue injections the complex was detected in vivo in the brainstem by light microscopy and electron microscopy via silver enhancement. This method does not require membrane permeabilization and so ultrastructure is maintained. Azido-CTB offers new possibilities to enhance the utility of CTB as a neuronal tracer and delivery vehicle by modification using ‘click’ chemistry

Deuchars, J

Deuchars, S

Guo, Y

Haigh, J

Poole, E

Turnbull, WB

Webb, ME

Williamson, DJ

Zhou, D

English

Haigh, JL

Deuchars, SA

Leeds Beckett Repository

A versatile cholera toxin conjugate for neuronal targeting and tracing

White Rose Research Online

This journal is©The Royal Society of Chemistry 2020 Chem. Commun.Cite this:DOI: 10.1039/d0cc01085eA versatile cholera toxin conjugate for neuronaltargeting and tracing†Jessica L. Haigh, ab Daniel J. Williamson,a Emma Poole, a Yuan Guo, cDejian Zhou, a Michael E. Webb, a Susan A. Deuchars, b Jim Deuchars *band W. Bruce Turnbull *aTracing of neurons plays an essential role in elucidating neuralnetworks in the brain and spinal cord. Cholera toxin B subunit (CTB)is already widely used as a tracer although its use is limited bythe need for immunohistochemical detection. A new constructincorporating non-canonical azido amino acids (azido-CTB) offersa novel way to expand the range and flexibility of this neuronaltracer. Azido-CTB can be detected rapidly in vivo following intra-muscular tongue injection by ‘click’ chemistry, eliminating the needfor antibodies. Cadmium selenide/zinc sulfide (CdSe/ZnS) core/shellnanoparticles were attached to azido-CTB by strain-promotedalkyne–azide cycloaddition to make a nano-conjugate. Followingtongue injections the complex was detected in vivo in the brainstemby light microscopy and electron microscopy via silver enhancement.This method does not require membrane permeabilization and soultrastructure is maintained. Azido-CTB offers new possibilities toenhance the utility of CTB as a neuronal tracer and delivery vehicleby modification using ‘click’ chemistry.Cholera toxin produced by Vibrio cholerae is usually an infec-tious agent of the gut, but the toxin also has the potential to bedeveloped for more versatile tracing and targeting of neurons.It is formed of two main parts: the catalytically active A subunitand the non-toxic pentamer of B subunits (CTB) that facilitatecell entry via binding ganglioside GM1.1,2 GM1 is ubiquitouslyexpressed in vertebrates but is found in high relative abundance inthe nervous system, including in the periphery of motor neurons atthe neuro-muscular junction; for this reason CTB has been usedextensively as a neuronal tracer.3–6 It is usually detected viaantibody-mediated detection using fluorescence or 30-Diamino-benzidine (DAB) immunohistochemistry,7–9 but direct fluorophoreconjugates are also available10,11 as well as CTB–horseradishperoxidase (HRP) conjugates for detection via peroxidasechemistry.4,12 The ability to detect the tracer directly withoutthe need of antibodies is advantageous. It does not require anamplification step and so the labelling process is quicker, andcheaper as no antibodies are required. Detecting proteinswithout immunohistochemistry also provides more scope forlabelling multiple proteins without the need to consider cross-reactivity of primary and secondary antibodies as well asremoving issues with antibody batch variability and limitedshelf-life. Existing fluorescein–CTB conjugates are prone tobleaching, CTB–HRP conjugates are not compatible withimmunofluorescence methods and neither enables easy detec-tion by electron microscopy as membranes must be permeabi-lised to enable detection.One method of detecting proteins without using antibodiesis to use a bioorthogonal reaction such as copper-catalysedazide–alkyne cycloaddition (CuAAC) commonly referred to as‘click chemistry’.13–15 As the azide group is small it can beincorporated into probe molecules without interfering withtheir behaviour in live organisms, while providing the oppor-tunity for subsequent conjugation to fluorescent probes. Alter-natively, strain-promoted azide–alkyne cycloaddition (SPAAC)allows preparation of conjugates under copper-free conditionsthat are suitable for use in live cells and animals.16–18 Azide-functionalised amino acids can be incorporated into proteinsusing the methionine surrogate azidohomoalanine (Aha).19,20In methionine-depleted cultures, Aha is activated by methionyl-tRNA synthetase of methionine auxotroph Escherichia coli andreplaces methionine in translated proteins. In this communi-cation we report a novel azido-CTB toxoid and demonstrate itsutility for neuronal tracing in vivo. Azido-CTB can be made inany lab with protein production facilities and a variety ofmolecules may be readily conjugated in a site-specific mannerto suit individual needs.The isosteric replacement of methionine with Aha at thesurface of CTB required the creation of a new mutant toxoid.Three sequential rounds of site-directed mutagenesis were usedto replace all the native methionine residues with leucinesa School of Chemistry and Astbury Centre for Structural Molecular Biology,University of Leeds, Leeds LS2 9JT, UK. E-mail: w.b.turnbull@leeds.ac.ukb School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds,Leeds LS2 9JT, UKc School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, UK† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0cc01085eReceived 11th February 2020,Accepted 23rd April 2020DOI: 10.1039/d0cc01085ersc.li/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 28 April 2020. Downloaded on 5/5/2020 12:56:46 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView JournalChem. Commun. This journal is©The Royal Society of Chemistry 2020(M37L, M68L, M101L) and a surface exposed lysine residue withmethionine (K43M) (Fig. S1, ESI†). As the K43M mutation is onthe non-binding face of the protein (Fig. 1a), future chemicalmodifications at this site were predicted not to interfere withCTB–GM1 binding. Methionine auxotroph E. coli B834 Gold(DE3) cells were transformed with the new construct, pSAB2.3(Azido), and azido-CTB was overexpressed using a definedgrowth medium supplemented with Aha.19–21 The azido-CTBwas purified using Ni-affinity chromatography and size exclu-sion chromatography before analysis by SDS-PAGE and massspectrometry (Fig. S2, ESI†). For the wild type CTB protein, thetetra-mutant CTB is sufficiently stable to migrate as a pentamerduring SDS-PAGE unless the sample has been boiled prior toloading (Fig. 1b). While the native CTB normally gives rise to aprotein band around 45 kDa on a gel, azido-CTB is morenegatively charged than native CTB which likely explains whyit ran faster (apparent mass o40 kDa).The ability of the purified azido-CTB protein to enter motorneurons, transport in a retrograde manner to the brainstemand to be detected in vivo via CuAAC, was tested using tongueinjections in mice. Azido-CTB successfully traced the motorneurons of the hypoglossal nucleus that innervate the tongue; itwas detected using CuAAC with a biotinylated alkyne (Kerafast)followed by Alexa-555 labelled streptavidin (streptavidin-555) inparaformaldehyde-fixed brain tissue (Fig. 2b–cii). Streptavidinlabelling (shown in red) overlapped with immunohisto-chemical detection for CTB using an anti-CTB primary antibodyand secondary antibody labelled with Alexa-488), it wasdetected in the same cells and to the same extent (Fig. 2a–ci).Control brainstem slices with no azido-CTB injection under-going the click reaction and detection with streptavidin-555gave no positive cells, verifying that the signal in injected tissueis from azido-CTB (Fig. 2d). Azido-CTB can therefore be used asa neuronal tracer. If used in conjunction with immunohisto-chemistry to identify other proteins such as ion channels andreceptor subunits within the tissue, it will reduce limitationsto the primary antibody species that can be used since CTB isdetected without antibodies. Furthermore, azido-CTB has thepotential to be used to make a fluorescently tagged tracer fordelivery with any fluorescent molecule containing an alkyne group.To further extend the flexibility of azido-CTB, we usedits azide moiety for conjugation with nanoparticles prior toneuronal tracing. Correlative light and electron microscopyprovides a powerful and complementary alternative to immuno-histochemistry for studying sub-cellular localisation of probes.In this context, targeted nanoparticles can be used as nucleationsites for deposition of silver to facilitate their detection withoutthe need to permeabilise the tissue. We therefore investigated theconjugation of CdSe/ZnS core/shell nanoparticles to azido-CTBFig. 1 Design and structure of azido-CTB. (a) Crystal structure of CTB(red) bound to GM1 (black). Residue K43 (yellow). (b) SDS-PAGE gel(12% acrylamide) of azido-CTB elution fraction after Ni-NTA column (leftto right: molecular weight markers; boiled azido-CTB; non-boiled sampleshowing pentameric azido-CTB. (c) Cartoon showing cycloaddition ofazido-CTB with (d) a biotin-functionalised alkyne.Fig. 2 Azido-CTB as a neuronal tracer detected by CuAAC. (a) Wholebrainstem section stained with anti-CTB antibody (green), hypoglossalnucleus shown in white box. (b and c) Hypoglossal nucleus stained withanti-CTB antibody (green, b-ci), CuAAC (red, b-cii) and merged (b-ciii).(d) Control brainstem slice from non-injected mouse stained with thenuclear marker DAPI (blue, di), CuAAC (red, dii) and merged (diii). Scalebars: a = 500 mm; b = 50 mm; c = 10 mm; d = 100 mm.Communication ChemCommOpen Access Article. Published on 28 April 2020. Downloaded on 5/5/2020 12:56:46 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineThis journal is©The Royal Society of Chemistry 2020 Chem. Commun.and their ability to target neurons in vivo. The nanoparticles werefirst functionalised with a ligand mixture of dihydrolipoic acid(DHLA)-PEG11-cyclooctyne and a DHLA-PEGB15-OMe spacer viacap-exchange at a total ligand : nanoparticle molar ratio of3000 : 1, with 10% or 25% of the ligands being DHLA-PEG11-cyclooctyne ligand (Fig. 3).22–24 Azido-CTB was then directlyconjugated to the nanoparticles by SPAAC as confirmed by gelelectrophoresis (ESI,† Fig. S3). Site-specific biorthogonal label-ling of CTB with the nanoparticles means that the bindingface of CTB remains unobstructed and thus endocytosis ofthe complex should be unaltered, unlike other studies usingrandom derivations.25The CTB-functionalised nanoparticles (2 mL of 2.4 mM) wereinjected via the tongue in mice to assess whether the nano-conjugates could be endocytosed and transported in a retro-grade direction in vivo. Silver enhanced brainstem slicesrevealed that the nano-conjugates were in neurons of thehypoglossal nucleus after injection (Fig. 4a). Staining was foundoptimal when using azido-CTB conjugated to nanoparticlesprepared using 25% of the active DHLA-PEG11-cyclooctyneligand as shown in Fig. 3. Given the reduced amount ofCTB–nanoparticles delivered versus azido-CTB, there are fewerlabelled cells. These nanoparticles were not detected in miceinjected with just nanoparticles (Fig. 4b), indicating that theycannot transport in a retrograde manner without CTB. Thesilver enhanced slices were then prepared for EM where punctacorresponding to the metal deposits were found to be mainlylocalised to lysosomes (Fig. 4c). While CTB is known to travelin a retrograde manner to the Golgi,26,27 the appearance ofCTB–nanoparticle conjugates in the lysosome is also in agree-ment with other studies,28,29 and may be the final fate of CTBafter longer periods in vivo. Nonetheless, the easy application ofazido-CTB as a nanoparticle-conjugate could further improveits use as a neuronal tracer for EM analysis. It does not requirecellular permeabilisation, and thus avoids damage to cellularmembranes, which occurs when using agents such as TritonX-100 or ethanol to allow penetrance of antibodies. The easydetection of CTB–nanoparticle conjugates via correlated lightmicroscopy and EM, thus provides a much wider scope forpossible experiments with tissue. These experiments highlightthat alkynated substances can be readily and efficiently ‘‘clicked’’onto azido-CTB prior to injection, increasing the possibility forrapid testing of CTB as a powerful delivery vehicle to carry differentcargoes into neurons.In summary, azido-CTB presents a useful tool for diversifyingand enhancing its use as both a tracer and a delivery vehiclecompared to current commercially available CTB conjugates dueto ease of directed conjugation via CuAAC and SPAAC. Azido-CTBmay be further developed to create interchangeable conjugatedproteins via click chemistry; it could deliver multiple cargoes atonce, or cargo along with a reporter dye or even a nanoparticle.This has been proven with CTB–nanoparticle conjugates pro-duced from azido-CTB; where the nanoparticles were detectableafter injection in vivo. Azido-CTB presents a diverse tool fortracing that is subsequently detected both with fluorescenceFig. 3 Preparation of CTB–nanoparticle-based neuronal tracers fordetection by electron microscopy. Azido-CTB was conjugated via SPAACto dihydrolipoic acid-PEG11-cyclooctyne-functionalised CdSe/ZnS nano-particles prepared with 10% (n : m = 1 : 9) or 25% (n : m = 1 : 3) of the activeligand. It is assumed that the final ligand composition on the nanoparticlesis comparable to that of the mixture of DHLA ligands used in theirpreparation.Fig. 4 Use of CTB–nanoparticles as neuronal tracers for detection byelectron microscopy. (a) Hypoglossal nucleus (dotted lines) having under-gone silver enhancement, inset shows enlarged positive neurons.(b) Control brainstem slice from uninjected mouse having undergone silverenhancement. (c) Electron microscopy of hypoglossal nucleus cell (ci) andlysosomes inside that cell (cii). Labelling is confined to these structures. Scalebars: a and b = 100 mm; a inset = 25 mm; ci = 0.5 mm; cii = 0.2 mm.ChemComm CommunicationOpen Access Article. Published on 28 April 2020. Downloaded on 5/5/2020 12:56:46 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineChem. Commun. This journal is©The Royal Society of Chemistry 2020and EM, for use in live and fixed tissue, and for use in extendedtime-lapse experiments. Moreover, other theranostic functionscan be readily incorporated via conjugating to other functionalnanoparticles (e.g. magnetic nanoparticles for magnetic reso-nance imaging;30 gold nanorods for photothermal treatmentand/or photoacoustic imaging).31,32 With the increasing array ofmolecules and nanoparticles able to be ‘clicked’ to azides, thereis a wide scope of opportunity for azido-CTB to be utilised forefficient and rapid attachment of novel tracers, cargoes and/orincorporating with multiple theranostic functions.The authors thank the BBSRC for funding through DoctoralTraining Partnership BB/J014443/1, and research grant BB/M005666/1, and EPSRC for funding through Doctoral TrainingPartnership EP/M50807X/1, with equipment support from theWellcome Trust grants WT094232MA and 094232/Z/10/Z.Conflicts of interestThere are no conflicts to declare.References1 T. Beddoe, A. W. Paton, J. Le Nours, J. Rossjohn and J. C. Paton,Trends Biochem. Sci., 2010, 35, 411–418.2 E. A. Merritt, S. 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A versatile cholera toxin conjugate for neuronal targeting and tracing

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