Matthew J Ovens

Andrew J Davies

Marieangela C Wilson

Clare M Murray

Andrew P Halestrap
email: a.halestrap@bristol.ac.uk

AR-C155858 is a potent inhibitor of Monocarboxylate Transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10

Keywords: Erythrocytes, lactate transport, MCT1, MCT2, MCT4, chimeric transporters, Xenopus oocytes

AR-C155858 is a potent inhibitor of monocarboxylate

transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10

Introduction

There are 14 members of the monocarboxylate transporter (MCT) family (SLC16) encoded by the human and mouse genomes [START_REF] Halestrap | The SLC16 gene family -from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond[END_REF]. Of these only MCT1, MCT2, MCT3 and MCT4 have been demonstrated to catalyse the bidirectional proton-linked transport of short chain monocarboxylates such as L-lactate and pyruvate across the plasma membrane of mammalian cells [START_REF] Bröer | Characterization of the monocarboxylate transporter 1 expressed in Xenopus laevis oocytes by changes in cytosolic pH[END_REF][START_REF] Bröer | Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes[END_REF][START_REF] Grollman | Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast[END_REF][START_REF] Dimmer | The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells[END_REF][START_REF] Manning Fox | Characterisation of human Monocarboxylate Transporter 4 substantiates its role in lactic acid efflux from skeletal muscle[END_REF]. MCT1 is expressed in most tissues and facilitates lactic acid uptake for oxidation in heart and red skeletal muscle, and for gluconeogenesis in the liver and kidney of some species [START_REF] Kim-Garcia | Molecular characterization of a membrane transporter for lactate, pyruvate, and other monocarboxylates -implications for the Cori Cycle[END_REF][START_REF] Mccullagh | Role of the lactate transporter (MCT1) in skeletal muscles[END_REF][START_REF] Jackson | Cloning of the monocarboxylate transporter isoform MCT2 from rat testis provides evidence that expression in tissues is species-specific and may involve post-transcriptional regulation[END_REF][START_REF] Halestrap | Lactate transport in heart in relation to myocardial ischemia[END_REF][START_REF] Halestrap | The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation[END_REF]. MCT1 is also used for lactic acid efflux by some cells that are exclusively glycolytic such as red blood cells and by all cells under hypoxic conditions [START_REF] Halestrap | The SLC16 gene family -from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond[END_REF][START_REF] Halestrap | Lactate transport in heart in relation to myocardial ischemia[END_REF][START_REF] Poole | N-Terminal protein sequence analysis of the rabbit erythrocyte lactate transporter suggests identity with the cloned monocarboxylate transport protein MCT1[END_REF]. MCT2 is a higher affinity transporter [START_REF] Bröer | Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes[END_REF] whose expression is more restricted and highly species dependent [START_REF] Halestrap | The SLC16 gene family -from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond[END_REF]. In some species it is the dominant MCT isoform in kidney and liver where it facilitates lactic acid uptake for gluconeogenesis [START_REF] Jackson | Cloning of the monocarboxylate transporter isoform MCT2 from rat testis provides evidence that expression in tissues is species-specific and may involve post-transcriptional regulation[END_REF][START_REF] Garcia | cDNA cloning of MCT2, a second monocarboxylate transporter expressed in different cells than MCT1[END_REF]. It is also expressed in neurons, especially at the post-synaptic density, and has been proposed to provide the uptake pathway for the oxidation of lactate produced by the more glycolytic astrocytes [START_REF] Pierre | Monocarboxylate transporters in the central nervous system: distribution, regulation and function[END_REF]15]. MCT3 expression is confined to the basal membrane of the retinal pigment epithelium and choroid plexus epithelia [16,17], but detailed information on its substrate and inhibitor specificity is lacking [START_REF] Grollman | Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast[END_REF]. MCT4 is a lower affinity transporter [START_REF] Dimmer | The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells[END_REF][START_REF] Manning Fox | Characterisation of human Monocarboxylate Transporter 4 substantiates its role in lactic acid efflux from skeletal muscle[END_REF] and is primarily expressed in highly glycolytic cells such as white muscle fibres where it is used to facilitate lactic acid efflux from the tissue [START_REF] Wilson | Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3[END_REF][START_REF] Juel | Lactate transport in skeletal muscle -role and regulation of the monocarboxylate transporter[END_REF]. Most cells can up-regulate MCT4 expression under hypoxic conditions when glycolytic flux is enhanced and this is mediated through transcriptional control by hypoxia inducible factor 1α (HIF-1α) [START_REF] Ullah | The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1 alpha-dependent mechanism[END_REF].

In view of the importance of plasma membrane lactic acid transport in metabolism, it would be desirable to have a specific inhibitor of each isoform that could probe their individual metabolic roles. A range of inhibitors of MCTs have been described including the α-cyanocinnamate derivatives, stilbene disulphonates such as di-isothiocyanostilbene disulphonate (DIDS), phloretin, bioflavenoids such as quercetin and organomercurial reagents such as p-chlomercuribenzene sulphonate (see [START_REF] Halestrap | The SLC16 gene family -from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond[END_REF][START_REF] Poole | Transport of Lactate and Other Monocarboxylates Across Mammalian Plasma Membranes[END_REF][START_REF] Meredith | The SLC16 monocaboxylate transporter family[END_REF]). However, none of these is specific for inhibition of MCTs and thus they cannot be used with confidence in metabolic studies. This reservation applies particularly to αcyano-4-hydroxycinnamate that some workers have used on the assumption that it is a specific MCT inhibitor (see for example [START_REF] Sonveaux | Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice[END_REF][START_REF] Fang | The H+-linked monocarboxylate transporter (MCT1/SLC16A1): a potential therapeutic target for high-risk neuroblastoma[END_REF][START_REF] Erlichman | Inhibition of monocarboxylate transporter 2 in the retrotrapezoid nucleus in rats: a test of the astrocyte-neuron lactate-shuttle hypothesis[END_REF]). However, this agent is at least two orders of magnitude more potent at inhibiting the mitochondrial pyruvate carrier than MCT1 [START_REF] Halestrap | Pyruvate and lactate transport into human erythrocytes. Evidence for the involvement of the chloride carrier and a chloride independent carrier[END_REF][START_REF] Halestrap | Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by α-cyano-4-hydroxycinnamate[END_REF][START_REF] Halestrap | The mitochondrial pyruvate carrier -Kinetics and specificity for substrates and inhibitors[END_REF][29]. Recently, a new class of specific and extremely high affinity inhibitors of MCT1 have been discovered by AstraZeneca [START_REF] Murray | Monocarboxylate transporter MCT1 is a target for immunosuppression[END_REF][START_REF] Guile | Potent blockers of the monocarboxylate transporter MCT1: Novel immunomodulatory compounds[END_REF][START_REF] Ekberg | The specific monocarboxylate transporter-1 (MCT-1) inhibitor, AR-C117977, induces donorspecific suppression, reducing acute and chronic allograft rejection in the rat[END_REF]. These compounds were originally identified as potent inhibitors of T-lymphocyte proliferation that act as immunosuppressants and were subsequently shown to bind to MCT1 and MCT2 but not MCT4 [START_REF] Murray | Monocarboxylate transporter MCT1 is a target for immunosuppression[END_REF]. Since T-lymphocyte activation and proliferation is accompanied by a large (up to fourteen-fold) stimulation of glycolysis, it would appear that inhibiting MCT1 and so preventing the efflux of the resulting lactic acid is responsible for the compounds' immunosuppressive activity [START_REF] Murray | Monocarboxylate transporter MCT1 is a target for immunosuppression[END_REF].

In this paper we seek to characterise the mode of action of one of these potent MCT1 inhibitors, AR-C155858. We have studied the concentration dependency of AR-C155858 inhibition of lactate transport into rat red blood cells mediated by endogenous MCT1, as well as its effects on the activity of MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes. We show that AR-C155858 inhibits MCT1 and MCT2 with similar potency but is inactive against MCT4, and that inhibition is exerted by the drug binding to a site on MCT1 accessible from the cytosol. The use of MCT1/MCT4 chimeric transporters reveals that this binding site is contained within transmembrane helices 7-10 of MCT1. In rat red blood cells a detailed analysis of the inhibition of lactate transport by AR-C155858 has enabled us to determine the turnover number (k cat ) of MCT1 (12.2 s -1 at 6 o C) and the K i for AR-C155858 (2.3 nM).

EXPERIMENTAL

Materials

Xenopus laevis toads were obtained from Xenopus Express (Haute-Loire, France) and oocytes harvested as described previously [START_REF] Manoharan | The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity[END_REF]. All reagents were obtained from Sigma (Poole, UK) unless otherwise stated. Polyclonal antibodies against the C-terminal 16 amino acids of rat MCT1, MCT2 and MCT4 and against a 17 amino acid sequence between TM7 and TM8 of MCT1 were raised in rabbits as described previously [START_REF] Jackson | Cloning of the monocarboxylate transporter isoform MCT2 from rat testis provides evidence that expression in tissues is species-specific and may involve post-transcriptional regulation[END_REF][START_REF] Wilson | Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3[END_REF][START_REF] Poole | Studies of the membrane topology of the rat erythrocyte H+/lactate cotransporter (MCT1)[END_REF]. Anti-rabbit secondary antibodies for immunofluorescence microsocopy were from Jackson ImmunoResearch. L-[ 14 C]-Lactate was obtained from GE healthcare and [ 14 C]-pyruvate from Perkin Elmer. AR-C155858 (whose structure is shown as an inset to Figure 1) was obtained from AstraZeneca and made up as a 10 mM stock in DMSO.

Methods

Generation of MCT chimeras of rat MCT1 and MCT4 was performed by PCR using pfu Taq polymerase (Roche). The two appropriate segments of MCT1 and MCT4 were produced with a region of overlapping sequence that was used to splice the two together to produce the desired chimera. For the MCT1/4 and MCT4/1 chimeras the switch occurs at the end of the loop between TMs 6 and 7 which contains the conserved sequence LDLS. For the MCT1/4TM11 and MCT4/1TM11 chimera the switch occurs at the beginning of TM11 containing the conserved sequence FSSA. However for the C-terminal chimeras MCT1/4c and MCT4/1c, where the switch occurs at the beginning of the C-terminus, there is no conserved sequence and thus a region of complimentarity was manufactured using modified primers [START_REF] Choi | Role of extracellular domains in PBAN/pyrokinin GPCRs from insects using chimera receptors[END_REF]. A C-terminal truncation of MCT1 was produced using PCR to amplify the MCT1 sequence minus the C-terminus and this was subsequently inserted into the oocyte expression vector pGHJ with the addition of a stop codon in the vector downstream of the MCT sequence. Sequences for all primers used are given in Supplementary data Table 1 and were designed to be between 15 and 30 bases in length. Typically, for an MCT fragment, thermocycling was performed using the following parameters: 30 s @ 95°C, 30 s @ 55°C and a 1.5 min extension at 72°C for 30 cycles. Subsequently, appropriate fragments of MCT1 and MCT4 with complimentary sequence in the crossover region were combined using PCR with primers to the 5'-and 3'-UTR of MCT1 and MCT4. Here, thermocycling was performed using the following parameters: 30 s @ 95°C, 30 s @ 55°C and a 1 min extension at 72°C for 10 cycles followed by; 30 s @ 95°C, 30 s @ 50°C and a 1 min extension at 72°C for 25 cycles. The MCT chimeras were subsequently ligated into the pGHJ oocyte expression vector. For the MCT1/4 and MCT4/1 C-terminal chimeras the C-terminal regions were brought into the correct reading frame by a single base deletion and addition respectively using site-directed mutagenesis with a QuikChange kit (Stratagene, UK) as described previously [START_REF] Wilson | Studies on the DIDS binding site of monocarboxylate transporter 1 suggest a homology model of the open conformation and a plausible translocation cycle[END_REF]. Confirmation that chimera constructs had been correctly engineered was provided by sequencing (The Sequencing Service, Dundee).

Measurement of MCT1 activity in rat erythrocytes L-lactate transport into rat erythrocytes was measured by monitoring the change in extracellular pH with a pH sensitive electrode as described previously [START_REF] Poole | Reversible and irreversible inhibition, by stilbenedisulphonates, of lactate transport into rat erythrocytes -identification of some new high-affinity inhibitors[END_REF][START_REF] Wilson | Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4 -The ancillary protein for the insensitive MCT2 is embigin (Gp70)[END_REF]. The cells were used at 7% or 3.5% haematocrit in lightly buffered saline medium supplemented with 5 µM DIDS and 100 µM acetazolamide to prevent bicarbonate / CO 2 mediated proton movements [START_REF] Poole | Reversible and irreversible inhibition, by stilbenedisulphonates, of lactate transport into rat erythrocytes -identification of some new high-affinity inhibitors[END_REF][START_REF] Wilson | Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4 -The ancillary protein for the insensitive MCT2 is embigin (Gp70)[END_REF]. The erythrocytes were pre-incubated for 1 hour at room temperature with or without AR-C155858 at the required concentration prior to assaying lactate transport. This was performed at 6 o C with substrate uptake initiated by addition of 10 mM L-lactate. Initial rates of transport were calculated by first order regression analysis of the time course of pH change and converted into nmoles H + per minute by determining the pH change induced by small additions of standardised NaOH.

Measurement of MCT transport activity in Xenopus oocytes cRNA was prepared by in vitro transcription from the appropriate linearised pGHJ plasmid (mMessage mMachine, Ambion, Texas, USA) and injected into Xenopus laevis oocytes as described previously [START_REF] Manoharan | The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity[END_REF]. For most assays 20 ng cRNA was injected, but for [ 14 C] kinetic assays injection quantity was adjusted to ensure that uptake was linear with time. Further details can be found in Supplementary Table 2. Controls received the equivalent volume (9.2 nl) of water. Oocytes were then cultured in OR3 medium for 72 hrs with fresh medium each day. Rates of L-lactate transport by wild-type or chimeric MCTs were determined as described previously, either by following the change in intracellular pH using the ratiometric pH-sensitive fluorescent dye 2'-7'-bis(carboxyethyl)-5-6-carboxy-fluorescein (BCECF) A c c e p t e d M a n u s c r i p t [START_REF] Manning Fox | Characterisation of human Monocarboxylate Transporter 4 substantiates its role in lactic acid efflux from skeletal muscle[END_REF][START_REF] Wilson | Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4 -The ancillary protein for the insensitive MCT2 is embigin (Gp70)[END_REF] or by measuring uptake of [ 14 C] substrate (L-lactate or pyruvate) [START_REF] Manoharan | The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity[END_REF]. For measurement of AR-C155858 sensitivity, 10 oocytes were placed in a 6-well plate containing 5 ml uptake buffer (75 mM NaCl, 2 mM KCl, 0.82 mM MgCl 2 , 1 mM CaCl 2 , 20 mM MES, pH 6.0) and allowed to preincubate for the required time (usually 45 min) with or without AR-C155858 as required. Five oocytes were removed and placed into 50 µl uptake buffer containing L-[ 14 C]-lactate (0.5 mM, 7.4 MBq/ ml) with or without AR-C155858 at the required concentration. Incubation at room temperature (22-25°C) was continued for the period over which uptake was linear with time; this varied with the construct employed as detailed in Supplementary Table 2. The oocytes were then rapidly washed five times with ice-cold uptake buffer and after the final wash each egg was transferred into a scintillation vial and homogenized in 100 µl 2 % (w/v) SDS by vigorous vortex mixing. Scintillation fluid (10 ml Emulsifier-Safe, Perkin Elmer) was then added and [ 14 C] assayed by scintillation counting.

For the determination of K m values for pyruvate and L-lactate, oocytes were equilibrated in incubation buffer (75 mM NaCl, 2 mM KCl, 0.82 mM MgCl 2 , 1 mM CaCl 2 , 20 mM Tris/HEPES, pH 7.4) for 5 min and then 4 oocytes incubated with 40µl uptake buffer containing [ 14 C]-labelled Llactate or pyruvate (7.4 MBq/ml) at a final concentration of 0.2, 0.5, 1, 2, 5, 20, 50, and 75 mM. Incubation was continued for the period over which uptake was linear with time (detailed in Supplementary Table 2), following which oocytes were washed and prepared for scintillation counting as above. Net MCT1-mediated uptake of L-lactate was determined by subtracting the uptake by water injected oocytes determined at the same time.

MCT expression at the plasma membrane was confirmed by immunofluorescence microscopy of oocytes sectioned by embedding in chicken liver as described previously [START_REF] Manoharan | The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity[END_REF][START_REF] Friesema | Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter[END_REF].

RESULTS

Determining the K i of AR-C155858 for MCT1 in rat erythrocytes

Rat erythrocytes support very rapid lactate transport that is mediated by three mechanisms. The majority is via MCT1 but a small proportion may occur via the anion exchanger AE1 and by free diffusion of the undissociated lactic acid [START_REF] Halestrap | Pyruvate and lactate transport into human erythrocytes. Evidence for the involvement of the chloride carrier and a chloride independent carrier[END_REF][START_REF] Deuticke | Discrimination of three parallel pathways of Llactate transport in the human erythrocyte membrane by inhibitors and kinetic properties[END_REF]. Transport mediated by AE1 can be blocked by 5 µM DIDS, a concentration that has minimal effect on MCT1 [START_REF] Poole | Reversible and irreversible inhibition, by stilbenedisulphonates, of lactate transport into rat erythrocytes -identification of some new high-affinity inhibitors[END_REF][START_REF] Wilson | Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4 -The ancillary protein for the insensitive MCT2 is embigin (Gp70)[END_REF]. Using a pH electrode to determine the extracellular increase in pH that accompanies the proton-linked transport of 10 mM Llactate into erythrocytes at 6 o C we have investigated the inhibition of MCT1 by AR-C155858. It was first established that inhibition by low (nM) concentrations of AR-C155858 reached equilibrium within 30-45 min incubation at room temperature (data not shown). Thus for detailed inhibitor titrations with increasing concentrations of AR-C155858 we used 60 min incubation of erythrocytes at both 3.5 and 7 % haematocrit. After incubation with inhibitor at room temperature, cell suspensions were cooled to 6 o C before measurement of L-lactate transport and determination of initial rates. In Figure 1 we show that the concentration dependence of inhibition is almost linear up to 80% inhibition as predicted for a very tight binding inhibitor whose concentration is less than the concentration of the target protein (MCT1). By performing parallel experiments at two cell densities (3.5% and 7% haematocrit) the data can be fitted by non-linear regression analysis to the equation for a tight binding non-competitive inhibitor using the total inhibitor concentration and the haematocrit as the 2 x-variables [START_REF] Shearman | The concentration of the mitochondrial pyruvate carrier in rat liver and heart mitochondria determined with α-cyano-β-(1-phenylindol-3yl)acrylate[END_REF][START_REF] Davidson | Partial Inhibition by Cyclosporin A of the swelling of liver mitochondria in vivo and in vitro induced by sub-micromolar [Ca2+] but not by butyrate Evidence for two distinct swelling mechanisms[END_REF]. This allowed us to determine values (± S.E. for the fit shown) for the K i (2.3 ± 1.4 nM), the number of binding sites (Et) for AR-C155858 inhibition (1.29 ± 0.09 nmoles per ml packed cells) and the turnover number (K cat ) of MCT1 in rat erythrocytes (12.2 ± 1.1 s -1 ).

Time and concentration dependence of MCT inhibition by AR-C155858 in Xenopus oocytes

In order to perform accurate comparisons of the affinity of the different MCT isoforms towards AR-C155858 we expressed MCT1, MCT2 and MCT4 in Xenopus laevis oocytes. It was first necessary to ensure that the time of incubation of oocytes with inhibitor was sufficient to allow compound binding to the MCT to reach equilibrium. In Figure 2 A c c e p t e d M a n u s c r i p t that, as with the erythrocytes, 45 min incubation with inhibitor was sufficient to give maximal inhibition. As might be predicted, with 1 µM AR-C155858 inhibition occurred much more quickly and was almost maximal at 5 min. This was confirmed by BCECF assay of transport as shown in the inset to Fig. 2. From these data we chose to use a 45 min incubation time for comparing the relative potency of AR-C155858 as an inhibitor of MCT1, MCT2 and MCT4. In Figure 3 we show that the concentration dependence of MCT1 follows a linear trend up to 30 nM (70% inhibition) and then curves off towards 100% inhibition at 100 nM. Again, this behaviour is that predicted for a very high affinity inhibitor and thus consistent with the K i for AR-C155858 of 2.3 nM determined using rat erythrocytes. By contrast, oocytes expressing MCT4 at levels supporting similar rates of lactate transport revealed no significant inhibition by 1 µM AR-C155858 and even at 10 µM AR-C155858 inhibition was only about 20%. In oocytes expressing MCT2 the initial rate of transport was less than for MCT1 and MCT4, consistent with the poorer expression of MCT2 at the plasma membrane as reported previously [START_REF] Wilson | Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4 -The ancillary protein for the insensitive MCT2 is embigin (Gp70)[END_REF]. This is a consequence of MCT2 binding less well to the endogenous basigin expressed by Xenopus oocytes than does MCT1 and MCT4 [START_REF] Wilson | Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4 -The ancillary protein for the insensitive MCT2 is embigin (Gp70)[END_REF][START_REF] Kirk | CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression[END_REF]. Nevertheless, it is clear that MCT2 is potently inhibited by AR-C155858, and the 70% inhibition seen at 10 nM is followed by a gradually increasing inhibition can only be explained by a K i value of significantly less than 10 nM.

Extrapolation of the linear portion of the plot shown for MCT1 inhibition to zero carriermediated rate (i.e. water injected rate) provided an estimated concentration of MCT1 in the incubation of 40 nM. Each egg was incubated in 10 µl so this represents 0.4 pmol of MCT1 per egg. The rate of L-lactate uptake was about 95 pmol per egg per min which gives a turnover number of 4 s -1 for MCT1 in oocytes at 22 o C with 0.5 mM L-lactate. Since the initial slope of the inhibitor plot for MCT2 cannot be accurately determined it is not possible to determine an accurate turnover number for this isoform. Nevertheless, linear extrapolation of the plot would give a maximal value for the concentration of MCT2 to be about 15 nM, corresponding to a maximal expression of MCT2 of 0.15 pmol per egg. This would yield a minimum estimate of the turnover number MCT2 in oocytes at 22 o C with 0.5 mM L-lactate of 5 s -1 which is similar to MCT1.

AR-C155858 probably binds to MCT1 from the cytosolic side

The slow time-dependence of inhibition by AR-C155858 could reflect slow binding to a site on the external surface or perhaps more likely, a slow permeation of the inhibitor into the cell where it binds to an internal site on MCT1. In order to discriminate between the two possibilities we microinjected 9.2 nl of 1 mM AR-C155858 into oocytes, corresponding to a total internal concentration of about 23 µM (assuming an internal volume of 0.4 µl per egg [START_REF] Stegen | Swelling-induced taurine release without chloride channel activity in Xenopus laevis oocytes expressing anion channels and transporters[END_REF] and ignoring binding to intracellular components). Each oocyte (20 in total) was immediately transferred into 5 ml of pH6 transport buffer and then left for 5 min prior to washing once and determining the rate of [ 14 C] L-lactate uptake. As a control, the same quantity of inhibitor (184 nl corresponding to 20 oocyte's worth of inhibitor) was added to 5 ml buffer (36.8 nM final) prior to addition of 20 oocytes and 5 min incubation. These oocytes were subsequently washed and transport determined as for AR-C155858injected oocytes. As an additional control, some oocytes were injected with 9.2 nl of DMSO. The data of Figure 4 show that the rate of L-lactate uptake was about 50% inhibited in the AR-C155858injected oocytes whilst the same quantity of inhibitor added outside was without effect. It might also be argued that the slow time course of inhibition could reflect internalisation of MCT1. However, the immunofluorescence data of Figure 4 confirm that the expression of MCT1 at the plasma membrane was unchanged in oocytes incubated with 1 µM AR-C155858 for 1 hour to give full inhibition of transport.

AR-C155858 inhibition involves transmembrane helices 7-10 in the C-terminal half of MCT1

Since MCT1 is inhibited by AR-C155858 whilst MCT4 is not, we made MCT1/4 chimeras to establish the region on MCT1 to which the inhibitor binds. The structures of these MCT1/4 chimeras are shown schematically in panel A of each relevant Figure . We first created chimeras in which the N-terminal half and large intracellular loop were of MCT1 and the C-terminal half was from MCT4. This chimera (MCT1/4) was expressed at the plasma membrane of oocytes and A c c e p t e d M a n u s c r i p t transported L-lactate albeit at a greatly reduced rate (Figure 5B). However, like MCT4, it was insensitive to inhibition by AR-C155858 (Figure 5C). We also made the reverse chimera -MCT4/1. It too was expressed at the plasma membrane although there appeared to be a significant amount of the chimera that was in a vesicular compartment and not at the plasma membrane (Figure 5B). Nevertheless, this MCT4/1 chimera showed a sensitivity to AR-C155858 similar to MCT1 (Figure5C). Thus our data imply that the binding site of MCT1 for AR-C155858 is contained within the C-terminal half of the molecule, beyond the intracellular loop between TMs 6 and 7.

In order to define the binding site of the inhibitor more precisely we created additional chimeras. We first made chimeras in which either the C-terminal tail of MCT1 was removed, or swapped for that of MCT4 as shown schematically in Figure 6A. Both the MCT1trnc and MCT1/4c were expressed at the plasma membrane of oocytes, although a significant amount of MCT1trnc remained intracellular. Nevertheless, this chimera still transported lactate with rates similar to that seen with normal MCT1 and MCT4. Although we did not perform detailed inhibitor titrations we showed that both the MCT1/4c chimera and MCT1trnc were strongly inhibited by 0.1 µM AR-C155858, like MCT1 itself. These data imply that the binding site for AR-C155858 does not require the C-terminal tail of MCT1. In order to establish which of the C-terminal TM helices might be important for inhibitor binding we compared the C-terminal TM sequences of MCT1, MCT2 and MCT4 and found that it was within TM 11 that MCT4 (inhibitor-insensitive) exhibited the greatest difference to MCT1 and MCT2 (inhibitor-sensitive). Thus we made chimeras in which the switch between MCT1 and MCT4 was performed at the start of TM11. Unfortunately, as shown in the inset to Figure 7B, the MCT1/4-TM11 chimera was not expressed properly at the plasma membrane and no enhancement of L-lactate transport over water injected eggs was detected. However, the MCT4/1TM11 chimera was expressed at the plasma membrane and did transport L-lactate, albeit very slowly (Figure 7B). Subtraction of the very low background rate of lactate transport by uninjected oocytes, which was inhibited by AR-C155858, revealed that the residual lactate transport activity mediated by MCT4/1TM11 was insensitive to inhibition by AR-C155858. This suggests that the binding site for AR-C155858 does not involve TMs 11 and 12 as we had initially reasoned and must therefore involve TMs 7-10.

Kinetic properties of the MCT1/4 and MCT4/1 chimeras

The relatively low rates of transport seen with the MCT4/1 and MCT1/4 chimeras may reflect a high K m for L-lactate and thus low rates of transport when measured at 0.5 mM L-lactate. In order to investigate this possibility, and to provide some clues as to how the two halves of the MCT might be involved in substrate binding, the K m values of the chimeras for L-lactate and pyruvate were determined (Table 1). The MCT4/1 chimera exhibited a K m for L-lactate of 15 -25 mM, depending on whether the BCECF or radioactive assay was used, whilst both techniques gave a K m value for pyruvate of 8-9 mM. These K m values are similar to that of MCT4 in the case of L-lactate but intermediate between MCT1 and MCT4 for pyruvate. In the case of the MCT4/1 chimera K m values were too high to be determined radioactively, but using BCECF they were found to be 209 ± 72 mM for L-lactate and 27 ± 5 mM for pyruvate. Thus with this chimera also, the K m for pyruvate was intermediate between MCT1 and MCT4 whilst the K m for L-lactate was much higher than that for either isoform.

DISCUSSION

Recently, a new class of specific and extremely potent inhibitors of MCT1 have been discovered by AstraZeneca that were reported to show no binding to MCT4 and exhibit lower affinity binding to MCT2 [START_REF] Murray | Monocarboxylate transporter MCT1 is a target for immunosuppression[END_REF][START_REF] Guile | Potent blockers of the monocarboxylate transporter MCT1: Novel immunomodulatory compounds[END_REF][START_REF] Ekberg | The specific monocarboxylate transporter-1 (MCT-1) inhibitor, AR-C117977, induces donorspecific suppression, reducing acute and chronic allograft rejection in the rat[END_REF]. The K d values for these inhibitors binding to endogenous MCT1 in rat and human cells was found to be in the low nanomolar region or less, as was also found for MCT1 expressed in Ins-1 cells that contain little or no endogenous MCT1 [START_REF] Murray | Monocarboxylate transporter MCT1 is a target for immunosuppression[END_REF][START_REF] Guile | Potent blockers of the monocarboxylate transporter MCT1: Novel immunomodulatory compounds[END_REF]. Similar K d values were determined for MCT1 expressed in Yeast [START_REF] Murray | Monocarboxylate transporter MCT1 is a target for immunosuppression[END_REF] and the K i value of AR-C155858 for MCT1 we have determined in rat erythrocytes of 2.3 ± 1.4 nM is entirely consistent with the K i of 1.2nM for binding of AR-C155858 to human erythrocyte MCT1 derived from radioligand binding experiments [START_REF] Guile | Optimization of Monocarboxylate Transporter 1 Blockers through Analysis and Modulation of Atropisomer Interconversion Properties[END_REF]. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

The turnover number for MCT1 can be calculated using AR-C155858

The inhibitor titration in rat erythrocytes has allowed us to determine the concentration of MCT1 in these cells to be 1.29 ± 0.09 nmoles per ml packed cells. Since the number of erythrocytes in 1 ml of packed blood is known to be about 10 10 [START_REF] Halestrap | Pyruvate and lactate transport into human erythrocytes. Evidence for the involvement of the chloride carrier and a chloride independent carrier[END_REF] it is possible to calculate that there are approximately 80,000 molecules of MCT1 per rat erythrocyte. This value is quite similar to the value of 46,000 ± 11,000 determined previously by [ 3 H] inhibitor binding [START_REF] Murray | Monocarboxylate transporter MCT1 is a target for immunosuppression[END_REF] and represents about 8% of the value for the anion exchanger AE1 [START_REF] Halestrap | Pyruvate and lactate transport into human erythrocytes. Evidence for the involvement of the chloride carrier and a chloride independent carrier[END_REF]. Using the rate of L-lactate transport and the concentration of MCT1 we calculated the turnover number (K cat ) to be 12.2 ± 1.1 s -1 at 6 o C. Since we have previously determined the temperature dependence of MCT1 [29] we are able to calculate that the rate of L-lactate transport by rat erythrocytes at room temperature (22 o C) will be about 7 fold higher than at 6 o C. This would bring the predicted turnover number for MCT1 in rat erythrocytes to about 85 s -1 which is similar to the value of 35 s -1 determined for the glucose transporter GLUT1 in rat erythrocytes at 20 o C [START_REF] Helgerson | Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the alternating conformation carrier model for sugar transport[END_REF][START_REF] Carruthers | Facilitated diffusion of glucose[END_REF] but about 5-fold less than the K cat determined for endogenous human GLUT1 in erythrocytes or when over-expressed in Xenopus oocytes [START_REF] Nishimura | Kinetics of GLUT1 and GLUT4 glucose transporters expressed in Xenopus oocytes[END_REF].

When expressed in Xenopus oocytes MCT1 also showed time-dependent inhibition by AR-C155858 (Fig. 2) with the extent of inhibition being linear with respect to AR-C155858 concentration up to about 70% inhibition (Fig. 3). This is consistent with a very low (nM) K i value determined in erythrocytes. By extrapolation of the linear portion of the inhibitor titration we were able to estimate that the amount of MCT1 expressed at the plasma membrane was about 0.4 pmol per oocyte. From this value and the rate of lactate transport the turnover number of MCT1 in oocytes at 22 o C was calculated to be 4 s -1 . However, this value was determined at 0.5 mM L-lactate and thus cannot be compared directly with the value of 85 s -1 calculated for rat erythrocytes at 22 o C with 10 mM L-lactate. The K m for MCT1 in erythrocytes and oocytes is about 3.5 mM [START_REF] Manning Fox | Characterisation of human Monocarboxylate Transporter 4 substantiates its role in lactic acid efflux from skeletal muscle[END_REF]29] and thus from Michaelis Menten kinetics the rate of transport at 10 mM L-lactate would be predicted to be six-fold higher than at 0.5 mM. This would increase the turnover number of MCT1 in oocytes to 24 s -1 , which is about 30% of the value determined for MCT1 in erythrocytes. However, we have shown previously that the rate of L-lactate uptake by oocytes determined radioactively underestimates the true transport rate determined with BCECF [START_REF] Manning Fox | Characterisation of human Monocarboxylate Transporter 4 substantiates its role in lactic acid efflux from skeletal muscle[END_REF]. This is because of an unstirred layer effect that slows diffusion of [ 14 C]-L-lactate taken up into the body of the oocyte. Thus, overall there is reasonable agreement between the values measured in erythrocytes and oocytes.

AR-C155858 is a potent inhibitor of MCT1 and MCT2 but not MCT4

In agreement with competition binding studies of MCTs expressed in Yeast for related compounds [START_REF] Murray | Monocarboxylate transporter MCT1 is a target for immunosuppression[END_REF], we have shown that AR-C155858 inhibits both MCT1 and MCT2 but not MCT4 when expressed in oocytes (Fig. 3). Although we were unable to determine an accurate K i value for inhibition of MCT2 by AR-C155858, we can conclude from the shape of the inhibitor titration that the value must be significantly less than 10 nM. In previous studies of inhibitor binding to MCT1 and MCT2 expressed in Yeast K d values of 4.9 and 100 nM respectively were determined (unpublished data). For MCT1 this represents a reasonable agreement with the K i values for endogenous MCT1 in rat and human cells derived here and previously [START_REF] Stegen | Swelling-induced taurine release without chloride channel activity in Xenopus laevis oocytes expressing anion channels and transporters[END_REF]. However, this is not the case for MCT2 where the yeast binding data suggest that the affinity of MCT2 for AR-C155858 is more than an order of magnitude less than for MCT1. One explanation for this difference may be the absence of either basigin or embigin when MCTs are expressed in yeast, and this might affect inhibitor binding. In this context it may be noted that inhibition of MCT1 and MCT2 by the organomercurial reagent p-chloromercuribenzene sulphonate also depends on the associated ancillary protein [START_REF] Wilson | Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4 -The ancillary protein for the insensitive MCT2 is embigin (Gp70)[END_REF]. Our data would also imply that interpretation of metabolic studies in which AR-C122982 has been used to discriminate between MCT1-and MCT2-mediated lactate transport [START_REF] Rae | Metabolic effects of blocking lactate transport in brain cortical tissue slices using an inhibitor specific to MCT1 and MCT2[END_REF] may not be straightforward.

The binding site for AR-C155858 involves TMs 7-10 of MCT1 and probably faces the cytosol

The use of MCT1/MCT4 chimeric transporters reveals that the binding site for AR-C155858 is contained within the C-terminal half of the transporter. Thus MCT1/4, like MCT4, was insensitive to inhibition by AR-C155858 whilst MCT4/1 was inhibitor-sensitive like MCT1 (Fig. 5). The C- A c c e p t e d M a n u s c r i p t terminal tail of MCT1 appears to have no major effect on the sensitivity to inhibition by AR-C155858 since potent inhibition by AR-C155858 was still observed when it was either removed or replaced with the tail of MCT4 (Fig. 6). However, the chimera containing TMs 1-10 of MCT4 and TMs 11 and 12 plus the C-terminal tail of MCT1 was insensitive to AR-C155858 (Fig. 7). These data demonstrate that the binding site for AR-C155858 is contained, at least in part, within TMs 7-10 of MCT1. The slow time dependence of inhibition by AR-C155858 (Fig. 2) could be explained either by the inhibitor having to enter the cell before binding to an intracellular site on MCT1 or by internalisation of MCT1 upon inhibitor binding. However, we showed that no MCT1 internalisation occurred even at full inhibition and that the inhibitor worked well if microinjected into oocytes (Fig. 4). These data imply that the binding site for AR-C15585 probably faces the cytosol where our molecular model of MCT1 predicts TMs 7 and 10 to form part of the inward facing substrate binding pocket [START_REF] Manoharan | The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity[END_REF][START_REF] Wilson | Studies on the DIDS binding site of monocarboxylate transporter 1 suggest a homology model of the open conformation and a plausible translocation cycle[END_REF]. This is also consistent with earlier observations that F360 in TM 10 is involved in substrate binding since its mutation to cysteine allows MCT1 to accommodate the larger monocarboxylate, mevalonate [START_REF] Kim-Garcia | Molecular characterization of a membrane transporter for lactate, pyruvate, and other monocarboxylates -implications for the Cori Cycle[END_REF][START_REF] Kim | cDNA cloning of MEV, a mutant protein that facilitates cellular uptake of mevalonate, and identification of a point mutation responsible for its gain in function[END_REF]. Furthermore, we have previously shown that D302 and R306 in TM8 are critical residues in the translocation cycle. However, analysis of the kinetics of MCT1/4 and MCT4/1 chimeras suggest that substrate binding may not be exclusively located within the Cterminal half of MCT1. Thus both the C-and N-terminal halves of the MCT influence the kinetics of transport (Table 1). This is entirely consistent with our recent molecular model of MCT1 that predicts that the substrate binding pocket and translocation channel involve helices from both the Cand N-terminal domains with critical roles for K38 in TM1 and D302 plus R306 in TM8 [START_REF] Manoharan | The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity[END_REF][START_REF] Wilson | Studies on the DIDS binding site of monocarboxylate transporter 1 suggest a homology model of the open conformation and a plausible translocation cycle[END_REF].

Table 1 K m values of MCT chimeras for L-lactate and pyruvate. The K m values reported were derived using either [ 14 C]-lactate uptake or by monitoring changes in intracellular pH with BCECF as described in the Experimental Section. Data are given ± S.E. derived from the fit of the mean data to the Michaelis Menten equation by non-linear least squares analysis. The n values given represent the number of separate eggs used for each substrate concentration. *BCECF data for MCT1 and MCT4 are taken from [START_REF] Manning Fox | Characterisation of human Monocarboxylate Transporter 4 substantiates its role in lactic acid efflux from skeletal muscle[END_REF]. No [ 14 C]-L-lactate uptake data are provided for the MCT4/1 chimera because the very high K m values required the use of high substrate concentrations for which the specific activity became too low to measure uptake of [ A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

LEGENDS TO FIGURES

Figure 1 Inhibition of L-lactate uptake into rat erythrocytes by AR-C155858. Rat erythrocytes were freshly isolated and resuspended at the specified haematocrit as outlined under "Experimental procedures". Cells were pre-incubated for 1 hour at room temperature in the presence or absence of the specified concentration of AR-C155858 (structure shown). Transport was measured by continuous monitoring of the extracellular pH and calibration of pH changes in terms of proton uptake was achieved by addition of standardised NaOH (10 µM final) just before addition of 10 mM L-lactate to initiate transport. Initial rates of transport were calculated by first order regression analysis of the time course of pH change. The data were then fitted by non-linear least squares inhibition to the equation for a tight binding non-competitive inhibitor using inhibitor concentration and haematocrit as the two x-variables [START_REF] Shearman | The concentration of the mitochondrial pyruvate carrier in rat liver and heart mitochondria determined with α-cyano-β-(1-phenylindol-3yl)acrylate[END_REF][START_REF] Davidson | Partial Inhibition by Cyclosporin A of the swelling of liver mitochondria in vivo and in vitro induced by sub-micromolar [Ca2+] but not by butyrate Evidence for two distinct swelling mechanisms[END_REF]. The derived values (± S.E. of the fit shown) for the K i and concentration of binding sites were 2.3 ± 1.4 nM, 1.29 ± 0.09 nmoles per ml packed cells respectively.

Figure 2 MCT1 expressed in Xenopus oocytes is inhibited by AR-C155858 in a time-and concentration-dependent manner Xenopus oocytes were injected with the appropriate cRNA and and after 72 hours expression were pre-incubated in pH 6 oocyte transport buffer in the presence or absence of 0.1µM or 1µM AR-C155858 for the times shown. The uptake of 0.5 mM L-[ 14 C]-lactate uptake was then determined after 2.5 min over which period it was found to be linear with time. Data are shown as means ± S.E.M of 10 separate oocytes. The inset shows the uptake of 20mM Llactate measured at pH7.4 using the pH-sensitive dye BCECF. Data are presented for the same oocyte in the absence of inhibitor and then after 20 minutes superfusion with 0.1µM and 1µM AR-C155858. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 20 Nov 2009 as manuscript BJ20091515 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091515A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 20 Nov 2009 as manuscript BJ20091515 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091515

  the rate of [ 14 C]-lactate uptake into MCT1expressing oocytes was determined after incubation with 0.1 µM AR-C155858 for 15, 30, 45 and 60 min. For each time point mean data (± S.E.M.) for 10 separate oocytes are presented. The data show Biochemical Journal Immediate Publication. Published on 20 Nov 2009 as manuscript BJ20091515 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091515

Biochemical

  Journal Immediate Publication. Published on 20 Nov 2009 as manuscript BJ20091515 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091515

Biochemical

  Journal Immediate Publication. Published on 20 Nov 2009 as manuscript BJ20091515 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091515

Biochemical

  Journal Immediate Publication. Published on 20 Nov 2009 as manuscript BJ20091515 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091515

Figure 3

 3 Figure 3 When expressed in Xenopus oocytes MCT1 and MCT2, but not MCT4, are sensitive to inhibition by AR-C155858. Xenopus oocytes were injected with appropriate cRNA or water and after 72 hours expression they were incubated with the concentration of AR-C155858 shown for 45 min prior to measurement of L-[ 14 C]-lactate uptake over 2.5 min. Data are shown as the means ± S.E.M of 15-40 separate oocytes for each inhibitor concentration. The inset images show the expression of the relevant MCT in oocytes sections revealed using immunoflourescence microscopy with C-terminal antibodies to the relevant MCT. Arrows indicate the location of the plasma membrane. Note that for MCT2 a significant proportion of the MCT remains in an intracellular compartment.

Figure 4

 4 Figure 4 Microinjection of AR-C155858 inhibits MCT1 expressed in Xenopus oocytes MCT1was expressed in Xenopus oocytes for 72 hours prior to inhibitor treatment and assay of L-[ 14 C]lactate uptake over 2.5 min. For addition of AR-C155858 internally, 20 oocytes were individually injected with 9.2nl of 1mM AR-C155858 or DMSO as control, incubated for 5 minutes in 5 ml pH 6 transport buffer and washed once prior to transport assay. For incubation with the equivalent amount of AR-C155858 added externally, 20 aliquots (9.2nl) of 1mM AR-C155858 were added to 5 ml pH6 transport buffer (final concentration 35 nM) and incubated with the oocytes for 5 min prior to a single wash and transport assay as above. Uptake was corrected for the uptake by water-injected eggs under the same conditions and are presented as means ± S.E.M (error bars) of 18-20 separate oocytes. The 2 inset images show the plasma membrane expression of MCT1 in control oocytes and those incubated for 1 hour with 1µM AR-C155858 revealed by immunofluorescence microscopy.

Figure 5

 5 Figure 5The binding site of MCT1 for AR-C155858 resides within the C-terminal half of the transporter. Panel A shows a schematic of the MCT1/4 and MCT4/1 chimeras used with MCT1 sequence being shown in black and MCT4 sequence in grey. The numbered arrows indicate the position the switch was made between MCT1 and MCT4. Native and chimeric MCTs were expressed in oocytes for 72 hours prior to incubation with AR-C155858 after which assay of [ 14 C]-Llactate uptake was determined as described in Figs.2 and 3. Note that different times of uptake were

Figure 6

 6 Figure 6The C-terminus of MCT1 is not involved in AR-C155858 sensitivity Panel A shows a schematic of the MCT1/4 and MCT4/1 chimeras used with MCT1 sequence being shown in black and MCT4 sequence in grey. The numbered arrows indicate the position the switch was made between MCT1 and MCT4. Native and chimeric MCTs were expressed in oocytes for 72 hours prior to incubation with or without 0.1 µM AR-C155858 for 45 min, after which assay of [ 14 C]-Llactate uptake was determined over 2.5 min as described in Figs.2 and 3. Panel B provides mean data (± S.E.M for 10 oocytes) on the absolute rate of transport of each chimera in the absence and presence of inhibitor. The inset images show the plasma membrane expression of each native and chimeric MCT revealed by immunofluorescence microscopy.

Figure 7 A

 7 Figure 7 A region within TMs 7-10 of MCT1 is required for sensitivity to AR-C155858 Panel A shows a schematic of the MCT1/4 and MCT4/1 chimeras used with MCT1 sequence being shown in black and MCT4 sequence in grey. The numbered arrows indicate the position the switch was made between MCT1 and MCT4. Native and chimeric MCTs were expressed in oocytes for 72 hours prior to incubation with or without 0.1 µM AR-C155858 for 45 min, after which assay of [ 14 C]-L-lactate uptake was determined over 1 hour. This prolonged uptake period was required because of the low rates of transport being measured. Panel B provides mean data (± S.E.M for 10 oocytes) on the absolute rate of transport of each chimera in the absence and presence of inhibitor. The inset images show the plasma membrane expression of each native and chimeric MCT revealed by immunofluorescence microscopy.

  Fig. 1

Fig. 2 Biochemical

 2 Fig. 2

  Fig. 3

Fig. 4 Lactate

 4 Fig. 4

Fig. 6 A

 6 Fig. 6

  14 C]-lactate accurately.

		K m value (mM) using [ 14 C]-substrate	K m value (mM) using BCECF
		Lactate	Pyruvate	Lactate	Pyruvate
	MCT1	5.7 ± 1.2 (16) 1.2 ± 0.2 (16)	3.5*	1*
	MCT4	12.9 ± 2 (16) 103 ± 28 (n24)	28*	150*
	MCT1/4	26.5 ± 5.7 (16) 8.9 ± 2 (n16)	15.8 ± 2.6 (7)	8.5 ± 1.9 (9)
	MCT4/1	-	-	209.2 ± 71.9 (7) 27.3 ± 5.2 (8)

Biochemical Journal Immediate Publication. Published on 20 Nov 2009 as manuscript BJ20091515 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091515

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements

This work was funded by a Project Grant (079792/z/06/) from The Wellcome Trust. MJO was supported by a PhD studentship from the Medical Research Council. We thank Agnieszka Bierzynska for skilled technical support.


Dr Chris Tselepis
email: c.tselepis@bham.ac.uk

Samuel J Ford

Andrew T Mckie

Wolfgang Vogel

Heinz Zoller

Robert J Simpson

Javier Diaz Castro

Tariq H Iqbal

Douglas G Ward

Characterisation of the Transition Metal Binding Properties of Hepcidin Running Head; Hepcidin-metal binding

Keywords: Hepcidin, iron, metal, copper, zinc Biochemical

Accumulating evidence suggests that hepcidin, a 25 residue peptide hormone, is the master regulator of iron metabolism. Further evidence suggests that the five N-terminal amino acids are crucial for mediating its biological function. With a histidine residue at position 3, this region also has the potential to bind divalent metal ions. To characterise this hepcidin-metal interaction in detail this study utilises electrospray mass spectrometry to measure the binding of a range of metal ions to wild type and mutant human and murine hepcidins. In addition the biological effects of these point mutations were tested on Caco-2 and HEK 293T human cell lines and in mice. Our results show that hepcidin-25 can form complexes with copper, nickel and zinc, though we failed to detect any hepcidin-25 binding to either ferric or ferrous ions. The greatest affinity observed was between hepcidin-25 and copper with a dissociation constant << 1 µM. Substituting the histidine at position 3 in human hepcidin-25 and comparably the asparagine at position 3 in murine hepcidin-25 with alanine markedly diminished the affinity for copper. The amino acid substitutions also decreased the biological activity of hepcidin-25; namely repression of ferroportin protein levels and hypoferraemia. In summary the high affinity of hepcidin for copper suggests that hepcidin could bind copper in vivo and this may be of biological relevance.

INTRODUCTION

Overwhelming evidence has identified the mature 25 amino acid hepatic anti-microbial peptide hepcidin as the master regulator of iron metabolism in man [START_REF] Ganz | Regulation of iron acquisition and iron distribution in mammals[END_REF][START_REF] Viatte | Hepcidin, the iron watcher[END_REF]. It is now also clear that there are several pathways involved in the regulation of this peptide hormone including inflammation, infection, erythropoietic demand, hypoxia and body iron status [START_REF] Zhang | Iron homeostasis: recently identified proteins provide insight into novel control mechanisms[END_REF]. Whilst exactly which intracellular signalling pathways are responsible for regulating hepcidin remains to be unravelled, it is clear that hepcidin mediates its biological activity by interacting with the cellular iron efflux protein ferroportin which is present on most cell types [START_REF] Nemeth | Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization[END_REF]. This hepcidin interaction results in the internalisation and degradation of ferroportin decreasing cellular iron efflux [START_REF] Nemeth | Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization[END_REF][START_REF] De Domenico | The molecular mechanism of hepcidin-mediated ferroportin down-regulation[END_REF]. There is also emerging evidence that the process of cellular iron efflux is also likely to be dependent upon the multicopper oxidases ceruloplasmin and hephaestin [START_REF] De Domenico | Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin[END_REF] which are thought to function as ferroxidases [START_REF] Chen | Decreased hephaestin expression and activity leads to decreased iron efflux from differentiated Caco2 cells[END_REF][START_REF] Chen | Decreased hephaestin activity in the intestine of copper-deficient mice causes systemic iron deficiency[END_REF][START_REF] Vulpe | Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse[END_REF].

The hepcidin gene encodes an 84 amino acid pre-propeptide which is cleaved by furin to generate the biologically active 25 residue peptide (hepcidin-25). Hepcidin forms a distorted β-sheet stabilised by 4 intramolecular disulphide bonds spanning residues 7 to 23 leaving a flexible 6residue N-terminal region [START_REF] Hunter | The solution structure of human hepcidin, a peptide hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis[END_REF][START_REF] Jordan | Hepcidin revisited: Disulfide connectivity, dynamics, and structure[END_REF]. In addition to hepcidin-25, two shorter forms, hepcidin-20 and hepcidin-22 have been identified where the N-terminal 5 and 3 amino acids are missing respectively. These are thought to represent breakdown products of hepcidin-25 [START_REF] Kemna | Hepcidin: from discovery to differential diagnosis[END_REF]. Hepcidin-20 has no effect on cellular iron efflux but does appear in-vitro to exert anti-bacterial and antifungal properties, albeit at supra physiological concentrations [START_REF] Krause | LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity[END_REF][START_REF] Park | Hepcidin, a urinary antimicrobial peptide synthesized in the liver[END_REF]. This suggests that the first five amino terminal amino acids of hepcidin are essential for mediating its role in regulating iron metabolism and this has been confirmed by the elegant studies of Ganz and colleagues which showed that sequential N-terminal amino acid deletion resulted in a stepwise loss of ferroportin binding and biological activity with >50% loss upon removal of the first three amino acids (aspartic acid, threonine and histidine) [START_REF] Nemeth | The Nterminus of hepcidin is essential for its interaction with ferroportin: structure-function study[END_REF][START_REF] Rivera | Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs[END_REF].

Melino et al have shown that the N-terminal region of hepcidin-25 is capable of binding divalent copper and nickel ions via an 'Amino Terminal Cu -Ni' (ATCUN) binding motif comprising the three N-terminal residues [START_REF] Melino | A metal-binding site is present in the amino terminal region of the bioactive iron regulator hepcidin-25[END_REF]. However, this study largely used truncated forms of hepcidin-25 and utilised both high concentrations of hepcidin and metal ion. In addition these studies were unable to provide information on affinity or stoichiometry. Studies by Farnaud et al suggest that hepcidin may bind iron, although this interaction was only ever observed under reducing conditions and not with disulphide bonded hepcidin (generally thought to be the biologically active form) [START_REF] Farnaud | Modelling of a metal-containing hepcidin[END_REF][START_REF] Farnaud | Identification of an iron-hepcidin complex[END_REF]. These suggestions that hepcidin might bind metal ions, in particular copper, are interesting as disturbances of copper metabolism have been shown to lead to altered iron homeostasis [START_REF] Chen | Decreased hephaestin expression and activity leads to decreased iron efflux from differentiated Caco2 cells[END_REF][START_REF] Chen | Decreased hephaestin activity in the intestine of copper-deficient mice causes systemic iron deficiency[END_REF][START_REF] Vulpe | Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse[END_REF]. Although these defects are generally thought to be caused by decreased activity of the ferroxidases hephaestin and ceruloplasmin, an interaction between copper and hepcidin had not been considered. Thus the aim of this study is to further characterise metal ion binding to hepcidin and, using mutation analysis, determine the critical amino acids within hepcidin responsible for binding. If hepcidin does bind metal ions with high affinity then this could have major implications in the regulation of human iron metabolism.

EXPERIMENTAL

Materials

All peptides were synthesised by Alta Bioscience (University of Birmingham, UK) and provided as linear (reduced) peptides. These were allowed to spontaneously oxidise by dissolving at 0.1 mg/ml in 6M urea, 30 mM MOPS (pH 7.0) and incubating overnight at room temperature with stirring as previously described [START_REF] Ward | SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labelled hepcidin as an internal standard[END_REF]. Complete disulphide bridge formation was accompanied by an 8 Dalton decrease in mass in MALDI-TOF mass spectra. The folded hepcidin was purified by C18 RP-HPLC in 0.1% TFA/acetonitrile and lyophilised. Hepcidin concentrations were determined by BCA assay calibrated with bovine serum albumin (Pierce). The sequences of the synthetic hepcidin peptides are as follows with amino acid substitutions underlined; Human hepcidin-25: DTHFPICIFCCGCCHRSKCGMCCKT Human H3A hepcidin: DTAFPICIFCCGCCHRSKCGMCCKT Human hepcidin-20: ICIFCCGCCHRSKCGMCCKT Murine hepcidin-25: DTNFPICIFCCKCCNNSQCGICCKT Murine N3A hepcidin: DTAFPICIFCCKCCNNSQCGICCKT Hepcidin-25 was purified from urine after informed consent had been obtained from patients as approved by the ethics research committee of Innsbruck Medical University (protocol Nr. UN3479). The purification was based on the method of Park et al [START_REF] Park | Hepcidin, a urinary antimicrobial peptide synthesized in the liver[END_REF]. Briefly, urine was centrifuged at 3000 g for 30 min, passed through a 0.45 µm filter and diluted 1:10 with water before loading onto a 50 ml weak cation exchange column (GE Healthcare, Vienna, Austria). After washing with 5 column volumes of 25 mM ammonium acetate, hepcidin was eluted with 5 % acetic acid. The eluate was lyophilized, dissolved in 0.1 % TFA and applied to a 10 × 250 mm C18 column (218TP510, Grace Vydac, USA) equilibrated in 0.1 % TFA, 2 % acetonitrile (ACN). Peptides were separated with an acetonitrile gradient of 3.6 % ACN increment/min for 5 min followed by a 1 % ACN increment for 25 min and a 3.5 %/min increase over 10 minutes followed by a 4 %/min increment for the final 5 min of the run at a flow rate of 1.5 ml/min. Hepcidin elution was monitored by MALDI-TOF mass spectrometry.

Hepcidin Mass Spectrometry

Mass spectra were acquired on a 7-T LTQ FT instrument (Thermo Fisher Scientific, Germany). Peptides were dissolved in 10 mM ammonium acetate, pH 6.8, and directly infused into the mass spectrometer at a flow rate of ~ 300 nL min -1 using a Triversa Nanospray source (Advion Biosciences, NY). The nanoelectrospray voltage was +1.7 kV. Data was collected for 1 minute per sample. The fraction of hepcidin with metal ion bound was estimated from the relative peak heights of the triply charged ions of free hepcidin and hepcidin-metal complex. Where peaks corresponding to hepcidin with 1, 2 or 3 metal ions bound were present the heights of all 3 were added together to calculate the fraction of 'complex' i.e. hepcidin-metal complex containing at least one metal ion.

Effect of hepcidin on ferroportin expression in Caco-2 cells

The cell line Caco-2 was routinely cultured in Dulbecco's modified eagles medium (Gibco, USA) with 10% foetal calf serum supplemented with 100 units/ml penicillin and 0.1mg/ml streptomycin. Upon reaching 14 days post confluence cells were challenged with either growth medium alone (control) or with i) human hepcidin-25 peptide, ii) human H3A hepcidin-25 or iii) hepcidin-20 A c c e p t e d M a n u s c r i p t 4 supplementation at concentrations of 1µM. Following 48hrs stimulation cells were lysed and processed for Western blotting with a rabbit polyclonal antibody to ferroportin (1000 dilution; Alpha Diagnostics Inc., San Antonio, TX1) and a mouse monoclonal β-actin antibody (1:5,000 dilution; Abcam ab8226-100); the latter was employed for normalisation of protein loading. All immunoreactive bands were then subject to densitometry using NIH Image 1.62 software.

Effect of hepcidin in a ferroportin-GFP assay HEK 293T cells (2 x 10 5 ) were seeded in six well plates 24 hrs prior to transfection. Cells were then transiently transfected with the wild-type FPN-GFP plasmid construct pBABS, (created by recombination of the pENTR221 clone IOH26826 (Invitrogen, Lofer, Austria) containing the wild-type human ferroportin ORF with the vector pcDNA6.2/N-EmGFP-DEST (Invitrogen)), using the liposomal transfection reagent Effectene (Qiagen) following the manufacturer`s instructions. After 16 hours transient transfection, cells were then incubated in the presence or absence of 0.5µM hepcidin for a further 16 hours. For flow cytometry cells were detached from the culture plates with cell dissociation solution (Sigma, Vienna, Austria) and gated for GFP expression using untransfected cells as negative control. Hepcidin induced ferroportin GFP degradation is expressed as mean fluorescence of 50,000 cells expressed as a ratio of mean fluorescence in hepcidin treated vs. untreated FPN-GFP expressing cells (control).

Animal model for testing the effect of hepcidin peptides 6 week old CD1 male mice (weights 28-32g) (Charles Rivers, Margate, Kent), were subject to intra-peritoneal injection with either i) 0.15M NaCl (control) ii) murine hepcidin-25 or iii) murine N3A hepcidin-25 (25µg/mouse). At 4 hrs post injection mice were anaesthetised, sacrificed by cervical dislocation and blood collected by cardiac puncture. Serum iron analysis was performed using a serum iron assay kit (Thermo Electron Corporation, Louisville, USA) according to the manufacturers' instruction. All animal experiments were performed under the authority of a UK Home Office licence. Mice were fed RM1 diet (Scientific Diet Supplies [SDS], Witham, Essex, United Kingdom). Experiments were performed in triplicate.

RESULTS

Hepcidin mass spectrometry:

The monoisotopic m/z value of the triply charged ion at 930.023 is the most abundant signal generated from synthetic human hepcidin-25 sprayed into the mass spectrometer at 5 µM (Figure 1A). Addition of sub-stoichiometric CuSO 4 generates a peak corresponding to the triply charged hepcidin-copper complex at m/z 950.329 (Figure 1B). The m/z change between the monoisotopic triply charged peaks of the free hepcidin and hepcidincopper complex equates to the complex being 60.9 Daltons heavier than the free hepcidin. The most abundant isotope of copper (69%) has an atomic mass of 62.9. Thus there is a loss of 2 Daltons and 2 positive charges when the complex is formed indicting that 2 H + ions are lost during complex formation. Addition of equimolar copper to the hepcidin-25 is sufficient for all of the hepcidin to exist as the hepcidin-copper complex (Figure 1C).

Copper binding to human hepcidin:

To further characterise copper binding to human hepcidin, mass spectra were obtained using 5 µM hepcidin and 0-50 µM CuSO 4 (Figure 2A). Human hepcidin-25 binds copper with an affinity such that at 5 µM hepcidin and 5 µM Cu 2+ no free hepcidin is detectable. In contrast only ~20 % of hepcidin-20 exists as a copper complex at 50 µM Cu 2+ clearly demonstrating that the high-affinity copper binding requires the presence of the N- A c c e p t e d M a n u s c r i p t terminal 5 residues of hepcidin. Changing the histidine residue at position 3 to an alanine residue also reduces the affinity of hepcidin for copper with 15-20 µM Cu 2+ being required for 50% complex formation with H3A hepcidin. The very high-affinity of hepcidin-25 for copper was further explored by decreasing the hepcidin concentration to 1 µM and adding copper in 0.2µM increments (Figure 2B). Stoichiometric copper was sufficient for complete complex formation (in several independent repeats saturation was obtained at 0.8 Cu 2+ per hepcidin suggesting an overestimation of the hepcidin concentration by ~25%). This experiment indicates that the dissociation constant for Cu 2+ binding to hepcidin-25 is well below 1 µM. A more accurate estimate is difficult to obtain as the mass spectra became unreliable below 1 µM hepcidin.

Copper binding to murine hepcidin-25 and mutant N3A hepcidin: The affinity of mouse hepcidin for copper is substantially lower than that of human hepcidin with ~8 µM CuSO 4 required for 50% complex formation. (Figure 3) As with human hepcidin the residue at position 3 appears to be involved in the binding as when this asparagine is replaced with alanine there is an approximate 5fold decrease in the affinity for Cu 2+ affinity (~40 µM Cu 2+ required for 50% complex formation with N3A hepcidin) (Figure 3).

Binding of other transition metal ions to human hepcidin:

We tested the ability of 200 µM iron (FeSO 4, Fe 2+ and FeCl 3 , Fe 3+ ), zinc (ZnSO 4, Zn 2+ ) and nickel (NiSO 4, Ni 2+ ) to form complexes with 5 µM human hepcidin-25. Neither Fe 2+ nor Fe 3+ showed any evidence of complex formation, whereas Zn 2+ , and Ni 2+ both formed complexes with hepcidin. Ni 2+ binds with much lower affinity than copper (K d of ~150 µM) and Zn 2+ with lower affinity still (Figure 4). Hepcidin did not appear to bind more than one Zn 2+ or Ni 2+ ion whereas at 200 µM Cu 2+ hepcidin-metal complexes containing 2 or 3 metal ions were detectable (data not shown).

Metal binding to urinary hepcidin:

The most intense signal in the mass spectrum of hepcidin-25 purified from human urine was a triply charged ion at m/z 930.022 i.e. identical to that of synthetic hepcidin-25. No hepcidin-copper complex was observed in the absence of exogenous CuSO 4 addition. This is as expected because the hepcidin purification included HPLC in acid conditions which would have removed any bound metal ions. As with the synthetic peptide, addition of stoichiometric CuSO 4 resulted in the disappearance of the hepcidin peak and the appearance of a peak corresponding to triply charged hepcidin-copper complex at m/z 950.325. The theoretical and experimentally determined masses of all the hepcidin peptides used in this study and their copper complexes are presented in Table 1. Theoretical masses were calculated from published hepcidin sequences and adjusted for the 4 internal disulphide bonds present in folded hepcidin [START_REF] Hunter | The solution structure of human hepcidin, a peptide hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis[END_REF][START_REF] Jordan | Hepcidin revisited: Disulfide connectivity, dynamics, and structure[END_REF]). The calculated isotopic distribution (based on the empirical formula of hepcidin including 4 disulphide bonds) and measured isotopic distribution for hepcidin-25 and hepcidin-25 complexed with copper are shown in Figure 5. The close agreement between theoretical and measured m/z values and isotopic distributions demonstrate that we are measuring monomeric hepcidin with or without a single metal ion bound. We also used collision induced dissociation to obtain MS/MS spectra of both free hepcidin and the hepcidin-copper complex. Similar fragmentation patterns were obtained in both cases although in the complex the y22 ion (residues 4-25) predominated whereas the y21 ion (residues 5-25) was the most intense fragment of free hepcidin (data not shown). Titration of 1µM urinary hepcidin with increasing concentrations of CuSO 4 (Figure 6) produced a binding curve very similar to that obtained with

Effect of hepcidin peptides on ferroportin expression in Caco-2 and HEK293T cells:

To determine if substitution of histidine for alanine influenced the biological activity of hepcidin the intestinal cell line Caco-2 was challenged with hepcidin-25, H3A hepcidin-25 or hepcidin-20 followed by determination of ferroportin levels by Western blotting. In agreement with previous studies, challenging with hepcidin-25 but not hepcidin-20 caused a decrease in ferroportin levels (P<0.05) However, substitution of histidine in position 3 for alanine (H3A hepcidin-25) abrogated the ability of hepcidin to decrease ferroportin expression (Figure 7A-B). In support of this observation utilising the well characterised HEK-293T ferroportin-GFP assay we observed a statistically significant (P<0.05) decrease in GFP fluorescence with both human urinary purified hepcidin-25 and synthetic hepcidin 25 but not with H3A hepcidin-25 (Figure 7C).

Effect of hepcidin peptides on murine serum iron levels:

To determine the biological importance of asparagine at position 3 in murine hepcidin-25 both wild type and N3A hepcidins were injected into wild type CD1 male mice (Figure 8). As expected wild type hepcidin caused a significant hypoferraemic effect after 4hrs compared to control mice that received saline alone (p<0.05). Injecting mice with the murine N3A hepcidin-25 peptide did not induce a significant hypoferraemia.

DISCUSSION

We have used electrospray FTICR mass spectrometry to characterise the binding of several transition metal ions to hepcidin. This technique has previously been used to study metal ion binding to peptides and small proteins [START_REF] Brewer | Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatin 3 and 5 by electrospray ionization mass spectrometry[END_REF][START_REF] Nousianen | Calcium and peptide binding to folded and unfolded Human hep-25[END_REF] and the use of non-denaturing conditions has allowed us to investigate the specificity, affinity and stoichiometry of metal ion binding to hepcidin. Hepcidin-metal complexes were observed with copper, nickel and zinc with a gradient of highest to lowest affinity as follows Cu 2+ > Ni 2+ > Zn 2+ . Hepcidin binds copper with a dissociation constant << 1 µM. Our data is consistent with previous work in terms of the metal specificity and the affinity of the ATCUN motif for copper [START_REF] Brewer | Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatin 3 and 5 by electrospray ionization mass spectrometry[END_REF]23]. Indeed, the pioneering work of Lau et al [23] demonstrated that both the ATCUN motif in albumin and also a short synthetic peptide containing an ATCUN motif bind copper with sub-femtomolar dissociation constants.

Most of our data were obtained using synthetic hepcidin, however, as there has been controversy with regards the structure of hepcidin [START_REF] Nemeth | The Nterminus of hepcidin is essential for its interaction with ferroportin: structure-function study[END_REF][START_REF] Rivera | Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs[END_REF] and whether synthetic hepcidin and 'native' hepcidin have identical properties we examined this metal-hepcidin interaction with hepcidin purified from human urine. Results presented herein show that hepcidin purified from human urine can bind copper with high affinity excluding the possibility that such an interaction is only a property of synthetic hepcidin. However, using synthetic hepcidin allowed us to make several variants of hepcidin to further characterise copper binding. Hepcidin-20 showed vastly reduced copper binding indicating that the high affinity site resides within residues 1-5. Moreover, and as expected, the histidine at position 3 plays a crucial role and substituting this with alanine greatly decreases the affinity of hepcidin for copper. The sequence of hepcidin is highly conserved across 23 mammalian species and the N-terminal region is particularly highly conserved. The entire sequence is identical in 9 primates but the N-terminal 6 residues are identical in all the mammalian hepcidins except rodents (rat and mouse) where histidine 3 is replaced by asparagine. We find that this amino acid substitution results in greatly reduced copper affinity although the asparagine does support some copper binding as evidenced by the further decrease in copper affinity when the asparagine is replaced with alanine.

Introducing alanine at position 3 in human hepcidin not only decreases metal binding but also biological activity as evidenced by decreased ferroportin expression in both Caco-2 and HEK-293T cells. With respect to the murine hepcidin data, we again demonstrate decreased copper binding upon substitution of asparagine at position 3 with alanine, and this amino acid change also appears to decrease biological activity, as evidenced by a lack of a hypoferraemic response in mice. However, we cannot exclude the possibility that the mutant hepcidin retains some biological activity not detected under these experimental conditions. Furthermore, detailed studies would be required to determine whether the loss of metal binding is responsible for this effect, whether the amino acid substitutions cause changes in the structural/biophysical properties of hepcidin or whether the presence of the bulky histidine/asparagine side chain plays a crucial role in determining biological activity.

The data presented in this study support the report of Melino et al where hepcidin-metal binding (copper and nickel) was observed using spectroscopic methods [START_REF] Melino | A metal-binding site is present in the amino terminal region of the bioactive iron regulator hepcidin-25[END_REF]. However, we failed to demonstrate any interaction between hepcidin and ferric or ferrous iron as previously reported by Farnaud et al [START_REF] Farnaud | Modelling of a metal-containing hepcidin[END_REF][START_REF] Farnaud | Identification of an iron-hepcidin complex[END_REF]. We speculate that this is due to a difference in the form of hepcidin used in the studies, with our study using the biologically active fully folded form of hepcidin as opposed to the reduced linear form of hepcidin used by Farnaud et al.

Given the affinities determined in this study and the circulating levels of the various transition metals in human serum, the only interaction which is likely to be physiologically relevant is that of copper binding to the N-terminal region of hepcidin. The vast majority of plasma copper is in complex with ceruloplasmin, however, free copper may be as high as 0.2 µM in healthy individuals and elevated to 1µM in patients with Wilson's disease [24] i.e. high enough to promote complex formation with hepcidin. The residues comprising the copper binding site are also crucial to hepcidin's ability to regulate iron metabolism [START_REF] Nemeth | The Nterminus of hepcidin is essential for its interaction with ferroportin: structure-function study[END_REF][START_REF] Rivera | Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs[END_REF]. It is likely that copper binds to this region of hepcidin in a similar manner to other reported ATCUN-copper interactions [23]: coordination of the metal ion by the N-terminal amine group, the amide groups of residues 2 and 3 and the imidazole group of the histidine. The resulting conformational restraints on the Nterminal region of hepcidin (which appears to be disordered in NMR and crystal structures [START_REF] Hunter | The solution structure of human hepcidin, a peptide hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis[END_REF][START_REF] Jordan | Hepcidin revisited: Disulfide connectivity, dynamics, and structure[END_REF]) would alter its ability to bind to ferroportin and/or other in vivo binding partners.

In summary, we have shown that hepcidin has a very high affinity for copper ions in vitro. We also show that the amino acid residues essential for copper binding are also essential for biological activity. Further work is required to investigate whether copper-hepcidin binding plays a role in iron and/or copper homeostasis. Table 1. Theoretical and measured masses of hepcidin peptides and their complexes with copper. We show the monoisotopic masses calculated from the amino acid sequence and the predicted m/z ratios of the triply charged ions of these peptides. All calculated masses include adjustment for the presence of 4 internal disulphide bonds. The calculated masses for the copper complexes include loss of two H + ions during complex formation. We also show the m/z ratios determined by FTICR mass spectrometry and the monoisotopic molecular masses calculated from these. The sequences of the hepcidin peptides are shown with amino acid substitutions underlined. The hepcidin-20 used in this study incorporated a stable isotope labeled phenylalanine residue indicated by an asterisk (plus 10 Da).

Biochemical

  Journal Immediate Publication. Published on 29 Jan 2010 as manuscript BJ20091521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091521 A c c e p t e d M a n u s c r i p t 3

Biochemical

  Journal Immediate Publication. Published on 29 Jan 2010 as manuscript BJ20091521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091521

  Biochemical Journal Immediate Publication. Published on 29 Jan 2010 as manuscript BJ20091521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091521

Biochemical

  Journal Immediate Publication. Published on 29 Jan 2010 as manuscript BJ20091521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091521 A c c e p t e d M a n u s c r i p t 6 synthetic hepcidin (Figure 3) showing near complete binding of Cu 2+ at all CuSO 4 concentrations. This shows that native human hepcidin can bind Cu 2+ with a dissociation constant << 1 µM.

Biochemical

  Journal Immediate Publication. Published on 29 Jan 2010 as manuscript BJ20091521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091521 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 29 Jan 2010 as manuscript BJ20091521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091521 A c c e p t e d M a n u s c r i p t 10 conformations of cardiac troponin C: Electrospray ionisation and fourier transform ion cyclotron resonance mass spectrometry. Eur. J. Mass Spectrom. 8, 471-481 23. Lau, S., Kruck, T. and Sarkar, B. (1974) A peptide molecule mimicking the copper(II) transport site of human serum albumin. A comparative study between the synthetic site and albumin. J Biol Chem. 249, 5878-5884 24. McMillin, G., Travis, J. and Hunt, J. (2009) Direct measurement of free copper in serum or plasma ultrafiltrate. Am J Clin Pathol. 131, 160-165 Biochemical Journal Immediate Publication. Published on 29 Jan 2010 as manuscript BJ20091521 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20091521 A c c e p t e d M a n u s c r i p t

BiochemicalFigure 1 .

 1 Figure 1. FTICR mass spectra of human hepcidin-25 and hepcidin-copper complexes.

Figure 2 .Figure 3 .

 23 Figure 2. Cu 2+ binding to wild type and mutated human hepcidin.

Figure 4 .Figure 5 .

 45 Figure 4. Ni 2+ and Zn 2+ binding to human hepcidin

Figure 6 .

 6 Figure 6. Cu 2+ binding to 1µM hepcidin purified from human urine.

Figure 7 .

 7 Figure 7. Effect of human hepcidin peptides on ferroportin in Caco-2 and HEK293T cells.

Figure 8 .

 8 Figure 8. Effect of murine hepcidin peptides on murine serum iron levels.

Table 1 .

 1

	Peptide	Mass	m/z of 3 +	Experimentally	Mass calculated
		calculated	ion	determined m/z	from
		from	calculated	of 3 + ion	experimental
		sequence	from		data (Da)
		(Da)	sequence

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

ACKNOWLEDGEMENTS

DGW is funded by a Birmingham Science City Fellowship. The mass spectrometry was performed with assistance from Cleidiane Zampronio in the School of Biosciences, University of Birmingham.