Specificity of the ergothioneine transporter natively expressed in HeLa cells

Biochemical and Biophysical Research Communications 513 (2019) 22e27

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Specificity of the ergothioneine transporter natively expressed in HeLa cells Robert A.J. Tucker, Irwin K. Cheah, Barry Halliwell* Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 28 Medical Drive, Singapore

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 February 2019 Accepted 22 February 2019 Available online 28 March 2019

Ergothioneine is a biologically important compound that has been shown to be transported by the organic cation transporter novel type 1 (OCTN1). Following this discovery, a variety of alternate functions for OCTN1 have been suggested including an integral function in the extra-neuronal cholinergic system. The present study reaffirms the primacy of ergothioneine over these alternate substrates using natively expressed OCTN1 in HeLa cells. Besides the general transport inhibitors, quinidine, verapamil and pyrilamine no other putative substrate inhibited ergothioneine transport significantly, with only a slight inhibition demonstrated by carnitine. Even compounds structurally similar to ergothioneine failed to inhibit ergothioneine uptake, suggesting high selectivity of OCTN1. Ergothioneine was found to be avidly accumulated even at low concentrations (300 nM) by HeLa cells. © 2019 Elsevier Inc. All rights reserved.

Keywords: Ergothioneine OCTN1 Transporter Uptake SLC22A4

1. Introduction Given the growing acceptance of ergothioneine (ET) as a biologically important agent with potential therapeutic applications [1e4] it is increasingly important to clearly establish the role of the organic cation transporter (novel) type 1 (OCTN1). OCTN1 was discovered in 1997 by Tamai and co-workers [5], which they later declared to be a multispecific pharmacokinetic transporter [6]. In 2005 OCTN1 was proposed to be a specific ET transporter by Grundemann et al. [7] who demonstrated its high selectivity for this compound over other previously reported and chemically similar substrates. Following this suggestion, there have been numerous reports that OCTN1 may serve alternate purposes. Most notably is the work published by the Indiveri group [8], using proteoliposomes containing OCTN1 from Escherichia coli overexpression. It was claimed to transport choline, acetylcholine and acetylated cations at an efficiency higher than that of ET. Such findings have served as the basis for the assertion that OCTN1 is a physiological acetylcholine exporter (and choline importer) and is intimately involved in the non-neuronal cholinergic system [9]. It was suggested that anti-inflammatory effects associated with OCTN1 are mediated through acetycholine export. OCNT1 was claimed to be the missing link in the non-neuronal

* Corresponding author. E-mail address: [email protected] (B. Halliwell). https://doi.org/10.1016/j.bbrc.2019.02.122 0006-291X/© 2019 Elsevier Inc. All rights reserved.

acetylcholine system; however Wessler and Kirkpatrick had previously identified organic cation transporter 1 and 2 (OCT1,2) as facilitating acetylcholine export in non-neuronal tissues [10]. Indiveri et al. formed their hypothesis using proteoliposomes, systems which they claim permit better resolution of kinetic and molecular functions over that of cell culture [11]. However results obtained with their system demonstrate very poor ET transport when compared with all other models of OCTN1 function. It seems odd that they demonstrated more effective uptake of tetraethylammonium (TEA) (6-fold greater) over ET [8] in direct contradiction to earlier work published by Grundemann et al. [7]. OCTN1 has also been claimed to transport cytarabine [12]. However the authors failed to demonstrate any competition between ET and cytarabine and suggested that OCTN1 has a distinct nucleoside binding site that does not overlap with the ET binding site. By contrast Tschirka et al. [13] suggested that this result was an artifact of the transfection system, as they were unable to find any transport of cytarabine, deoxycytidine, gemcytibine or deoxyadenosine in their OCTN1 model. Recently, Bai et al. [14] suggested that OCTN1 contributes to the placental transport of sulpiride based upon a small reduction in sulpiride uptake when co-incubated with a 4-fold greater concentration of ET; however they failed to demonstrate any inhibition of sulpiride uptake in BeWo cells on application of an excess of ET. Evidence exists that gabapentin and ET interact to activate OCTN1, i.e. pre-incubation of cells with ET stimulates uptake of gabapentin and vice versa. It was even suggested that gabapentin is

R.A.J. Tucker et al. / Biochemical and Biophysical Research Communications 513 (2019) 22e27

exchanged for ET to enhance ET reuptake at the apical surface of the kidney [15]. Ingestion of ET-rich shiitake mushrooms was found to increase renal clearance of gabapentin by 19%, prolonging the time to reach maximal gabapentin plasma concentration; however no other pharmacokinetic parameters of gabapentin were modified [16]. Tschirka et al., [13] however, identified gabapentin as a weak substrate for OCTN1. OCTN1 has also been implicated as a metformin transporter by Nakamichi et al. [17]; however, evidence has since been accumulated to suggest otherwise. Arner et al. [18] demonstrated that OCTN1 knockdown did not impair biguanide driven lipolysis, and likewise Tschirka et al. [13] demonstrated the lack of metformin transport by OCTN1 in their overexpression model. With all the confusion surrounding the substrates of OCNT1 and its physiological role, more studies are needed to re-establish the role of this compound in the body and rule out any potentially important alternate functions. The work here examined the kinetics of ET uptake by OCTN1 natively expressed in HeLa cells, previously demonstrated to express OCTN1 [19] as well as investigating the competitive inhibition of ET uptake by various other compounds.

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solution (50% ethanol, 0.5% sodium dodecyl sulfate) was added and the plate was incubated on a rocker for 15min to solublize the crystal violet. Absorbance measurements were taken at 590 nm. 2.3. Establishing Michaelis-Menten curve HeLa cells were plated in 6-well plates (as previously described) and washed once. Cells were incubated with 2 mL of ET solution of varying concentration (500, 250, 120, 70, 30, 20, 10 and 5 mM) for 5min then washed and extracted as previously described. 2.4. Ergothioneine/substrate competition studies Cells were plated in 6-well plates and incubated with 2 mL of 20 mM ET solution together with varying concentrations of the possible inhibitors for 10min. Cells were washed and extracted as previously stated. Neostigmine was added at a concentration of 100 mM in the acetylcholine experiment to prevent ester hydrolysis. Some inhibitors were found to be toxic at high concentrations including verapamil, pyrilamine and quinidine and hence concentrations were kept below 200 mM where no toxicity was observed.

2. Materials and methods

2.5. Liquid chromatography tandem mass spectrometry analysis

2.1. Materials

Samples were centrifuged at 20,000g for 10min and supernatants were transferred to 2 mL glass vials (Agilent CrossLab) and evaporated at 37
C under a stream of nitrogen. Samples were resuspended in 100 mL ultrapure water and mixed for 10s. The contents of each vial were transferred to silanized glass inserts and analyzed by liquid chromatography mass spectrometry, (LC-MS/ MS; 6460-QQQ, Agilent Technologies) as previously published [2].

Pyrilamine maleate, verapamil hydrochloride, metformin hydrochloride, tetraethylammonium chloride (TEA), L-carnitine, acetyl-L-carnitine, choline chloride, acetylcholine chloride, gabapentin, quinidine and neostigmine chloride were purchased from Sigma-Aldrich, (MA, USA). L-ergothioneine, hercynine, S-methylergothioneine, d9-hercynine, and d9-L-ergothioneine were kindly provided by Tetrahedron (Paris, France). All chemicals used were either cell culture or analytical grade. HeLa and HEK293 cell lines were acquired from ATCC (VA, USA). Cell culture materials including Dulbecco's modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Biowest (Nuaille, France). TrypLe express was from Life Technologies (CA, USA). 2.2. Establishing uptake and efflux HeLa and HEK293 cells (passage number less than 10) were maintained in DMEM containing 5% FBS. Cells were plated at 800,000 cells/well in 6-well plates and allowed to adhere overnight or until confluent. Cells were rinsed once with phosphate buffered saline (PBS) and either 0.3 or 20 mM ET in unsupplemented DMEM was added to each well and the cells incubated for the durations stated for each experiment. To collect cell lysate for analysis, the medium was removed and the cells washed with ice cold 0.5x PBS. 1 mL of methanol was added to each well together with 6 mL of internal standard solution (to a final concentration of 400 mM d9ET, 200 mM d9-hercynine). The methanol was collected and the extraction was repeated with another 1 mL (sans internal standard). Pooled samples for each well were retained for analysis. To measure efflux, cells were pre-loaded with ET, washed 4x and incubated with 2 mL of unsupplemented DMEM for the duration of the experiment. To halt the experiment the cells were washed once with ice cold 0.5x PBS and extracted with methanol as previously described. Following methanol extraction, plates were stained with crystal violet for normalization of cell counts. In brief, 2 mL crystal violet solution (0.5% crystal violet, 10% methanol in PBS) was added to each well and placed on a rocker for 15min. Wells were gently washed twice with ultrapure water and dried. 2 mL of solublizing

2.6. Statistical analysis All graphs and statistical analysis were carried out using GraphPad Prism (version 7, CA, USA). Data are represented as the mean ± standard deviation (SD), with n numbers given in corresponding legends. Experiments were conducted twice on 2 separate days. Inhibition efficiency of ET uptake by a given inhibitor was stated as an inhibition coefficient, calculated as Km/Ki from data fitted with a competitive inhibition model. A number less than one suggests that an inhibitor interacts more avidly with the transporter than ET; the opposite is true for inhibition coefficients greater than one. In the case of gradient comparison, the extra sum of squares F test was used. P  0.05 was taken as significance. 3. Results In this study we re-examined the kinetics of ET transport and tested the ability of various suggested substrates of OCNT1 to inhibit ET uptake. Using a native ET transporter expressed in the human HeLa cell line ET was found to be taken up with a Km of 51 mM and Vmax of 118 pmol/min per million cells (Fig. 1B). HeLa cells were found to accumulate ET faster than HEK293 cells in line with the fact that HEK293 cells express little OCNT1 [20] (Fig. 1C). Even low concentrations (300 nM) led to considerable intracellular content of ET (Fig. 1A). Using a HeLa volume estimate of 1540mm3 an intracellular concentration of 23 mM can be calculated. This is much greater than the exterior. As the rate of efflux was only 67% of the uptake rate despite the internal concentration being much higher than the extracellular concentration, it suggests that the transport of ET is highly selective in the inward direction over that of efflux. We next examined the ability of compounds claimed by various groups to be substrates for OCTN1 to inhibit ET uptake (Fig. 2A,B,C).

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Fig. 1. A) Uptake of ET at a concentration of 300 nM. Initiation of efflux is indicated by the dotted line. Estimated intracellular ET concentration at initiation of efflux (HeLa volume estimated at 1540mm3), estimated intracellular concentration 23 mM. Efflux experiment conducted by aspirating ET containing media at 270min, washing and replacement with DMEM not containing ET. B) Michaelis-Menten curve, ET uptake (5 min transport) Km 47.87 mM, Vmax 114.5 pmol/min per million cells. C) HEK293 and HeLa cells incubated in 20 mM ergothioneine solution. All data are shown as mean ± SD, n ¼ 3.

Fig. 2. Compound vs 20 mM ET (except neostigmine and hercynine which used 100 mM ET). Inhibition coefficient calculated as Km/Ki from data fitted with a competitive inhibition model. A number less than one suggests that an inhibitor interacts more avidly with the transporter than ET; conversely the opposite is true for inhibition coefficients greater than one, undefined means no notable inhibition. Inhibition coefficients: A) Acetylcholine: undefined, no notable inhibition, acetylcarnitine: 145, choline: undefined, no notable inhibition, carnitine: 37.4. B) Metformin: undefined, pyrilamine: 1.52, gabapentin: ET uptake was observed to increase (positive slope, significantly different from zero, p ¼ 0.0011), quinidine: 0.821. C) Neostigmine: undefined, S-methyl ergothioneine: 249, verapamil: 0.728, hercynine: 6892, tetraethylammonium chloride: undefined. All data are shown as mean ± SD, n ¼ 4.

Strong inhibition of ET transport was only seen with pyrilamine, quinidine and verapamil. Carnitine demonstrated an inhibition coefficient of 33, the only other compound shown to compete at a

less than 100-fold lower effectiveness. No other compounds tested demonstrated any significant inhibition of ET transport out to 10x concentration over ET.

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4. Discussion Following the discovery that OCTN1 transports ET [7] numerous substrates and alternate physiological roles have been suggested for OCTN1. Earlier studies have examined this [13,26] however this work was performed using overexpression systems, which may not always recapitulate all the aspects required for successful assay of substrates that compete with ET, e.g. transporters could be expressed in the wrong conformation or sometimes inserted in the wrong orientation in the plasma membrane or certain intracellular regulatory factors could be missing. In general our data support the conclusions of Grundemann et al. [7], Grigat et al. [21] and Tschirka et al. [13] in suggesting that OCNT1 is primarily an ET transporter. 4.1. Choline, acetylcholine, acetylcarnitine Pochini et al. [8] posited from proteoliposome experiments that OCNT1 is primarily a permease that handles choline, acetylcholine and acetylcarnitine. They demonstrated a Michaelis constant for acetylcholine import of 1 mM with an estimate for choline uptake being around this value. Considering a plasma choline concentration of around 10 mM [22] and with acetylcholine level being vanishingly small due to plasma esterase action it would be unlikely that OCNT1 could contribute to the uptake of these compounds in vivo. Furthermore, in vivo OCT1 and OCT2 have already been shown to facilitate acetylcholine transport in extra-neuronal tissues [23,24] Our experiments demonstrated no inhibition by choline or acetylcholine, revealing little, if any, competition with ET for uptake using a cellular model. Acetylcarnitine was suggested as a low efficiency substrate for OCTN1 following a demonstration that 1 mM acetylcarnitine caused a 50% reduction in acetylcholine uptake [25]. This is in contrast with OCNT2 which transports acetylcarnitine with a Michaelis constant of 6.57 mM [26]. Considering a plasma concentration of less than 40 mM [27], it would be hard to imagine a transporter with > 1 mM Km contributing to physiological transport of acetylcarnitine. No convincing evidence exists to suggest that OCTN1 contributes to the transport of these compounds in a physiologically relevant manner. Acetylcarnitine demonstrated an inhibition coefficient of 124, suggesting that any acetylcarnitine transport by OCTN1 is not physiologically relevant. 4.2. Gabapentin, metformin, neostigmine Various reports have suggested that OCNT1 is involved in the transport of gabapentin and metformin [15,28]. Tshikira et al. [13] demonstrated a weak enhancement of gabapentin uptake by OCTN1 overexpression, but suggested it was only very weak (100x weaker than for ET). In this report no inhibition of ET uptake was seen with either metformin or gabapentin, with gabapentin actually demonstrating an enhancement of ET uptake, in line with previous observations. It may be, as suggested by Ref. [15], that Lamino acid transporter mediated uptake of gabapentin enhances ET uptake via electroneutral substrate exchange/trans-stimulation. Neostigmine also failed to demonstrated significant inhibition of ET uptake. Nakamichi et al. [29] first suggested that OCTN1 facilitated metformin uptake. However this assertion has not been confirmed. Arner et al. [18] demonstrated that OCTN1 knockdown did not impair biguanide driven lipolysis, and likewise Tschirka et al. [13] demonstrated no metformin transport by OCTN1. 4.3. Carnitine, tetraethylammonium chloride TEA was the first designated substrate for OCTN1 and carnitine was the first claimed physiological substrate. Grundemann et al. [7]

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calculated an uptake efficiency over 100x lower for both carnitine and TEA over ET. In our investigation carnitine was the only substrate outside of verapamil, pyrilamine and quinidine with a inhibition coefficient less than 100, demonstrating slight inhibition (inhibition coefficient of 37.4) of ET uptake. This weak inhibition further demonstrates the fact that OCTN1 is a very poor carnitine transporter. TEA demonstrated no significant inhibition. 4.4. Ergothioneine metabolites: hercynine and S-methyl ergothioneine Despite their closely related structures neither hercynine nor Smethyl ergothioneine interfered with ET uptake. The presence of ET was not found to interfere with the uptake of either compound and vice-versa. This suggests that OCTN1 is highly selective for ET over related metabolites. This is interesting in the case of hercynine, a well established ET breakdown product [30] suggesting that cells can accumulate ET, but retain hercynine much less avidly. 4.5. Pyrilamine, quinidine and verapamil These compounds were the most efficient inhibitors of OCNT1 previously identified by Grigat et al. [21] and Yabuuchi et al. [6]; who demonstrated that these compounds are not competing for uptake, but inhibiting the transporter. Our studies confirmed these previous findings in a native OCTN1 system. Verapamil is a very potent, promiscuous inhibitor that has been observed to inhibit a wide range of transporters including OCT1, OCT2, OCTN1, OCTN2, GLUT1, P-glycoprotein pumps (PGP pumps) along with its pharmacological target the L-type calcium channel [6,31,32]. Quinidine is a type I Na channel blocker with off target effects inhibiting PGP pumps as well as OCNT2 [31,33]. Pyrilamine is an antihistamine/ anticholinergic for which exists a putative pyrilamine transporter. The transporter has been demonstrate to facilitate cation exchange in the brain. Curiously, verapamil and quinidine demonstrate inhibition of pyrilamine uptake via the pyrilamine transporter underlining their promiscuity as transport blockers [34]. It is possible that plasma concentrations of these drugs when used therapeutically could inhibit ET transport (peak plasma concentrations of verapamil 0.6 mM [35], quinidine 10 mM [36] versus around 1 mM for ET [37]), but it is hard to predict what the consequences of this might be. 4.6. Kinetics of ergothioneine transport The transport kinetics reported in our natively expressed ET model are similar to those previously reported by Grundemann et al. [7] and Grigat et al. [21] in their HEK293 overexpression systems (Km 21 mM) and we demonstrated that HEK293 cells poorly accumulate ET. This finding is consistent with a previous report that HEK293 cells express little OCTN1 [20] and therefore should be expected to accumulate ET very poorly. It is interesting to note that FBS contains between 0.5 and 1 mM ET [38], making FBS-containing media a source of between 25 and 100 nM ET depending on the FBS concentration. The fact that ET is accumulated even at low concentrations means that any cell line expressing OCTN1 will contain a moderate amount of ET (we observed initial concentrations between 6 and 11 pmol/million cells for HeLa cells and 1e2 pmol/million cells for HEK293 cells) even before ET supplementation. This suggests that the low mM plasma concentration found in plasma [37] is enough to drive ET into any cell expressing OCTN1. Researchers need to be aware of the possible presence of ET in their cells.

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5. Summary In recent years numerous groups have claimed that OCTN1 functions to transport a wide array of compounds. Our study shows that it seems unlikely that OCTN1 plays any role in the nonneuronal acetylcholine system, neither is it likely that OCTN1 plays a role in the transport of metformin or any significant role in the transport of carnitine or acetylcarnitine. In agreement with Urban et al. [15] gabapentin was found to enhance ET uptake, possibly in an electroneutral exchange process and could conceivably enhance ET retention in the body. ET transport is not notably interfered with compounds of very similar chemical structure, (Smethyl ergothioneine and hercynine) suggesting high selectivity for ET. The only substrates found to inhibit the transport of ET were 3 highly promiscuous transport inhibitors whose actions have been previously demonstrated by Yabuuchi et al. [6] and Grigat et al. [21]. Conflicts of interest All authors declare no conflicts of interest Acknowledgments We wish to acknowledge Tetrehedron, (Paris, France) for kindly supplying ET, S-methyl ergothioneine, hercynine and deuterated standards. We are grateful to the National Medical Research Council Singapore (Individual Research Grant NMRC/1264/2010/082/12) and Tan Chin Tuan Foundation for research support. References [1] I.K. Cheah, B. Halliwell, Ergothioneine; antioxidant potential, physiological function and role in disease, Biochim. Biophys. Acta (BBA) - Mol. Basis Dis. 1822 (5) (2012) 784e793, https://doi.org/10.1016/j.bbadis.2011.09.017. [2] R.M.Y. Tang, I.K.M. Cheah, T.S.K. Yew, B. Halliwell, Distribution and accumulation of dietary ergothioneine and its metabolites in mouse tissues, Sci. Rep. 8 (1) (2018) 1e15, https://doi.org/10.1038/s41598-018-20021-z. [3] B.N. Ames, Prolonging healthy aging: longevity vitamins and proteins, Proc. Natl. Acad. Sci. U.S.A. 115 (43) (2018) 201809045, https://doi.org/10.1073/ PNAS.1809045115. [4] B. Halliwell, I.K. Cheah, R.M.Y. Tang, Ergothioneine - a diet-derived antioxidant with therapeutic potential, FEBS (Fed. Eur. Biochem. Soc.) Lett. 592 (2018) 3357e3366, https://doi.org/10.1002/1873-3468.13123. [5] I. Tamai, H. Yabuuchi, J.I. Nezu, Y. Sai, A. Oku, M. Shimane, A. Tsuji, Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1, FEBS (Fed. Eur. Biochem. Soc.) Lett. 419 (1) (1997) 107e111, https:// doi.org/10.1016/S0014-5793(97)01441-5. [6] H. Yabuuchi, I. Tamai, J.-I. Nezu, K. Sakamoto, A. Oku, M. Shimane, Y. Sai, A. Tsuji, Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pH-dependent transport of organic cations, J. Pharmacol. Exp. Therapeut. 289 (2) (1999) 768e773. [7] D. Grundemann, S. Harlfinger, S. Golz, A. Geerts, A. Lazar, R. Berkels, N. Jung, A. Rubbert, E. Schomig, Discovery of the ergothioneine transporter, Proc. Natl. Acad. Sci. U.S.A. 102 (14) (2005) 5256e5261, https://doi.org/10.1073/ pnas.0408624102. [8] L. Pochini, M. Scalise, M. Galluccio, L. Amelio, C. Indiveri, Reconstitution in liposomes of the functionally active human OCTN1 (SLC22A4) transporter overexpressed in Escherichia coli, Biochem. J. 439 (2) (2011) 227e233, https:// doi.org/10.1042/BJ20110544. [9] L. Pochini, M. Scalise, M. Galluccio, G. Pani, K.A. Siminovitch, C. Indiveri, The human OCTN1 (SLC22A4) reconstituted in liposomes catalyzes acetylcholine transport which is defective in the mutant L503F associated to the Crohn's disease, Biochim. Biophys. Acta Biomembr. 1818 (3) (2012) 559e565, https:// doi.org/10.1016/j.bbamem.2011.12.014. [10] I. Wessler, C.J. Kirkpatrick, Acetylcholine beyond neurons: the non-neuronal cholinergic system in humans, Br. J. Pharmacol. 154 (8) (2008) 1558e1571, https://doi.org/10.1038/bjp.2008.185. [11] M. Scalise, L. Pochini, N. Giangregorio, A. Tonazzi, C. Indiveri, Proteoliposomes as tool for assaying membrane transporter functions and interactions with xenobiotics, Pharmaceutics 5 (3) (2013) 472e497, https://doi.org/10.3390/ pharmaceutics5030472. [12] C.D. Drenberg, A.A. Gibson, S.B. Pounds, L. Shi, D.P. Rhinehart, L. Li, S. Hu, G. Du, A.T. Nies, M. Schwab, N. Pabla, W. Blum, T.A. Gruber, S.D. Baker, A. Sparreboom, OCTN1 is a high-affinity carrier of nucleoside analogues, Cancer Res. 77 (8) (2017) 2102e2111, https://doi.org/10.1158/0008-

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