Biochimica et Biophysica Acta 1858 (2016) 2617–2624 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbamem Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter Jacek Grebowski, Maciej Studzian, Grzegorz Bartosz, Lukasz Pulaski ⁎ Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, 12/16 Banacha St., 90-237 Lodz, Poland article info abstract Article history: The need for large amounts of reproducibly produced and isolated protein arises not only in structural studies, Received 8 January 2016 but even more so in biochemical ones, and with regard to ABC transporters it is especially pressing when faced Received in revised form 13 June 2016 with the prospect of enzymatic/transport activity studies, substrate screening etc. Thus, reliable heterologous ex- Accepted 22 June 2016 pression systems/model organisms for large and complex proteins are at a premium. We have verified the appli- Available online 24 June 2016 cability of the recently established novel eukaryotic expression system, using Leishmania tarentolae as a host, for Keywords: human ABC protein overexpression. We succeeded in overexpressing human ABCB6, a protein with controversial fi ABC transporters subcellular localization and multiple proposed cellular functions. We were able to demonstrate its ef cient ex- Membrane protein expression pression in the expected subcellular locations as well as biochemical activity of the overexpressed protein Leishmania (ATPase activity and porphyrin-like substrate transport). This activity was absent in cells overexpressing the cat- alytically inactive variant of ABCB6 (K629M). We demonstrate the possibility of applying a cost-effective expres- sion system to study the activity of membrane transporters from the ABC superfamily. © 2016 Elsevier B.V. All rights reserved. 1. Introduction polymorphisms in ABCB6 gene can be more common than previously expected [10]. Individuals that are Lan-negative do not show any ap- ATP-binding cassette (ABC) transporters move structurally diverse parent clinical phenotype, arguing against a vital role of ABCB6 in compounds across membranes: in humans, among other functions, porphyrin synthesis. However, ABCB6 mutations have unexpectedly they protect normal tissues against toxins and confer multidrug resis- been identified as causal for a number of non-hematological genetic tance on malignant cells [1–3]. ABC proteins are divided into seven diseases including coloboma [11], dyschromatosis universalis [12] and subfamilies, based on domain organization and sequence homology. pseudohyperkalemia [13]. ABCB6 belongs to the ABCB subfamily, which includes P-glycoprotein Leishmania tarentolae is a trypanosomatid protozoan parasite of the (ABCB1/ MDR1) causing multidrug resistance of cancer cells. Despite Mauritanian gecko, easily cultured in vitro as a protein expression extensive studies, the physiological function of several ABC transporters host [14]. There are established vectors and procedures for transfection remains unknown until now — one of more controversial proteins in as well as selection of transgenic strains that allow efficient production this respect is ABCB6 [4]. It was demonstrated that the putative mito- of recombinant proteins. L. tarentolae cells are rich in glycoproteins, chondrial/endosomal ABC transporter ABCB6 activates de novo heme which may constitute even N10% of the total protein. Moreover, oli- and porphyrin biosynthesis and elevates intracellular protoporphyrin gosaccharide structures are similar to those appearing in mammalian IX (PPIX) levels [5–7], whereas the loss of one Abcb6 allele in embryonic cells, with N-linked galactose and fucose residues [14,15]. This may stem cells impairs porphyrin synthesis [8]. ABCB6 is also expressed in make them especially suitable for expression of ABC transporters which red blood cells: after upregulation during erythroid differentiation, it are often strongly glycosylated. The advantages of the L. tarentolae ex- is expressed in cell membranes of mature erythrocytes at relatively pression system include facile handling of cultures, an optimal culture high levels [4]. ABCB6 protein was identified as the antigen of Langereis temperature of 26 °C, rapid growth rate (with a doubling time of approx- (Lan) blood group. Lan-negativity is believed to be very rare and imately 6 h in agitated culture), high cell densities (up to 109 cells/ml) manifests in approximately 0.005% of the Caucasian population [9], with promising potential for scale-up as well as the stability of transgenic although recent populational and genetic studies show that missense strains [16–18]. The aim of this study was to investigate the possibility of expression of human ABCB6 protein (as a model ABC transporter) using L. tarentolae as Abbreviations: NTC, nourseothricin; BHI, brain-heart infusion; HRP, horseradish a heterologous host and to characterize localization and function of the peroxidase. ⁎ Corresponding author. transporter in a stable transgenic strain. To evaluate the potential role of E-mail address: [email protected] (L. Pulaski). ABCB6 and other transporters in cellular physiology, it is important to http://dx.doi.org/10.1016/j.bbamem.2016.06.022 0005-2736/© 2016 Elsevier B.V. All rights reserved. 2618 J. Grebowski et al. / Biochimica et Biophysica Acta 1858 (2016) 2617–2624 establish reliable models of functional protein. While other heterologous marker) of ~7.1 kbp (ABCB6) and ~4.6 kbp (control) were purified by expression systems were used in the past to biochemically characterize gel extraction. Parasite cells in logarithmic growth phase were cen- ABC transporters, including Pichia pastoris [19],insect[20] or mammalian trifuged (2500 ×g, 5 min), resuspended in Eppendorf Hypoosmolar [6] cell lines, their main disadvantages (which partially led to limited suc- Buffer (Eppendorf, Hamburg, Germany) at final concentration of cess in their application to ABCB6 functional studies) are respectively: 1×108 cells/ml and kept on ice for 10 min prior to the addition of complicated membrane isolation procedure, complex transgenic strain 2.5 μg of linearized expression cassette. Following electroporation derivation and relatively high cost. In all these respects, the L. tarentolae (Eppendorf Multiporator, 2 mm cuvette, 1000 V, 160 μs) and system was shown to be superior [21,22], also for human membrane 10 min incubation on ice, cells were resuspended in fresh BHI culture proteins [23]. Here, we present a proof of concept for applying this medium and grown in suspension for the next 24 h. Single colony- convenient and cost-effective Eukaryotic host to study the biochemistry derived, NTC resistant strains were then selected after 9 days of growth of ABCB6, a clinically relevant and poorly characterized membrane on solid media (BHI-agar containing 100 μg/ml NTC) and maintained in transporter. suspension culture with constant antibiotic concentration. Genomic in- tegration of the expression cassette in control and ABCB6 transfected 2. Materials and methods strains was verified by diagnostic PCR reaction with 3 sets of primers (Fig. 1): 2.1. Materials and cell culture 1) ABCB6 specific primers (annealing temperature of 60 °C): ′ ′ L. tarentolae cells, empty expression vector (pLEXSY-sat2) and all cul- ABCB6-F 5 -CTGGTCTCCTGCTCCTCTG-3 ′ ′ ture materials were purchased from Jena Bioscience (Jena, Germany). ABCB6-R 5 -GCGCATACTTTGTCACTGAGC-3 Cells were grown at 26 °C in static suspension cultures in LEXSY BHI liquid 2) ssu locus integration primers (annealing temperature of 60 °C): medium supplemented with porcine hemin (5 μg/ml), penicillin/strepto- SSU-F 5′-GATCTGGTTGATTCTGCCAGTAG-3′ mycin (Pen-Strep, 100 U/ml) and nourseothricin (NTC, 100 μg/ml). APRT-R 5′-TATTCGTTGTCAGATGGCGCAC-3′ Genomic DNA from transgenic L. tarentolae strains was isolated 3) Nourseothricin resistance cassette integration primers (annealing using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). For ABCB6 temperature of 53 °C): immunostaining, anti-ABCB6 antibody (clone 24.39; Santa Cruz Bio- SAT-F 5′-CCTAGTATGAAGATTTCGGTGATC-3′ technology, Santa Cruz, CA, USA) was used. An antibody derived SSU-R 5′-CTGCAGGTTCACCTACAGCTAC-3′ against the Leishmania major major surface glycoprotein Gp63 (clone [96-126], Abcam, Cambridge, UK) was used as a plasma mem- 2.3. Preparation of microsomal and plasma membrane fractions brane marker since it was previously demonstrated in the literature [24] to detect the L. tarentolae ortholog (which is a larger protein mi- Lysis and membrane fractioning of transgenic L. tarentolae strains grating at ca. 75 kDa MW). As secondary antibodies, HRP-conjugated (empty vector control and ABCB6) was made by a modification of the goat anti-mouse IgG (ThermoFisher Scientific, Waltham, MA, USA) method of Chen et al. [25]. All preparations were made at 4 °C. Briefly, was used for Western blotting and Alexa Fluor 555-conjugated goat cultures containing approximately 5 × 1010 cells were harvested anti-mouse IgG2a antibody (Life Technologies, Carlsbad, CA, USA) was (2500 ×g, 5 min), washed twice with buffer A (10 mM Tris-HCl pH = used for immunofluorescence. 7.4, 0.3 M sucrose, 0.2 mM EDTA, 0.2% BSA) and lysed in hypoosmolar Sf9-ABCB6 membranes, overexpressing human ABCB6 from a buffer B (10 mM Tris-HCl, pH = 7.4) with glass homogenizer (approx- baculoviral vector, were a kind gift from Dr. G. Szakács (Budapest) [20]. imately 30 full strokes).