Molecular Biotechnology https://doi.org/10.1007/s12033-018-0095-2

ORIGINAL PAPER

Molecular Cloning and Biochemical Characterization of Iron Superoxide Dismutase from Leishmania braziliensis Camila C. B. Brito1 · Fernando V. Maluf2 · Gustavo M. A. de Lima2,4 · Rafael V. C. Guido2 · Marcelo S. Castilho1,3 

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Leishmaniasis is one of the most important neglected tropical diseases, with a broad spectrum of clinical manifestations. Among the clinical manifestations of the disease, cutaneous leishmaniasis, caused by species of Leishmania braziliensis, presents wide distribution in Brazil. In this work, we performed the cloning, expression, and purification of the enzyme superoxide dismutase of Leishmania braziliensis (LbSOD-B2) considered a promising target for the search of new compounds against leishmaniasis. In vitro assays based on pyrogallol oxidation showed that LbSOD-B2 is most active around pH 8 and hydrogen peroxide is a LbSOD-B2 inhibitor at low millimolar range ­(IC50 = 1 mM). Keywords  Leishmania braziliensis · Superoxide dismutase · Heterologous expression · Kinetic assay

Introduction Leishmaniasis, one of the seven most important neglected tropical diseases, has a broad spectrum of clinical manifestations [1, 2], including visceral leishmaniasis (VL), cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), which can cause disability, mutilation, and even death [3, 4]. The annual incidence of CL was estimated to be near one million cases in 2015 [5] and Brazil ranks among the ten countries with the highest number of cases [6]. Among the 20 parasite species that are pathogenic to humans, Leishmania braziliensis is the most prevalent in Brazil, where it is responsible for both CL and MCL forms [7, 8]. Until 2030, it is estimated that US $13 million per year will be spent with the treatment of patients with CL [9]. However, the interest of big pharma industries to develop novel therapeutic * Marcelo S. Castilho [email protected] 1



Programa de pós‑graduação em Biotecnologia, Universidade Estadual de Feira de Santana, Feira de Santana, BA, Brazil

2



Sao Carlos Institute of Physics, University of Sao Paulo, Av. Joao Dagnone, 1100 Jardim Santa Angelina, São Carlos, SP 13563‑120, Brazil

3

Faculdade de Farmácia, Universidade Federal da Bahia, Salvador, BA 40170‑290, Brazil

4

MAX IV Laboratory, Lund University, PO Box 118, 221 00 Lund, Sweden



strategies to fight this disease has been scarce [10]. Even the DNDi has been more concerned with VL than CL, according to its pipeline report (https​://www.dndi.org/wp-conte​nt/ uploa​ds/2017/05/DNDi_Leish​mania​sisPi​pelin​e_2017.pdf). Although it is reasonable to assume that drugs that cure VL will have a positive effect on CL patients, different pharmacokinetic requirements, as well as delivery issues, might limit their usefulness [11]. To make matter worse, pentavalent antimonials ­(Sb5+) are prescribed to most patients, regardless of the clinical form of the disease or the parasite infecting species [12, 13]. This simplistic therapeutic regimen, along with the high incidence of side effects (nausea, lethargy, hepatotoxicity, and cardiotoxicity), makes it hard for the patient to complete the treatment course [6, 14]. As a consequence, some parasite species have already shown reduced susceptibility to this class of drugs [15, 16]. Therefore, there is an urgent need to develop novel drugs to fight leishmaniasis. An alternative to overcome these problems is to explore biochemical pathways that have enough differences to allow selective modulation, whereas the human counterpart remains active. The protozoan parasites have developed an efficient antioxidant system that is essential for the parasite survival [17] whose first step is controlled by superoxide dismutase (SOD, E.C.1.15.1.1) [18, 19]. There are three isoforms of SOD in Leishmania braziliensis [3] that are responsible for the dismutation of superoxide into hydrogen peroxide and molecular oxygen: FeSOD-A

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isoform is located in the mitochondria, where it is involved in cellular respiration; FeSOD-B1 and FeSOD-B2 isoforms are found in the parasite glycosome [3], where they participate in metabolic pathways such as glycolysis, fatty acid oxidation, and lipid biosynthesis [20]. The glycosome enzymes are also important for protection against oxidative cellular damage [20]. Although SOD is also found in the human host, there are significant differences between their active sites, such as the metal-ion responsible for the catalytic activity (Fe in Leishmania braziliensis vs. Cu/Zn and Mn in the human host) [21]. Such differences supported the search for selective inhibitors of SOD from several Leishmania species [8, 22, 23]. Phthalazine derivatives, for instance, show ­IC50 values for Fe-SOD tenfold lower than for Cu/Zn-SOD [21]. In addition, it has been reported that inhibition of the LbSOD enzyme affects the survival of the parasite in the host [8, 24]. Considering these facts, herein we describe the cloning, expression, and biochemical characterization of FeSOD-B2 of Leishmania braziliensis as a promising target for drug discovery efforts to fight cutaneous leishmaniasis.

Materials and Methods LbSOD‑B2 Gene Cloning The genomic sequence encoding the superoxide dismutase from Leishmania braziliensis (LbrM_32_2010) (LbSOD-B2), available in http://www.ncbi.nlm.nih.gov, was employed to design the primers. Briefly, DNA fragment containing the sequence that codes for LbSOD-B2 was amplified with the Phusion system (New England Biolabs®) using the forward and reverse primers: CAG​GGC​ GCC​ATGC ​ CGT ​ TCT ​ GTG ​ CCC ​ AG​ and ACCC ​ GAC ​ GCG ​ GT​ TAC​AAC​TGG​CTA​GAT​GCA​AAGTC, respectively, which were acquired from Exxtend (https​://www.exxte​nd.com.br/ site/). The required nucleotide sequence for the use of the cloning system employed (LIC) is underlined in the primer sequences shown above. The template DNA was extracted from genomic DNA from Leishmania (viannia) braziliensis cells (MHOM/BR1997/LTCP11245), provided by Dra. Luciana S. Cardoso (UFBA-Brazil). The following parameters were used for the PCR amplification step: initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 43 °C for 30 s and extension at 72 °C for 110 s as final step of reaction. The product of this reaction was loaded on a 1% agarose gel for electrophoretic separation (100 V/ 1 h), purified directly from the gel matrix using the kit Wizard® SV Gel and PCR Clean-Up System (Promega), and quantified spectrophotometrically at 260 nm wavelength (Nanodrop 200, Thermo Scientific). The expression vector (pETM11)

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and the purified insert (LbSOD) were incubated with T4 DNA polymerase (1:3) for 30 min at 25 °C. The product of that reaction was employed to transform E. coli (DH10β) competent cells, by heat-shock protocol [25], which were grown in Luria Bertani medium (tryptone 1%, sodium chloride 1%, yeast extract 0,5%, and bacteriological agar 1,5%) supplemented with 30 µg/mL kanamycin (16 h at 37 °C). Colony PCR was employed to confirm the presence of the plasmid within the recombinant cells [26]. The positive clones were heat-shock transformed into expression cells [E. coli BL21 (DE3)], which were grown in Luria Bertani medium supplemented with 30 µg/mL kanamycin for 16 h at 37 °C and then kept at − 80 °C in glycerol.

LbSOD‑B2 Heterologous Expression and Chromatographic Purification Recombinant E. coli BL21 (DE3) cells, which have the pETM11-LbSOD plasmid, were cultured at 37 °C/180 rpm in Luria Bertani medium supplemented with 30 µg/mL kanamycin until O ­ D600 reached 0.6–0.9. At this moment, 1 mM IPTG (final concentration) was added to the culture and the temperature was reduced to 20 °C. After 16 h, the cells were harvested by centrifugation (2800×g, 4 °C, 30 min) and resuspended in lysis buffer (PBS 50 mM, NaCl 100 mM pH 7) supplemented with 1 mM phenylmethanesulphonylfluoride (PMSF). Next, cells were incubated with lysozyme 0.5 mg/mL for 30 min and then disrupted by sonication (10 × 15 s bursts with 30 s intervals between each burst, 9 W). These steps were carried out in ice-bath. The soluble fraction was clarified by centrifugation (16,000×g, 20 min, 4 °C) and then loaded onto a His-Trap HP column (GE Healthcare), pre-equilibrated with buffer A (PBS 50 mM pH 7.0, NaCl 100, 20 mM imidazol). Then, contaminants were eluted with 20 column volumes of buffer A. Next, LbSODB2 was eluted within an increasing gradient of imidazole (50–500 mM) in buffer A. Purification steps were monitored by UV absorbance measurement at 280 nm and the level of protein purity was confirmed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) 12%. The fractions containing the protein were pooled together and dialyzed to buffer B (PBS 50 mM, NaCl 100 mM pH 7.0). Protein concentrations were determined spectrophotometrically using a theoretical extinction coefficient of (55,775/M/cm)  mol/L/cm at 280  nm calculated using ExPASy (http://web.expas​y.org/protp​aram/). Next, LbSOD-B2 was subjected to TEV protease digestion (1 mg per 20 mg LbSOD-B2), 4 °C, overnight. After the proteolysis step, LbSOD-B2 was loaded again onto His-Trap column, pre-equilibrated with buffer B, to separate cleaved from uncleaved His-tag LbSOD-B2 and TEV protease. Cleaved LbSOD-B2 was eluted with five column volumes of this same buffer. The other proteins were

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eluted with buffer B supplemented with 500 mM imidazole. The cleaved His-tag LbSOD-B2 was concentrated (10  kDa MWCO Amicon Ultra devices, Millipore) to 10 mg/mL and stored in 30% glycerol at − 80 °C.

Determination of Superoxide Dismutase activity LbSOD-B2 activity was evaluated using indirect assay that relies on the pyrogallol oxidation [27]. Briefly, the capacity of LbSOD-B2 (0.1 µM) to inhibit the auto-oxidation of pyrogallol (0.2–2.5  mM) was evaluated in buffer A (50 mM Tris–HCl (pH 8.0) containing 1 mM EDTA). The reaction was monitored for 60 s at wavelength of 320 nm in a UV–Vis spectrophotometer (Shimadzu®). SOD activity was calculates as ( ) SOD activity (inhibition rate %) = A0 − A1 ∕A0 × 100, where A0 corresponds to the absorbance measure without the presence of the enzyme and A1 corresponds to absorbance in the presence of SOD. The experiments were performed in triplicate and the values were used for non-linear regression analysis using the GraphPad Prism®.

Effect of pH in the LbSOD‑B2 Activity The effect of pH over LbSOD-B2 activity was evaluated in the following pH 4.0–10 range [buffers: sodium acetate (pH 4.0; 4.5; 5.0); sodium phosphate (pH 6.0; 7.0); bicine (pH 8.0); Hepes (pH 7.0;7.5; 8.0); Ches (pH 9.0; 9.5; 10); Tris–HCl (pH 7.5; 8.0; 8.5)]. Briefly, each buffer at the final concentration of 50 mM was incubated with LbSODB2 (1 µM) for 30 min, 4 °C. After this period, the solution was diluted (10×) in buffer 50 mM Tris–HCl buffer containing 1 mM EDTA pH 8 and the SOD activity was determined by the pyrogallol oxidation assay as described above.

Results and Discussion Cloning, Expression, and Purification of LbSOD‑B2 The gene encoding LbSOD-B2 was PCR amplified, from Leishmania braziliensis genomic DNA, using the forward and reverser primers designed for ligation-independent cloning strategy [28, 29]. This strategy afforded a 588-bp product (Fig. 1a), which was inserted into pETM11 vector following treatment T4 DNA polymerase to create overhangs. The recombinant plasmid was heat-shock transferred to DH10β E. coli cells, as evidenced by colony PCR (Fig. 1b). E. coli BL21 (DE3) cells were transformed with plasmid LbSOD-pETM11 and LbSOD-B2 expression was induced with IPTG 1 mM at 20 °C for 16 h. Higher temperatures afford only insoluble LbSOD-B2 (data not shown). The LIC vector employed in this work (pETM11) allowed the expression of the recombinant LbSOD-B2 fused to Histag and, for that reason, the protein purification was carried out by an affinity chromatography step (Fig. 2a). Considering that histidine residues (pKa 6.0) must be deprotonated to interact with the immobilized ­Ni+2 ions from the resin, the buffer selected for the purification step had pH 7 (sodium phosphate 50 mM containing NaCl 100 mM). The initial use of low concentrations of imidazole allowed the removal of proteins present in the purification supernatant and thus it was possible to elute pure LbSOD-B2 using a higher concentration of imidazole (500 mM) (Fig. 2b). Once His-tag can alter kinetics and structural features of LbSOD-B2, the biological assays were carried out after this

LbSOD‑B2 Activity in the Presence of Hydrogen Peroxide The effect of different concentrations of hydrogen peroxide (0.5–3.5 mM) over LbSOD-B2 activity was evaluated as follows: hydrogen peroxide was incubated for 5 min with LbSOD-B2 (1 µM) and then the solution was diluted (10×) in 50 mM Tris–HCl buffer containing 1 mM EDTA pH 8 and the SOD activity was determined by the pyrogallol oxidation assay as described above.

Fig. 1  Cloning steps to insert LbSOD gene into pETM11 expression vector. a 1% agarose gel electrophoresis. Lane 1: DNA marker (M); lane 2: PCR amplification product with base pairs (bp) equivalent to LbSOD gene; b colony PCR of the recombinant clones (DH10β E. coli cells): DNA marker (M). 1–3: recombinant clones of LbSOD pETM11

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Fig. 2  Chromatographic purification profile of LbSOD-B2. a Purification chromatogram using 20–500  mM imidazole gradient. b SDS-PAGE 12% with fractions collected during the chromatography (23  kDa) 1: molecular weight standard (kDa); 2: purification supernatant; 3: fraction 20 mM imidazole; 4: 50 mM imidazole; 5–8: 500  mM imidazole. c SDS-PAGE 12% with samples before (2) and after (1) cleavage LbSOD-B2

tag was removed by proteolytic cleavage with TEV protease (Tobacco etch virus) (Fig. 2c), whose recognition site had already been engineered into pETM11 expression vector. The advantages of using TEV include its high specificity, catalytic activity at low temperatures and in the presence of several protease inhibitors, such as PMSF, which was used in the purification steps of this work [30]. In addition, TEV maintains its catalytic activity over a wide pH range (4–9) [31] that guarantees protease activity under conditions that maximize the stability of the protein of interest (LbSODB2). The overall yield of the purification steps is 20 mg of LbSOD-B2 per liter of culture medium.

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Cu-ZnSOD [27]. As the pH increases, there is a decrease in the sensitivity of the enzyme, but 93% of pyrogallol oxidation inhibition is still observed at pH 9. Therefore, we decided to determine which LbSOD-B2 concentration would afford a suitable signal-to-noise ratio. These assays, carried out in Tris–HCl buffer (pH 8), show that in the range of 0.1–0.2 µM the reaction rate depends only on the concentration of the enzyme. Thus, it seems that steady-state conditions can be assumed and that it follows classical Michaelian kinetics behavior (Fig. 3). In order to guarantee that these conditions last for the time of the assays, we employed 0.1 µM of LbSOD-B2 for subsequent studies. Next, the effects of different concentrations of pyrogallol over LbSOD-B2 activity were evaluated (Fig. 4). We observed that the LbSOD-B2 ability to block pyrogallol oxidation decreases inasmuch as its concentration levels up and it plateaus at just above 2 mM. Next, we investigated the effect of pH over LbSOD-B2 to guarantee that a suitable signal to noise would be achieved, thus saturating pyrogallol concentration (0.4 mM) were employed in this study. The results show that low pH values

Fig. 3  Inhibition of pyrogallol oxidation according to LbSOD-B2 concentration. All measurements were carried out in triplicate using 0.4 mM pyrogallol in pH 8 (50 mM Tris–HCl 1 mM EDTA)

Determination of Superoxide Dismutase Activity Several studies [27, 32, 33] have shown that indirect SOD assays based on pyrogallol oxidation are too slow (low signal-to-noise ratio), if concentrations below 0.2 mM are employed and have reduced sensitivity when concentrations above 0.4 mM are used, due to rapid production of superoxide radicals in the medium. In fact, the optimal pyrogallol concentration depends on the protein catalytic efficiency as well as the medium pH [27]. For instance, at pH 7.9 the autoxidation of pyrogallol is inhibited by 99% by bovine

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Fig. 4  Activity of LbSOD-B2 in increasing concentrations of pyrogallol. All measurements were carried out in triplicate. Buffer: 50 mM Tris–HCl 1 mM EDTA pH 8; 0.1 µM LbSOD.

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for LbSOD-B2, described in this work, is the first step to change this scenario. Our data suggest that activity assays should be carried out in pH 8 for maximal enzymatic activity, whereas in physiologic conditions (pH 7.4) at least 50% of LbSOD-B2 activity is conserved. Finally, the inhibition of LbSOD-B2 by hydrogen peroxide, described herein, can be considered as proof-of-concept that the assay conditions are suitable to screen for LbSOD-B2 inhibitors. Acknowledgements  The authors are grateful for the academic support of PPGBiotec-UEFS and the Grants from CNPq 306277/2014-0, FAPESB BOL0688/2014, and FAPESP 2013/07600-3. Fig. 5  Inhibition profile of LbSOD in the presence of increasing concentrations of ­H2O2 (0.5–3.5  mM). Buffer: 50  mM Tris–HCl 1  mM EDTA pH 8; 0.4  mM pyrogallol. 0.1  µM LbSOD. Measurements were performed in triplicate

(sodium acetate, pH 4) have a higher detrimental effect towards activity than high pH values (CHES, pH 10) (< 10% activity in pH 4 vs still 40% of activity in pH 10). Meier and Coworkers [34] have also shown the dependence of Propionibacterium shermanii FeSOD on pH. However, P. shermanii SOD stability decreases with increasing pH values. Finally, after determining the best conditions for the catalytic activity of LbSOD-B2, we investigated the inhibitory effect of ­H2O2, a FeSOD inhibitor [35], against the target enzyme. Hydrogen peroxide causes an irreversible inactivation of superoxide dismutase enzyme by interaction with iron, the metal cofactor [18]. Our results (Fig. 5) show that ­H2O2 is a low millimolar inhibitor of LbSODB2 ­(IC50 = 1 mM). These data are in good agreement with the ­H2O2 inhibitory activity observed against Plasmodium falciparum SOD [36]. The inhibition mechanism is due to a Fenton reaction in which the ­Fe2+ found in LbSOD-B2 active site reduces ­H2O2 to generate O ­ H− and the radical 2+ 3+ ∙ Hydroxil ­(Fe  + H2O2 → Fe  +  OH + OH−) [19]. Our results indicate that the cloning, expression, and purification steps were useful to obtain pure LbSOD-B2. Moreover, we identified the best conditions to evaluate the enzymatic activity of the purified enzyme as well as standardized an inhibition assay. The assay is useful to screen LbSOD-B2 inhibitor candidates as new lead compounds for Leishmaniasis.

Conclusion Fe-Superoxide dismutase is an important enzyme for the detoxification of radicals in Leishmania braziliensis; however, the enzyme has been poorly investigated as a drug target. The design of a heterologous expression system

Compliance with ethical standards  Conflict of interest  The authors confirm that this article content has no conflicts of interest.

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