The physiological concentration of ferrous iron (II) alters the inhibitory effect of hydrogen peroxide on CD45, LAR and PTP1B phosphatases
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Hydrogen peroxide is an important regulator of protein tyrosine phosphatase activity via reversible oxidation. However, the role of iron in this reaction has not been yet elucidated. Here we compare the influence of hydrogen peroxide and the ferrous iron (reagent for Fenton reaction) on the enzymatic activity of recombinant CD45, LAR, PTP1B phosphatases and cellular CD45 in Jurkat cells. The obtained results show that ferrous iron (II) is potent inhibitor of CD45, LAR and PTP1B, but the inhibitory effect is concentration dependent. We found that the higher concentrations of ferrous iron (II) increase the inactivation of CD45, LAR and PTP1B phosphatase caused by hydrogen peroxide, but the addition of the physiological concentration (500 nM) of ferrous iron (II) has even a slightly preventive effect on the phosphatase activity against hydrogen peroxide.
KeywordsFerrous iron Hydrogen peroxide CD45 LAR PTP1B
Protein tyrosine phosphatases (PTPs) are responsible for the regulation of tyrosine phosphorylation status controlling numerous cellular processes, such as cellular growth, differentiation, metabolism, cell–cell communication and immune response (Tonks 2006). One of the key representatives of PTPs is CD45, which controls many cellular processes and its overactivity is involved in autoimmune disorders, allergic response and carcinogenesis (Hermiston et al. 2009; Tan et al. 2000; Dios et al. 2005; Huntington and Tarlington 2004). CD45 is abundantly expressed in the Jurkat cell line (Zamoyska 2007). Phosphatases PTP1B is involved in pathogenesis of type 2 diabetes and obesity (Bence et al. 2006) and is a target in breast cancer treatment (Aceto and Bentires-Alj 2012). LAR phosphatase have also been implicated in metabolic regulation and cancer (Chagnon et al. 2004). CD45 and LAR are receptor-like PTPs predominantly found in the plasma membrane, while PTP1B is intracellular (cytosolic) phosphatase localized in a variety of intracellular compartments, such as cytosol, plasma membrane or endoplasmic reticulum (Andersen et al. 2001).
The hallmark defining the PTP superfamily is the strictly conserved active site sequence C(X)5R within the catalytic domain, which constitutes the phosphate-binding pocket of the enzyme (Tabernero et al. 2008). The cysteine residue inside the signature motif exists in the thiolate anion form, and is highly prone to oxidation (Pagliarini et al. 2004). Oxidation of the cysteine residue leads to the formation of a reversible form of sulfenic acid residue, while a highly oxidizing environment can induce further oxidation yielding physiologically irreversible sulfinic and sulfonic acid residues, all of which consequently cause inactivation of the enzyme (Ostman et al. 2011). Oxidative stress, defined as excessive reactive oxygen species (ROS) formation, may induce inactivation of protein tyrosine phosphatases. Inactivation via oxidation was suggested as a mechanism of protein tyrosine phosphatases regulation (Persson et al. 2004).
A unique biochemical and structural characteristic of the PTPs catalytic cysteine engendered a hypothesis that these enzymes might be direct targets of ROS chemistry. Many PTPs are shown to be oxidized transiently in response to various cellular stimuli. ROS such as hydrogen peroxide, function as second messengers and can regulate tyrosine phosphorylation-mediated signaling pathways (Finkel 2003).
Interestingly, the cysteine residue, which is essential for PTPs activity, is regarded as a main target for the hydroxyl radical. Comparison of the kinetic data for reactions of the hydroxyl radical with different amino acids (Fig. 1b), allows us an observation of the difference in oxidation. Although the variation in the rate constants is relatively small, the aromatic and sulfur-containing amino acids would be expected to be damaged more rapidly with the highest selectivity to cysteine residue (Davies 2005). Does selective diversity of the hydroxyl radical in biological systems (including PTPs) really exist? The high reactivity of hydroxyl radical is supposed to limit its ability to diffuse and cause active site—specific inactivation.
Hydrogen peroxide may relatively easily cross the cell membrane in response to insulin or epidermal growth factor, and control the cellular activity of protein tyrosine phosphatases therein (Rhee et al. 2000). Hydrogen peroxide is able to oxidize the catalytic cysteine residue to sulfenic acid, which can be reversibly reduced by various cellular reducing agents (Goldstein et al. 2005). The effect of hydrogen peroxide on activity of PTPs was already studied and described (Ross et al. 2007; Chiarugi and Cirri 2003). But the effect of the presence of ferrous iron (II), physiologically relevant reagent, was not been yet elucidated. The trace amounts of iron are presented in many cells. The potentially toxic labile iron exists in cells as a transit pool known as labile iron pool (LIP). It was estimated that the physiological concentration of LIP in Jurkat cells is around 3 ± 0.5 µM and that the treatment with hydrogen peroxide increased the cytosolic LIP levels (Al-Qenaei et al. 2014). Other studies showed that the estimated values of LIP for resting erythroid and myeloid cells are in the range of 0.2–1.5 µM (Epsztejn et al. 1997). Here we decided to compare the impact of hydrogen peroxide on recombinant phosphatases CD45, LAR, PTP1B and cellular CD45 from Jurkat cells in presence and in absence of ferrous iron at concentrations close to physiological.
Materials and methods
Cell line and cell culture
The human Jurkat T cell line, clone E.6-1, was obtained from European Collection of Cell Culture (ECACC, UK). The cells were cultured at 37 °C in RPMI 1640 medium supplemented with 10 % fetal bovine serum, 100 μg/mL penicillin/streptomycin and 2 mM l-glutamine. The culture was maintained at 37 °C and in an atmosphere containing 5 % CO2. RPMI 1640 medium and supplements were obtained from Sigma–Aldrich. The cell culture density was kept at 1 × 106 cells/mL. At least every two days the medium was replaced with the fresh one, and the cells were counted and reseeded to maintain the recommended density. Concentration of protein in Jurkat cell lysate was measured using the Bradford colorimetric method. The Bradford method is based on Coomassie Brilliant Blue G-250 absorbance shift in the presence of protein. Binding to the protein being assayed under acidic conditions, the red dye is converted into the blue derivative. The amount of protein in the sample is proportional to the amount of bound dye, and thus to increase of an absorbance at 595 nm. Based on prepared standard concentrations of bovine serum albumin, concentration of protein in samples was calculated. Cells were suspended in FBS free RPMI 1640 medium before treatment with inhibitors.
Cell viability assay with MTT
The Jurkat cells (1 × 106 cells/mL) untreated (control) or treated with solution of hydrogen peroxide, FeSO4, or hydrogen peroxide together with FeSO4 (Fenton reaction) after the appropriate incubation time were suspended in solution of 0.5 mg/mL MTT(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) in RPMI 1640 without phenol red. The 100 µL samples were incubated for 2–4 h at 37 °C in 96-well plates. When the purple precipitate was clearly visible under the microscope, 100 µL of DMSO was added to each well and the plate with cover was left in the dark for 2–4 h. The absorbance at 540 nM was determined using a microplate reader.
Determination of PTP CD45 activity in cell lysate
The Jurkat cells (density at 1 × 106 cells/mL) were untreated (control) or treated with solution of hydrogen peroxide, FeSO4, or hydrogen peroxide together with FeSO4 (Fenton reaction) and incubated for 1 h at 37 °C in 24-well plates. The cells were rinsed twice with TBS, suspended at the density of 1 × 107 cells/mL in Lysis buffer pH 7.4 with 0.5 % NP-40, 25 μg/mL leupeptin, 25 μg/mL pepstatin, 2 μg/mL aprotinin, 1 mM PMSF, vortexed briefly and placed on ice for 15 min. The cells were then solubilized by forcing the lysates through a 19-gauge needle (0.686 mm inner diameter) 20 times and centrifuged at 12000×g at 4 °C for 5 min. The supernatants were transferred to test tubes and assayed immediately. The day prior to the assay, the 96-well microplates were coated with CD45 capture antibodies (8 μg/mL in PBS) and incubated overnight at room temperature. After washing the wells, cell lysate was added, and the plate was placed on a rocking platform at 30 rpm for 3 h at room temperature. Lysates were aspirated from the wells and PTP activity was measured colorimetrically using 200 μM tyrosine phosphate specific substrate (phosphopeptide DADEY(PO3)LIPQQG in 10 mM HEPES buffer pH 7.4) and malachite green. The phosphopeptide substrate was dephosphorylated by active CD45 to generate unphosphorylated peptide and free phosphate. The free phosphate was then detected by a sensitive dye binding assay using malachite green and molybdic acid. The increase in absorbance at 620 nM was measured with the microplate reader. The activity of CD45 was determined by calculating the rate of phosphate release. CD45 capture antibody, tyrosine phosphate substrate DADEY(PO3)LIPQQG, malachite green and molybdic acid were purchased from R&D Systems. Detergent NP-40, protease inhibitors (leupeptin, pepstatin, aprotinin) and phenylmethylsulfonylfluoride (PMSF) were purchased from Sigma–Aldrich.
Recombinant CD45, LAR and PTP1B activity assay
Human recombinant CD45 protein tyrosine phosphatase (PTP catalytic domain) was obtained from Sigma–Aldrich. Human LAR phosphatase (PTP catalytic domain) was obtained from Calbiochem. Human PTP1B phosphatase was purchased from Prospec. The solution of the recombinant protein tyrosine phosphatase CD45, LAR and PTP1B was prepared in 10 mM HEPES buffer pH 7.4. The final concentration of phospahatses in reaction samples was 0.8 μg/mL (10 nM). The CD45, LAR and PTP1B enzymes was untreated (control) or treated with solution of hydrogen peroxide, FeSO4, or hydrogen peroxide together with FeSO4 in different concentrations and) in the presence or absence of 1 mM EDTA. The assay was performed in 96-well microplates, and the final volume of each sample was 200 μL. The enzymatic activity of CD45, LAR and PTP1B was measured using 1 mM chromogenic substrate para-nitrophenyl phosphate (pNPP) in 10 mM HEPES buffer pH 7.4, at 37 °C. Phosphatase hydrolyzed pNPP to para-nitrophenol and inorganic phosphate. Para-nitrophenol is an intensely yellow colored soluble product under alkaline conditions. The increase in absorbance (due to para-nitrophenol formation) is linearly proportional to enzymatic activity concentration (with excessive substrate, i.e. zero-order kinetics) and was assessed at 405 nM on a microplate reader Jupiter (Biogenet) using DigiRead Communication Software (Asys Hitech GmbH).
NBD-Cl modification assay
Sulfenic acid-labeling reagent 7-chloro-2-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) was purchased from Sigma. NDB-Cl react with both sulfenic acid and thiols forming adducts with different spectra. The amount of modified CD45, LAR, PTP1B thiol adduct with NBD (Cys-S-NBD adduct) was measured after 30 min incubation with NDB-Cl (0.6 mM in a 0.5 mL of sample) as absorbance in 347 and 420 nM with spectrophotometer.
The experiments were performed at least three times. The data were applied and analyzed with GraphPad Prism (GraphPad Software v.4). Statistical analyses were performed using ANOVA combined with Tukey’s test, or t test. The data were expressed as mean ± SD. Differences between means were considered significant for P < 0.05.
To asses the effect of ferrous iron (II) and hydrogen peroxide we measured the enzymatic activity of recombinant CD45, LAR and PTP1B phosphatases under the cell-free conditions and CD45 phosphatase in Jurkat cells. The enzymes and cells were treated with solution of hydrogen peroxide, iron (II) sulfate, or both solutions together in different concentrations. Iron (II) sulfate (FeSO4) in aqueous solutions undergoes dissociation to ferrous iron (II) and sulfate ion (SO4 2−).
Comparison of the effect of hydrogen peroxide and ferrous iron on activity of recombinant CD45 phosphatase
In first step we decided to assess the effect of different concentrations of hydrogen peroxide on enzymatic activity of recombinant CD45 (data not shown) for calculation of IC50 value to plan the range of concentrations of hydrogen peroxide to be used in our studies. We calculated IC50 value for hydrogen peroxide as 8 µM, which is compatible with previous literature (Groen et al. 2005; Rider et al. 2003).
Effect of ferrous iron on CD45 in Jurkat cells
The cell viability after 1 h of treatment of Jurkat cells with 10 µM hydrogen peroxide was not significantly changed (98 %), whereas the effect of the Fenton reaction on cell viability was slightly higher (92 %) (Fig. 3b).
Effect of ferrous iron and hydrogen peroxide on recombinant phosphatase CD45, LAR and PTP1B
We demonstrated that both the hydrogen peroxide and ferrous iron (II) induce the inactivation of recombinant CD45, LAR and PTP1B and that the inhibitory effect was concentration dependent. Incubation of recombinant phosphatases with solution of 20 µM iron (II) sulfate significantly lowered the enzymatic activity, while 500 nM iron (II) sulfate had virtually no effect on enzymatic activity (Fig. 4a).
We observed also difference in the inactivation level between the phosphatases. PTP1B was considerably more sensitive for higher concentrations of iron (II) sulfate than hydrogen peroxide, while it was conversely for CD45 (Fig. 4a). We found that 20 µM iron (II) sulfate increased the effect of hydrogen peroxide on enzymatic activity of PTP1B. Interestingly, in our studies, we discovered that the low concentration (500 nM) of iron (II) sulfate decreases the inhibitory effect of hydrogen peroxide, paradoxically preventing PTPs from inactivation by hydrogen peroxide (Fig. 4a).
To examine the oxidation status of cysteine residues in studied PTPs we performed the thiol-labeling assay with NBD-Cl. We have measured the amount of NBD-Cl adducts with CD45, LAR and PTP1B thiol groups. As observed for CD45 and LAR phosphatase the amount of non-oxidized thiol groups was lower after treatment with hydrogen peroxide comparing to control and iron (II) sulfate and was increasing in addition of ferrous iron (Fig. 4b). The amount of non-oxidized thiol groups in PTP1B was lower after treatment with iron (II) sulfate than control and hydrogen peroxide. Moreover, the inhibitory effect of hydrogen peroxide in presence of iron (II) sulfate was significantly enhanced (Fig. 4b).
Electrostatic potential of CD45, LAR and PTP1B active sites analysis
The similar electrostatic potential calculations were performed for the active sites of LAR and PTP1B. We showed that the surface of LAR and PTP1B active sites are largely positively charged and there are only slight differences found between surfaces of active site surrounding (Fig. 5b, c).
Here we showed that ferrous iron (II) can induce inactivation of recombinant CD45, LAR, PTP1B and cellular CD45 phosphatase in Jurkat cells. We found that the effect of ferrous iron (II) was concentration dependent.
We demonstrate that the physiological concentrations of ferrous iron (II) has lower inhibitory effect on CD45, LAR and PTP1B phosphatase than hydrogen peroxide. Moreover, in our studies, the addition of low concentration of ferrous iron to hydrogen peroxide paradoxically exhibits a slightly preventive effect.
The weaker effect of low concentrations of ferrous iron (II) than hydrogen peroxide was observed also for CD45 phosphatase in Jurkat cells (Fig. 3a). We suggest that ferrous iron (II) due to electrostatic potential of PTPs active site, is not able to reach the intracellular target, while hydrogen peroxide would easily cross the cell membrane of Jurkat cells and penetrate to PTP active site inducing inactivation of enzymatic activity. The fact that the impact of ferrous iron with hydrogen peroxide in the presence of EDTA in Jurkat cells was similar to the effect of hydrogen peroxide confirmed the paradoxically protective role of the low amounts of ferrous iron on the activity of phosphatase CD45. EDTA has virtually no impact on the enzymatic activity of recombinant CD45 (Fig. 2b), but enhances the inhibitory effect of hydrogen peroxide in presence of ferrous iron in Jurkat cells, probably by chelating the extracellular ferrous iron derived from the cell treatment and allowing free penetration by hydrogen peroxide. The other explanation may be the formation of iron chelate complex (FeEDTA) leading to the initiation of other strong oxidants or lipid peroxidation (Bucher et al. 1983a, b) and consequently to the oxidation of CD45 phosphatase (Conrad et al. 2010).
We found that addition of ferrous iron (II) to hydrogen peroxide has slightly decreases the viability of Jurkat cells (Fig. 3b). Further studies are required to investigate this problem. The hydroxyl radical form from hydrogen peroxide in presence of ferrous iron is highly reactive and may react unspecifically with all structures of cells inducing damage of DNA and proteins. It has previously been noted that the viability of Jurkat cells can be decreased by oxidative DNA damage in cells caused by hydroxyl radicals generated in the Fenton reaction (Riviere et al. 2006; Tenopoulou et al. 2005).
The comparison of the effect of ferrous iron and hydrogen peroxide on activity of recombinant CD45, LAR and PTP1B showed that the impact of ferrous was concentration and phosphatase dependent. The addition of higher concentrations of ferrous iron to hydrogen peroxide led to increase of inactivation, especially of PTP1B phosphatase.
In conclusion, hydrogen peroxide produced upon activation of many cell surface receptors is considered as a major regulator of PTPs in biological systems. Here, we demonstrate that ferrous iron (II) may be considered as inhibitor of PTPs. In this studies, we present how different concentration of iron may alter inhibitory effect of hydrogen peroxide. We also propose that probably electrostatic potential of proteins surface protects enzymes from the chemistry of elements present in the surrounding environment.
This work was supported by MN Grant No. 01-0172/08/259 from Medical University of Gdansk and Ministry of Science and Higher Education. MG gratefully acknowledges to Polish National Science Center Grant No. 2012/07/N/NZ1/00012. MW acknowledges ST46 from Medical University of Gdansk. JAT acknowledges support from NSERC (Canada) and the Allard Foundation.
Compliance with ethical standards
Conflict of interest
The authors declare no competing financial interest.
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