Biochemical Evidence for Lead and Mercury Induced Transbilayer

May 9, 2013 - Human phospholipid scramblase 1(hPLSCR1) is a transmembrane protein involved in bidirectional scrambling of plasma membrane phospholipid...
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Biochemical Evidence for Lead and Mercury Induced Transbilayer Movement of Phospholipids Mediated by Human Phospholipid Scramblase 1 Ashok Kumar Shettihalli and Sathyanarayana N. Gummadi* Applied and Industrial Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India S Supporting Information *

ABSTRACT: Human phospholipid scramblase 1(hPLSCR1) is a transmembrane protein involved in bidirectional scrambling of plasma membrane phospholipids during cell activation, blood coagulation, and apoptosis in response to elevated intracellular Ca2+ levels. Pb2+ and Hg2+ are known to cause procoagulant activation via phosphatidylserine exposure to the external surface in erythrocytes, resulting in blood coagulation. To explore its role in lead and mercury poisoning, hPLSCR1 was overexpressed in Escherichia coli BL21 (DE3) and purified using affinity chromatography. The biochemical assay showed rapid scrambling of phospholipids in the presence of Hg2+ and Pb2+. The binding constant (Ka) was calculated and found to be 250 nM−1 and 170 nM−1 for Hg2+ and Pb2+, respectively. The intrinsic tryptophan fluorescence and far ultraviolet circular dichroism studies revealed that Hg2+ and Pb2+ bind to hPLSCR1 and induce conformational changes. hPLSCR1 treated with protein modifying reagent N-ethylmaleimide before functional reconstitution showed 40% and 24% inhibition in the presence of Hg2+ and Pb2+, respectively. This is the first biochemical evidence to prove the above hypothesis that hPLSCR1 is activated in heavy metal poisoning, which leads to bidirectional transbilayer movement of phospholipids.

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INTRODUCTION Lead and mercury are considered major environmental toxicants throughout the world, and much effort is directed toward reducing environmental pollution caused by them.1,2 According to Centre for Disease Control and Prevention (CDC), lead poisoning is defined as a blood lead level (BLL) exceeding 5 μg/dL in children.3 Exposure to lead causes various deleterious effects on the hematopoietic, renal, reproductive, and central nervous systems, mainly through increased oxidative stress.4 Chronic lead poisoning is characterized by persistent vomiting, encephalopathy, lethargy, delirium, convulsion, and coma.5 Lead exposure on the peripheral nervous system causes peripheral neuropathy, which results in reduced motor activity due to a loss of the myelin sheaths of nerves impairing the transduction of nerve impulses.6 The renal functional abnormality caused by lead exposure is due to degenerative changes in the tubular epithelium along with the formation of nuclear inclusion bodies of lead protein complexes leading to Fanconi’s syndrome.7,8 The lead toxicity causes oxidative stress through the generation of reactive oxygen species (ROS) and the depletion of antioxidant reserves in the cell caused by the formation of covalent attachments between the lead moiety and the sulfhydryl groups of antioxidant enzymes and intracellular glutathione.9 The oxidative stress also causes lipid peroxidation on lipid membranes. The free radicals © XXXX American Chemical Society

generated by lead poisoning capture electrons from the lipids present inside the cell membranes and damage the cell.8,10 Lead also acts as a substitute for other bivalent cations like Ca2+, Mg2+, and Fe2+ and monovalent cations like Na+ affecting the various vital cellular processes such as intra- and intercellular signaling, cell adhesion, protein folding, apoptosis, ionic transportation, enzyme regulation, and release of neurotransmitters.11 According to the recent recommendation by the U.S. Environmental Protection Agency (EPA), the allowable or safe daily intake of methyl mercury is 0.1 μg of mercury per kilogram per day.12 Humans are exposed to three distinct but related forms of mercury, which include elementary mercury, inorganic mercury (less toxic Hg+ and more toxic Hg2+), and organic mercury (alkyl and aryl mercury).13 The major clinical toxicological effects of elemental mercury exposure include tremor, erethism, gingivostomatitis, peripheral neuropathy, acrodymia, and pneumonitis. The exposure to inorganic mercury causes acute tubular necrosis, gastroenteritis, acrodynia, urticaria, and vasication.13,14 The clinical toxicological effects of organic mercury exposure are tubular necrosis, acrodynia, paresthesia, ataxia, and visual and hearing loss.13,15 Received: March 5, 2013

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Bio Lab (Ipswich, MA, USA). 1-Oleoyl-2-[6-(7-nitrobenz-2-oxa-1,3diazol-4-yl)amino] caproyl-sn-glycero-3-phosphocholine (NBD-PC) and 1-oleoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproylsn-glycero-3-phosphoserine (NBD-PS) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Bio-Beads SM-2 adsorbent, protein molecular weight markers, and Chelex 100 resin were from Bio-Rad Laboratories (Hercules, CA, USA). The nickel-nitrilotriacetic acid (Ni2+-NTA) matrix was purchased from Qiagen (Valencia, CA, USA). All other chemicals were procured from Himedia, India. Reconstitution and Functional Scramblase Assay in Proteoliposomes. hPLSCR1 was overexpressed in E. coli BL 21 (DE3) and purified to homogeneity (Supporting Information, Materials and Methods and Figure S1). hPLSCR1 was reconstituted into proteoliposomes containing exogenous PLs (10% PS and 90% PC) at 4.5 μmol of total PLs as described earlier.32−34 Briefly, egg PC and brain PS were dried under N2, solubilized in 10 mM HEPES/NaOH, pH 7.5, 100 mM NaCl, and 1% (w/v) Triton X-100, and 200 μg of hPLSCR1 was added. The detergent was removed slowly using SM2 biobeads (0.1 g/L). Similarly, liposomes were prepared following the same procedure without hPLSCR1. The liposomes and proteoliposomes were collected by ultracentrifugation using an MLA130 rotor (Beckman Coulter ultracentrifuge, USA) and washed 3−4 times with the assay buffer (10 mM HEPES/NaOH, pH 7.5, and 100 mM NaCl). The vesicles were extruded to make uniform sizes using a 0.1 μm polycarbonate membrane filter. These liposomes and proteoliposomes were labeled with 0.3 mol % of NBD−PC/NBD−PS (Avanti Polar Lipids, USA) to form outside labeled vesicles as described earlier.35 A schematic representation of the scramblase assay is shown in Supporting Information, Figure S2. The scramblase activity was measured for the labeled proteoliposomes with liposomes as control using a Perkin-Elmer LS-55 fluorescence spectrophotometer as described earlier.35 Briefly, labeled liposomes and proteoliposomes were incubated for 5 h at 37 °C in the presence of 4 mM EGTA or varying concentrations of Ca2+ or other heavy metal ions to be tested. An aliquot of liposomes and proteoliposomes (∼25−50 μL) was transferred to a stirred fluorescence cuvette and diluted to 2 mL with assay buffer. The time-dependent fluorescence (excitation at 470 nm, emission at 530 nm) was monitored continuously at 22 °C with constant low-speed stirring. After the fluorescence intensity was stabilized (∼100 s), sodium dithionite (freshly prepared in 1 M Tris base, pH 10) was added to a final concentration of 20 mM, and the fluorescence was monitored for the next 500 s. The difference in residual (nonquenchable) fluorescence observed for samples preincubated at 37 °C in the presence versus absence of metal ions is attributed to the metal ion induced scrambling of NBD−PC or NBD− PS located in the outer leaflet of proteoliposomes. The scramblase activity was calculated using the following formula:

The severity of mercury poisoning is due to the fact that it targets the thiol containing enzymes forming covalent linkage with thiol groups leading to inactivation of enzymes and also causing oxidative stress.16,17 The severity of Hg2+ poisoning comes from the fact that it oxidizes the sulfhydryl groups of the key transmembrane enzyme transporter Na+/K+-ATPase or sodium pump, which is involved in maintaining the electrochemical Na+ and K+ gradients across the cell membranes.18,19 In human blood, about 90−95% of lead and mercury get accumulated in erythrocytes.20,21 It has also been reported that exposure of erythrocytes to lower doses of Hg2+ (0.25−5 μM) and Pb2+ (1−5 μM) changes the shape of erythrocytes from biconcave normocytes to echinocytes and spherocytes, accompanied by microvesicle (MV) generation.21,22 Maintenance of plasma membrane (PM) asymmetry is one of the important properties of the cell. Exposure of phosphatidylserine (PS) in the presence of elevated Ca2+ levels is one of the important signaling events in apoptosis.23 It has been reported that exposure to Pb2+ and Hg2+ can also translocate PS to the outer leaflet of the erythrocyte cell membrane resulting in apoptosis.24 This in turn serves as a site for the assembly of a prothrombinase and tenase complex, leading to thrombin generation and blood clotting.25 Furthermore, increased adhesion of PS-expressing erythrocytes to endothelial cells causes vasoocclusion. So far, the exact mechanism of PS translocation is not clear. Human phospholipid scramblase 1 (hPLSCR1), a type II membrane protein localized to plasma membrane, is known to scramble phospholipids (PL) when the intracellular calcium levels rise by 1000-fold.26 Hence, membrane asymmetry is destroyed, which leads to blood coagulation and apoptosis.27 hPLSCR1 has an EF-hand like Ca 2+ binding motif [273DADNFGIQFPLD284], located in close proximity to the predicted transmembrane helix (Ala291-Gly309), including the cysteine palmitoylation motif, nuclear localization signal, and DNA binding motif.28 The EF-hand is a helix−loop−helix structural domain or motif found in a large family of calcium binding proteins, in which the Ca2+ ions are coordinated by ligands (amino acid residues) within the loop.29,30 These five domains are well conserved in all other homologues (hPLSCR2−hPLSCR4) of hPLSCR1.26,31 In view of this, we hypothesize that PS translocation to the outer surface, upon lead and mercury poisoning, could be attributed to the binding of these metal ions to hPLSCR1 that induces scramblase activity. To address this, we performed in vitro biochemical reconstitution and the scramblase assay with hPLSCR1. In addition, we performed biophysical characterization of lead and mercury interaction with hPLSCR1.

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Scramblase activity(% of NBD − PL translocated) = (Fmetal ion − Fcontrol) × 100 where Fmetal ion is the relative fluorescence activity in the presence of metal ion, and Fcontrol is the relative fluorescence activity in the presence of EGTA. Treatment of hPLSCR1 with Protein Modifying Reagents. hPLSCR1 was treated with protein modifying reagents for cysteine, histidine, and arginine following the method reported in earlier studies.33,36 Briefly, freshly prepared stock solutions (typically 100 mM) of diethyl pyrocarbonate (DEPC) in water, N-ethylmaleimide (NEM), and phenyl glyoxal (PG) in assay buffer were added to hPLSCR1 to yield the desired molar ratio of amino acid residue to inhibitor (1:10) and incubated at 37 °C for 30 min before reconstitution. The scramblase activity was measured for the labeled proteoliposomes as described previously. The proportion of functional scramblase activity eliminated by treatment with protein modifying reagents (% inhibition) was calculated as

EXPERIMENTAL PROCEDURES

Chemicals and Reagents. Egg phosphatidylcholine (PC), brain phosphatidyl serine (PS), phenylmethanesulfonyl fluoride (PMSF), diethyl pyrocarbonate (DEPC), phenyl glyoxal, N-ethylmaleimide, sodium dithionite (Na2S2O4), molecular biology grade calcium chloride (CaCl2), N-lauroyl sarcosine (NLS), ULTROL-grade Triton X-100, and protein estimation kit (BCA kit) were obtained from Sigma (St. Louis, USA). Mercury(II) chloride (HgCl2) and lead(II) chloride (PbCl2) were from Merck (Darmstadt, Germany), Stains-all and terbium(III) chloride were obtained from Sigma-Aldrich (St. Louis, MO, USA). E. coli DH5α and BL21 (DE3) strains were from ATCC (Manassas, VA, USA), cDNA of hPLSCR1 was from Invitrogen (Carlsbad, CA, USA), pET-28b(+) from Novagen (Billerica, MA, USA), and DNA molecular weight markers, Taq polymerases, restriction enzymes, and d-NTPs were obtained from New England

%inhibition = [1 − (A inhibitor /A mock )] × 100 where Ainhibitor is the scramblase activity of hPLSCR1 treated with inhibitor, and Amock is the scramblase activity of untreated hPLSCR1. B

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Fluorescence Spectroscopy. Intrinsic tryptophan fluorescence of protein was measured using a Perkin-Elmer LS-55 fluorescence spectrofluorometer with a sample compartment maintained at 25 °C. The buffer used was 20 mM Tris-HCl, pH 7.5, and 0.2 M NaCl. The spectra were recorded by exciting the sample at 295 nm and emission from 300−500 nm with a scanning speed of 100 nm min−1. Metal ions were added to the desired concentration, and all the spectra were corrected by subtracting the buffer blanks for data evaluation. Terbium luminescence assays were performed as described earlier.37 Terbium binding to hPLSCR1 in the presence of heavy metal ions was monitored on a JASCO-6500 spectrofluorometer with the excitation wavelength set at 295 nm. The protein solution (2 μM) after saturation with 40 μM Tb3+ was titrated against increasing concentrations of metal ions. The increase in right-angle light scattering of the protein (1 μM) with the increasing concentration of metal ions was measured at the first harmonic of the excitation wavelength (295 nm) using Perkin-Elmer LS-55 fluorescence spectrofluorometer. Far-UV CD Spectra. The CD spectra were measured using a JASCO J-810 Spectropolarimeter (Easton, MD, USA) at 25 °C as described by earlier studies.37,38 Structural changes upon metal-binding were monitored by titrating 10 μM protein in 20 mM Tris-HCl, pH 7.5, and 0.1 M NaCl with varying concentrations of metal ions. All of the spectra were corrected by subtracting the buffer blanks for data evaluation. Because of the significantly higher absorption of the buffer blank, scans below 200 nm were not performed. Statistical Analysis. All of the data are expressed as the mean ± SD of three independent experiments. The difference between the two groups was evaluated using Student’s t test. Significance was acknowledged when the p value was less than 0.05.

Figure 1. Scramblase activity of hPLSCR1 in the presence of metal ions. Outside labeled liposomes with NBD−PC (A) and proteoliposomes with either NBD−PC (B) or NBD−PS (C) were incubated for 5 h at 37 °C in the presence of 4 mM EGTA (trace a), 2 mM Ca2+ (trace b), 5 μM Hg2+ (trace c), or 5 μM Pb2+ (trace d). The reaction was stopped with 4 mM EGTA. Distribution of NBD−PC or NBD− PS in the inner leaflet was determined by quenching with 20 mM dithionite. The ordinate denotes NBD fluorescence normalized to t = 0; abscissa denotes fluorescence acquisition time (seconds). All of the experiments were performed at least three times, and the values reported are the mean values (p < 0.0001).

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RESULTS Overexpression and Purification of hPLSCR1. To investigate the in vitro effect of heavy metals on the biological activity of hPLSCR1, the protein was overexpressed and purified as 6X His tag protein in the BL-21 (DE3) strain of E.coli following an earlier reported procedure.32,39 The detailed purification procedure and the gel image are provided in the Supporting Information. Effect of Hg2+ and Pb2+ on Scrambling Activity. In the control experiments using liposomes without hPLSCR1, no scramblase activity was noticed both for NBD−PC (Figure 1A) and NBD−PS (data not shown). The purified hPLSCR1 was reconstituted into proteoliposomes and labeled exogenously with NBD−PC/NBD−PS. After incubation in the presence of either EGTA (control) or metal ions, the distribution of NBD− PC or NBD−PS between outer to inner membrane leaflets was determined. The percentage of NBD−PC sequestered inside due to scrambling activity was found to be 11, 13.5, and 18.6 for Ca2+, Hg2+, and Pb2+, respectively (p < 0.0001) (Figure 1B). Similarly, the percentage of NBD−PS sequestered inside was found to be 9.8, 10.6, and 14 for Ca2+, Hg2+, and Pb2+, respectively (p < 0.0001) (Figure 1C). It was observed that scramblase activity for both NBD−PC and NBD−PS was higher in the presence of Hg2+ and Pb2+ compared to Ca2+. These data strongly suggest that the mechanism of PS translocation to the cell surface is hPLSCR1-mediated during lead and mercury poisoning. Effect of Metal Ion Concentration on Scramblase Activity. To confirm whether the extent of clinical toxicity depends on the concentration of metal ions, we carried out biochemical reconstitution and a functional assay of human phospholipid scramblase1 (hPLSCR1) with varying concentrations of metal ions using NBD−PC/NBD−PS labeled vesicles. In this study, we used a higher concentration of Ca2+ (in mM range) compared to a lower concentration of Hg2+ and

Pb2+ (in μM range) because of the following reasons: (i) it is known that scrambling activity gets activated by a 1000-fold rise in Ca2+ during cell activation26 and that scrambling activity was not observed at the micromolar range; and (ii) the toxic effect of Hg2+ and Pb2+ can occur in cells in the micromolar range,21,22 and we wanted to test whether scrambling activity can be attained at a low concentration range. The scramblase activity was enhanced at higher concentration of metal ions, and no significant increase in the activity was found beyond 3 mM Ca2+, 7.5 μM Hg2+, and Pb2+ (Figure 2A and B). The scramblase activity observed at these concentrations was 11, 15, and 18% for NBD-PC (Figure 2A) and 10, 13, and 17% for NBD-PS (Figure 2B) in the presence of Ca2+, Hg2+, and Pb2+, respectively. No scramblase activity was noticed both for NBD−PS (Figure 2C) and NBD−PC (data not shown) in the control experiments, which were performed using empty liposomes in the presence of 10 mM EGTA, 4 mM Ca2+, and 10 μM Hg2+ and Pb2+. These data indicate that Hg2+ and Pb2+ show greater scramblase activity compared to that of calcium at lower concentrations. Effect of Protein Modifying Reagents on Scramblase Activity. Heavy metals are known to bind the metal-binding motif CXXC in many metal-binding proteins and conserved CPC motif found in the transmembrane region of all ATPases.40−42 As hPLSCR1 (with 18 cysteine residues) showed higher scramblase activity in the presence of heavy metals compared to that of Ca2+, we investigated the role of C

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Figure 3. Effect of protein modifying reagents on scramblase activity. hPLSCR1 was incubated with buffer alone (light gray bars), PG (1.6 mM, moderate dark gray bars), DEPC (1 mM, dark gray bars), or NEM (3.6 mM, intense dark gray bars) for 30 min at 37 °C before reconstitution into proteoliposomes. (A) Scramblase activity using outside labeled proteoliposomes with NBD−PC. (B) Scramblase activity using outside labeled proteoliposomes with NBD−PS. Proteoliposomes were incubated for 5 h at 37 °C in the presence of 4 mM EGTA (control), 2 mM Ca2+, 5 μM Hg2+, or 5 μM Pb2+. All of the experiments were performed at least three times, and the values reported are the mean values (p < 0.0001).

significant loss of scramblase activity both for NBD−PC and NBD−PS labeled proteoliposomes (Figure 3). Intrinsic Tryptophan Fluorescence Studies. The affinity of metal ions (Pb2+ and Hg2+) to hPLSCR1 was monitored by measuring the intrinsic tryptophan fluorescence. The emission maxima spectra of hPLSCR1 were centered around 340 nm (blue shift). We used this technique to monitor the changes in the microenvironment around tryptophan residues in hPLSCR1 upon Pb2+ and Hg2+ binding. Binding of Pb2+ and Hg2+ to hPLSCR1 led to a decrease in tryptophan fluorescence intensity, and the decrease was proportional to the concentration of the metal ion added (Figure 4A and B), suggesting a conformational change. The affinity of Pb2+ and Hg2+(Ka) to hPLSCR1 was calculated by a Scatchard plot using one-site binding nonlinear regression analysis (Prism 3.0, Graph Pad Software Inc., San Diego, CA, USA) (Figure 4C and D). The binding constant (Ka) was 250 nM−1 for Hg2+ and 170 nM−1 for Pb2+. To determine if the added metal ions (Hg2+/ Pb2+) bind to the Ca2+ binding site of the hPLSCR1, we used a fluorescence resonance energy transfer (FRET) assay. Tb3+ is used as a Ca2+ mimicking probe and is known to produce induced luminescence peaks at 492 and 547 nm after binding to the Ca2+ binding motif of proteins through fluorescence resonance energy transfer from nearby tryptophan and tyrosine residues.43 When excited at 295 nm in the presence of hPLSCR1, luminescence from Tb3+ at 492 and 547 nm increased with the increase in the concentration of Tb3+ and reached saturation at 40 μM (data not shown). When this

Figure 2. Effect of metal ion concentration on scramblase activity. Outside labeled proteoliposomes with either NBD−PC (A) or NBD− PS (B) were incubated for 5 h at 37 °C in the presence of 4 mM EGTA (control), 1 mM Ca2+, 2 μM Hg2+, or 2 μM Pb2+ (light gray bars), 2 mM Ca2+, 5 μM Hg2+, or 5 μM Pb2+ (moderate dark gray bars), 3 mM Ca2+, 7.5 μM Hg2+, or 7.5 μM Pb2+ (dark gray bars) and 4 mM Ca2+, 10 μM Hg2+, or 10 μM Pb2+ (intense dark gray bars). Outside labeled empty liposomes with NBD−PS (C) were used in the control experiments in the presence of 4 mM Ca2+, 10 μM Hg2+, or 10 μM Pb2+. All of the experiments were performed at least three times, and the values reported are the mean values (p < 0.0001).

cysteine, arginine, and histidine residues on scramblase activity using suitable protein modifying reagents. hPLSCR1 was treated with N-ethylmaleimide (NEM) for cysteine, diethyl pyrocarbonate (DEPC) for histidine, and phenyl glyoxal (PG) for arginine before reconstitution. Interestingly, hPLSCR1 treated with modifiers showed that Hg2+ and Pb2+ induced scramblase activity was significantly affected only upon NEM treatment (Figure 3). However, there was no significant change in Ca2+ induced scramblase activity. Inhibition of Hg2+ and Pb2+ induced scramblase activity was calculated to be 48 and 36% for proteoliposomes labeled with NBD−PC (Figure 3A). Similarly, for proteoliposomes labeled with NBD−PS, inhibition of Hg2+ and Pb2+ induced scramblase activity was found to be 40 and 24% (Figure 3B). The other modifiers did not show any D

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maintained at EMBL (Heidelberg, Germany) to quantify the relative secondary structural content of hPLSCR1. The relative α-helical content of hPLSCR1 decreased from 56% to 18% in the presence of 20 μM Hg2+ and to 14% in the presence of 80 μM Pb2+.

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DISCUSSION In resting cells, maintenance of plasma membrane (PM) asymmetry is one of the important properties of a cell. An increase in intracellular Ca2+ by 1000-fold due to cell activation, injury, or apoptosis causes rapid bidirectional movement of plasma membrane phospholipids between leaflets, resulting in exposure of PS and PE to the cell surface.23,26 Studies have shown that exposure to heavy metals induce PS translocation to the external surface of the erythrocyte cell membrane, which is an indicator for apoptosis.24 It has been found that upon exposure of erythrocytes to Pb2+ and Hg2+, the ATPase (energy dependent flippase) activity was inhibited, and the amount of PS translocation increased.21,22 At present, the effect of lead and mercury poisoning on PS translocation to the external surface of the membrane and the role of hPLSCR1 in this mechanism are not known. hPLSCR1 is known to be involved in rapid bidirectional, nonspecific scrambling of phospholipids across the lipid bilayer in response to increased cytosolic Ca2+ levels.26,28 It has also been demonstrated that scramblase activity can be induced by other divalent metal ions such as zinc, manganese, and magnesium.28 On the basis of these observations, we hypothesized that PS exposure was probably due to the activation of scramblase in response to heavy metals. To prove our hypothesis, we cloned, overexpressed, and purified hPLSCR1; reconstituted into proteoliposomes; and performed scramblase activity in the presence of lead and mercury. In addition, we studied the effect of protein modifying reagents on scramblase activity and characterized the metal ion induced conformational changes in the protein. Our results showed that both Hg2+ and Pb2+ induce scramblase activity mediated by hPLSCR1, leading to transbilayer movement of PLs similar to that of Ca2+ as reported earlier.28,32,39 Higher scramblase activity was observed in the presence of Hg2+ and Pb2+compared to that in Ca2+ for both NBD−PC and NBD−PS, which can be attributed to the difference in the affinity of hPLSCR1 toward metal ions. A similar difference in the ion selectivity for the activation of erythrocyte PL scramblase was observed in the presence of other divalent metal ions such as Mn2+, Zn2+, Sr2+, Ba2+, and Mg2+.28 The scrambling activity increased with an increase in the concentration of metal ions for both NBD−PC and NBD− PS with saturation at higher concentration. The results showed that hPLSCR1 translocated both NBD−PC and NBD−PS with relatively higher selectivity toward NBD−PC compared to that of NBD−PS. As reported earlier, Ca2+and its mimic Tb3+ are known to bind to the EF-hand like motif [273DADNFGIQFPLD284] of hPLSCR1.28,39 The hydrated ionic diameter of Pb2+ (590 pm) and Hg2+ (500 pm) is very close to the hydrated ionic diameter of Ca2+ (600 pm).44 Hence, there is a possibility that heavy metals also bind to the same motif, which was probed using the fluorescence resonance energy transfer (FRET) assay. The titration of the Tb3+− protein complex against increasing concentrations of metal ions led to the partial displacement of Tb3+ as indicated by decreased luminescence from Tb3+ at its emission wavelengths of 492 and 546 nm. This indicates that Hg2+ and Pb2+ partially compete with Tb3+ for binding to the same segment of the

Figure 4. Fluorescence measurements. (A) Effect of Hg2+ on the intrinsic tryptophan fluorescence of hPLSCR1. (B) Effect of Pb2+ on the intrinsic tryptophan fluorescence of hPLSCR1. (C) Scatchard plot for hPLSCR1 with Hg2+. Binding constant (Ka) calculated was 250 nM−1. (D) Scatchard plot for hPLSCR1 with Pb2+. The binding constant (Ka) calculated was 170 nM−1. (E) Tb3+ incorporation into hPLSCR1 competed by Hg2+ and Pb2+. Luminescence from proteinbound Tb3+ at emission wavelengths of 492 and 546 nm was measured. All of the experiments were performed at least three times, and the values reported are the mean values (p < 0.0001).

Tb3+−protein complex was titrated against increasing concentrations of metal ions, luminescence from Tb3+ at its emission wavelengths of 492 and 546 nm decreased (Figure 4E) implying that these metal ions partially compete with Tb3+ for binding to the same segment of the polypeptide. A distinct increase in right-angle scattering of protein solution (measured at the first harmonic of the excitation wavelength of 295 nm) was observed upon addition of metal ions, suggesting that metal ion binding is accompanied by selfaggregation of the protein (Figure 5A). A change in the conformation of hPLSCR1 upon Ca2+ binding has been revealed by earlier studies.32,37 CD Spectroscopy. To confirm whether hPLSCR1 undergoes similar conformational changes upon binding to metal ions Hg2+ and Pb2+, far-UV CD spectra of the protein were measured. The apo form of hPLSCR1 showed double negative minima at 208 and 222 nm (trace a in Figure 5B and C), indicating the presence of a large proportion (56.26%) of αhelical structure. The apparent decrease in the molar ellipticity was noticed upon addition of increasing concentrations of Hg2+ and Pb2+ (Figure 5B and C). We used K2D2, a web server E

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Figure 5. Metal ion induced conformational changes in hPLSCR1. (A) Right-angle light scattering of hPLSCR1 incubated in the presence of various Hg2+ or Pb2+ concentrations indicated by the abscissa. Error bars denote the mean ± SD (n = 2). (B) Trace a, far-UV CD of apo-hPLSCR1; trace b to e, far-UV CD of hPLSCR1 in the presence of 4, 6, 10, and 20 μM Hg2+. (C) Trace a, far-UV CD of apo-hPLSCR1; trace b to e, far-UV CD of hPLSCR1 in the presence of 15, 35, 45, and 80 μM Pb2+. All of the experiments were performed at least three times, and the values reported are the mean values (p < 0.0001).

it could be concluded that these metal ions might interact with the cysteine residues in addition to the EF-hand like Ca2+ binding motif. The conformational changes in hPLSCR1 induced by metal ions were studied using right-angle scattering and far-UV CD spectra. A distinct increase in right-angle scattering of protein solution upon addition of metal ions suggests that metal ion binding was accompanied by self-aggregation of the protein, which is in agreement with the earlier reports for Ca2+ induced aggregation required for scramblase activity.28 Far-UV CD spectra of the protein have revealed that hPLSCR1 exists predominantly in α-helical form in apo form, which was lost upon metal binding due to conformational changes. This is in agreement with similar spectral changes observed during metal ion binding to Ca2+-binding proteins and EF-hand peptides.43,45 In conclusion, we report for the first time Hg2+ and Pb2+ induced scramblase activity mediated by hPLSCR1 leading to transbilayer movement of PLs in proteoliposomes. Higher scramblase activity was observed in the presence of lower doses (in μM range) of Hg2+ and Pb2+compared to that of Ca2+ (in mM range) for both NBD−PC and NBD−PS. The FRET assay confirms the probable metal binding site (EF-hand like motif) of hPLSCR1. Protein modification studies suggest the involvement of cysteine residues as additional binding sites for heavy metals in addition to the EF-hand like motif. Further, a far-UV CD spectroscopic study revealed the metal induced conformational changes in the protein, which may be required for scramblase activity.

polypeptide. This is in agreement with the earlier reports suggesting that Ca2+ competes with protein-bound Tb3+ for binding to the same segment of the polypeptide.28 The binding affinity studies using intrinsic tryptophan fluorescence confirm the higher binding affinity of Hg2+ (Ka ∼ 250 nM−1) and Pb2+ (Ka ∼ 170 nM−1) for hPLSCR1 compared to Ca2+ (Ka ∼ 3.8 mM) as reported earlier.45 This higher affinity of Hg2+ and Pb2+ for hPLSCR1 could be attributed to the binding of these metal ions to additional segments of the protein based on the following reasons: (i) the metal-binding motif CXXC is present in many metal-binding and metal-transport proteins such as MerP and MerA, CadA, Atx1, CopA, superoxide dismutase, and six cytoplasmic metalbinding domains of the copper ATPases associated with Menkes and Wilson’s disease.41,42 Though this motif is not found in hPLSCR1, it contains many XCCX regions, which might also be involved in metal binding; (ii) hPLSCR1 also contains the 186CPC188 motif mainly found in the transmembrane region of all ATPases involved in transporting metals into the cell;40 (iii) earlier reports suggest that Hg2+ can bind to any nucleophilic functional group of the biological molecule indicating the role of serine, histidine, aspartate, glutamate, and cysteine in metal binding; and (iv) the affinity constant for Hg2+ binding to a reduced sulfur atom is very high,46 and lead inactivates antioxidant enzymes by forming covalent attachments with sulfhydryl groups.8−11,46,47 This shows the possible role of 18 cysteine residues of hPLSCR1 in metal binding. To validate whether these cysteine residues do take part in metal ion induced scrambling activity, we carried out protein modification studies. Interestingly, the scramblase activity of both NBD−PC and NBD−PS in the presence of Hg2+ and Pb2+ was significantly altered when NEM treated hPLSCR1 was used for reconstitution. However, Ca2+ induced scrambling activity was unaltered. Similarly, there was no significant alteration in the scramblase activity when hPLSCR1 was treated with other modifiers before reconstitution. Hence,

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ASSOCIATED CONTENT

S Supporting Information *

Details of the construction of the (His)6-hPLSCR1 expression vector, overexpression and purification of human phospholipid scramblase 1, and the principle of the scramblase assay. This F

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AUTHOR INFORMATION

Corresponding Author

*Tel: +91-44-22574114. Fax: +91-44-2257-4102. E-mail: [email protected] Funding

This work was supported by a research grant from the Department of Biotechnology, Government of India, New Delhi. S.N.G. acknowledges DST-FIST funding for CD spectroscopy. A.K.S. acknowledges BMSCE, Bengaluru and AICTE, Government of India, New Delhi for providing the fellowship. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS BLL, blood lead level; IPTG, isopropyl-β-D-thiogalactoside; EGTA, ethylene glycol tetraacetic acid; PMSF, phenylmethanesulfonyl fluoride; PC, egg phosphatidylcholine; PS, brain phosphatidyl serine; DEPC, diethyl pyrocarbonate; PG, phenyl glyoxal; NEM, N-ethylmaleimide; NLS, N-lauroyl sarcosine; NBD−PC, 1-oleoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl-sn-glycero-3-phosphocholine; NBD− PS, 1-oleoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphoserine

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