Membrane skeletal protein S-glutathionylation in human red blood

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Membrane skeletal protein S-glutathionylation in human red blood cells as index of oxidative stress Daniela Giustarini, Isabella Dalle Donne, Aldo Milzani, Daniela Braconi, Annalisa Santucci, and Ranieri Rossi Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00408 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

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Chemical Research in Toxicology

Membrane skeletal protein S-glutathionylation in human red blood cells as index of oxidative stress

Daniela Giustarini1, Isabella Dalle-Donne2, Aldo Milzani2, Daniela Braconi1, Annalisa Santucci1 and Ranieri Rossi1*

1Department

of Biotechnology, Chemistry and Pharmacy, (Department of Excellence 2018-2022)

University of Siena, Via A. Moro 2, I-53100, Siena, Italy 2Department

of Biosciences, (Department of Excellence 2018-2022) Università degli Studi di

Milano, via Celoria 26, I-20133 Milan, Italy

*Correspondence to: Ranieri Rossi, Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, I-53100, Siena, Italy, [email protected]

Keywords Glutathione, protein thiols, S-glutathionylation, erythrocytes, oxidative stress

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Table of Content Graphic


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Abstract Glutathione (GSH) is one of the most studied biomarkers of oxidative stress. Under oxidizing conditions GSH is transformed into its disulfide forms, glutathione disulfide (GSSG) and Sglutathionylated proteins (PSSG), which are considered reliable biomarkers of oxidative stress. In red blood cells (RBCs), the main targets of S-glutathionylation are hemoglobin and membraneassociated skeletal proteins, but S-glutathionylated hemoglobin (HbSSG) is much more studied as biomarker of oxidative stress than S-glutahionylated RBC membrane skeletal proteins. Here, we have investigated whether and how all these biomarkers are altered in human RBCs treated with a slow and cyclically intermittent flux of the oxidant tert-butyl hydroperoxide. To this aim, a new device for sample treatment and collection was developed. During and at the end of the treatment, GSH, GSSG and PSSG (discriminating between HbSSG and membrane PSSG) were measured by the use of spectrophotometer (for GSSG) and HPLC (for GSH, HbSSG and membrane PSSG). The main results of our study are the following: i) GSH decreased and GSSG increased, but only in the presence of the oxidant, and recovered their initial values at the end of the infusion; ii) the increase in total PSSG concentration was lower than that of GSSG, but it kept on throughout the experiments; iii) membrane skeletal proteins did not recover their initial values, whereas HbSSG levels recovered their initial values similarly to GSH and GSSG; d) membrane skeletal PSSG were more stable and also more abundant than HbSSG. Western blot analysis indicated spectrin, ankyrin, band 3, band 4.1 and 4.2 as the proteins most susceptible to S-glutathionylation in RBC membrane.

These results suggest that S-glutathionylated membrane skeletal proteins can be considered as a suitable biomarker of oxidative stress. Mostly when the oxidant insult is slight and intermittent, PSSG in RBC membrane are worth to be measured in addition to GSSG by virtue of their greater stability.



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Introduction Unbalanced production of reactive oxygen and nitrogen species (RONS) can lead to the onset of several different human diseases, such as atherosclerosis, cardiovascular disease, neurodegenerative diseases, diabetes mellitus and its complications.1 Information regarding the existence of oxidative stress status may be obtained from the analysis of the so-called discrete biomarkers of oxidative stress from various tissues, but mainly from biological fluids such as blood, due to its easy accessibility. Glutathione (GSH) is the most abundant antioxidant in mammals, and it plays a key role in cell resistance against oxidative and nitrosative damage by providing enzymes involved in the metabolism of RONS with reducing equivalents, by eliminating potentially toxic oxidation products, and by reducing oxidized protein thiols.2 Under oxidizing conditions, GSH is transformed into its disulfide form, i.e. GSSG. The availability of GSH under oxidative conditions is ensured by GSSG reductase which, together with NADPH, reduces GSSG back to GSH.3 GSSG can also react with exposed protein cysteinyl groups, forming protein mixed disulfides (also called S-glutathionylated proteins, PSSG) via transsulfuration reactions (1).4

PSH + GSSG   PSSG + GSH

(1)

Because blood glutathione concentrations may reflect glutathione status in other less accessible tissues, measurement of both GSH and GSSG in blood has been considered essential as an index of whole-body glutathione status and, as a consequence, a good biomarker of oxidative stress.5-7 Additionally, considered that a significant amount of glutathione may be reversibly bound to proteins by reaction (1), several studies have investigated the formation of S-glutathionylated hemoglobin (HbSSG) under conditions of oxidative stress, such as diabetes mellitus, hyperlipidemia, and smoking,8-10 although this biomarker was not always found increased.11 Instead, there is little data available about membrane skeletal protein S-glutathionylation in human 4 ACS Paragon Plus Environment

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RBCs.


12-14

Additionally, it has never been established whether GSSG or PSSG represents a better

indicator of oxidative status in blood and which factors, sample manipulation included, can influence the levels of both. In this work we studied the extent of S-glutathionylation of both hemoglobin and membrane skeletal proteins in comparison to GSH oxidation in human RBCs. We used an in vitro model developed ad hoc with the purpose of administrating an oxidizing stimulus to RBCs by a slow and intermittent flux. The model was developed with the aim of better resembling what occurs in vivo when the RBCs are exposed temporarily and intermittently to oxidants localized in a specific organ/tissue. The membrane skeletal proteins mostly targeted by S-glutathionylation were also identified.

Experimental Procedures 2.1 Materials Monobromobimane (mBrB) was obtained from Calbiochem (Milan, Italy). HPLC grade solvents were purchased from Mallinckrodt-Baker (Milan, Italy). Mouse monoclonal anti-GSH antibody (101-A) was obtained from Virogen (Watertown, MA, USA). Goat anti-mouse IgG and mouse antiGAPDH, horseradish peroxidase conjugate, were obtained from Sigma-Aldrich (Milan, Italy). Precision plus protein standards molecular weight was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Luminata Crescendo reagent for chemiluminescence was obtained from Merck-Millipore (Milan, Italy). All other reagents were obtained from Sigma-Aldrich (Milan, Italy) unless otherwise indicated. Human blood (about 20 ml) was collected in K3EDTA from 4 healthy donors after oral consent.

Purification of RBCs and sample derivation for thiol/disulfide analyses RBCs were prepared by centrifugation of whole blood at 10,000×g for 20 s and three washings with phosphate buffered saline (PBS), pH 7.4, containing 5 mM glucose. The washed RBCs were 5 ACS Paragon Plus Environment


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suspended in PBS containing 5 mM glucose to a hematocrit value of 10% and were treated by a slow and cyclically intermittent flux of tert-butyl hydroperoxide (t-BOOH, 20 mM in saline) by a home-made device (Fig. 1). The flux of t-BOOH was 0.7 mol/min. The RBC solution was continuously exposed to the oxidant for 120 min but the sample was cyclically turned away from the oxidant for 4 min. At the indicated times, aliquots of RBCs were taken from the treatment vessel (1) or the detection points (2 and 3) for GSH, GSSG and PSSG detection. At each time, 1 ml of RBCs was treated with 5 mM (final concentration) N-ethylmaleimide (NEM) for 30 s and then centrifuged at 10,000×g for 30 s in order to remove supernatant. Fifty microliters of RBCs were then 1:4 (vol/vol) treated with trichloroacetic acid (TCA, 10% (w/v)). These samples were used for GSH and GSSG analyses. The rest of RBCs was used for PSSG analyses.

Analysis of GSH and GSSG in RBCs

GSH and GSSG were measured in RBCs according to Giustarini et al.15 Briefly, the acidified samples were centrifuged for 2 min at 10,000×g and both GSH and GSSG were measured in the clear supernatants. GSH analysis was carried out by loading 0.05 ml of supernatant onto HPLC and the GS-NEM conjugate was revealed by a diode-array detector at 265 nm. GSSG was measured spectrophotometrically by the GSH recycling method with some modifications.15

Analysis of S-glutathionylated proteins in RBCs and membrane skeletal proteins by HPLC The rest of packed erythrocytes were hemolysed by addition of 1 ml of 5 mM phosphate buffer, pH 6.5, containing 2 mM NEM and centrifuged at 20,000×g for 15 min at 4°C. Supernatants were used for cytosolic analyses.16 For the analyses of membrane skeletal proteins, the pellets were resuspended with a glass rod in 5 mM phosphate buffer, pH 6.5, containing 1 mM NEM and centrifuged at 20,000×g for 15 min at 4°C; this step was repeated three times.17 For the cytosolic analyses, supernatants were passed through gel-filtration columns (PD10 desalting columns, 6 ACS Paragon Plus Environment

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equilibrated with 50 mmol/L phosphate buffer, pH 7.4) to remove low-molecular weight thiols and disulfides. The protein fraction was incubated with 0.5 mM dithiothreitol (DTT) and, after 15 min, 2 mM (final concentration) mBBr was added. After incubation for 15 min at room temperature in the dark, samples were deproteinized with 5% (w/v, final concentration) TCA and loaded on the HPLC. For protein membrane analysis, 20 l of the pellets were treated with 5 mM DTT and mBBr, and analyzed by HPLC. The rest of the pellet was used for analysis by western blotting.

Detection of protein S-glutathionylation by Western blotting Protein samples were mixed with 2× SDS sample buffer, without reducing agents and supplemented with 5 mM NEM, to block unreacted thiol groups and then subjected to SDS-PAGE. In a parallel set of samples, to confirm that S-glutathionylated proteins were modified via mixed disulfide formation, 5 mM DTT was added to the SDS sample buffer. Electrophoresis was carried out using 10% resolving gels. Gels were stained with Coomassie blue. The molecular weight of membrane skeletal proteins was estimated from the calibration curve of Precision plus protein standards ranging from 20 to 250 kDa. Protein samples were electroblotted onto nitrocellulose membrane. After washes and membrane blocking as described,18 immunological evaluation of protein Sglutathionylation was performed incubating the blocked membrane for 2 h with monoclonal mouse anti-GSH antibody (1:1,000 dilution) in 5% commercial non-fat dried milk powder/PBST (10 mM Na+ phosphate, pH 7.2, 0.9% (w/v) NaCl, 0.1% (w/v) Tween 20) followed by a 1-h incubation with a 1:10,000 dilution of a horseradish peroxidase-conjugated anti-mouse antibody in PBST containing 5% (w/v) milk.18 For GAPDH (glyceraldehyde-3-phosphate dehydrogenase) detection, used as loading control normalization, membranes were first washed in PBST and then incubated with antiGAPDH antibody 1:50,000 in TBST (20 mM Tris, pH 7.6, 140 mM NaCl, 0.1% (v/v) Tween 20) containing 5% (w/v) bovine serum albumin for 3 h at room temperature. Immunoreactive bands were visualized through chemiluminescence using Luminata Crescendo. Images of membranes and



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gels were acquired with ImageQuant LAS4000 (GE Healthcare). Analysis of band areas was performed by Image-Quant TM TL analysis software (GE Healthcare).

Protein analysis Hemoglobin concentration was measured by means of a standard kit, based on the Drabkin method, according to the manufacturer's instructions, in samples hemolysed by 5 mM Na+/K+ phosphate buffer, pH 7.4. Membrane protein concentration was measured by the Bradford assay.19

An Agilent series 1100 HPLC (Agilent Technologies, Milan, Italy) equipped with diode array and a fluorescence detector was used for all determinations.

Statistics Data are the mean ± SD. Differences between means were evaluated by using ANOVA followed by Bonferroni post-test. A value of p band 3 > protein 4.1 (Fig. 5).

4. Discussion The main aim of the present study was to identify, in a model that can resemble in vivo slight oxidative stress conditions, the best biomarker of oxidative stress between GSSG and PSSG in human erythrocytes. It is widely known that GSH plays a key role in cell resistance to oxidative damage. We had previously demonstrated that GSH and its oxidized forms are powerful and reliable biomarkers of oxidative stress compared to other potential biomarkers (protein carbonyls, malondialdehyde).20 In fact, under normal conditions, GSH is oxidized to GSSG, which, in turn, is reduced back to GSH by GSSG reductase using NADPH as a cofactor.2 Generally, this system maintains very low intracellular GSSG levels and rarely the GSH/GSSG ratio is found to be

Membrane skeletal protein S-glutathionylation in human red blood

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