Oxidation of the active site cysteine residue of glyceraldehyde-3-phosphate dehydrogenase to the hyper-oxidized sulfonic acid form is favored under crowded conditions

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and ‘moonlighting ’ activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitro-sylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H 2 O 2 ) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H 2 O 2 - and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation.


Introduction
The oxidation of protein thiols to reversible products (e.g.disulfides, RS-SR'; sulfenic acids, RS-OH; S-nitrosylation, RS-NO; S-sulfhydration, RS-SH) plays a major role in both oxidant-induced cell signaling and, via further oxidation, irreversible loss of protein function.However, not all protein thiols are equally sensitive to oxidation.Under physiological conditions, only a limited number of protein cysteine (Cys) residues react directly and rapidly with hydrogen peroxide (H 2 O 2 ), an endogenous oxidant and signaling agent [1].This is because most reactions involving H 2 O 2 have high activation energies, and consequently efficient and rapid reaction occurs with only a limited number of molecules including transition metal centers and certain protein Cys residues, with these being predominantly in their ionized, more reactive, thiolate (RS -) form [2].In this context, the highly abundant cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [3], is one of the most reactive non-peroxidase targets for both H 2 O 2 , and also other oxidants (e.g.peroxynitrite/peroxynitrous acid; ONOO -/ONOOH) [4,5].Yet, the second-order rate constant for the reaction of GAPDH and H 2 O 2 is ~500 M − 1 s − 1 [6], which is many orders of magnitude lower than the second-order reaction rate constant determined for the highly specialized peroxide-removing cytosolic peroxiredoxin proteins (c.f.rate constants of up to 10 8 M − 1 s − 1 ) [7].These kinetic data indicate that other factors must play a key role for GAPDH to be a major oxidant target and mediator of redox signaling.
GAPDH contains an oxidant-sensitive catalytic thiol (Cys152 in the human enzyme) with a pK a value of ⁓5.8 (i.e. at physiological pH it exists mostly as the thiolate form) [8].However, it is clear that this is not the only factor that determines the reactivity of Cys residues, as some other proteins with low pK a Cys residues (e.g.glutaredoxin-1, PTP1B and papain) do not show an especially enhanced reactivity with H 2 O 2 [9].In the case of GAPDH, a proton relay, and the structure of the reaction site, appear to stabilize the transition state involved in the reaction with H 2 O 2 [10].Yet, despite the experimental evidence indicating that GAPDH is sensitive to oxidation, it is still debated how GAPDH is efficiently and selectively modified in the cytosol in the presence of antioxidant enzymes, and it has been proposed that compartmentalization and protein-protein complex formation may be important factors [10][11][12].
The intracellular environment is heavily crowded (up to 4 million proteins per μm 3 [13]) with an increasing number of studies reporting that macromolecular crowding affects biochemical interactions and reactions [11,12].For example, crowding may alter GAPDH activity directly, or alternatively modulate the oligomerization state of the enzyme to favor the more active tetrameric complex over the monomer and dimeric states of the protein [14].Such changes may modulate the oxidative pathways that GAPDH undergoes under crowded conditions compared to those that occur in dilute systems.) and nitric oxide ( • NO), which rapidly react (k > 10 9 M − 1 s − 1 ) to form ONOO -/ONOOH [15].These reaction mixtures were analyzed using SDS-PAGE (to examine structural changes), thiol assays (to examine total thiol loss) and LC-MS peptide mass mapping (to determine the nature and extent of modification at specific Cys residues).

Materials
All reagents, including GAPDH from rabbit muscle, were purchased from Sigma Aldrich (Søborg, Denmark) unless otherwise indicated.Solutions were prepared using ultrapure water.LC-MS grade solvents were obtained from VWR (Søborg, Denmark).

Protein oxidation
GAPDH (1 mg mL − 1 , 27.9 μM) was incubated with H 2 O 2 for 3 h, or with SIN-1 for 1 h, at 37 • C in 100 mM sodium phosphate buffer solution (pH 7.4) containing 0.1 mM DTPA.Oxidants were added as a bolus dose at 0.1-, 1-, 5-and 40-fold molar excesses to give final concentrations of 3, 30, 140 and 1120 μM.Oxidation under crowded conditions were performed using the same experimental conditions, but with stock solutions of GAPDH, H 2 O 2 or SIN-1 prepared in buffer solutions containing 120 or 300 mg mL − 1 dextran.Dissolved O 2 levels, and thus O 2 availability, are not affected by dextran [16].

Electrophoresis
Control and oxidized GAPDH samples, incubated in the absence or the presence of crowding agents, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions.Aliquots (12 μg of protein) were loaded onto NuPAGE™ 4-12% Bis-Tris gels (Thermo Fisher) and run at 120 V for 90 min using NuPAGE™ MOPS SDS running buffer.Gels were subsequently stained with Coomassie blue.

Quantification of protein thiols
Protein thiols were quantified using the ThioGlo-1 assay following the protocol reported in Hawkins et al. (2009) [17].Protein thiol levels were determined against a standard curve prepared with glutathione (GSH).Blank solutions included ThioGlo1 in 100 mM phosphate buffer (pH 7.4) containing 0.1 mM DTPA in the absence and the presence of up to 300 mg mL − 1 of dextran with a mean molecular mass of 9000 or 35, 000 Da.

LC-MS/MS analysis
Protein samples (20 μg) were dried down using a SpeedVac concentrator, and resuspended in 45 μL of a solution containing 90 mM iodoacetamide and 8 M urea in 50 mM Tris buffer (pH 8.0) to alkylate non-oxidized Cys residues in order to avoid their artifactual oxidation during sample preparation.After incubation for 30 min at 21 • C, 5 μL of SP3 solution (prepared by mixing two Sera-Mag Magnetic Carboxylate modified particles: 24152105050250 and 44152105050250, Cytiva, in a 1:1 ratio) were added to the samples followed by addition of 50 μL of absolute ethanol.This solution was incubated for 5 min in a thermomixer at 20 • C and 1000 rpm.The tubes were then placed in magnetic racks and incubated until the migration of the magnetic beads to the wall of the tubes was complete.The supernatant was discarded, and the beads washed twice with 180 μL of 80 % v/v ethanol/water.After the second wash, 0.1 μg of LysC in 25 μL 50 mM Tris buffer containing 4 M urea (pH 7.5) were added and incubated for 2 h at 37 • C before addition of 125 μL of 50 mM Tris buffer (pH 7.5) containing 0.15 μg of trypsin, or 0.15 μg of GluC.These samples were incubated overnight at 37 • C, before being subjected to solid phase extraction using activated Empore C18 reversed-phase discs (3 M, St. Paul, MN, USA), with the samples eluted using 50 μL of 0.5% v/v trifluoroacetic acid (TFA) in 80% v/v acetonitrile.The resulting peptides were separated by micro-liquid chromatography (Dionex Ultimate 3000 chromatography system, ThermoFisher Scientific) using a Kinetex® 2.6 μm XB-C18 100 Å column (150 × 0.5 mm) kept at 40 • C and flow rate of 30 μL min − 1 (gradient elution using 0.1% formic acid in H 2 O (solvent A) and 80% acetonitrile/ 0.1% formic acid in H 2 O (solvent B)).The LC system was connected online to an Impact II ESI-QTOF (Bruker Daltonics, Germany) mass spectrometer with the electrospray needle held at 4500 V, an end plate offset of 500 V, and a temperature of 200 • C. Nitrogen gas was used for both the nebulizer (0.7 Bar) and as the drying gas (6.0 L min − 1 ).
The resulting spectra were matched to GAPDH (UniProt accession code: P46406) using the search engine MaxQuant and PMI-Byos with oxidation (+16 Da) and di-oxidation (+32 Da) set as variable modifications on Cys, Met, Trp and Tyr residues, and tri-oxidation (+48 Da) and carbamidomethylation (+57 Da, from reaction of Cys residues with iodoacetamide) set as variable modifications on Cys.Additionally, carbamidomethylation of the sulfenic acid form (+73 Da) was searched for in all samples, as previous data have reported the addition of iodoacetamide to protein-SOH [18].Nitration (+45 Da) of Tyr residues was also set as a variable modification for SIN-1 treated samples.The settings utilized for the search engines included a peptide level false discovery rate of 1%, a maximum of 3 missed cleavages, and a maximum mass error of 20 ppm.The data were validated via manual confirmation of the MS/MS spectra against in-silico protein cleavage data generated using GPMAW software (version 9.1, Lighthouse Data, Denmark).
The % of modification at Cys150 (occupancy) was determined by calculating the ratio between the area of a particular precursor ion (with a particular charge state) from a modified peptide containing Cys150, and the sum of the peak areas for all identified peptides containing Cys150 (modified and non-modified) with the same charge state matching the same peptide sequence.Larger peptides having missed cleavages, which contained Cys150, were also accounted for when calculating the % occupancy values.The relative % of the sulfenic (R-SOH), sulfinic (R-SO 2 H), and sulfonic acid forms (R-SO 3 H) of M.R. Glover et al.Cys150 was determined by calculating the ratio between the area of the peptides containing Cys150 modified to the respective oxyacid, and the sum of the peak areas for all peptides containing modified Cys150 (i.e.abundance relative to all modified species).Areas were determined using Bruker QuantAnalysis software.

Statistical analysis
SDS-PAGE data are representative individual images of at least three independent experiments.Quantitative data are presented as mean ± SD from at least three individual experiments, with the figures created using GraphPad (v 8.0).Statistical significance was determined by oneway ANOVA with Holm-Šídák's post-hoc test (p < 0.05).

Results
GAPDH from rabbit muscle was employed due to its commercial availability, high sequence homology to human GAPDH (~95 % [19]), and the conserved nature of its catalytic site, with Cys150 in the rabbit isoform being analogous to Cys152 in the human protein (Fig. 1).Dextran was used as a crowding agent as it has been employed widely for such studies, has well-established chemical properties [20], and is not expected to react with H 2 O 2 (or other peroxides) and thereby act as scavenger.
To investigate possible changes on the molecular mass of GAPDH on incubation with H 2 O 2 , or SIN-1, SDS-PAGE analysis was performed under reducing and non-reducing conditions.As depicted in Fig. 2A, incubation with H 2 O 2 led to the formation of protein cross-links including protein dimers, trimers, tetramers and covalent oligomer species with higher molecular masses.The formation of these species increased as the oxidant-to-protein ratio increased from 0.1-fold to 40fold.The cross-links generated on exposure of GAPDH to H 2 O 2 are mostly reducible species (e.g.disulfide bonds) as determined from the considerable differences between the non-reducing (Fig. 2A) and reducing (Fig. 2B) gels.When oxidation was carried out under crowded conditions, a slight (non-significant) increase in the intensity of the cross-link bands was observed under non-reducing conditions compared to those observed from dilute solutions (Supplementary Fig. 1).
Incubation with SIN-1, which generates an equilibrium mixture of ONOO -/ONOOH (pK a 6.8 [21]) at pH 7.4, resulted in a greater yield of GAPDH cross-links than detected with H 2 O 2 , with these being a mixture of both reducible and non-reducible species (Fig. 2C and D and Supplementary Fig. 1).Furthermore, protein fragments and loss of the monomer protein band were detected with the 5-and 40-fold molar excess conditions.In contrast to the behavior observed with H 2 O 2 , the presence of dextran led to a lower monomer consumption and decreased formation of GAPDH cross-links as determined by densitometric analysis of lanes 9 (dilute condition) and 10 (crowded condition) (Supplementary Fig. 1).
As each GAPDH monomer unit possesses four Cys residues (Cys150, Cys154, Cys245 and Cys282), quantification of total (solvent accessible) protein thiols was determined following GAPDH incubation with H 2 O 2 using the probe ThioGlo1.As depicted in Fig. 3, the quantified thiol levels in control samples were below the theoretically expected value (i.e. ~111 μM).However, this is not surprising as Cys154, Cys245 and Cys282 are buried in the quaternary structure of GAPDH and may   therefore not be accessible to the probe.Similar effects have been reported for other thiol-reacting probes (e.g.DTNB), with buried Cys residues not reacting with the probe [22].Despite this, a significant thiol consumption was observed for GAPDH incubated with increasing molar excesses of H 2 O 2 .No significant differences were detected in the extent of overall thiol consumption under crowded versus dilute conditions.
To gain further insights into possible differences between individual Cys residues (and the products formed) and particularly for the oxidantsensitive Cys150 residue, peptide mass mapping analyses were carried out using LC-MS/MS.Due to the reversible nature of the initial products (e.g.sulfenic acids) no reducing agents were used in the sample preparation process (cf. the use of DTT in most proteomic analyses).Iodoacetamide was used to block any remaining thiols (to avoid artifactual oxidation during processing), and precipitation of the proteins on to magnetic beads allowed rapid and efficient removal of any remaining H 2 O 2 or SIN-1 from the samples.
Sequence coverages of between 75.7 and 82.3 % was observed for control and oxidized GAPDH samples under dilute and crowded conditions, with peptides containing the active site Cys150, and structural Cys154 and Tyr312 residues (Cys156 and Tyr314 in the human isoform) detected (Supplementary Tables 1 and 2 and Supplementary Figs. 2 and  3).Both Cys154 and Tyr312 participate in the proton relay with Cys150 reported for the human isoform [23], and are important for the structure of the active site (Fig. 1).A peptide containing native and oxidized Cys245 was also identified, but Cys282 was not detected in any of the samples.As depicted in Fig. 4, LC/MS analysis allowed detection of a (doublycharged) peptide containing both Cys150 and Cys154.This peptide was observed with multiple different modifications at Cys150 (but not Cys154) on oxidant exposure, with sulfenic (two-electron oxidation), sulfinic (four-electron oxidation) and sulfonic acids (six-electron oxidation) detected.The first of these is an unstable product and was detected at low levels (cf. the signal-to-noise ratio in Fig. 4B).The carbamidomethylated adduct of the sulfenic acid (potentially arising from the use of iodoacetamide to block unreacted thiol residues) was not detected in any of the samples.Sulfinic and sulfonic acids are considered irreversible oxidation products, though there is some evidence for biological reduction of sulfinic acids generated on peroxiredoxins by sulfiredoxins (not present in our experimental setup, thus the sulfinic acid is considered as an irreversible modification) [24].In contrast, Cys154 was detected predominantly as the carbamidomethylated (i.e.non-oxidized) species, however low levels of the sulfenic acid form at this residue were observed (vide infra).Along with the modifications observed at Cys150 and Cys154, low levels of modification were also observed at Cys245 (to the sulfinic and sulfonic acid forms; Supplementary Tables 1 and 2).Furthermore, oxidation of Met to the sulfoxide form (i.e.+16 Da) was detected at Met41, Met44, Met128, Met131, Met173, Met229, Met326 and Met329 (Supplementary Fig. 2, 3).
Incubation of GAPDH with a 40-fold molar excess of H 2 O 2 or SIN-1 resulted in a significant loss of parent (reduced) Cys150 under dilute conditions, with SIN-1 giving a higher extent of loss than H 2 O 2 (~95 and ~79 %, respectively), as expected from the lower rate constants for reaction of H 2 O 2 , compared to ONOO -/ONOOH, with free Cys (Fig. 5A and Supplementary Fig. 4) [9].In contrast, the loss of the parent (reduced) Cys154 residue was modest with percentages of modification of ~0.2 and ~2.5 % for GAPDH samples incubated with a 40-fold molar excess of H 2 O 2 and SIN-1, respectively (Fig. 5B).These data confirm the sensitivity of Cys150, but not Cys154, to oxidation.Interestingly, oxidation by H 2 O 2 in the presence of dextran resulted in a decreased extent of modification at Cys150, with this effect increasing with a higher concentration of dextran.A similar effect was seen with SIN-1 at the higher dextran concentration (Fig. 5A).In contrast, the extent of modification of Cys154 by H 2 O 2 increased from ~0.2 to ~0.5 % under dilute (PB) and crowded (D300) conditions respectively, and from ~2.5 to ~4.8 % for samples incubated with SIN-1 under dilute (PB) and crowded (D120) conditions, respectively (Fig. 5B).However, the extent of modification of Cys154 was lower (~1.1 %) at the highest concentration of dextran (i.e.300 mg mL − 1 ).The enhanced reactivity of SIN-1 in comparison to H 2 O 2 was supported by the detected yields of the sulfonic acid (Fig. 6), which were greater for SIN-1 when compared to H 2 O 2 (36.2 versus 17.2 %, respectively).
To get further insight into the products formed, and the possible effects of crowding on the oxidation of the key Cys150 residue, product quantification was performed using LC-MS/MS (Fig. 6A).These data showed a decreased fraction of oxidized products under crowded conditions (i.e. a higher abundance of native Cys150) indicating that less GAPDH monomers contained modifications at Cys150 (as assessed by the yields of the sulfenic, sulfinic and sulfonic acid species) under crowded conditions when compared to the results obtained under dilute conditions.Of the products detected by LC-MS/MS, the sulfinic acid form was the most abundant.However, analysis of the relative abundance of the different oxyacids showed a significantly increased extent of sulfonic acid formation (six-electron oxidation of Cys) at Cys150 in the presence of 300 mg mL − 1 dextran (Fig. 6D).For H 2 O 2 , the relative abundance of sulfonic acid at Cys150 increased from ~17 to ~31 % for dilute and crowded conditions, respectively.The increase for SIN-1 was less marked, but statistically different (~36 and ~42 % for dilute and crowded conditions, respectively).Furthermore, a decreased yield of the sulfenic and sulfinic acid forms was observed at Cys150 under crowded conditions when compared to dilute conditions for both H 2 O 2 and SIN-1 (Fig. 6B and C).With H 2 O 2 , the relative sulfenic acid abundance at Cys150 decreased from ~2.1 to ~0.8 %, and for SIN-1 from ~1.0 to ~0.3 % under dilute and crowded conditions, respectively (Fig. 6B).The relative abundance of the sulfinic acid form of Cys150 was also less abundant on exposure to H 2 O 2 under crowded compared to dilute conditions decreasing from ~81 to ~69 % respectively, and for SIN-1 oxidized samples decreasing from ~63 to ~58 % for dilute and crowded conditions, respectively (Fig. 6C).

Discussion
The oxidant-sensitive GAPDH enzyme is key for the metabolism and adaptation of mammalian cells as confirmed from the different functions reported for this protein (i.e.catalysis of the sixth step in glycolysis, and diverse non-metabolic activities including transcriptional gene regulation, maintenance of DNA integrity and inter-organelle trafficking, amongst others) [25].Recently, it has been described that oxidation of the active site Cys152 residue in the human isoform leads to GAPDH inactivation with this being essential for metabolic adaptation.Thus, oxidation and inactivation of GAPDH re-routes glucose metabolism from glycolysis to the pentose phosphate pathway increasing the reductive power of cells, which seems to be critical for cell adaptation to oxidative environments [4].However, despite this evidence, it is still under debate how the catalytic Cys residue in GAPDH is efficiently modified by biological oxidants (e.g.H 2 O 2 and ONOO -) when the second order reaction Native and oxidized samples of GAPDH prepared under dilute (100 mM sodium phosphate buffer solution (pH 7.4) containing 0.1 mM DTPA; PB) or crowded (PB containing 120 or 300 mg mL − 1 dextran 35,000; D120 and D300, respectively) conditions were digested and analyzed by LC-MS/MS as described in the Materials and methods.The % modifications at Cys150 and Cys154 were determined by calculating the ratio between the area of precursor ions from modified peptides and the sum of the peak areas for all identified peptides (i.e.modified and non-modified) containing these residues.The data are mean values ± SD from three independent experiments.Statistical differences are indicated as follows: *p < 0.05, **p < 0.01, and ****p < 0.0001.Note the different vertical (y-) axis scales of Panels A and B, due to the lower extent of modification at Cys154.rate constants determined for reaction of GAPDH with these oxidants are many orders of magnitude lower than the reaction constants reported for specialized peroxide-removing enzymes such as cytosolic peroxiredoxins.One possibility is that GAPDH oxidation is modulated by molecular crowding as encountered in the cell cytosol, where ~40% of the total intracellular volume is occupied by macromolecules generating a heavily-packed milieu [12].
Previous studies have reported that macromolecular crowding conditions generated by the addition of up to 300 mg mL − 1 bovine serum albumin, polyethylene glycol (PEG, ~20,000 Da), or dextran (~70,000 Da) does not affect the activity of either the monomeric or tetrameric forms of GAPDH [14,26].However, these studies also showed that crowding modulates GAPDH folding and shifts the monomer ↔ dimer ↔ tetramer equilibrium towards the homotetramer complex by promoting association interactions, with these observations being in agreement with the excluded volume phenomenon (see Ref. [12]).Consequently, as macromolecular crowding affects protein-protein interactions, as well as substrate binding and the rate of transition-state limited reactions to a greater extent than non-specific associations [20], and considering that GAPDH has a defined binding site for H 2 O 2 [23], it is rational to Fig. 6.Quantification and distribution of the native (reduced) and oxyacid products generated at Cys150 residue (Panel A), and relative abundance of the sulfenic (R-SOH), sulfinic (R-SO 2 H) and sulfonic acid (R-SO 3 H) forms generated at this site (Panels B, C and D, respectively) after incubation of GAPDH with a 40-fold molar excess of H 2 O 2 or SIN-1 under dilute (PB) or crowded (300 mg mL − 1 dextran Mw ~35,000 in PB; D300) conditions.The relative abundance of the different oxyacids (expressed as a %) was determined by calculating the ratio between the area of precursor ions from each specific modified species (i.e.R-SOH, R-SO 2 H or R-SO 3 H) and the sum of the peak areas for all modified peptides containing these residues combined.Data are mean ± SD from three independent experiments.Statistical differences are indicated as follows: *p < 0.05, **p < 0.01, and ****p < 0.0001.Note that the y-axis scale of Panel B has been adjusted to facilitate visualization of the data.Fig. 7. Schematic representation of the two-electron oxidation pathways of the active site and oxidant-sensitive Cys residue in GAPDH (enzyme represented in green).The two-electron oxidation of the reduced Cys (represented as enzyme-SH) leads to the formation of the reversible sulfenic acid (enzyme-SOH), which can either react with another thiol (represented as R-SH) to form a disulfide (enzyme-S-S-R) or with a second sulfenic acid (R-SOH) to form a reversible thiosulfinate (enzyme-S-S(=O)-R).In addition, the sulfenic acid can be further oxidized to irreversible sulfinic and sulfonic acid forms (enzyme-SO 2 H and enzyme-SO 3 H, respectively).An alternative pathway for the formation of the Cys (hyper)oxidized sulfonic acid form is via two-electron oxidation of the thiosulfinate to give thiosulfonates, which can be further oxidized to disulfide trioxide and then to the disulfone species, which eventually leads to cleavage of the S-S bond to give the R-SO 3 H species.
hypothesize that macromolecular crowding would modulate the oxidation of the active site Cys residue (Cys150 in the rabbit isoform).
To test our hypothesis we firstly investigated the effect of macromolecular crowding induced by dextran on the H 2 O 2 -induced crosslinking of GAPDH.As observed in Fig. 2A and B, crowding led to a slight increase in the formation of GAPDH cross-links on incubation with a 40-fold molar excess of H 2 O 2 , with most of the cross-links being reducible.These data suggest that two-electron oxidation of Cys to the sulfenic acid form (Cys-SOH), followed by the reaction of this species with a reduced Cys residue in a different GAPDH monomer (to give dimers or higher aggregates) is slightly favored under crowded systems (Fig. 7).In contrast, incubation of GAPDH with SIN-1 under crowded conditions led to a decreased extent of enzyme cross-linking and fragmentation, when compared to the results observed under dilute conditions (Fig. 2C and D).This is not surprising as an alternative pathway for ONOOH decomposition (half-life ~1 s [27]) is formation, in yields of 10-30 % of hydroxyl (HO • ) and nitrogen dioxide radicals ( • NO 2 ) [21,27].This implies that, unlike H 2 O 2 -mediated oxidation, the reactions induced by SIN-1 include a mixture of two-electron and one-electron processes.In this context, macromolecular crowding is known to differentialy affect transition-state limited reactions (e.g.two-electron oxidations) compared to diffusion-controlled reactions (e.g.radical reactions, including dimerization) by favoring the rate of the first, and decreasing the latter [12,20].Alternatively, dextran may react with a fraction of the HO • formed (as reported for PEG [28]), though this may not explain the observed differences in extent of cross-linking.
The effects of crowding on GAPDH oxidation were investigated further by determining the total protein thiol concentration using the ThioGlo-1 assay.A significant loss of protein thiols was observed with the highest concentration of H 2 O 2 (40-fold molar excess over GAPDH concentration), but no differences were observed between the samples oxidized under dilute and crowded conditions (Fig. 3).This data, however, provides only a partial picture of the fate of Cys residues as the thiol-reactive fluorescent probe ThioGlo-1 reacts primarily with solvent accessible native (reduced) Cys residues, whilst buried residues (e.g.Cys154) are unlikely to react with the probe.As a consequence, and due to the importance of the catalytic Cys150 and the structural Cys154 residues for GAPDH activity LC-MS analyses were carried out to examine the loss of these key residues and to determine the oxidation products formed at these sidechains.As depicted in Figs. 4 and 5, the Cys150 residue in GAPDH was significantly oxidized on incubation with H 2 O 2 and SIN-1, whereas only modest levels of modification were detected at Cys154, as expected from its buried location and lower reactivity [23].
The differences observed in the extent of oxidation of Cys150 under dilute versus crowded conditions, clearly suggest altered oxidation pathways.The reduced yield of sulfenic and sulfinic acids, along with the higher levels of the sulfonic acid formed from Cys150 (Fig. 6), may be rationalized by an effect of crowding on local oxidant-to-protein ratios, i.e. formation of hydrophilic nanodomains with higher local concentrations of H 2 O 2 or SIN-1 than the bulk concentration (see Ref. [12]).Alternatively, the enhanced oxidation may arise from an increased affinity of (for example) H 2 O 2 for residues in the binding-site cavity of GAPDH.This is supported by data indicating that macromolecular crowding affects substrate binding to a greater extent than other non-specific biomolecular associations [20].This explanation is consistent with the results of Peralta and coworkers (2015) who reported that GAPDH possesses a defined binding site, within the catalytic pocket, for H 2 O 2 which would help explain the enhanced reactivity of Cys150 with H 2 O 2 [23].Whether such interactions also explain the effects seen with SIN-1 is less clear, as it is unclear whether this binds to proteins and induces site-specific oxidation.However it is rational to speculate that the lesser effects of SIN-1 indicate that such interactions are more limited.
Although the current data are limited to the quantification of the reversible sulfenic acid, and irreversible sulfinic and sulfonic acids, formed from the catalytic and oxidant-sensitive Cys150 residue in GAPDH under dilute and crowded conditions, our results clearly illustrate that crowding favors over(hyper)-oxidation of this residue.As illustrated in Fig. 7, the increased prevalence of the sulfonic acid form of Cys150 (per molecule of GAPDH modified) may be a result of different reaction pathways.This is because two-electron oxidation of thiols can follow different mechanisms with formation of a number of unstable intermediaries, but over(hyper)-oxidation eventually leads to the formation of the sulfonic acid (six-electron oxidized) species.

Conclusions
The data presented here demonstrate that GAPDH oxidation, by two different oxidants is modulated under crowded systems.Oxidations carried out in the presence of dextran resulted in altered pathways with a decreased extent of loss of Cys150, but a higher yield of (irreversible) sulfonic acid formation as determined by LC-MS/MS.These novel findings suggest that crowding modulates the redox status of GAPDH, and therefore potentially its redox signaling activity and other functions within cells.To achieve a greater understanding of these events, both crowding and buffer compositions (e.g. the effects of bicarbonate) should also be considered [29].

Fig. 1 .
Fig. 1.Superposition of the crystal structures of human GAPDH (Uniprot code: P04406; PDB: 4WNC) and rabbit GAPDH (Uniprot code: P46406; PDB: 1J0X) using the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.Panel A: overview of the protein superposition with the proteins represented as monomer units with the human and rabbit GAPDH represented in grey and green, respectively, and the NAD + molecule that binds in proximity to the catalytic site in orange.Panel B: Zoom image of the active site of GAPDH with the catalytic Cys150 residue and the structural Cys154 residue highlighted in yellow, and the structural Tyr312 and Tyr318 highlighted in blue.For the zoomed image, the highlighted residues appear with the numbering of rabbit GAPDH and human GAPDH (in parenthesis).

Fig. 2 .
Fig. 2. Oxidation of GAPDH (1 mg mL − 1 ) mediated by H 2 O 2 (Panels A and B) and SIN-1 (Panels C and D) under dilute and crowded conditions, respectively, results in the formation of protein cross-links.Panel A and B correspond to SDS-PAGE of GAPDH incubated with H 2 O 2 run under non-reducing and reducing conditions, respectively.Panel C and D correspond to SDS-PAGE of GAPDH incubated with SIN-1 run under non-reducing and reducing conditions, respectively.For all gels, the samples loaded into the lanes was as follows: lanes 1 and 2, control in dilute and crowded conditions, respectively; lanes 3, 5, 7 and 9, incubation with 0.1, 1, 5 and 40-fold oxidant to protein molar excess under dilute conditions (i.e.phosphate buffer); lanes 4, 6, 8 and 10, incubation with 0.1, 1, 5 and 40-fold oxidant to protein molar excess under crowded conditions (i.e.phosphate buffer containing 120 mg mL − 1 dextran 35,000).The protein monomer (orange arrow), dimer and cross-links (blue arrows), and fragments (green arrows) are indicated with arrows.The images are representative of those from three independent experiments.

Fig. 3 .
Fig. 3. Quantification of the total protein thiols in control and oxidized GAPDH samples carried out using the ThioGlo 1 assay.GAPDH (27.9 μM) was incubated with 1-, 5-, and 40-fold molar excess of H 2 O 2 under dilute (100 mM sodium phosphate buffer solution (pH 7.4) containing 0.1 mM DTPA; PB) or crowded (PB containing 120 mg mL − 1 dextran 35,000 or dextran 9000) conditions.Data are mean ± standard deviations of at least three independent experiments carried out on different days.Asterisk (*) indicates statistical significance at the p < 0.05 level.

Fig. 5 .
Fig. 5. Quantification of the loss of the parent (reduced) Cys150 (Panel A) and Cys154 (Panel B) residues in GAPDH samples incubated with a 40-fold molar excess of H 2 O 2 or SIN-1.Native and oxidized samples of GAPDH prepared under dilute (100 mM sodium phosphate buffer solution (pH 7.4) containing 0.1 mM DTPA; PB) or crowded (PB containing 120 or 300 mg mL − 1 dextran 35,000; D120 and D300, respectively) conditions were digested and analyzed by LC-MS/MS as described in the Materials and methods.The % modifications at Cys150 and Cys154 were determined by calculating the ratio between the area of precursor ions from modified peptides and the sum of the peak areas for all identified peptides (i.e.modified and non-modified) containing these residues.The data are mean values ± SD from three independent experiments.Statistical differences are indicated as follows: *p < 0.05, **p < 0.01, and ****p < 0.0001.Note the different vertical (y-) axis scales of Panels A and B, due to the lower extent of modification at Cys154.
In this context, we hypothesized that macromolecular crowding might modulate the H 2 O 2 -induced oxidation of GAPDH.In the present work, we have explored the extent of loss of parent Cys residues in GAPDH and hyper-(over)oxidation of the catalytic Cys to higher oxyacids (i.e.sulfinic, RSO 2 H, and sulfonic acid, RSO 3 H, forms) in GAPDH under both dilute and crowded conditions.To test this hypothesis, samples of GAPDH were exposed to a range of molar excesses of H 2 O 2 , in the absence or presence of different concentrations of crowding agents of different molecular mass.Comparative studies were also carried out with 3-morpholinosydnonimine (SIN-1), a thermo-labile compound that releases superoxide (O 2•-