S-glutathionylation of human glyceraldehyde-3-phosphate dehydrogenase and possible role of Cys152-Cys156 disulfide bridge in the active site of the protein

Highlights

• Human recombinant GAPDH is S-glutathionylated in the presence of H₂O₂ and GSH.

• Formation of mixed disulfide between the catalytic Cys152 and GSH inactivates GAPDH.

• GAPDH-SSG reacts with adjacent Cys156 to form Cys152-Cys156 disulfide bond.

• The mutation C156S prevents the disulfide bond formation and breakdown of GAPDH-SSG.

• The mutation C156S decelerate reactivation of S-glutathionylated GAPDH by GSH and Grx.

Abstract

Background

We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of H₂O₂ and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated.

Methods

Human recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH.

Results

S-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S.

Conclusions

S-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1.

General significance

The described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.

Introduction

S-glutathionylation is a posttranslational modification of proteins at cysteine residues, resulting in the formation of a mixed disulfide (Protein-S-SG) between a cysteine residue of a protein and the cysteine residue of glutathione. Currently, it has been demonstrated that many proteins are capable of being S-glutathionylated [[1], [2], [3]]. This reaction proceeds spontaneously, as well as it can be catalyzed by enzymes [4]. Spontaneous modification with reduced glutathione (GSH) has been demonstrated for proteins containing cysteine residues subjected to oxidation (hemoglobin, beta-actin, tyrosine phosphatase, peroxiredoxin 2, and glyceraldehyde-3-phosphate dehydrogenase) [[5], [6], [7], [8], [9], [10]]. In this case, GSH reacts with sulfenic acid derivatives of the cysteine residues resulting in the mixed disulfide, which prevents the formation of irreversible oxidation products (sulfinates and sulfonates). Consequently, one of the function of this modification is the protection of proteins from the irreversible oxidation. At the same time, S-glutathionylation of various proteins and enzymes (carbonic anhydrase III, PTP1B, STAT3, c-jun, NF-kB, caspase 3, and others) can change their properties [11]. The redox sensing and ROS forming properties of pyruvate dehydrogenase, oxoglutarate dehydrogenase, and complex I were shown to be modulated by S-glutathionylation [[12], [13], [14]]. Deglutathionylation of proteins can take place spontaneously at a high GSH/GSSG ratio, or catalyzed by glutaredoxins [[15], [16], [17]] as well as by thioredoxins [[18], [19], [20]]. Diversity of proteins undergoing S-glutathionylation and the reversibility of this modification suggest that it can be involved in regulation of different cell functions depending on the redox state of the cell. For this reason, S-glutathionylation attracts the attention of researchers.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme composed of four identical subunits, each containing an active site with two cysteine residues. One of them (Cys150 in rabbit or Cys152 in human GAPDH) takes part in catalysis, being involved in the formation of the intermediate covalent product with the substrate (glyceraldehyde-3-phosphate). The second cysteine (Cys154 in rabbit or Cys156 in human GAPDH) is not involved in catalysis, but it is highly conserved [21]. However, its function is still unclear. The catalytic cysteine residue easily oxidizes in the presence of relatively low H₂O₂ concentrations, which results in the inactivation of the enzyme [22,23]. As was mentioned above, proteins containing cysteine residues that are sensitive to oxidation are readily subjected to S-glutathionylation. This is confirmed by the fact that S-glutathionylated GAPDH was found in plant and animal tissues [1,24]. In our previous work, we demonstrated that rabbit muscle GAPDH is S-glutathionylated in the presence of H₂O₂ and reduced glutathione (GSH). As was shown by MALDI-TOF MS analysis, together with the mixed disulfide GAPDH-SSG, S-glutathionylation resulted in GAPDH species with the intrasubunit disulfide bridge between the catalytic Cys150 and the adjacent Cys154 [10]. The modification resulted in the inactivation of the enzyme. In contrast to the oxidized GAPDH, S-glutathionylated enzyme could be completely reactivated in the presence of dithiothreitol, or partially reactivated in the presence of GSH or thioredoxin/thioreductase system. The conclusion was made that the formation of the mixed disulfide with GSH at the catalytic cysteine residue together with the intramolecular disulfide bridge protect GAPDH from irreversible inactivation by H₂O₂, which allows a reversible inhibition of glycolysis and suggests the activation of pentose-phosphate pathway in response to oxidation stress. However, given that the formation of the mixed disulfide with GSH is sufficient to protect the protein against irreversible oxidation, the physiological relevance of the disulfide bridge in the active site of GAPDH during S-glutathionylation remained unclear. It should be noted that the CTTNC motif in the active site of GAPDH is conserved in the vast majority of species, and it is very likely that the formation of the disulfide bridge between two active site cysteines during S-glutathionylation should have some biological sense. The following assumptions are possible: 1) the disulfide bridge provides more efficient protection of GAPDH from the irreversible inactivation than the mixed disulfide GAPDH-SSG; 2) formation of the disulfide bridge changes the geometry of the active site, which facilitates the access of GSH or specific proteins (glutaredoxin or thioredoxin) to the active site, providing more efficient reduction of the enzyme; 3) since GAPDH in the animal cell is known to be involved in a number of non-glycolytic functions [25], the formation of the disulfide bridge in the active site of GAPDH may change the tertiary structure of the protein and influence the interaction of GAPDH with other proteins and ligands (for example, nucleic acids), which could be of importance for redox signaling. To understand the role of the disulfide bridge in the active site of GAPDH, it was necessary to compare the properties of two S-glutathionylated enzymes: wild-type GAPDH and GAPDH with the mutation C156S excluding the formation of the intrasubunit disulfide bridge. The goal of the present work was to obtain human recombinant wild-type GAPDH (hGAPDH) and GAPDH with C156S substitution (hGAPDH_C156S), to compare structural changes caused by S-glutathionylation of these proteins, and to study the ability of the modified proteins to reactivate in the presence of GSH, glutaredoxin and thioredoxin.