Posttranslational Modification of Human Glyoxalase 1 Indicates Redox-Dependent Regulation

Gerd Birkenmeier,Ralf Hoffmann,Claudia Birkemeyer,Christin Stegemann,Robert Günther,Klaus Huse,Jörg Hoheisel

doi:10.1371/journal.pone.0010399

Gerd Birkenmeier, Ralf Hoffmann + Show 5 more

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https://doi.org/10.1371/journal.pone.0010399

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Abstract

BackgroundGlyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) are ubiquitously expressed cytosolic enzymes that catalyze the conversion of toxic α-oxo-aldehydes into the corresponding α-hydroxy acids using L-glutathione (GSH) as a cofactor. Human Glo1 exists in various isoforms; however, the nature of its modifications and their distinct functional assignment is mostly unknown.Methodology/Principal FindingsWe characterized native Glo1 purified from human erythrocytes by mass spectrometry. The enzyme was found to undergo four so far unidentified posttranslational modifications: (i) removal of the N-terminal methionine 1, (ii) N-terminal acetylation at alanine 2, (iii) a vicinal disulfide bridge between cysteine residues 19 and 20, and (iv) a mixed disulfide with glutathione on cysteine 139. Glutathionylation of Glo1 was confirmed by immunological methods. Both, N-acetylation and the oxidation state of Cys19/20, did not impact enzyme activity. In contrast, glutathionylation strongly inhibited Glo1 activity in vitro. The discussed mechanism for enzyme inhibition by glutathionylation was validated by molecular dynamics simulation.Conclusion/SignificanceIt is shown for the first time that Glo1 activity directly can be regulated by an oxidative posttranslational modification that was found in the native enzyme, i.e., glutathionylation. Inhibition of Glo1 by chemical reaction with its co-factor and the role of its intramolecular disulfides are expected to be important factors within the context of redox-dependent regulation of glucose metabolism in cells.

Highlights

Glyoxalases (Glo1, E.C. 4.4.1.5, and glyoxalase 2 (Glo2), E.C.3.1.2.6) constitute an ubiquitous detoxification system that protects against cellular damage caused by reactive 2-oxo-aldehydes such as methylglyoxal (MGO)
Purified Glyoxalase 1 (Glo1) from human erythrocytes was subjected to analysis by direct-infusion ESI-FTICR (Figure 1A)
This mass difference would be in accordance with the two possible monomers of the enzyme that differ at position 111, carrying either alanine or glutamic acid

Summary

Introduction

Glyoxalases (Glo, E.C. 4.4.1.5, and Glo, E.C.3.1.2.6) constitute an ubiquitous detoxification system that protects against cellular damage caused by reactive 2-oxo-aldehydes such as methylglyoxal (MGO). Cells with increased glucose metabolism are suggested to protect themselves against cellular damage by MGO through up-regulation of Glo activity. In expression studies, cellular effects expected from reduced Glo activity and Glo knockdown such as increased MGO concentration revealed rather discordant findings [6,7]. Within this context, GSH was proposed to exhibit other functions beyond its mere anti-oxidative properties as enzyme co-factor and redox-regulator [8]. Glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2) are ubiquitously expressed cytosolic enzymes that catalyze the conversion of toxic a-oxo-aldehydes into the corresponding a-hydroxy acids using L-glutathione (GSH) as a cofactor. Human Glo exists in various isoforms; the nature of its modifications and their distinct functional assignment is mostly unknown

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