# Park7 (DJ-1), a Parkinson-associated glyoxalase/deglycase: characterization of enzymatic activity in biological samples using fluorescence-based liquid chromatography

Nicolas Mathas, Emmanuelle Braud, Erwan Galardon, Lucie Larigot, Mélanie Etheve-Quelquejeu, Marie-Agnès Sari, Beatrice Le Grand, Etienne Blanc, Xavier Coumoul, Julien Dairou

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Nicolas Mathas, Emmanuelle Braud, Erwan Galardon, Lucie Larigot, Mélanie Etheve-Quelquejeu, et al. Park7 (DJ-1), a Parkinson-associated glyoxalase/deglycase: characterization of enzymatic activity in biological samples using fluorescence-based liquid chromatography. Biochimie, 2025, 235, pp.113-121. 10.1016/j.biochi.2025.06.005. hal-05343900

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# Park7 (DJ-1), a Parkinson-associated glyoxalase/deglycase: characterization of enzymatic activity in biological samples using fluorescence-based liquid chromatography

Nicolas Mathas<sup>a</sup>, Emmanuelle Braud<sup>a</sup>, Erwan Galardon<sup>a</sup>, Lucie Larigot<sup>b,c</sup>, Mélanie Etheve-Quelquejeu<sup>a</sup>, Marie-Agnès Sari<sup>a</sup>, Beatrice Le Grand<sup>b,c</sup>, Etienne B. Blanc<sup>b,c</sup>, Xavier Coumoul<sup>b,c</sup>, Julien Dairou<sup>a,*</sup>

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## ARTICLE INFO

**Article history:**
Received 16 April 2025
Received in revised form
2 June 2025
Accepted 11 June 2025
Available online 14 June 2025

Handling Editor: Dr B Friguet

**Keywords:**
DJ-1
Methylglyoxal
Guanosine fluorescein
Guanosine dansyl
Enzyme assay
Electrophile stress

## ABSTRACT

Glycation is an inevitable nonenzymatic covalent reaction between nucleophilic groups in proteins and nucleotides, and endogenous reducing sugars or dicarbonyls (e.g., methylglyoxal) leading to protein and/or nucleotide covalent modifications. This process initiates a series of dehydrations, oxidations and rearrangements that lead to a myriad of products called advanced glycation end products (AGEs). Previously, we showed that Park7 (or DJ-1) is a protein deglycase that repairs methylglyoxal-glycated biomolecules by acting on early glycation intermediates and releasing the repaired biomolecule and lactate. Moreover, the PARK7 gene is associated with recessive and sporadic forms of Parkinson's disease (PD). Thus, over the last few years, the function of Park7 has become an important research topic for understanding the etiology of PD. In this context, the development of sensitive enzymatic assays is crucial to quantify the activity of Park7 and develop specific modulators. Here, we describe a new method for measuring Park7 activity based on the separation and quantification of glycated and unglycated fluorescent nucleotides by Liquid Chromatography (LC). Kinetic and mechanistic analyses using human recombinant Park7 confirmed the reliability of this approach. In addition, this assay was further validated using cellular lysates. Our results indicate that this novel Park7 assay is simple, sensitive, and specific.

© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

A major factor contributing to cellular and organismal aging is the accumulation of advanced glycation end products (AGEs) on

proteins, lipids and nucleic acids [1,2]. The glycation process is initiated by a non-enzymatic reaction between sugar-derived aldehydes and protein or nucleic acid nucleophiles such as amino or thiol groups. This reaction was discovered by the French chemist Louis-Camille Maillard in 1912 and the major glycating agents are reducing sugars (glucose, fructose, ribose, and their phosphorylated derivatives) and glyoxals such as methylglyoxal (MGO, CH3CO-CHO). Glyoxals are formed as by-products of glucose metabolism and are responsible for approximately 65 % of cellular glycating events. The condensation reaction between the aldehyde group of glyoxals and amino acids begins with the rapid formation of a hemithioacetal with cysteine or aminocarbinols with arginine or lysine (Fig. 1). Similarly, nucleic acids undergo permanent glycation by MGO with the most susceptible nucleotides being guanosine and deoxyguanosine. Subsequently, a series of

**Abbreviations:** pNPA, 4-Nitrophenyl acetate; pNP, 4-nitrophenol; 5-DG, 5-dansyl-guanosine; 5-FG, 5'-fluorescein-guanosine; DiFMUAc, 6,8-Difluoro-4-methylumbelliferyl acetate; DiFMU, 6,8-Difluoro-4-methylumbelliferyl; AGEs, Advanced glycation end products; aSyn, Alpha-synuclein; DMF, Dimethylformamide; DMSO, Dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's medium; HR-MS, High-resolution mass spectrometry; IC50, Inhibitory concentration 50; LC, Liquid Chromatography; MS, Mass spectrometry; MGO, Methylglyoxal; PD, Parkinson disease; ppm, Parts per million.

* Corresponding author. Campus Saint-Germain-des-Prés, 45 rue des Saints-Pères, 75006, Paris, France.

E-mail address: julien.dairou@u-paris.fr (J. Dairou).

<footnote><sup>a</sup>Université Paris Cité, CNRS, Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, 45 rue des Saints Pères, F-75006, Paris, France</footnote>

<footnote><sup>b</sup>Université Paris Cité, 45 rue des Saints Pères, F-75006, Paris, France</footnote>

<footnote><sup>c</sup>INSERM, UMR-S1124, HealthFex, 45 rue des Saints Pères, F-75006, Paris, France</footnote>
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** The formation of various adducts by the reaction between the C1 of MGO and proteins or DNA (red arrow), and the repair process catalyzed by the glyoxalase/deglycase activity of Park7 (DJ-1) (green arrow).</figcaption></figure>

dehydrations, oxidations and rearrangements yields a myriad of products, including Schiff bases, Amadori products, AGEs and protein and/or DNA crosslinks [3].

Recently, our team reported that the protein Park7 is a major protein deglycase that repairs MGO-glycated proteins and nucleotides [4-8]. We have characterized the activity of Park7, which catalyzes the repair of guanines damaged by MGO and prevents mutations. The repair of DNA glycation damage by Park7 ("guanine glycation repair") is as critical to cells as the repair of oxidized DNA by the "guanine oxidation repair" system [6]. Furthermore, we reported that Park7 repairs MGO-glycated cysteine, arginine, and lysine, and subsequently releases repaired proteins and also lactate [8]. Park7 may be the only enzyme known to repair both proteins and nucleic acids (Fig. 1). Since Maillard adducts are its substrates, we have renamed Park7 and its counterparts "Maillard deglycases" [4].

Park7 displays several enzymatic activities including glyoxalase and deglycase activities; however, several independent groups have shown that the deglycase activity of Park7 is a secondary consequence of its glyoxalase activity, acting on methylglyoxal in the presence of reversible glycation products [9-11]). These groups have used a diverse array of methods and model systems and have come to the same conclusion, which contradicts the reports from our group. Nevertheless, even though the nature of its physiological substrates is still a matter of controversy [9-14], Park7 protects and/or repairs various biomolecules against glycation by glyoxals [5,6,8,15-19] and detoxifies reactive compounds produced during glycolysis [20,21]. Thus, Park7 plays a crucial role in protecting against glycation-induced damage.

Interestingly, the PARK7 gene is associated with recessive and sporadic forms of Parkinson's disease (PD) [22]. PD is the second most common neurodegenerative disease with complex etiology and variable pathology [23]. Most cases originate from a combination of environmental and genetic factors that are not yet fully understood. Nevertheless, alpha-synuclein (aSyn) misfolding and aggregation play a central role in PD [24]. Among the possible contributors to PD pathology, several recent studies implicate glycation as an important process in the pathogenesis of PD. Thus, it has been shown that aSyn itself can be glycated, at least under

experimental conditions. Recently, Outeiro and colleagues established that the glycation process enhanced aSyn toxicity in vitro and in vivo [2,25-27]. Using glycation inhibitors, this team demonstrated that normal clearance of aSyn was reestablished probably due to a reduced aggregation; moreover, motor phenotypes in Drosophila were alleviated. However, there are only a few studies regarding the role of Park7 in glycation and its connection to PD pathogenesis, likely due to the lack of a reliable Park7 activity assay. Indeed, currently available activity tests are based on related enzymatic activities of Park7 such as the hydrolytic activity or on its deglycase function. However, these assays are not suitable for complex biological samples, such as cellular and tissue extracts, due to non-specific interactions and low sensitivity.

To better understand the role of Park7 and to develop specific effectors (activators or inhibitors), it is crucial to use a simple, sensitive and quantitative assay. Recently, a colorimetric assay was developed based on the hydrolytic activity of Park7 given that cysteine-proteases can act on both amide and ester substrates. Indeed, by using 4-nitrophenyl acetate (pNPA) or 6,8-difluoro-4-methylumbelliferyl acetate (DiFMUAc) as an *in vitro* substrate for esterase activity, colorimetric assays showed that Park7 possesses esterase activity as monitored by the release of 4-nitrophenol (pNP) or 6,8-difluoro-4-methylumbelliferyl (DiFMU) induced by the hydrolysis of the ester bond [28].

This assay is very useful for recombinant Park7 activity measurement but is incompatible with biological samples (cellular and tissue extracts) due to the presence of endogenous esterases. However, most studies on Park7, particularly those focused on *ex vivo* and *in vivo* extracts, have used LC-based method. Briefly, Park7 activity is assessed on the hemi-aminoacetal formed *in situ* upon reaction of MGO and guanosine (or other nucleophiles) as substrates. The disappearance of the hemi-aminoacetal and the concomitant formation of guanosine and lactate, due to Park7 activity, are monitored using LC. This approach is an efficient method for detecting Park7 deglycase activity but has low detection sensitivity.

Here, we report the development of a new LC-based assay to measure Park7 activity both in recombinant purified enzyme preparations and biological samples. This assay is based on the
separation and quantification by reverse-phase LC of a chromophore-labeled glycated guanosine using fluorescein or dansyl (5-FG or 5-DG) and its glycated product. The accuracy, specificity and biological relevance of the assay were confirmed through kinetic and mechanistic analyses using well-characterized Park7 inhibitors and cell extracts containing different amounts of Park7. We further demonstrate that Cys106 is the active residue required for this activity. Overall, this assay is well-suited for screening and kinetic characterization of Park7 inhibitors and activators, providing a reliable method for measuring glyoxalase/deglycase activity in biological samples.

## 2. Materials and methods

### 2.1. Human DJ-1 enzyme production and purification

Park7 was purified following previously described protocols [4–6,8]. Briefly, a Park7 expression strain was cultured in LB medium until it reached an optical density of A600 = 0.6, after which protein expression was induced using 1 mM isopropyl β-D-1-thiogalactopyranoside for a period of 4 h. The bacterial cells were then harvested and resuspended in 30 mL of extraction buffer, composed of 30 mM Tris (pH 8), 20 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol (DTT), and a protease inhibitor cocktail tablet (cOmplete™). The suspension was sonicated on ice for 6 min (pulses of 10 s on, 30 s off), and the resulting lysate was centrifuged at 100,000×g for 30 min, with the supernatant retained for purification. The initial purification step involved anion-exchange chromatography using a DEAE-Sephacel (Cytivia) column, equilibrated in the same Tris-based extraction buffer at 4 °C. The proteins were eluted using a linear gradient of 20–400 mM NaCl. A second purification step was performed using hydroxyapatite chromatography (Bio-Gel HTP, Bio-Rad), with the column equilibrated under identical conditions. Elution was carried out using a gradient of 0–250 mM sodium phosphate (pH 8). Following purification, Park7 was dialyzed overnight at 4 °C against 50 mM sodium phosphate buffer (pH 8), 20 mM NaCl, 0.5 mM EDTA, and 1 mM DTT. The protein concentration was determined using the Bradford assay, and its purity was assessed via SDS-PAGE and/or western blotting with an anti-Park7 antibody (SAB4500248, Sigma). The purified protein was stored at –80 °C for further use.

### 2.2. Synthesis of 5-fluorescein-guanosine (5-FG) and 5-dansyl-guanosine (5-DG) (Scheme 1)

Unless otherwise specified, all chemicals and reagents were obtained from commercial suppliers and used without further purification. Pyridine and dimethylformamide (DMF) were dried over calcium hydride.

**Synthesis of 5-FG:** 5'-amino-5'-deoxyguanosine (33 mg, 0.117 mmol), prepared as described in Niewiadomski et al. [29], was dissolved in anhydrous DMF (2.4 mL). After cooling to 0 °C, fluorescein isothiocyanate (45.5 mg, 0.117 mmol) was added. The reaction mixture was stirred at room temperature for 24 h and freeze-dried. The product was purified using reverse-phase HPLC. The mobile phase consisted of solvent A (water with 0.1 % trifluoroacetic acid) and solvent B (acetonitrile). The purification was performed with a linear gradient from 0 % of B to 100 % of B to afford 5-FG as an orange solid. ¹H NMR (500 MHz, DMSO-d₆): δ 10.70 (s, 1H), 10.09 (s, 2H), 10.0 (s, 1H), 8.33 (s, 1H), 8.09-8.07 (m, 2H), 7.74 (dd, 1H, *J* = 8.5 Hz, *J* = 2 Hz), 7.16 (d, 1H, *J* = 8.5 Hz), 6.67 (d, 2H, *J* = 2 Hz), 6.60-6.55 (m, 4H), 6.51 (s, 2H), 5.74 (d, 1H, *J* = 5.5 Hz), 4.56 (t, 1H, *J* = 5.5 Hz), 4.14 (dd, 1H, *J* = 5 Hz, *J* = 3.5 Hz), 4.11-4.09 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆): δ 180.7, 168.4, 159.4, 156.4, 153.7, 151.8, 151.2, 147.2, 141.3, 135.9, 129.5, 128.9,

126.4, 123.9, 116.4, 116.3, 112.5, 109.7, 102.2, 86.6, 82.3, 72.8, 71.2, 46.2. HRMS (ESI) *m/z* calculated for C₃₁H₂₄N₇O₉S, [M – H]: 670.1361, found: 670.1376.

**Synthesis of 5-DG:** A solution of 70 mg (0.22 mmol) of N-(4-aminobutyl)-5-(dimethylamino)-1-naphthalenesulfonamide and 5 µL of triethylamine in 1 mL of DMF were added dropwise to 39 mg (0.22 mmol) of thiocarbonyldiimidazole in 0.5 mL of DMF. After stirring for 1 h at room temperature, 70 mg (0.2 mmol) of 5'-amino-5'-deoxyguanosine was added, and the mixture was stirred for 7 h at 65 °C. After filtration through a short bed of Celite column, the solvent was removed under vacuum. The resulting oil was washed with diethyl ether and acetonitrile, and dried to yield a yellow powder (48 mg, 34 %). ¹H NMR (500 MHz, DMSO-d₆): δ 8.45 (d, 1H, *J* = 8.3Hz), 8.30 (d, 1H, *J* = 8.3Hz), 8.09 (d, 1H, *J* = 7.2 Hz), 7.91 (m, 2H), 7.61 (m, 2H), 7.43 (br, 1H), 7.25 (d, 1H, *J* = 7.2Hz), 6.51 (br, 2H), 5.67 (d, 1H, *J* = 8.3 Hz), 5.47 (m, 1H), 5.21 (m, 1H), 4.48 (m, 1H), 4.03 (m, 1H), 3.92 (m, 2H), 3.51 (m, 1H), 3.23 (m, 1H), 2.83 (s, 6H), 2.76 (m, 2H), 1.33 (m, 4H). ¹³C NMR (125 MHz, DMSO-d₆ + 3 % D₂O): δ 183.6, 157.5, 154.2, 151.9, 151.6, 136.6, 136.4, 129.8, 129.5, 129.5, 128.6, 128.3, 124.1, 119.6, 117.4, 115.6, 86.7, 83.4, 73.2, 71.6, 45.6, 44.7, 43.7, 42.6, 27.2, 26.4. HRMS (ESI) *m/z* calculated for C₂₇H₃₄N₉O₆S₂, [M – H]: 644.2073, found 644.2082.

### 2.3. LC-based separation and quantitation of a chromophore-labeled guanosine and its glycated product

The purity and identity of the chromophore-labeled guanosine 5-FG and 5-DG were initially assessed by reverse-phase liquid chromatography using a Nexera Shimadzu system interfaced with LabSolutions software. Samples were injected into an Phenomenex C18 column (150 mm × 4.6 mm, 3 µm particle size) at 40 °C. The mobile phase consisted of solvent A (water with 0.1 % formic acid) and solvent B (acetonitrile with 0.1 % formic acid). Separation was performed using an isocratic flow (75 % A/25 % B) at a rate of 0.3 mL/min 5-FG and its glycated products were monitored by fluorescence emission at λ = 530 nm after excitation at λ = 485 nm and quantified by integration of the peak area integration using a calibration curve. Similarly, 5-DG and its glycated product were detected at λ = 537 nm after excitation at λ = 375 nm.

### 2.4. Kinetic analysis and inhibition of Park7 by Isatin

Assays were performed in a 96-well ELISA plates in a total volume of 50 µl containing of Park7 buffer (50 mM Phosphate buffer, 1 mM EDTA, pH 7.2), MGO (up to 1000 µM), 5-FG or 5-DG (up to 100 µM), and purified Park7 (up to 5 µg) or cell extracts (up to 250 µg total protein extract). In our original protocol, all components (MGO, 5-FG or 5-DG, and Park7 or protein extract) were incubated simultaneously. After a variable incubation time (0–120 min), the reaction is stopped by adding 50 µL of perchloric acid (7 % final concentration), the plate is centrifuged and 10 µL of supernatant is injected into the LC system. Regarding the inhibition experiment with Isatin (a bicyclic aromatic compound derived from indole), we based our protocol on the study by Shinya Tashiro et al. [30]. In this study, Isatin was preincubated with recombinant Park7 for 10 min at 37 °C before initiating the enzymatic reaction. Similarly, in our protocol, Park7 was incubated with eight different concentrations of Isatin (up to 100 µM). The reaction was then carried out under the same conditions as described for activity assays. Isatin was dissolved in dimethyl sulfoxide (DMSO). No effect of 1 % DMSO on the assay was observed.

### 2.5. Lysates of E. Coli

Bacterial lysates overexpressing Park7 were obtained during
the enzyme production experiment, prior to the purification steps. The same protocol was applied using a plasmid allowing the overexpression of the Park7 C106S mutant. Production yields were identical for the WT and mutant form [4–6,8].

## 2.6. SH-SY5Y cell culture

SH-SY5Y cell line (neuroblastoma cell line) were cultured as monolayers in 100-mm Petri dishes at 37 °C in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Paris, France) supplemented with 10% fetal bovine serum and penicillin/streptomycin. At 90% confluence, cells were washed with PBS, lysed in 50 mM phosphate buffer (pH 8) containing 1 mM EDTA, 0.2% Triton X-100, and protease inhibitors. Lysates were sonicated and centrifuged at 13,000×g for 15 min. Supernatants (total protein extracts) were collected, and protein concentration was determined using the Bradford assay with bovine serum albumin as the standard.

CRISPR/Cas9 gene editing was performed to generate PARK7/DJ-1 knock out in the SHSY-5Y cell line. In brief, PARK7

TrueGuide™ Synthetic sgRNA (ThermoFisher Scientific, sequence: CTGGCCTCATGACATCTACA) and TrueCut™ Cas9 Protein v2 (ThermoFisher Scientific) were transfected into SHSY-5Y cells using Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (ThermoFisher Scientific). Optimization to obtain the highest editing efficiency was performed considering the optimal cell density and molar ratio of gRNA to Cas9 protein. Clonal isolation of cell lines after transfection was performed by serial dilutions, followed by an expansion period. A PARK7/DJ1 KO cell line was then selected after determination of RNA and protein level using real-time RT-PCR and Western Blot, respectively. For West Blot analysis, we used Stain-Free gel technology (Bio-Rad) enabling detection of protein bands on transfer membranes without using colorimetric or fluorescent stains.

## 2.7. Statistical analysis

All values are expressed as means ± standard deviation (SD) from at least three independent experiments. Statistical analyses

<figure><img src="image_4.png" /><figcaption>**Scheme 1.** Synthesis of 5-FG and 5-DG.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** LC chromatograms of 5-FG (pink) and mixture of 5-FG and MGO (black).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 3. LC chromatograms of 5-FG (pink), 5-FG + MGO (black), 5-FG + MGO + Park7 C106S (Blue) and 5-FG + MGO + Park7 WT (brown).</figcaption></figure>

were performed using paired t-tests and analysis of variance (ANOVA).

# 3. Results and discussion

## 3.1. Human DJ-1 enzyme production and purification

The fully native human recombinant Park7 protein was used in this study. As shown in supplementary material (Fig. S1), a highly purified Park7 protein was obtained with a molecular mass of 25 kDa, which aligns with its predicted molecular weight. Western-blot experiments using anti-Park7 antibody further confirmed the successful expression and purification of DJ-1 from E. coli (Fig. S1). The average yield of purified DJ-1 was approximately 20 mg of Park7 per liter of cell medium.

## 3.2. Synthesis of 5-FG and 5-DG

As described in Scheme 1, 5-FG was synthesized by condensation between 5'-amino-5'-deoxyguanosine prepared as reported by Niewiadomski et al. [29] and fluorescein isothiocyanate. For the preparation of 5-DG, N-(4-aminobutyl)-5-(dimethylamino)-1-naphthalenesulfonamide was first activated in the presence of thiocarbonyldiimidazole (TCDI) and subsequently reacted with 5'-amino-5'-deoxyguanosine. The structure of the two compounds were confirmed by NMR (¹H and ¹³C) and HRMS. The result of these analyzes are presented in supplementary material (Figs. S2a, S2b, S3a and S3b).

## 3.3. LC-based separation and quantitation of a chromophore-labeled guanosine and its glycated product

To evaluate the activity of Park7 using LC, we designed a fluorescein- or dansyl-conjugated guanosine (see ‘Synthesis of 5-fluorescein-guanosine (5-FG) and 5-dansyl-guanosine (5-DG) in M&M and Scheme 1), which can be easily glycated by MGO [6]. We hypothesized that 5-FG and its glycated derivatives should be effectively separated by reverse-phase LC and specifically quantified by detecting the fluorescence of the fluorescein or dansyl moiety. As shown in the chromatograms of Fig. 2, the injection of a 5-FG solution produces a single peak with a retention time of 11.2 min. In vitro incubation of 5-FG with MGO and subsequent

injection, results in the appearance of two new predominant peaks, corresponding to two glycated derivatives with retention times of 9.9 and 14.4 min, respectively. The identity of each peak was confirmed by mass spectrometry, with the measured masses matching those expected: 5-FG (m/z = 671) and its MGO-glycated derivatives (m/z = 743).

These results confirm that the two glycated forms of 5-FG are isomers. Similar results were obtained with 5-DG (retention times of 8.5 and 11.3 min). Support for the formation of two isomers of aminocarbinols by the reaction MGO and 5-FG or 5-DG comes from ¹³C NMR experiments showing that the new signals obtained upon addition of MGO to 5-DG nicely match the expected values for aminocarbinols and not those of other possible adducts (Fig. S4).

The percentage of glycation was determined by integrating each peak's area under the curve (AUC) using the formula:

$$ \% \text{ of Glycation} = \frac{\sum \text{AUC of glycated } 5 - \text{FG}}{\sum \text{AUC of } 5 - \text{FG and glycated } 5 - \text{FG}} $$

To maximize the glycation of guanosine in the shortest time, we tested various experimental conditions. Table 1 summarizes the glycation percentage of 5-FG as a function of different reagent concentrations (5-FG and MGO).

To achieve a glycation rate of approximately 70 %, the optimal conditions were determined as 25 µM 5-FG and 250 µM MGO incubated for 1 h.

## 3.4. Effect of Park7 incubation of 5-FG glycation

We then tested the effect of Park7 on 5-FG glycation. As shown in Fig. 3, incubation with Park7 completely eliminates the glycated forms of 5-FG (brown line), restoring its native state, thereby counteracting the effect of MGO. In contrast, the catalytically inactive Park7 C106S mutant has no effect on 5-FG glycation (blue line).

We evaluated the effect of different Park7 concentrations (WT and C106S mutant) by measuring the percentage of 5-FG glycation normalized to the control (5-FG + MGO) as a function of the amount of recombinant protein added (Fig. 4).

As expected, the level of glycated 5-FG decreases linearly with incubation time in the presence of Park7 (Fig. 5A). The initial rate
<table><caption>**Table 1**
Effect of reagent concentrations and reaction time on glycation of 5-FG.</caption><thead><tr><th rowspan="2">ratio</th><th rowspan="2">MGO/5-FG (µM/µM)</th><th colspan="4">% glycation</th></tr><tr><th>0 min</th><th>30 min</th><th>60 min</th><th>120 min</th></tr></thead><tbody><tr><td>5–1</td><td>500/100</td><td>34</td><td>70</td><td>82</td><td>85</td></tr><tr><td>10–1</td><td>500/50</td><td>31</td><td>66</td><td>80</td><td>85</td></tr><tr><td>20–1</td><td>500/25</td><td>26</td><td>66</td><td>81</td><td>84</td></tr><tr><td>40–1</td><td>500/12.5</td><td>26</td><td>65</td><td>81</td><td>85</td></tr><tr><td>5–1</td><td>250/50</td><td>20</td><td>51</td><td>67</td><td>76</td></tr><tr><td>10–1</td><td>250/25</td><td>18</td><td>48</td><td>71</td><td>76</td></tr><tr><td>20–1</td><td>250/12.5</td><td>19</td><td>52</td><td>70</td><td>76</td></tr><tr><td>40–1</td><td>250/6.25</td><td>17</td><td>45</td><td>71</td><td>77</td></tr><tr><td>5–1</td><td>125/25</td><td>13</td><td>31</td><td>49</td><td>63</td></tr><tr><td>10–1</td><td>125/12.5</td><td>15</td><td>29</td><td>54</td><td>62</td></tr><tr><td>20–1</td><td>125/6.25</td><td>20</td><td>28</td><td>58</td><td>61</td></tr><tr><td>40–1</td><td>125/3.125</td><td>17</td><td>27</td><td>56</td><td>62</td></tr><tr><td>5–1</td><td>62.5/12.5</td><td>17</td><td>20</td><td>34</td><td>48</td></tr><tr><td>10–1</td><td>62.5/6.25</td><td>23</td><td>18</td><td>39</td><td>47</td></tr><tr><td>20–1</td><td>62.5/3.125</td><td>15</td><td>16</td><td>45</td><td>47</td></tr><tr><td>40–1</td><td>62.5/1.6</td><td>16</td><td>22</td><td>44</td><td>45</td></tr><tr><td>5–1</td><td>31.25/6.25</td><td>12</td><td>15</td><td>23</td><td>34</td></tr><tr><td>10–1</td><td>31.25/3.125</td><td>14</td><td>16</td><td>25</td><td>22</td></tr><tr><td>20–1</td><td>31.25/1.6</td><td>16</td><td>13</td><td>35</td><td>34</td></tr><tr><td>40–1</td><td>31.25/0.8</td><td>13</td><td>20</td><td>32</td><td>32</td></tr></tbody></table>

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Measurement of the % 5-FG glycation in presence of Park7 enzyme using the LC-based assay.</figcaption></figure>

of the reaction ($v_i$) was determined from the plot of AUC versus reaction time (Fig. 5B). The assay was found to be linear between 1 and 20 µg of recombinant purified enzyme.

The detection limit for 5-FG or glycated 5-FG was estimated to

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Analysis of the inhibitory effect of Isatin on Park7 using LC-based assays.</figcaption></figure>

be 10 fmol. These values are comparable to those obtained with 5-DG, with the exception of detection sensitivity, which was lower for 5-FG (10 fmol) compared to 5-DG (1 fmol).

### 3.5. Inhibition of Park7 by Isatin

Isatin has been identified as a potent inhibitor of Park7 [30]. In the LC-based Park7 assay, different concentrations of Isatin were tested (Fig. 6): as expected, the Park7 activity decreased as the concentration of Isatin increased. The half-maximal inhibitory concentration (IC50) was determined to be 14 µM. These results are consistent with previously reported data showing an IC50 of 13 µM, using a spectroscopic assay with phenylglyoxal as a substrate [30].

The reproducibility and robustness of the assay were further evaluated by comparing IC50 values obtained for Isatin on successive days and by determining the Z' statistic, as described by Zhang et al. [31]. A Z' value of 0.69 was obtained, confirming the validity and reliability of the assay. Overall, our data demonstrate that the LC-based assay is suitable for kinetic analysis of Park7 and for identifying and characterizing Park7 effectors (activators or inhibitors).

### 3.6. Measurement of Park7 activity in biological samples

As reported above, the LC-based Park7 assay is versatile and can be applied to various settings where the determination of glyoxalase/deglycase activity of purified recombinant Park7 is required. However, measuring Park7 activity in complex biological samples would be also of great significance.

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** A. LC chromatograms of mixture of 5-FG and MGO and Park7 WT (8 µM) with different incubation times (up to 45 min). B. plot of AUC versus time of reaction.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Measurement of the % 5-FG glycation in *E. coli* extracts using the LC-based assay.</figcaption></figure>

Our first approach involved bacterial lysates from *E. coli* over-expressing either the wild-type Park7 (WT) or its C106S mutant form. The Park7 activity of these lysates was evaluated using LC, and the percentage of 5-FG glycation was plotted as a function of the total protein amount.

The results are presented in Fig. 7.

A significant reduction in 5-FG glycation was observed with 3 µg or more of protein extract from *E. coli* overexpressing Park7 WT, whereas no significant effect was detected in extracts from the mutant form. A dose-dependent effect on glycation reversion was observed for the WT enzyme. Interestingly, for high quantities of Park7 C106S lysate (above 100 µg), a slight glycation protection effect was noted, despite the lack of Park7 enzymatic activity. This suggests the presence of endogenous glyoxalase/deglycase activities in *E. coli*. Another possibility is that high levels of extract displace the glycation equilibrium of 5-FG.

These findings indicate that our LC-based approach is suitable for assessing Park7 activity in biological bacterial samples, thereby extending its applicability beyond purified recombinant enzymes. We then conducted experiments on mammalian cells.

### 3.7. Measurement of endogenous Park7 activity in SH-SY5Y extracts

In order to assay our test with a mammalian system, we knocked-out the expression of Park7 in a mammalian cell, the SH-

<figure><img src="image_11.png" /><figcaption>**Fig. 9.** Quantification of 5-DG glycation in the presence of MGO and SH-SY5Y cell lysates. The percentage of 5-DG glycation was measured after incubation with MGO (500 µM) for 1 h, either alone or in the presence of 250 µg of protein from SH-SY5Y WT or SH-SY5Y Park7 KO cells lysates. Data represent mean ± SEM from six independent experiments.</figcaption></figure>

SY5Y cell line, using the CrispR-Cas9 system. The control experiments with this SH-SY5Y Park7 KO cell line demonstrated satisfactory results, as qRT-PCR confirmed the absence of PARK7 transcripts (data not shown), and Western blot analysis showed a complete lack of Park7 protein in the KO cell extracts (Fig. 8A). Stain-Free gel technology is the most commonly used method to control differences in protein loading by independently quantifying housekeeping proteins. As shown in Fig. 8B, the loading levels are equivalent between the WT and KO lysates.

The glycation protection activity of 5-DG was then assessed on 5 million SH-SY5Y cell (WT or KO) lysate by LC, and the percentage of 5-DG glycation was calculated. The results are derived from six independent experiments for each condition and are presented in Fig. 9. The glycation of 5-DG (100 µM) in the presence of MGO (500 µM), without lysate, results in approximately 65 % glycation after 1 h. This is comparable to the results obtained for 5-FG. In the presence of 250 µg of protein from SH-SY5Y WT lysate, the glycation rate decreases to 21 %. Glycation is also reduced with Park7 KO lysate, but only to 35 %. The difference between the two lysates is statistically significant, with a p-value of 0.003, confirming that the glycation protection effect is indeed partly dependent on the presence of Park7.

Our system for measuring the glycation protection activity of guanosine, conjugated with a fluorophore and based on LC separation, demonstrated its effectiveness in complex matrices,

<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Western blot detection of Park7 using a polyclonal antibody against Park7. Protein Marker (PM) is indicated in kDa.</figcaption></figure>
including eukaryotic cell extracts. Notably, this protection occurs independently of the Park7 protein, as glycation inhibition was also observed in SH-SY5Y KO lysates for Park7. This can be explained by the presence of other anti-glycation systems, particularly those mediated by glyoxalase enzymes. Consequently, our LC-based assay is also suitable for measuring glyoxalase activity.

## 4. Conclusion

Glycation has emerged as a crucial factor in the progression of various degenerative diseases, particularly Parkinson's disease (PD). The glyoxalase/deglycase system plays a fundamental role in mitigating the harmful effects of glycation, yet the lack of reliable and sensitive enzymatic assays has hampered progress in understanding the function of Park7 (DJ-1) and its therapeutic potential. In this study, we have developed a novel LC-based assay with fluorescence detection, enabling precise quantification of Park7 enzymatic activity through the separation of fluorescently labeled glycated-guanosine derivatives.

Our approach provides several advantages over previously available methods. First, it offers high sensitivity and specificity, allowing for robust kinetic and mechanistic analyses of Park7 activity, not only in purified recombinant protein preparations but also in complex biological samples, such as cell lysates. The assay successfully quantified the protective effects of Park7 against glycation, as demonstrated by experiments using wild-type and knockout SH-SY5Y cell lines. Furthermore, the inhibition studies with Isatin confirmed that this assay is suitable for identifying and characterizing Park7 effectors, paving the way for future drug discovery efforts targeting this enzyme.

Beyond its relevance for enzymology, this assay represents a valuable tool for investigating the role of Park7 in PD and other glycation-related diseases. Given the increasing evidence implicating glycation in α-synuclein aggregation and neurotoxicity, this method could be applied to explore the interplay between glycation, Park7 function, and PD pathology in greater depth. Moreover, its applicability to biological samples makes it a promising platform for biomarker identification and high-throughput screening of glyoxalase/deglycase modulators.

### CRediT authorship contribution statement

**Nicolas Mathas:** Validation, Resources, Investigation, Formal analysis. **Emmanuelle Braud:** Resources, Investigation, Formal analysis. **Erwan Galardon:** Resources, Formal analysis. **Lucie Larigot:** Resources, Formal analysis. **Mélanie Etheve-Quelquejeu:** Writing – review & editing. **Marie-Agnès Sari:** Writing – review & editing. **Beatrice Le Grand:** Resources, Formal analysis. **Etienne B. Blanc:** Resources, Formal analysis. **Xavier Coumoul:** Writing – review & editing. **Julien Dairou:** Writing – original draft, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no conflict of interest.

### Acknowledgements

The authors acknowledge the support of the French National Health Safety Agency (ANSES number: EST 2019/1/25); the Foundation for Medical Research (FRM number: ENV202109013715); French Association for Parkinson's Disease (Subvention France Parkinson 2020-22) and the ANR FRANCE (French National Research Agency) for its financial support of the "Appel à projets générique 2021" project no. ANR-21-CE44-0022-01. We also

acknowledge the technical platform "Service de spectrométrie de Masse du LCBPT" for provision of LCMS facilities.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2025.06.005.

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