Fig. 3

Amidolysis domains of GATD3A and DJ-1 are required for deglycation of nucleotides and amino acids. A Genesis of advanced glycation end products (AGE). The Maillard reaction involving glyoxal results in non-enzymatic glycation of nucleotides (e.g., guanine) and amino acids (e.g., arginine), which is reversed by GATD3A/DJ-1 deglycation. AGE are formed by subsequent dehydration and crosslinking of nucleotides and amino acids with Maillard adducts, leading to associated pathologies. B Glutamine amidotransferase active sites in GATD3A and DJ-1, showing high structural homology and conservation of key predicted catalytic residues, E18 and C106 in DJ-1, and E62 and C176 in GATD3A. C Luciferase-linked glutamate detection assay demonstrating the capacity of GATD3A and DJ-1 to hydrolyze glutamine. Glutaminase is used as a positive control. Amidolytic activity is reduced with mutation of conserved catalytic cysteine residues C176 and C106 in GATD3A and DJ-1, respectively. Representative results of n = 3 assays are shown. D Immunoreactivity assay of glyoxal (blue) or methylglyoxal (MGO) (red) glycated plasmid DNA or γ-globulin protein. Following 2 h of incubation with either MGO or GO, samples were treated with recombinant DJ-1 (4HR DJ-1), GATD3A (4HR GATD3A), GATD3A C176A (4HR GATD3A C176A), or a no enzyme control (4HR) for a further 2 h. Optical density (O.D.) of immunodetected 1,2-dicarbonyl and AGE (images in Additional file 1, Fig. S1D) is calculated for three replicate experiments and normalized to the no enzyme control (4HR) (p < 0.01, two-tailed Student’s t test, n = 3, error bars = SEM). The 0HR timepoint was omitted from analysis due to high variability in glycation levels. Glyoxal—GO, Methylglyoxal—MGO. Full blots are shown in SI2. E Detection of glutathione-dependent glyoxylases GLO-1 and GLO-2 and deglycases DJ-1 and GATD3A in mouse heart mitochondria (M) and post-mitochondrial cytosol (C) (N = 3 mice). COXIV is used as a mitochondrial control, GAPDH is used as a cytosolic control. Extended analysis is included in Additional file 1, Fig. S4A