Figure 6.

DJ-1 mitigates glycation by decreasing MGO concentration. (A) 10 mM MGO was pretreated with 100 μM His-tagged DJ-1 for 2.5 h at 37 °C in pH 7.3 20 mM PBS before addition of Ni-NTA resin for 30 min at room temperature to bind the His-tagged DJ-1. The resulting supernatant (PT-MGO stock) was then collected using spin columns and (B) used in peptide glycation reactions. These glycation reactions were conducted using 1 mM peptide 1 and a 1:10 dilution of the PT-MGO stock and were incubated for 3 h at 37 °C in 20 mM PBS at pH 7.3 before being analyzed by LC-MS. (C) Left: Distribution of glycation products observed by LC-MS using PT-MGO stocks obtained after incubation of MGO with no enzyme, DJ-1^WT, or DJ-1^C106A; middle: determined concentrations of MGO in the PT-MGO stocks, as measured by a quantitative HPLC assay; right: distribution of glycation products observed by LC-MS using new MGO stocks prepared at the previously determined concentrations. A nondirectional (two-tailed) one-way ANOVA using Dunnett’s multiple comparison test was used to determine statistically significant differences in total glycation concentrations, p < 0.0001(****).