A Chemical Mechanistic Path Leads the Way to Cellular Argpyrimidine

Argpyrimidine (APY) is a methylglyoxal-derived advanced glycation end-product (AGE) that has been associated with multiple diseases. As APY formation occurs without an enzyme, it remains exceptionally difficult to pinpoint where APY is likely to be found, both on individual proteins and in cells. In this study, we used a peptide model system and mass spectrometry analysis to investigate the chemical mechanism through which APY forms from methylglyoxal (MGO), a biologically relevant glycating agent. Consistent with other proposed APY formation mechanisms, our results show that that another AGE, tetrahydropyrimidine (THP) is a direct precursor to APY. However, our results rule out previously proposed reductone or oxidative decarboxylation mechanisms. Instead, we show that a formal oxidation step is not required, and that formate, not CO2 is released. We further show the potential for a nearby residue such as Tyr to assist in the APY formation mechanism by acting as a general base. These experiments revealed that phosphorylated Tyr or Ser residues could also promote equivalent levels of APY formation, despite introducing additional negative charges that we previously showed to impede glycation. Guided by these mechanistic insights and newly defined role for phosphorylated residues on glycation substrates, we performed quantitative bottom-up proteomics analysis for MGO-treated cells. Gene ontology analysis for AGE-modified proteins revealed significant enrichment of phosphorylation-related terms (e.g. kinase activity or protein phosphorylation) for APY, while other Arg post-translational modifications did not. Collectively, these data define a chemical mechanistic path to APY and suggest significant crosstalk between cellular phosphorylation and glycation events including APY formation.