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 forms 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 arises from methylglyoxal (MGO), a biologically relevant
glycating agent. Consistent with other proposed APY formation mechanisms,
our results identify AGE species with a mass change of [M + 144],
presumably including tetrahydropyrimidine (THP), as 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 is released instead
of CO2. 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 also reveal that phosphorylated
Tyr or Ser residues can 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
a newly defined role for phosphorylated residues on glycation substrates,
we performed quantitative bottom-up proteomics analysis for MGO-treated
cells. Gene ontology and functional annotation clustering analyses
for APY-modified proteins suggested a correlation with phosphorylation-related
terms (e.g., kinase activity or protein phosphorylation), which was
validated using synthetic phosphopeptide substrates. Collectively,
these data define a chemical mechanistic path to APY and suggest significant
crosstalk between cellular phosphorylation and glycation events including
APY formation.