Discovery of Glycation-Derived Cross-links at Arginine

Glycation cross-links account for more than 40% of all known advanced glycation end products (AGEs) and are correlated with many age-related diseases. Despite much interest, cross-linking AGEs (xl-AGEs) remain poorly understood, as they have been challenging to discover, prepare, and quantify. Here, we describe a peptide platform that is ideally suited for the study of xl-AGEs, which not only facilitates direct comparisons between the prevalence of known xl-AGEs and other AGEs but also enables the discovery of previously unknown xl-AGEs. In this study, we use this platform to discover the first known Arg-Arg xl-AGEs, a pair of <u><b>m</b></u>ethylglyoxal-derived dihydroxy<u><b>i</b></u>mi<u><b>d</b></u>azolidine hemi<u><b>a</b></u>cetal cross<u><b>l</b></u>ink, or MIDAL, isomers. We show that MIDAL can become the major AGE, exceeding levels of all other AGEs, for substrates in which two Arg glycation sites are optimally positioned. We further demonstrate that MIDAL is readily and reversibly generated in biocompatible conditions, persisting with a half-life of more than 3 days. We also demonstrate that MIDAL can form in living mammalian cells, suggesting that it has the potential to be a dynamic, physiologically relevant and functional xl-AGE. This work therefore offers important insights about MIDAL formation and describes a versatile platform to enable the study of xl-AGEs under a variety of conditions. We expect that it will be highly useful for further discovery of biologically relevant glycation cross-links that are yet to be identified.