The chemical language of protein glycation

Abstract

Glycation is a non-enzymatic post-translational modification (PTM) that is correlated with many diseases, including diabetes, cancer and age-related disorders. Although recent work points to the importance of glycation as a functional PTM, it remains an open question whether glycation has a causal role in cellular signaling and/or disease development. In this Review, we contextualize glycation as a specific mechanism of carbon stress and consolidate what is known about advanced glycation end-product (AGE) structures and mechanisms. We highlight the current understanding of glycation as a PTM, focusing on mechanisms for installing, removing or recognizing AGEs. Finally, we discuss challenges that have hampered a more complete understanding of the biological consequences of glycation. The development of tools for predicting, modulating, mimicking or capturing glycation will be essential for interpreting a post-translational glycation network. Therefore, continued insights into the chemistry of glycation will be necessary to advance understanding of glycation biology.

Figure 1.. The landscape of post-translational modifications.

The ~70 classes of post-translational transformations curated on dbPTM (e.g. phosphorylation, acetylation, ADP-ribsosylation, etc…), were manually annotated as enzymatic or non-enzymatic. The transformations compiled on dbPTM produce more than 600 discrete protein modifications. The majority of these PTMs occur enzymatically (grey). Of the ~30% that transpire without an enzyme, most arise from carbon stress (blue), or oxidative stress from reactive oxygen species (ROS), reactive nitrogen species (RNS) or reactive sulfide species (RSS) (purple). Some modifications produced by carbon stress, like lact(o)ylation, produce a single modification, while others produce multiple modifications (e.g. carbamylation). Compared to the rest of carbon stress (14 modifications), glycation produces an unusually diverse array of discrete protein modifications (38 modifications). *denotes a PTM that can occur either enzymatically or non-enzymatically; only modifications resulting from non-enzymatic transformations are depicted in the right panel inset.

Figure 2.. Mechanisms of non-enzymatic carbon stress.

a) Glycation, or non-enzymatic glycosylation, is defined as the reaction of amino (top) or guanidino (bottom) groups with aldehydes. top Glycation of lysine with aldehydes proceeds through rapid and reversible Schiff base formation. Rearrangment to the Amadori product generates a stable AGE. bottom Glycation of arginine with α-oxoaldehydes also proceeds through initial reversible steps prior to rearrangements that produce stable AGEs. b) Most other types of non-enzymatic carbon stress, including acetylation, butyrylation, crotonylation, glutarylation, malonylation, palmitoylation, and propionylation, occur through acyl-transfer chemistry from acyl-CoA donors (top). Phosphoglyceration (middle) and lact(o)ylation (bottom) use atypical acyl donors (acid phosphate or glutathione thioester, respectively) and are mechanistically distinct from the reactions of glycation.

Figure 3.. Biologically relevant glycating agents.

a) Hexose or pentose sugars, drawn in their open-chain forms, which contain free carbonyls that can react in glycation. The corresponding cyclic hemiacetals do not participate in glycation. Multiple pathways produce smaller metabolites containing b) aldehydes or c) α-oxoaldehydes that are potent glycating agents.

Figure 4.. A catalog of advanced glycation end-products (AGEs).

a) AGEs that have been reported to form on lysine residues. b) AGEs that have been reported to form on arginine residues. c) AGEs that have been reported to form crosslinks. Structure names are: fructoselysine (1) Nε-carboxymethyllysine (2), Nε-carboxyethyllysine (3), methylglyoxal-derived hydroimidazolones-1, -2, and -3 (4, 5, 6), methylglyoxal-derived dihydroxyimidazolidine (7), glyceraldehyde-derived pyridinium (8), glycolaldehyde-pyridine (9), glycolic acid lysine amide (10), pyralline (11), glyoxal-derived hydroimidazolone-1 (12), glyoxal-derived dihydroxyimidazolidine (13), Nω-carboxyethylarginine (14), Nω-carboxymethylarginine (15), X-alkyl formyl glycosyl pyrrole (16), G-alkyl formyl glycosyl pyrrole (17), imidazolone (18), argpyrimidine (19), tetrahydropyrimidine (20), 3-deoxyglucosone-imidazolone (21), pentosidine (22), pentosinane (23), glucosepane (24), mercaptomethylimidazole crosslinks between cysteine and arginine (25), methylglyoxal-lysine dimer (26), glyoxal-lysine dimer (27), glyoxal-derived imidazolium crosslink (28), methylglyoxal-derived imidazolium cross-link (29), N-6-{2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-5-[(2S)-2,3-dihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene}-l-lysinate (30), N-6-{2-{[(4S)-4-ammonio-5-oxido-5-oxopentyl]amino}-5-[(2S,3R)-2,3,4-trihydroxybutyl]-3,5-dihydro-4H-imidazol-4-ylidene}-l-lysinate (31), 3-deoxyglucosone-lysine dimer (32), glyoxal lysine amide (33), glyoxal lysine amidine (34), crossline (35), vesperlysine A (36), vesperlysine B (37), vesperlysine C (38), 2-(2-Furoyl)-4(5)-(2-furanyl)-1H-imidazole (39). Structures indicated in gray are those for which there is only limited structural characterization (34), or that have only been structurally characterized following strongly acidic conditions (16, 17, 36, 37, 38) or borohydride reduction (3, 10, 33, 32) (Supplementary Table 1). The reported cysteine adducts carboxymethyl- or carboxyethyl-cysteine (CMC or CEC) are not shown, as they have only been characterized following total acid hydrolysis.

Figure 5.. Connecting AGEs through chemical mechanism.

Many prior studies on AGE formation mechanisms have used amino acid monomers or derivatives under forcing conditions. Here we profile AGE formation mechanisms with an emphasis on those that have been reported or postulated for peptide or protein substrates under mild conditions. a) The lysine side chain (N^𝜀) or protein N-terminus (not shown) condenses with monoaldehydes like glucose, glycolaldehyde, or glyoxal to form a Schiff base. In the case of glucose, subsequent Amadori rearrangement produces FL (1). CML has been shown to form from FL through an oxidative fragmentation mechanism, though an exact mechanism remains unknown. CML is also suggested to be generated from the reaction of glycolaldehyde or glyoxal with lysine through a distinct (unknown) mechanism. b) Reaction of the arginine side chain (N^𝜀 and N^ω) with methylglyoxal proceeds through addition that forms MGH-DH (7), then elimination and tautomerization to form MGH-1 (4), and hydrolysis to yield CEA (14), as has been demonstrated for peptide substrates. A similar pathway engaging N^ω and N^δ on Arg amino acids produces MGH-3 (6), which can also form CEA (14). THP (20) is thought to be generated from MGH-1 and MGH-3, though the mechanism is unknown. Additionally, there are several competing proposals about the APY formation mechanism. These include the formation of APY from MGH-3 (proposal 1), or THP (proposal 2), or via the formation of an MGO reductone prior to reaction with Arg (proposal 3). Solid lines: mechanisms that have been defined. Dashed lines: known precursor to product, but the mechanism remains unknown. Dotted lines: limited evidence for precursor to product and an unknown mechanism.

Figure 6.. PTMs are integrated into cellular signaling cascades.

a) Enzymatic PTMs like phosphorylation are defined by the enzymes that ‘write’ (kinases) or ‘erase’ (phosphatases) them, along with ‘readers’ (adapter proteins) that propagate their signal. b) As a non-enzymatic PTM, glycation has no ‘writer’, leaving open many questions about the mechanisms through which it is removed or recognized.

Figure 7.. Glycation can participate in cellular signaling.

Activation of pro-inflammatory signaling pathways occurs when the cell surface receptor for AGE (RAGE) recognizes extracellular AGE-modified proteins, along with other ligands (top right). Glycation of KEAP1 releases the NRF2 transcription factor, thereby preventing its ubiquitination and degradation, which activates the antioxidant response (top left). Glycation of histones remodels chromatin architectures, thereby altering gene transcription (bottom left).