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A new glycation product 'norpronyl-lysine,' and direct characterization of cross linking and other glycation adducts: NMR of model compounds and collagen.

ABSTRACT: NMR is ideal for characterizing non-enzymatic protein glycation, including AGEs (advanced glycation endproducts) underlying tissue pathologies in diabetes and ageing. Ribose, R5P (ribose-5-phosphate) and ADPR (ADP-ribose), could be significant and underinvestigated biological glycating agents especially in chronic inflammation. Using [U-¹³C]ribose we have identified a novel glycoxidation adduct, 5-deoxy-5-desmethylpronyl-lysine, 'norpronyl-lysine', as well as numerous free ketones, acids and amino group reaction products. Glycation by R5P and ADPR proceeds rapidly with R5P generating a brown precipitate with PLL (poly-L-lysine) within hours. ssNMR (solid-state NMR) ¹³C-¹³C COSY identifies several crosslinking adducts such as the newly identified norpronyl-lysine, in situ, from the glycating reaction of ¹³C₅-ribose with collagen. The same adducts are also identifiable after reaction of collagen with R5P. We also demonstrate for the first time bio-amine (spermidine, N-acetyl lysine, PLL) catalysed ribose 2-epimerization to arabinose at physiological pH. This work raises the prospect of advancing understanding of the mechanisms and consequences of glycation in actual tissues, in vitro or even ex vivo, using NMR isotope-labelled glycating agents, without analyses requiring chemical or enzymatic degradations, or prior assumptions about glycation products.

SUBMITTER: Bullock PT

PROVIDER: S-EPMC3953948 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Advanced-Glycation-Endproducts (AGEs) are known to be a major cause of impaired tissue material properties. In collagen fibrils, which constitute a major building component of human tissue, these AGEs appear as fibrillar cross-links. It has been shown that when AGEs accumulate in collagen fibrils, a process often caused by diabetes and aging, the mechanical properties of the collagen fibril are altered. However, current knowledge about the mechanical properties of different types of AGEs, and their quantity in collagen fibrils is limited owing to the scarcity of available experimental data. Consequently, the precise relationship between the nano-scale cross-link properties, which differ from type to type, their density in collagen fibrils, and the mechanical properties of the collagen fibrils at larger scales remains poorly understood. In our study, we use coarse-grained molecular dynamics simulations and perform destructive tensile tests on collagen fibrils to evaluate the effect of different cross-link densities and their mechanical properties on collagen fibril deformation and fracture behavior. We observe that the collagen fibril stiffens at high strain levels when either the AGEs density or the loading energy capacity of AGEs are increased. Based on our results, we demonstrate that this stiffening is caused by a mechanism that favors energy absorption via stretching rather than inter-molecular sliding. Hence, in these cross-linked collagen fibrils, the absorbed energy is stored rather than dissipated through friction, resulting in brittle fracture upon fibrillar failure. Further, by varying multiple AGEs nano-scale parameters, we show that the AGEs loading energy capacity is, aside from their density in the fibril, the unique factor determining the effect of different types of AGEs on the mechanical behavior of collagen fibrils. Our results show that knowing AGEs properties is crucial for a better understanding of the nano-scale origin of impaired tissue behavior. We further suggest that future experimental investigations should focus on the quantification of the loading energy capacity of AGEs as a key property for their influence on collagen fibrils.

Project description:Background Although diabetes attenuates abdominal aortic aneurysms (AAAs), the mechanisms by which diabetes suppresses AAAs remain incompletely understood. Accumulation of advanced glycation end- (AGEs) reduces extracellular matrix (ECM) degradation in diabetes. Because ECM degradation is critical for AAA pathogenesis, we investigated whether AGEs mediate experimental AAA suppression in diabetes by blocking AGE formation or disrupting AGE-ECM cross-linking using small molecule inhibitors. Methods and Results Male C57BL/6J mice were treated with streptozotocin and intra-aortic elastase infusion to induce diabetes and experimental AAAs, respectively. Aminoguanidine (AGE formation inhibitor, 200 mg/kg), alagebrium (AGE-ECM cross-linking disrupter, 20 mg/kg), or vehicle was administered daily to mice from the last day following streptozotocin injection. AAAs were assessed via serial aortic diameter measurements, histopathology, and in vitro medial elastolysis assays. Treatment with aminoguanidine, not alagebrium, diminished AGEs in diabetic AAAs. Treatment with both inhibitors enhanced aortic enlargement in diabetic mice as compared with vehicle treatment. Neither enhanced AAA enlargement in nondiabetic mice. AAA enhancement in diabetic mice by aminoguanidine or alagebrium treatment promoted elastin degradation, smooth muscle cell depletion, mural macrophage accumulation, and neoangiogenesis without affecting matrix metalloproteinases, C-C motif chemokine ligand 2, or serum glucose concentration. Additionally, treatment with both inhibitors reversed suppression of diabetic aortic medial elastolysis by porcine pancreatic elastase in vitro. Conclusions Inhibiting AGE formation or AGE-ECM cross-linking enhances experimental AAAs in diabetes. These findings support the hypothesis that AGEs attenuate experimental AAAs in diabetes. These findings underscore the potential translational value of enhanced ECM cross-linking as an inhibitory strategy for early AAA disease.

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A new glycation product 'norpronyl-lysine,' and direct characterization of cross linking and other glycation adducts: NMR of model compounds and collagen.

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