Modification of proteins in vitro by physiological levels of glucose - Pyridoxamine inhibits conversion of Amadori intermediate to advanced glycation end-products through binding of redox metal ions

Hyperglycemic conditions of diabetes accelerate protein modifications by glucose leading to the accumulation of advanced glycation end- products ( AGEs). We have investigated the conversion of protein- Amadori intermediate to protein- AGE and the mechanism of its inhibition by pyridoxamine ( PM), a potent AGE inhibitor that has been shown to prevent diabetic complications in animal models. During incubation of proteins with physiological diabetic concentrations of glucose, PM prevented the degradation of the protein glycation intermediate identified as fructosyllysine ( Amadori) by C-13 NMR using [ 2-C-13]- enriched glucose. Subsequent removal of glucose and PM led to conversion of protein-Amadori to AGE N-epsilon- carboxymethyllysine ( CML). We utilized this inhibition of post- Amadori reactions by PM to isolate protein- Amadori intermediate and to study the inhibitory effect of PM on its degradation to protein-CML. We first tested the hypothesis that PM blocks Amadori-to- CML conversion by interfering with the catalytic role of redox metal ions that are required for this glycoxidative reaction. Support for this hypothesis was obtained by examining structural analogs of PM in which its known bidentate metal ion binding sites were modified and by determining the effect of endogenous metal ions on PM inhibition. We also tested the alternative hypothesis that the inhibitory mechanism involves formation of covalent adducts between PM and protein-Amadori. However, our C-13 NMR studies demonstrated that PM does not react with the Amadori. Because the mechanism of interference with redox metal catalysis is operative under the conditions closely mimicking the diabetic state, it may contribute significantly to PM efficacy in preventing diabetic complications in vivo. Inhibition of protein- Amadori degradation by PM also provides a simple procedure for the isolation of protein-Amadori intermediate, prepared at physiological levels of glucose for relevancy, to study both the biological effects and the chemistry of post- Amadori pathways of AGE formation

Mechanism of perturbation of integrin-mediated cell-matrix interactions by reactive carbonyl compounds and its implication for pathogenesis of diabetic nephropathy

Perturbation of interactions between cells and the extra-cellular matrix (ECU) of renal glomeruli may contribute to characteristic histopathological lesions found in the kidneys of patients with diabetic nephropathy. However, the mechanism by which the diabetic conditions may affect cell-ECM interactions is unknown. Existing hypotheses suggest a role of glucose in direct modification of ECM. Here, we have demonstrated that carbonyl compound methylglyoxal (MGO) completely inhibited endothelial cell adhesion to recombinant α3 noncollagenous 1 domain of type IV collagen mediated via a short collagenous region containing RGD (Arg-Gly-Asp) sequence as well as binding of purified αvβ3 integrin to this protein. Specific MGO adducts of the arginine residue were detected within RGD sequence using mass spectrometry. Modification by carbonyl compounds glyoxal or glycolaldehyde had similar but smaller effects. MGO strongly inhibited adhesion of renal glomerular cells, podocytes, and mesangial cells to native collagen IV and laminin-1 as well as binding of collagen IV to its major receptor in glomerular cells, α1β1 integrin. In contrast, modification of these proteins by glucose had no effect on cell adhesion. Pyridoxamine, a promising drug for treatment of diabetic nephropathy, protected cell adhesion and integrin binding from inhibition by MGO. We suggest that in diabetes, perturbation of integrin-mediated cell-matrix interactions occurs via the modification of critical arginine residues in renal ECM by reactive carbonyl compounds. This mechanism may contribute to the development of diabetic nephropathy. © 2005 by the American Diabetes Association.link_to_subscribed_fulltex

Collagen IVα345 dysfunction in glomerular basement membrane diseases. I. Discovery of a COL4A3 variant in familial Goodpasture's and Alport diseases

Diseases of the glomerular basement membrane (GBM), such as Goodpasture's disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Companion Papers II and III. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge, and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies