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DAF in diabetic patients is subject to glycation/inactivation at its active site residues.

ABSTRACT: Decay accelerating factor (DAF or CD55) is a cell associated C3 and C5 convertase regulator originally described in terms of protection of self-cells from systemic complement but now known to modulate adaptive T cell responses. It is expressed on all cell types. We investigated whether nonenzymatic glycation could impair its function and potentially be relevant to complications of diabetes mellitus and other conditions that result in nonenzymatic glycation including cancer, Alzheimer's disease, and aging. Immunoblots of affinity-purified DAF from erythrocytes of patients with diabetes showed pentosidine, glyoxal-AGEs, carboxymethyllysine, and argpyrimidine. HPLC/MS analyses of glucose modified DAF localized the sites of AGE modifications to K125 adjacent to K126, K127 at the junction of CCPs2-3 and spatially near R96, and R100, all identified as being critical for DAF's function. Functional analyses of glucose or ribose treated DAF protein showed profound loss of its regulatory activity. The data argue that de-regulated activation of systemic complement and de-regulated activation of T cells and leukocytes could result from non-enzymatic glycation of DAF.

SUBMITTER: Fluckiger R

PROVIDER: S-EPMC5884443 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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DAF in diabetic patients is subject to glycation/inactivation at its active site residues.

Flückiger Rudolf R Cocuzzi Enzo E Nagaraj Ram H RH Shoham Menachem M Kern Timothy S TS Medof M Edward ME

Molecular immunology 20170905

Decay accelerating factor (DAF or CD55) is a cell associated C3 and C5 convertase regulator originally described in terms of protection of self-cells from systemic complement but now known to modulate adaptive T cell responses. It is expressed on all cell types. We investigated whether nonenzymatic glycation could impair its function and potentially be relevant to complications of diabetes mellitus and other conditions that result in nonenzymatic glycation including cancer, Alzheimer's disease, ...[more]

PMID: 28886871

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Project description:Hyperglycaemia, triose phosphate decomposition and oxidation reactions generate reactive aldehydes in vivo. These compounds react non-enzymatically with protein side chains and N-terminal amino groups to give adducts and cross-links, and hence modified proteins. Previous studies have shown that free or protein-bound carbonyls inactivate glyceraldehyde-3-phosphate dehydrogenase with concomitant loss of thiol groups [Morgan, Dean and Davies (2002) Arch. Biochem. Biophys. 403, 259-269]. It was therefore hypothesized that modification of lysosomal cysteine proteases (and the structurally related enzyme papain) by free and protein-bound carbonyls may modulate the activity of these components of the cellular proteolytic machinery responsible for the removal of modified proteins and thereby contribute to a decreased removal of modified proteins from cells. It is shown that MGX (methylglyoxal), GO (glyoxal) and glycolaldehyde, but not hydroxyacetone and glucose, inhibit catB (cathepsin B), catL (cathepsin L) and catS (cathepsin S) activity in macrophage cell lysates, in a concentration-dependent manner. Protein-bound carbonyls produced similar inhibition with both cell lysates and intact macrophage cells. Inhibition was also observed with papain, with this paralleled by loss of the active site cysteine residue and formation of the adduct species S-carboxymethylcysteine, from GO, in a concentration-dependent manner. Inhibition of autolysis of papain by MGX, along with cross-link formation, was detected by SDS/PAGE. Treatment of papain and catS with the dialdehyde o-phthalaldehyde resulted in enzyme inactivation and an intra-molecular active site cysteine-lysine cross-link. These results demonstrate that reactive aldehydes inhibit cysteine proteases by modification of the active site cysteine residue. This process may contribute to the accumulation of modified proteins in tissues of people with diabetes and age-related pathologies, including atherosclerosis, cataract and Alzheimer's disease.

Project description:In two-component signal transduction, response regulator proteins contain the catalytic machinery for their own covalent phosphorylation and can catalyze phosphotransfer from a partner sensor kinase or autophosphorylate using various small molecule phosphodonors. Although response regulator autophosphorylation is physiologically relevant and a powerful experimental tool, the kinetic determinants of the autophosphorylation reaction and how those determinants might vary for different response regulators and phosphodonors are largely unknown. We characterized the autophosphorylation kinetics of 21 variants of the model response regulator Escherichia coli CheY that contained substitutions primarily at nonconserved active site positions D + 2 (CheY residue 59) and T + 2 (CheY residue 89), two residues C-terminal to conserved D57 and T87, respectively. Overall, the CheY variants exhibited a >10(5)-fold range of rate constants (kphos/KS) for reaction with phosphoramidate, acetyl phosphate, or monophosphoimidazole, with the great majority of rates enhanced versus that of wild-type CheY. Although phosphodonor preference varied substantially, nearly all the CheY variants reacted faster with phosphoramidate than acetyl phosphate. Correlation between the increased positive charge of the D + 2 and T + 2 side chains and faster rates indicated electrostatic interactions are a kinetic determinant. Moreover, sensitivities of rate constants to ionic strength indicated that both long-range and localized electrostatic interactions influence autophosphorylation kinetics. The increased nonpolar surface area of the D + 2 and T + 2 side chains also correlated with an enhanced autophosphorylation rate, especially for reaction with phosphoramidate and monophosphoimidazole. Computer docking suggested that highly accelerated monophosphoimidazole autophosphorylation rates for CheY variants with a tyrosine at position T + 2 likely reflect structural mimicry of phosphotransfer from the sensor kinase histidyl phosphate.

Dataset Information

DAF in diabetic patients is subject to glycation/inactivation at its active site residues.

Publications

DAF in diabetic patients is subject to glycation/inactivation at its active site residues.

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