title: Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells.
abstract: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) plays a key regulatory function in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion. Previous studies documented metabolic reprogramming in stored red blood cells (RBCs) and oxidation of GAPDH at functional residues upon exposure to pro-oxidants diamide and H2O2 Here we hypothesize that routine storage of erythrocyte concentrates promotes metabolic modulation of stored RBCs by targeting functional thiol residues of GAPDH. Progressive increases in PPP/glycolysis ratios were determined via metabolic flux analysis after spiking (13)C1,2,3-glucose in erythrocyte concentrates stored in Additive Solution-3 under blood bank conditions for up to 42 days. Proteomics analyses revealed a storage-dependent oxidation of GAPDH at functional Cys152, 156, 247, and His179. Activity loss by oxidation occurred with increasing storage duration and was progressively irreversible. Irreversibly oxidized GAPDH accumulated in stored erythrocyte membranes and supernatants through storage day 42. By combining state-of-the-art ultra-high-pressure liquid chromatography-mass spectrometry metabolic flux analysis with redox and switch-tag proteomics, we identify for the first time ex vivo functionally relevant reversible and irreversible (sulfinic acid; Cys to dehydroalanine) oxidations of GAPDH without exogenous supplementation of excess pro-oxidant compounds in clinically relevant blood products. Oxidative and metabolic lesions, exacerbated by storage under hyperoxic conditions, were ameliorated by hypoxic storage. Storage-dependent reversible oxidation of GAPDH represents a mechanistic adaptation in stored erythrocytes to promote PPP activation and generate reducing equivalents. Removal of irreversibly oxidized, functionally compromised GAPDH identifies enhanced vesiculation as a self-protective mechanism in ex vivo aging erythrocytes.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO; and.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Reisz
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO; and.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Wither
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO; and.
authors.forename: Monika
authors.initials: M
authors.lastname: Dzieciatkowska
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO; and.
authors.forename: Travis
authors.initials: T
authors.lastname: Nemkov
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO; and.
authors.forename: Aaron
authors.initials: A
authors.lastname: Issaian
authors.affiliation: New Health Sciences Inc, Boston, MA.
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: New Health Sciences Inc, Boston, MA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Dunham
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO; and.
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Hill
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO; and.
authors.forename: Kirk C
authors.initials: KC
authors.lastname: Hansen
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO; and.
authors.forename: Angelo
authors.initials: A
authors.lastname: D'Alessandro
mesh_terms: D001793:Blood Preservation; D004912:Erythrocytes / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000737:chemistry* / Q000378:metabolism*; D006019:Glycolysis; D006801:Humans; D055432:Metabolomics; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D017382:Reactive Oxygen Species; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D005947:Glucose