Increased Methylation of Deamidated Asparagines and Aspartates in Stored Red Blood Cells from Glucose 6-Phosphate Dehydrogenase-Deficient Blood Donors
Authors: D'Alessandro Angelo , Nemkov Travis , Reisz Julie , Dzieciatkowska Monika , Culp-Hill Rachel , Stefanoni Davide , Yoshida Tatsuro , Dunham Andrew , Kanias Tamir , DuMont Larry J. , Busch Michael Paul , Eisenmesser Elan Z , Hansen Kirk C , Fu Xiaoyun , Thomas Tiffany , Karafin Matthew , Hod Eldad A. , Francis Richard O , Spitalnik Steven L , Zimring James C. 
Published: October 2019

Abstract
Blood transfusion is a life-saving intervention for millions of recipients worldwide every year. However, refrigerated storage of red blood cells (RBCs) for up to 42 days promotes impairments in energy and redox homeostasis, which impact RBC hemolytic propensity and post-transfusion performance of the storage-damaged RBC. Since mature RBCs are devoid of de novo protein synthesis - owing to the lack of organelles and nuclei - they evolved metabolic mechanisms to cope with oxidative stress. Two of these involve (i) activation of the pentose phosphate pathway (PPP), which generates reducing equivalents (NADPH) to preserve glutathione homeostasis and recharge NADPH-dependent antioxidant enzymes; and (ii) recycling of oxidatively damaged proteins via methylation of dehydrated and deamidated aspartate and asparagine residues, a process that consumes methionine as the main methyl group donor. Thus, we hypothesize that the latter mechanism is relevant to routine blood bank storage, especially in glucose-6-phosphate dehydrogenase (G6PD)-deficient donors. In this routinely accepted donor population (~10% of donors of African descent), mutations of G6PD, the rate-limiting enzyme of the PPP, result in instability and decreased enzymatic activity (e.g., <1% residual activity in the Mediterranean variant).
Within the framework of the REDS-III RBC-Omics study, packed RBC units (n=599) were stored under blood bank conditions for 10, 23 and 42 days and profiled for oxidative hemolysis (following AAPH insult) and time-dependent metabolic dysregulation of the trans-sulfuration pathway. The rate of methionine consumption positively correlated with storage age and oxidative hemolysis. In a separate cohort, RBCs from G6PD-deficient and G6PD-normal donors were also collected and metabolically profiled during storage. The G6PD-deficient donors had higher pre-storage methionine levels as compared against G6PD-sufficient donors, although one week of storage was sufficient to significantly flip this ratio (p<0.01). To ensure that the phenomenon was driven by storage-induced oxidative insults, RBCs were also stored under normoxic, hyperoxic (SO2 >95%), or hypoxic conditions (SO2 = 20%, 10%, 5% of < 3%) for up to 42 days. Of note, hypoxia decreased methionine consumption rates and increased S-adenosylmethionine/S-adenosylhomocysteine ratios in comparison to normoxic control RBCs, while opposite trends were observed in response to oxidative stress. In parallel, RBCs were incubated under normoxic or hypoxic conditions for 16h, in presence or absence of a generic methyltransferase inhibitor, adenosine dialdehyde, or hydrogen peroxide (0.1 or 0.5%). 13C,15N-Methionine uptake and consumption rates were faster in response to pro-oxidant insults and were prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and 13C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and the formation of methyl-Asp on key structural proteins and enzymes, including band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of GAPDH) and/or residues interacting with the N-terminal cytosolic domain of band 3. Hypoxic incubation with hydrogen peroxide phenocopied normoxic conditions in the presence of pro-oxidant insults, suggesting that the beneficial role of hypoxia in preventing methionine consumption and protein methylation is attributable to decreased oxidative stress. G6PD-deficient donor RBCs had significantly (p<0.0001) higher levels of methylated-deamidated Asn residues in hemoglobin, band 3, and GAPDH, as compared to stored G6PD-normal donor RBCs.
Although methylation of basic amino acid residues is an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated Asn residues is a non-epigenetic post-translational sensor of oxidative stress and refrigerated storage in anucleate human RBCs. This damage repair mechanism is exacerbated in RBCs from G6PD-deficient donors, as a compensatory mechanism for the lack of normal PPP function.

Disclosures
D'Alessandro: Hemanext inc: Membership on an entity's Board of Directors or advisory committees; Omix Technologies inc: Equity Ownership. Nemkov:Omix Technologies inc: Equity Ownership. Yoshida:Hemanext inc: Employment. Dunham:Hemanext inc: Employment. DuMont:Hemanext inc: Membership on an entity's Board of Directors or advisory committees. Hansen:Omix Technologies inc: Equity Ownership. Spitalnik:Hemanext inc: Membership on an entity's Board of Directors or advisory committees. Zimring:Rubius Therapeutics: Membership on an entity's Board of Directors or advisory committees.