Methylation of protein aspartates and deamidated asparagines as a function of blood bank storage and oxidative stress in human red blood cells

Abstract

Background Being devoid of de novo protein synthesis capacity, red blood cells (RBCs) have evolved to recycle oxidatively damaged proteins via mechanisms that involve methylation of dehydrated and deamidated aspartate and asparagine residues. Here we hypothesize that such mechanisms are relevant to routine storage in the blood bank. Study design and Methods 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 and time-dependent metabolic dysregulation of the trans-sulfuration pathway. Results In these units methionine consumption positively correlated with storage age and oxidative hemolysis. Mechanistic studies show that this phenomenon is favored by oxidative stress or hyperoxic storage (SO2 >95%), and 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 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. Conclusion While methylation of basic amino acid residues serves as an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated asparagines is a non-epigenetic post-translational sensor of oxidative stress and refrigerated storage in anucleated human RBCs.

Hypoxic storage of red blood cells improves metabolism and post‐transfusion recovery

“…This phenomenon is paralleled by the accumulation of reactive oxygen species, which is aggravated by a progressive loss in the RBCs capacity to cope with oxidant stress . As a result, RBCs face a supraphysiological oxidant insult, ultimately resulting in the irreversible oxidation of critical structural and functional proteins such as hemoglobin, band 3, and peroxiredoxin 2 . While RBCs are naturally equipped to cope with oxidant stress, most of these mechanisms either fail or are overwhelmed as a function of storage duration.…”

“…While RBCs are naturally equipped to cope with oxidant stress, most of these mechanisms either fail or are overwhelmed as a function of storage duration. Examples include 1) the progressive consumption and slower capacity to synthesize de novo reduced glutathione; 2) the incapacity of the pentose phosphate pathway (PPP) to sustain the generation of nicotinamide adenine dinucleotide phosphate (NADPH) which is necessary to recycle oxidized glutathione; 3) the failure/limited activity of purine oxidation salvage reactions in the mature erythrocyte; 4) the consumption of other antioxidant thiols (e.g., methionine), which is in part explained by the consumption of these metabolites by oxidant damage‐repair enzymes; and, 5) the failure of proteasomal‐dependent degradation of irreversibly modified proteins …”

Postpartum breast cancer progression is driven by semaphorin 7a mediated invasion and survival

“…Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on a Thermo nanoEasy LC II coupled to a Q Exactive HF. MS acquisition parameters are detailed previously(85). Raw files were searched with Proteome Discoverer 2.2 against the Mus Musculus, Homo Sapiens, and Bos Taurus uniprotKB database in Mascot.…”

Translating red cell “omics” into new perspectives in transfusion medicine: mining the gems in the data mountains

“…The RBC‐Omics investigators have now published seven significant and interrelated papers in Transfusion . These can be topically arranged as follows: (1) design of the REDS‐III RBC‐Omics program and performance of pilot and method optimization studies, and execution of a quality assurance program; (2) in vitro hemolysis testing of donated RBCs from recalled RBC‐Omics donors (focusing on methodology, reproducibility, and changes in hemolysis measures through storage of RBC components from the recalled donors); (3) in vitro hemolysis testing of RBCs relative to previous donation intensity, including donations by so‐called “super donors”; (4) general metabolomics studies of donated RBCs throughout the storage interval; (5) intermediary metabolism of methionine and trans‐sulfuration in stored RBCs; and (6) design of the methodology for GWAS of RBC donors . Although the last paper focused entirely on methodology, it is anticipated that some GWAS results using these methods will be available soon…”

“…Perhaps the most exciting result from these early publications is that the metabolomics data described above inspired experiments leading to the elucidation of a novel, and potentially important, pathway linking oxidative stress and protein oxidation to intermediary metabolism involving sulfur‐containing compounds, such as methionine (i.e., trans‐sulfuration) . Reisz et al used metabolomics methods, along with metabolic tracing using stable isotopes, sophisticated proteomics, specific enzyme inhibitors, and molecular modeling, to describe this model in detail (published in the December 2018 issue of Transfusion) . In particular, it suggests mechanisms whereby RBCs, which lack organelles, may repair oxidative damage to proteins involved in RBC structure, enzymatic function, and anti‐oxidant pathways.…”