Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality.

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.

Protein-L-isoaspartate O-methyltransferase is required for in vivo control of oxidative damage in red blood cells.

Red blood cells have the special challenge of a large amount of reactive oxygen species (from their substantial iron load and Fenton reactions) combined with the inability to synthesize new gene products. Considerable progress has been made in elucidating the multiple pathways by which red blood cells neutralize reactive oxygen species via NADPH driven redox reactions. However, far less is known about how red blood cells repair the inevitable damage that does occur when reactive oxygen species break through anti-oxidant defenses. When structural and functional proteins become oxidized, the only remedy available to red blood cells is direct repair of the damaged molecules, as red blood cells cannot synthesize new proteins. Amongst the most common amino acid targets of oxidative damage is the conversion of asparagine and aspartate side chains into a succinimidyl group through deamidation or dehydration, respectively. Red blood cells express an L-Isoaspartyl methyltransferase (PIMT, gene name PCMT1) that can convert succinimidyl groups back to an aspartate. Herein, we report that deletion of PCMT1 significantly alters red blood cell metabolism in a healthy state, but does not impair the circulatory lifespan of red blood cells. Through a combination of genetic ablation, bone marrow transplantation and oxidant stimulation with phenylhydrazine in vivo or blood storage ex vivo, we use omics approaches to show that, when animals are exposed to oxidative stress, red blood cells from PCMT1 knockout undergo significant metabolic reprogramming and increased hemolysis. This is the first report of an essential role of PCMT1 for normal RBC circulation during oxidative stress.

Red blood cell metabolism in Rhesus macaques and humans: comparative biology of blood storage.

Macaques are emerging as a critical animal model in transfusion medicine, because of their evolutionary similarity to humans and perceived utility in discovery and translational science. However, little is known about the metabolism of Rhesus macaque red blood cells (RBC) and how this compares to human RBC metabolism under standard blood banking conditions. Metabolomic and lipidomic analyses, and tracing experiments with [1,2,3-13C3]glucose, were performed using fresh and stored RBC (sampled weekly until storage day 42) obtained from Rhesus macaques (n=20) and healthy human volunteers (n=21). These results were further validated with targeted quantification against stable isotope-labeled internal standards. Metabolomic analyses demonstrated inter-species differences in RBC metabolism independent of refrigerated storage. Although similar trends were observed throughout storage for several metabolic pathways, species- and sex-specific differences were also observed. The most notable differences were in glutathione and sulfur metabolites, purine and lipid oxidation metabolites, acylcarnitines, fatty acyl composition of several classes of lipids (including phosphatidylserines), glyoxylate pathway intermediates, and arginine and carboxylic acid metabolites. Species-specific dietary and environmental compounds were also detected. Overall, the results suggest an increased basal and refrigerator-storage-induced propensity for oxidant stress and lipid remodeling in Rhesus macaque RBC cells, as compared to human red cells. The overlap between Rhesus macaque and human RBC metabolic phenotypes suggests the potential utility of a translational model for simple RBC transfusions, although inter-species storage-dependent differences need to be considered when modeling complex disease states, such as transfusion in trauma/hemorrhagic shock models.