Sec14l4 SNP Mice Have Increased Susceptibility to Oxidative Hemolysis through Modulation of G6PD and the Pentose Phosphate Pathway

Abstract

Red blood cell function and recovery following transfusion is influenced by a variety of factors including sex, race, sickle cell trait and single nucleotide polymorphisms (SNPs). In collaboration with the REDS III program, we performed a genome wide association study identifying SNPs that are associated with in vitro osmotic, oxidative and spontaneous storage hemolysis stress. Sec14L4, was one of 27 genes identified with enhanced hemolysis, specifically following oxidative insult and later confirmed by an independent study to be associated with decreased hemoglobin increments following transfusion. SEC14L4 is an understudied, highly conserved phosphatidylinositol transfer protein that is predicted to reside with the Golgi Apparatus. While the function of SEC14L4 is not understood, it is clear that polymorphisms in this gene have clinical impact and understanding its role in red cell biology and transfusion is critical. We generated a novel humanized Sec14L4 SNP and knockout mouse model to assess RBC metabolism, function and transfusion outcomes following storage. While Sec14l4 KO mice have complete loss of SEC14L4 protein expression, the SNP has an approximate 50% decrease in expression, suggesting the mutation decreases protein and/or mRNA stability. Interestingly, knockout mice experience anemia with decreased hemoglobin levels and lower hematocrit at baseline. Following 12 days of storage (equivalent to 42 days of human blood storage) Sec14l4 knockout RBCs have increased hemolysis and are more susceptible to lysis following treatment with the oxidant diamide. Tracing experiments with 1,2,313C3-glucose from Sec14l4 SNP mouse RBCs reveals decreased levels of glucose-6-phosaphate dehydrogenase (G6PD) and depression of metabolic fluxes through the pentose phosphate pathway with lower levels of 13C3-6-phosphogluconate and nicotinamide adenine dinucleotide phosphate (NADP+), and reduced ADP and ATP. Compensatory increases in glycolytic flux was also observed. G6PD is the rate liming enzyme of the pentose phosphate pathway which is the sole producer of NADPH in the red blood cell. NADPH is an essential cofactor for glutathione reductase and peroxidase, the primary erythrocyte antioxidant enzymes. Red blood cells deficient for G6PD undergo acute hemolysis if exposed to oxidative stress due to decreased antioxidant capacity. These data suggest that red blood cells deficient in SEC14L4 phenocopy G6PD deficient red cells, possibly explaining increased susceptibility to oxidative insult. Lipidomic evaluation also reveled increased accumulation of oxidized lipids during storage (12-HETE and 15-HETE). Oxilipins are markers of poor post-transfusion recovery in stored mouse and human red blood cells. Together these data indicate that SEC14L4 is an important player in red blood cell stability during storage, oxidative stress, and following transfusion. How a Golgi resident protein influences the adult red cell is not yet understood but suggests a role for SEC14L4 in early erythrocyte development which has an effect over the lifetime of the cell, with the result of increased stability and antioxidant capacity. In support of this hypothesis, Sec14l4 is highly expressed in the bone marrow with decreasing expression as the red cell terminally differentiates. Future work will confirm proteomic, metabolomic and lipidomic findings and focus on how SEC14L4 may be regulating the G6PD pathway in mature red cells.