Functional characterization of Sec14L4 in hudep-2 and mouse knockout models reveals its essential role in erythropoiesis.

Abstract Background Red blood cell (RBC) transfusion is a common intervention for hospitalized patients, particularly those with sickle cell disease or thalassemia. To ensure transfusion quality, the FDA mandates <1% hemolysis during cold storage and ≥75% post-transfusion recovery (PTR) within 24 hours. However, RBCs undergo structural and biochemical changes during storage that compromise their viability. Oxidative injury to RBCs during storage leads to membrane damage and hemolysis. In a large, multi-ancestry genome-wide association study (GWAS) involving over 12,000 healthy donors, the Sec14-like lipid-binding 4 (Sec14L4) protein, which encodes a phosphatidylinositol transfer protein, was associated with increased hemolysis associated with oxidative stress. Sec14L4 is predicted to be a phosphatidyl inositol transfer protein based on sequence similarity to yeast Sec14. The functional role of Sec14L4 in RBC biology, however, remains poorly understood. Aim Our aim is to determine the role of Sec14L4 in erythropoiesis, RBC function and integrity. Methods Using CRISPR/Cas9, we generated mouse and HUDEP-2 SEC14L4 knockout (KO) models to assess in vivo and in vitro erythropoiesis and RBC function. The kinetics of differentiation of bone marrow erythroid precursors were tracked using flow cytometry. Additionally, HUDEP-2 wild-type (WT) and SEC14L4-KO cells were differentiated in vitro over 10 days (D) and analyzed using flow cytometry and microscopy to measure changes in differentiation, apoptosis, mitochondrial content, hemoglobinization and cell cycle. Results SEC14L4 deletion in HUDEP-2 erythroid cells resulted in impaired erythroid differentiation, with decreased late-stage erythroblasts following 7 days of differentiation. This occurs concomitantly with increases in apoptosis, cell cycle arrest and increased numbers of multinucleated enlarged cells. Cells lacking Sec14L4 have increased expression of CD71, a transferrin receptor that promotes cellular iron uptake and regulates hemoglobinization. As a result, we observed decreased hemoglobin in SEC14L4 KO cells as evidenced by benzidine staining. Mitochondrial clearance decreases in Sec14l4 KO cells, leading to higher reactive oxygen species (ROS). Analysis of the membrane reveals abnormal structure. Interestingly, Sec14L4 KO mice display abnormal membrane morphology with increased formation of echinocyte-shaped RBCs, while hematologic parameters remained normal, and there were no defects in erythroid maturation. Analysis of RBCs from two patients with SNPs in SEC14L4 also revealed similar morphologic abnormalities with spiked RBCs membranes after 7 days of cold storage. Conclusion This study is the first to characterize the role of Sec14L4 in erythropoiesis. Our findings reveal that SEC14L4 deficiency in a HUDEP-2 model disrupts erythroid maturation, proliferation, apoptosis, hemoglobinization, and may contribute to oxidative stress through impaired mitochondrial clearance. These insights may provide a mechanistic foundation for understanding how mutations in SEC14L4 affect RBC storage and function, with implications for transfusion medicine. Future work will focus on the function of Sec14L4 within the maturing erythrocyte to elucidate the molecular mechanisms underlying defective erythropoiesis.