ATP11C Encodes a Major Flippase in Human Erythrocyte and Its Genetic Defect Causes Congenital Non-Spherocytic Hemolytic Anemia

Flippases are members of the P-IV ATPase family of proteins, and contribute to localization of phosphatidylserine (PS) in inner leaflet by its ATP-dependent active transport from outer to inner leaflet of lipid bilayer of erythrocyte membranes. It is critical that erythrocytes maintain PS in the inner monolayer to ensure their 120-day survival in circulation since externalization of PS will be recognized as an eat-me signal, resulting in phagocytosis by splenic macrophages. In the present study, we identified that ATP11C gene encodes a major flippase in human erythrocytes. A 13 years-old boy was referred to our hospital for consultation for work up of an undiagnosed congenital hemolytic anemia. Since extensive biochemical and molecular analysis failed to identify hemoglobin, erythrocyte membranes and enzyme abnormalities for  the pathogenesis of hemolysis, we performed the whole exome analysis by massively parallel sequencing. We identified that the proband is hemizyogous, and the mother is heterozygous for a missense mutation of ATP11C, c.1253C>A, corresponding to a single amino acid substitution, p.Thr418Asn. Flipping activity as measured by PS internalization was decreased to 10% in the red cells of the proband compared to a normal control, clearly demonstrating that ATP11C encodes a major flippase in the human erythrocyte membranes. The PS-positive erythrocytes were not significantly increased in the whole blood but only in the most dense senescent cells, suggesting that PS exposure did not occur until very late stages of lifespan. We showed that PS exposure mediated by Ca2+-stimulated phospholipid scrambling was not different between red cells of the proband and control. Taken together, our findings imply that suppressed scrambling activity rather than flippase activity is the major contributor to maintainance of PS in inner leaflet of normal red cells during their 120-day lifespan, and that PS exposure to cell surface as an ‘eat-me’ signal depends primarily on scramblase activity at the end of lifespan. Importantly, our study has enables us to identify the major flippase of human erythrocyte membrane.