Anti-ATP11C (aa 596-625) polyclonal antibody

Phosphatidylserine is localized exclusively to the inner leaflet of the membrane lipid bilayer of most cells, including erythrocytes. This asymmetric distribution is critical for the survival of erythrocytes in circulation since externalized phosphatidylserine is a phagocytic signal for splenic macrophages. Flippases are P-IV ATPase family proteins that actively transport phosphatidylserine from the outer to inner leaflet. It has not yet been determined which of the 14 members of this family of proteins is the flippase in human erythrocytes. Herein, we report that ATP11C encodes a major flippase in human erythrocytes, and a genetic mutation identified in a male patient caused congenital hemolytic anemia inherited as an X-linked recessive trait. Phosphatidylserine internalization in erythrocytes with the mutant ATP11C was decreased 10-fold compared to that of the control, functionally establishing that ATP11C is a major flippase in human erythrocytes. Contrary to our expectations phosphatidylserine was retained in the inner leaflet of the majority of mature erythrocytes from both controls and the patient, suggesting that phosphatidylserine cannot be externalized as long as scramblase is inactive. Phosphatidylserine-exposing cells were found only in the densest senescent cells (0.1% of total) in which scramblase was activated by increased Ca2+ concentration: the percentage of these phosphatidylserine-exposing cells was increased in the patient's senescent cells accounting for his mild anemia. Furthermore, the finding of similar extents of phosphatidylserine exposure by exogenous Ca2+-activated scrambling in both control erythrocytes and the patient's erythrocytes implies that suppressed scramblase activity rather than flippase activity contributes to the maintenance of phosphatidylserine in the inner leaflet of human erythrocytes.

Cellular membrane asymmetry is a hallmark characteristic of all eukaryotic cells. The balance of phospholipid composition within the cytoplasmic inner leaflet and the extracellular outer leaflet of the plasma membrane (PM) maintains cellular function and vitality. The proper exposure of particular phospholipids is necessary to maintain cellular signalling, controlled apoptosis, and vesicle transportation among other roles. Phospholipid asymmetry is coordinated by P4-type phospholipid transferases (flippases or ATPases). ATP11A, ATP11B, and ATP11C belong to class VI of the P4-flippase family (vertebrates) and are responsible for the movement of phosphatidylserine (PS) from the outer leaflet to the inner leaflet of the PM. To date, there is a lack of knowledge of the tissue specific expression of these three flippases on a whole-organism level in a vertebrate system. Here we have determined the spatial-temporal expression profiles of each gene in a zebrafish model using in situ hybridization and performed comparative phylogenetic analyses with other vertebrates. Our data reveals sequence similarity between vertebrate flippases and specific synteny of zebrafish and human chromosomes. Both atp11b and atp11c are maternally expressed in zebrafish, while zygotic expression analysis demonstrates tissue and temporal specificity for all three genes. atp11a is expressed in the neural crest cells as well as in the developing eye and ear, while atp11b is expressed early in the ventricular epithelial lining and later in the ear. atp11c is expressed in the anterior most rhombomeres of the hindbrain, pharyngeal arches, and liver. Our expression data suggests that each of the three flippases are integral for the development of specific tissues, and aberrant function of either could lead to visual, hearing, neural, or liver dysfunction.