ATP11C is a major flippase in human erythrocytes and its defect causes congenital hemolytic anemia

Abstract

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 Ca(2+) 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 Ca(2+)-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.

Figure 1.

Schematic representation of ATP11C, erythrocyte morphology, and genotyping for the ATP11C mutation. (A) Schematic of ATP11C and the site of a c.1253C>A missense mutation coding for Thr418Asn. (B) Phase-contrast microscopy images (×1,000; prepared from original images without any modifications) of Giemsa-stained blood from the healthy control and proband. No morphological abnormality was observed. (C) Wave data from direct sequencing of genomic DNA for exon 13, which includes the coding region of Thr418. The sequence of the anti-sense strand corresponding to the sense strand represented in (A) is displayed. Arrows indicate position of the mutations.

Figure 2.

Flipping activity of erythrocytes with the ATP11C mutation. (A) Primary NBD-derived fluorescence data from flow cytometry. Left: NBD-PS loaded onto erythrocyte membranes for 20 min without BSA treatment [20 min BSA (−)]. Right: time-dependent internalization of NBD-PS with BSA treatment to remove NBD-PS remaining in the outer leaflet. Events are indicated with arbitrary units. (B) Quantitation of the proportion of internalized NBD-PS calculated by mean fluorescence obtained from (A). The values were obtained by dividing internalized NBD-PS by loaded NBD-PS in each individual. NEM-treated cells from control (C + NEM), the patient’s (P + NEM), and maternal erythrocytes (M + NEM) were also analyzed to confirm the contribution of ATP11C to observed flipping activity. Primary flow data are shown in Online Supplementary Figure S2.

Figure 3.

PS cell surface exposure in circulating erythrocytes with the ATP11C mutation. Exposed PS was detected by Ca²⁺-dependent specific binding of FITC-annexin V. Unfractionated erythrocytes (total) and fractionated erythrocytes obtained from density gradient centrifugation (senescence) for the control, patient, and mother were suspended in isotonic buffer including 5 mM CaCl₂ before adding FITC-annexin V. Primary data obtained from flow cytometry analyses of FITC-derived fluorescence on erythrocytes are displayed. Events are shown with arbitrary units. The values in each panel indicate the proportion of PS-positive cells.

Figure 4.

PS cell surface exposure in Ca²⁺-loaded erythrocytes with the ATP11C mutation. Effect of Ca²⁺-stimulated scrambling on PS exposure in control and patient’s erythrocytes. Ca²⁺ was introduced by a Ca²⁺ ionophore, A23187, for 20 min at 37 °C. After removal of the Ca²⁺ ionophore, PS-exposed cells were analyzed by monitoring FITC-annexin V binding to erythrocytes.