© Ferrata Storti Foundation
Total Page：16
File Type：pdf, Size：1020Kb
Red Cell Biology & its Disorders ARTICLE ATP11C is a major flippase in human erythrocytes and its defect causes congenital Ferrata Storti EUROPEAN HEMATOLOGY Foundation hemolytic anemia ASSOCIATION Nobuto Arashiki,1 Yuichi Takakuwa,1 Narla Mohandas,2 John Hale,2 Kenichi Yoshida,3 Hiromi Ogura,4 Taiju Utsugisawa,4 Shouichi Ohga,5 Satoru Miyano,6 Seishi Ogawa,3 Seiji Kojima,7 and Hitoshi Kanno,4,8 1Department of Biochemistry, School of Medicine, Tokyo Women’s Medical University, Japan; 2Red Cell Physiology Laboratory, New York Blood Center, NY, USA; 3Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Japan; 4Department of Transfusion Medicine and Cell Processing, School of Medicine, Tokyo Women’s Medical University, Japan; 5Department of Pediatrics, Graduate School of Medicine, Yamaguchi University, Japan; 6Laboratory of DNA Information Analysis, Human Genome Center, Institute of Medical Science, The University of Tokyo, Japan; 7Department of Pediatrics, Graduate School of Medicine, Nagoya University, Japan; and 8Division of Genomic Medicine, Department of Advanced Biomedical Engineering and Haematologica 2016 Science, Graduate School of Medicine, Tokyo Women's Medical University, Japan Volume 101(5):559-565 ABSTRACT hosphatidylserine is localized exclusively to the inner leaflet of the membrane lipid bilayer of most cells, including erythrocytes. This Pasymmetric 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 active- ly 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 Correspondence: recessive trait. Phosphatidylserine internalization in erythrocytes with the [email protected] mutant ATP11C was decreased 10-fold compared to that of the control, functionally establishing that ATP11C is a major flippase in human erythro- cytes. Contrary to our expectations phosphatidylserine was retained in the inner leaflet of the majority of mature erythrocytes from both controls and Received: January 13, 2016. the patient, suggesting that phosphatidylserine cannot be externalized as Accepted: February 26, 2016. long as scramblase is inactive. Phosphatidylserine-exposing cells were Pre-published: March 4, 2016. found only in the densest senescent cells (0.1% of total) in which scram- 2+ blase was activated by increased Ca concentration: the percentage of doi:10.3324/haematol.2016.142273 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+-activat- Check the online version for the most updated ed scrambling in both control erythrocytes and the patient’s erythrocytes information on this article, online supplements, implies that suppressed scramblase activity rather than flippase activity and information on authorship & disclosures: contributes to the maintenance of phosphatidylserine in the inner leaflet of www.haematologica.org/content/101/6/559 human erythrocytes. ©2016 Ferrata Storti Foundation Introduction Material published in Haematologica is cov- ered by copyright. All rights reserved to the In human erythrocytes, phosphatidylserine (PS) is present exclusively in the inner Ferrata Storti Foundation. Copies of articles leaflet of the membrane lipid bilayer as a result of ATP-dependent active transport are allowed for personal or internal use. Permission in writing from the publisher is (flipping) of aminophospholipids (such as PS and phosphatidylethanolamine) from required for any other use. the outer to inner leaflet. PS interacts with spectrin, a cytoskeletal protein under- neath erythrocyte membranes, to maintain membrane deformability and mechani- cal stability of the erythrocytes1 and protects spectrin from glycation, which haematologica | 2016; 101(5) 559 N. Arashiki et al. decreases the membrane deformability necessary for tra- 1A).11,12 They contribute to localization of PS in the inner versing narrow capillaries and the splenic sinuses.2 More leaflet of erythrocyte membranes through ATP-dependent importantly, preventing surface exposure of PS is critical active transport of aminophospholipids (such as PS) from for erythrocyte survival: exposure of PS on the outer sur- the outer to inner leaflet.13-15 However, the flippase in face of the membrane at the end of the 120-day lifespan of human erythrocytes has not yet been definitively identi- erythrocytes is a phagocytic signal for splenic fied. Among the 14 family members, ATP8A1, ATP8A2, macrophages to remove the senescent cells.3-5 Indeed in ATP11A, and ATP11C were previously shown to trans- these cells, the Ca2+ concentration is elevated to activate port PS in the plasma membrane.12,16-19 ATP11C has been lipid scrambling, with consequent surface exposure of PS, implicated as one of the candidates in murine erythrocytes which is recognized as an “eat-me signal” by based on the finding that its mutation in mice resulted in macrophages.4-7 Besides being exposed on normal senes- anemia with stomatocytosis.20 However, there are differ- cent cells, PS is exposed prematurely by sickle erythro- ences between the characteristics of human and murine cytes and thalassemic erythrocytes, resulting in a short- erythrocytes, including their life span, cell volume and cell ened life span of the red blood cells and consequent hemoglobin content. Furthermore, recent studies have hemolytic anemia in these disorders.7-10 Maintenance, reg- documented significant differences in gene expression ulation, and disruption of the asymmetric PS distribution during human and murine erythropoiesis.21 For example, are, therefore, important for both erythrocyte survival and GLUT1 which is abundantly expressed in human erythro- death. While it has been well established that PS distribu- cytes is not expressed by murine erythrocytes. As such it tion is determined by flippase and scramblase activities, is important to identify the major flippase in human ery- the molecular identities of these activities in human ery- throcytes. throcytes have not been defined. Furthermore, the relative In the present study, we identified a point mutation in contributions of these two activities in maintaining the ATP11C through whole-exome sequencing of DNA from asymmetric distribution of PS under physiological and a male patient with mild hemolytic anemia without mor- pathological states are not well understood. phological abnormalities. Detailed analyses established Flippases are members of the P-IV ATPase family of pro- that this mutation is responsible for hemolysis and related teins composed of 10 transmembrane domains (Figure clinical features and that ATP11C is a major flippase in A B 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 mis- C sense mutation coding for Thr418Asn. (B) Phase-contrast microscopy images (×1,000; pre- pared from original images without any modifications) of Giemsa- stained blood from the healthy con- trol 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. 560 haematologica | 2016; 101(5) ATP11C, a major flippase in human erythrocytes human erythrocyte membranes. By defining the contribu- three times with 1 mL TBS-G including 1% BSA to remove tion of ATP11C to PS distribution when scramblase is A23187 from the erythrocyte membranes. Ten microliters of the inactive under physiologically low Ca2+ concentrations packed erythrocytes were suspended in 1 mL TBS-G including 5 2+ and when it is activated under elevated high Ca concen- mM CaCl2. Thereafter, 1 μL 0.25 mg/mL fluorescein isothio- trations, as in senescent cells, we established that sup- cyanate (FITC)-conjugated annexin V was added, and the fluores- pressed scramblase activity rather than flippase activity cence on erythrocytes quantified by FACS as described above. The contributes to the maintenance of PS in the inner leaflet of cut-off for identification of PS-positive cells was set at a fluores- human erythrocytes. cent signal value 20-fold higher than that detected in the absence of FITC-annexin V. Methods Results This study was approved by the Ethics Committee for Human Genome/Gene Analysis Research of Tokyo Women’s Medical Clinical history and analyses of a male patient with University (#223D). The healthy volunteer, the proband, and the congenital hemolytic anemia proband’s mother provided informed consent for blood sample collection and the blood was used in all of the studies outlined. A male proband was born at full-term after a clinically Suppliers of all reagents and laboratory instruments, and the com- normal pregnancy without any apparent anomalies. At position of buffer solutions are given in the Online Supplementary the age of 4 years, his mother noted that the boy had pig- Information.