Transbilayer Phospholipid Movements in ABCA1-Deficient Cells

Tangier disease is an inherited disorder that results in a deficiency in circulating levels of HDL. Although the disease is known to be caused by mutations in the ABCA1 gene, the mechanism by which lesions in the ABCA1 ATPase effect this outcome is not known. The inability of ABCA1 knockout mice (ABCA1−/−) to load cholesterol and phospholipids onto apoA1 led to a proposal that ABCA1 mediates the transbilayer externalization of phospholipids, an activity integral not only to the formation of HDL particles but also to another, distinct process: the recognition and clearance of apoptotic cells by macrophages. Expression of phosphatidylserine (PS) on the surface of both macrophages and their apoptotic targets is required for efficient engulfment of the apoptotic cells, and it has been proposed that ABCA1 is required for transbilayer externalization of PS to the surface of both cell types. To determine whether ABCA1 is responsible for any of the catalytic activities known to control transbilayer phospholipid movements, these activities were measured in cells from ABCA1−/− mice and from Tangier individuals as well as ABCA1-expressing HeLa cells. Phospholipid movements in either normal or apoptotic lymphocytes or in macrophages were not inhibited when cells from knockout and wildtype mice or immortalized cells from Tangier individuals vs normal individuals were compared. Exposure of PS on the surface of normal thymocytes, apoptotic thymocytes and elicited peritoneal macrophages from wildtype and knockout mice or B lymphocytes from normal and Tangier individuals, as measured by annexin V binding, was also unchanged. No evidence was found of ABCA1-stimulated active PS export, and spontaneous PS movement to the outer leaflet in the presence or absence of apoA1 was unaffected by the presence or absence of ABCA1. Normal or Tangier B lymphocytes and macrophages were also identical in their ability to serve as targets or phagocytes, respectively, in apoptotic cell clearance assays. No evidence was found to support the suggestion that ABCA1 is involved in transport to the macrophage cell surface of annexins I and II, known to enhance phagocytosis of apoptotic cells. These results show that mutations in ABCA1 do not measurably reduce the rate of transbilayer movements of phospholipids in either the engulfing macrophage or the apoptotic target, thus discounting catalysis of transbilayer movements of phospholipids as the mechanism by which ABCA1 facilitates loading of phospholipids and cholesterol onto apoA1

Endogenous PS externalization in apoptotic EBV-transformed human B lymphocytes.

<p>EBV-transformed normal (A–D) or Tangier (E–H) B lymphocytes were untreated (A&B, E&F) or treated with camptothecin (C&D, G&H) to induce apoptosis, stained with fluorescent annexin V, and examined by flow cytometry. Left panel, forward (FS) vs side (SS) light scatter plots, with cells of normal size and shape in the R2 gate and shrunken cells in the R1 gate. Right panel, fluorescence profile of cells in the R2 gate.</p

Identification and annexin V staining of macrophages in peritoneal lavage from wildtype or <i>ABCA1^−/−</i> mice.

<p>Cells from peritoneal lavage were stained with mAb F4/80 (or isotype control mAb) or with annexin V on ice and examined by flow cytometry at room temperature. A, forward (FS) vs side (SS) light scatter plot, with cells having typical macrophage light scatter characteristics in the M gate and Asmall@ cells in the S gate. B, median fluorescence intensity of cells in the M gate (black) or S gate (grey) following staining with mAb F4/80 or isotype control mAb. C&D, annexin V staining, in the presence (thin line) or absence (thick line) of Ca²⁺, of cells in the M gate from wildtype (C) or <i>ABCA1^−/−</i> (D) mice.</p

Internalization (translocation) of NBD-labeled phospholipids by normal human fibroblasts and fibroblasts from Tangier individuals.

<p>The outer leaflet of the plasma membrane of normal (circles) or Tangier (squares) fibroblasts was labeled with NBD-PC (open symbols) or NBD-PS (filled symbols). At various times samples were removed into BSA to extract outer leaflet probe and remaining inner leaflet probe was measured by flow cytometry and expressed as percent transported. Measurements were made at room temperature. (A) Viable fibroblasts, (B) spontaneously apoptotic fibroblasts. Dashed and dotted lines in B have been redrawn from A for transport by normal fibroblasts.</p

Internalization (translocation) of NBD-labeled phospholipids by peritoneal macrophages from wildtype and <i>ABCA1^−/−</i> mice.

<p>The outer leaflet of the plasma membrane of macrophages from wildtype (diamonds) or <i>ABCA1^−/−</i> (squares) mice was labeled with NBD-PC (open symbols) or NBD-PS (closed symbols). At various times samples were removed into dithionate to reduce outer leaflet probe and after 5 min remaining inner leaflet probe was measured by flow cytometry at room temperature and expressed as percent transported. Cells from wildtype (triangles) and <i>ABCA1^−/−</i> (circles) mice in the S gate in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000729#pone-0000729-g006" target="_blank">Figure 6</a> are shown for comparison.</p

Basal (or Ca²⁺-activated) NBD-PS externalization in normal and ABCA1-deficient cells.

<p>(A) Normal human or Tangier fibroblasts, (B) EBV-transformed normal or Tangier B lymphocytes, (C) Control or ABCA1-GFP-transfected HeLa cells, (D) thymocytes from wildtype or <i>ABCA1^−/−</i> mice, (E) normal human fibroblasts or (F) Tangier fibroblasts were allowed to internalize NBD-PS, dithionite added to reduce and render non-fluorescent externalized NBD-PS, and cellular fluorescence (unexternalized NBD-PS) measured continuously over time at room temperature. (A–D) normal/wildtype, filled symbols; ABCA1-deficient or replete (HeLa), open symbols. (D) untreated, circles; treated with Ca²⁺ and Ca²⁺ ionophore, squares. (E and F) presence (open triangles) or absence (closed triangles) of apoA1.</p

Known and potential transbilayer lipid transporters.

<p>The aminophospholipid translocase (left) and ABCA1 (right) proteins are drawn approximately to scale in outline forms taken from atomic structures of a P-type ATPase (the Ca²⁺ transporter;<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000729#pone.0000729-Toyoshima1" target="_blank">[54]</a>) and an ABC protein (BtuCD,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000729#pone.0000729-Locher1" target="_blank">[55]</a>). The molecular shape of the scramblase (center) is arbitrary, since the protein responsible for this activity is not known. One possibility, investigated here, is that the ABCA1 protein is actually the protein responsible for this activity; another possibility is that the ABCA1 protein transports PS from the inner to the outer leaflet, as depicted. An HDL particle (far right) is also drawn approximately to scale by extrapolation from the structure of other lipoprotein particles, with the apoA1 protein drawn as the belt-like helices enclosing the lipid core. The molecular shape of an unlipidated apoA1 protein is unknown.</p