Phospholipid Scramblase 1 Modulates FcR-Mediated Phagocytosis in Differentiated Macrophages.

Phospholipid Scramblase 1 (PLSCR1) was initially characterized as a type II transmembrane protein involved in bilayer movements of phospholipids across the plasma membrane leading to the cell surface exposure of phosphatidylserine, but other cellular functions have been ascribed to this protein in signaling processes and in the nucleus. In the present study, expression and functions of PLSCR1 were explored in specialized phagocytic cells of the monocyte/macrophage lineage. The expression of PLSCR1 was found to be markedly increased in monocyte-derived macrophages compared to undifferentiated primary monocytes. Surprisingly, this 3-fold increase in PLSCR1 expression correlated with an apparent modification in the membrane topology of the protein at the cell surface of differentiated macrophages. While depletion of PLSCR1 in the monocytic THP-1 cell-line with specific shRNA did not inhibit the constitutive cell surface exposure of phosphatidylserine observed in differentiated macrophages, a net increase in the FcR-mediated phagocytic activity was measured in PLSCR1-depleted THP-1 cells and in bone marrow-derived macrophages from PLSCR1 knock-out mice. Reciprocally, phagocytosis was down-regulated in cells overexpressing PLSCR1. Since endogenous PLSCR1 was recruited both in phagocytic cups and in phagosomes, our results reveal a specific role for induced PLSCR1 expression in the modulation of the phagocytic process in differentiated macrophages

Cell surface expression of PLSCR1 during monocyte differentiation into macrophages.

<p>THP-1 cells (A and B) were cultured for 72 h in the presence or absence of PMA, while primary monocytes (C and D) were differentiated in macrophages as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145617#pone.0145617.g001" target="_blank">Fig 1</a>. (A and C) Cell surface staining was performed at 4°C with anti-N-ter-PLSCR1 (1E9 mAb) followed by an Alexa488-anti-mouse IgG (left panels), anti-C-ter-PLSCR1 (rabbit pAb) followed by an Alexa555-anti-rabbit IgG (middle panels), or PE-conjugated Annexin-V (right panels). In upper panels of (A) and (C), MFIs are indicated on histograms, and in the lower panels, these results are quantified and expressed as a percentage of the mean value obtained for non-treated THP-1 cells (A) or monocytes (C). Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. (B and D) Representative biparametric analyses of the cell surface staining with anti-N-ter-PLSCR1 and anti-C-ter-PLSCR1 antibodies, and Annexin-V are shown for treated and untreated THP-1 cells (B), and monocytes and MDMs (D). In (B) and (D), the percentage of cells in each quadrant is indicated.</p

Cellular distribution of PLSCR1 during monocyte-to-macrophage differentiation.

<p><b>(</b>A) Monocytes (left images) were differentiated into macrophages (right images) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145617#pone.0145617.g001" target="_blank">Fig 1</a> and cell surface expression of PLSCR1 was detected by indirect immunofluorescence at 4°C using anti-C-ter-PLSCR1 (rabbit pAb) followed with Alexa555-anti-rabbit IgG (upper images), or anti-N-ter-PLSCR1 (1E9 mAb) followed with Alexa488-anti-mouse IgG (middle images). (B) THP-1 cells (upper images) were cultured for 72 h with or without PMA, while primary monocytes were differentiated into macrophages (lower images) as indicated previously. After fixation and permeabilization, PLSCR1 was detected by indirect immunofluorescence with anti-PLSCR1 (1E9 mAb). Scale bars, 10 μm.</p

Monocyte-to-macrophage differentiation and cell surface phosphatidylserine exposure in PLSCR1-depleted cells.

<p>THP-1 cells were transduced with lentiviruses expressing shRNA against either PLSCR1 or Luciferase used as a control, and then cultured for 72 h with or without PMA as previously. (A) Lysates from shRNA-transduced THP-1 cells were analyzed by Western blotting with anti-PLSCR1 (upper panels) and anti-ɣ-tubulin (lower panels). (B and C) Cell surface expression of CD14 and PS exposure. Treated or untreated shRNA-transduced THP-1 cells were stained with FITC-conjugated anti-CD14 antibodies (B) or PE-conjugated Annexin-V (C), and surface expression was measured by flow cytometry. Results are expressed as the percentage of the MFI relative to the non differentiated control shLuc-transduced cells. Values are the means of 3 independent experiments. Error bars represent 1 SD from the mean. Statistical significance was determined using Student's <i>t</i> test (non significant, p > 0.05).</p

A DNA Sequence Recognition Loop on APOBEC3A Controls Substrate Specificity

<div><p>APOBEC3A (A3A), one of the seven-member APOBEC3 family of cytidine deaminases, lacks strong antiviral activity against lentiviruses but is a potent inhibitor of adeno-associated virus and endogenous retroelements. In this report, we characterize the biochemical properties of mammalian cell-produced and catalytically active <i>E. coli</i>-produced A3A. The enzyme binds to single-stranded DNA with a K_d of 150 nM and forms dimeric and monomeric fractions. A3A, unlike APOBEC3G (A3G), deaminates DNA substrates nonprocessively. Using a panel of oligonucleotides that contained all possible trinucleotide contexts, we identified the preferred target sequence as TC (A/G). Based on a three-dimensional model of A3A, we identified a putative binding groove that contains residues with the potential to bind substrate DNA and to influence target sequence specificity. Taking advantage of the sequence similarity to the catalytic domain of A3G, we generated A3A/A3G chimeric proteins and analyzed their target site preference. We identified a recognition loop that altered A3A sequence specificity, broadening its target sequence preference. Mutation of amino acids in the predicted DNA binding groove prevented substrate binding, confirming the role of this groove in substrate binding. These findings shed light on how APOBEC3 proteins bind their substrate and determine which sites to deaminate.</p></div

The preferred consensus deamination target site of A3A is TC (A/G).

<p>A) rA3A was incubated with ssDNA substrates containing four different target site sequences. B) rA3A was incubated with ssDNA substrates containing different target site sequences.</p