Peter J. Sims

Calcium-dependent phospholipid scrambling by TMEM16F

In all animal cells, phospholipids are asymmetrically distributed between the outer and inner leaflets of the plasma membrane. This asymmetrical phospholipid distribution is disrupted in various biological systems. For example, when blood platelets are activated, they expose phosphatidylserine (PtdSer) to trigger the clotting system. The PtdSer exposure is believed to be mediated by Ca2+-dependent phospholipid scramblases that transport phospholipids bidirectionally, but its molecular mechanism is still unknown. Here we show that TMEM16F (transmembrane protein 16F) is an essential component for the Ca2+-dependent exposure of PtdSer on the cell surface. When a mouse B-cell line, Ba/F3, was treated with a Ca2+ ionophore under low-Ca2+ conditions, it reversibly exposed PtdSer. Using this property, we established a Ba/F3 subline that strongly exposed PtdSer by repetitive fluorescence-activated cell sorting. A complementary DNA library was constructed from the subline, and a cDNA that caused Ba/F3 to expose PtdSer spontaneously was identified by expression cloning. The cDNA encoded a constitutively active mutant of TMEM16F, a protein with eight transmembrane segments. Wild-type TMEM16F was localized on the plasma membrane and conferred Ca2+-dependent scrambling of phospholipids. A patient with Scott syndrome, which results from a defect in phospholipid scrambling activity, was found to carry a mutation at a splice-acceptor site of the gene encoding TMEM16F, causing the premature termination of the protein.

Molecular Cloning of Human Plasma Membrane Phospholipid Scramblase A PROTEIN MEDIATING TRANSBILAYER MOVEMENT OF PLASMA MEMBRANE PHOSPHOLIPIDS

The rapid movement of phospholipids (PL) between plasma membrane leaflets in response to increased intracellular Ca2+ is thought to play a key role in expression of platelet procoagulant activity and in clearance of injured or apoptotic cells. We recently reported isolation of a ∼37-kDa protein in erythrocyte membrane that mediates Ca2+-dependent movement of PL between membrane leaflets, similar to that observed upon elevation of Ca2+ in the cytosol (Bassé, F., Stout, J. G., Sims, P. J., and Wiedmer, T. (1996) J. Biol. Chem.271, 17205–17210). Based on internal peptide sequence obtained from this protein, a 1,445-base pair cDNA was cloned from a K-562 cDNA library. The deduced “PL scramblase” protein is a proline-rich, type II plasma membrane protein with a single transmembrane segment near the C terminus. Antibody against the deduced C-terminal peptide was found to precipitate the ∼37-kDa red blood cell protein and absorb PL scramblase activity, confirming the identity of the cloned cDNA to erythrocyte PL scramblase. Ca2+-dependent PL scramblase activity was also demonstrated in recombinant protein expressed from plasmid containing the cDNA. Quantitative immunoblotting revealed an approximately 10-fold higher abundance of PL scramblase in platelet (∼104 molecules/cell) than in erythrocyte (∼103 molecules/cell), consistent with apparent increased PL scramblase activity of the platelet plasma membrane. PL scramblase mRNA was found in a variety of hematologic and nonhematologic cells and tissues, suggesting that this protein functions in all cells.

Isolation of an Erythrocyte Membrane Protein that Mediates Ca2+-dependent Transbilayer Movement of Phospholipid

Elevation of intracellular Ca2+ in erythrocytes, platelets, and other cells initiates rapid redistribution of plasma membrane phospholipids (PL) between inner and outer leaflets, collapsing the normal asymmetric distribution. Consequently, phosphatidylserine and other lipids normally sequestered to the inner leaflet become exposed at the cell surface. This Ca2+-induced mobilization of phosphatidylserine to the surface of activated, injured, or apoptotic cells confers a procoagulant property to the plasma membrane, which promotes fibrin clotting and provides a signal for cell removal by the reticuloendothelial system. To identify the constituent of the membrane that mediates this Ca2+-dependent “PL scramblase” activity, we undertook purification and reconstitution of membrane component(s) with this activity from detergent extracts of erythrocyte ghosts depleted of cytoskeleton. Active fractions were identified by their capacity to mediate the Ca2+-dependent redistribution of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled PL between leaflets of reconstituted proteoliposomes. This PL scramblase activity co-eluted through multiple chromatographic steps with a single polypeptide of ∼37 kDa, which was purified to apparent homogeneity as resolved by silver staining. The activity associated with this protein band was inactivated by trypsin. The isolated protein reconstituted in proteoliposomes mediated nonselective, bidirectional transport of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-PL between membrane leaflets, with half-maximal activation between 20 and 60 μM Ca2+ (saturation >100 μM), mimicking the Ca2+-dependent transbilayer lipid movement intrinsic to the erythrocyte membrane.

Formation of K-region epoxides as microsomal metabolites of pyrene and benzo[a]pyrene

Abstract Epoxides of pyrene and benzo[a]pyrene have been detected for the first time as microsomal metabolites of these polycyclic hydrocarbons. The epoxides, which have been identified as the K-region derivatives, pyrene 4,5-oxide and benzo[a]pyrene 4, 5-oxide respectively, were formed by the NADPH-dependent mixed function oxidase of a rat-liver microsomal incubation system where the epoxide hydrase was inhibited. In the absence of hydrase inhibition, benzo[a]pyrene was converted into a metabolite with the Chromatographic properties of a K-region dihydrodiol, trans-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, which has not previously been described as a metabolite of this hydrocarbon. Pyrene 4,5-oxide and benzo[a]pyrene 4,5-oxide rearrange in acid to compounds with the properties of 4-pyrenol and 4-benzo[a]pyrenol respectively, are converted by microsomal epoxide hydrase into compounds indistinguishable from the corresponding 4, 5-dihydro-4,5-dihydroxy derivatives and react with glutathione to yield conjugates. Both epoxides are reactive towards polyguanylic acid. The significance of these results is discussed in relation to our hypothesis that polycyclie hydrocarbon carcinogenesis results from somatic mutations caused by epoxides that are formed from the hydrocarbons by metabolism.

Human CD59 is a receptor for the cholesterol-dependent cytolysin intermedilysin

Cholesterol is believed to serve as the common receptor for the cholesterol-dependent cytolysins (CDCs). One member of this toxin family, Streptococcus intermedius intermedilysin (ILY), exhibits a narrow spectrum of cellular specificity that is seemingly inconsistent with this premise. We show here that ILY, via its domain 4 structure, binds to the glycosyl-phosphatidylinositol–linked membrane protein human CD59 (huCD59). CD59 is an inhibitor of the membrane attack complex of human complement. ILY specifically binds to huCD59 via residues that are the binding site for the C8α and C9 complement proteins. These studies provide a new model for the mechanism of cellular recognition by a CDC.

Changes in cytosolic Ca2+ associated with von Willebrand factor release in human endothelial cells exposed to histamine. Study of microcarrier cell monolayers using the fluorescent probe indo-1.

A method for measuring fluorescence in anchored monolayers of human endothelial cells is described and used to demonstrate changes in the cytosolic free-calcium concentration ([Ca2+]c) in these cells exposed to histamine and thrombin; some endothelial responses to both agonists (e.g., mitogenesis) have been suggested to be Ca2+-mediated. Umbilical vein endothelial cells were cultured on microcarriers and loaded with the Ca2+ indicator, indo-1. Enzymatic cell detachment was avoided by monitoring the indo-1 fluorescence ratio (400/480 nm) of a stirred suspension of cell-covered microcarriers. Basal [Ca2+]c was estimated to be 70-80 nM. Thrombin induced a transient dose-dependent increase in [Ca2+]c, which was active-site dependent. Histamine stimulated a dose-dependent increase in [Ca2+]c, which was reversed by removal of histamine and inhibited competitively by the H1-receptor antagonist pyrilamine, but not by the H2-receptor antagonist cimetidine. Furthermore, histamine induced a dose-dependent secretion of von Willebrand factor, which paralleled the rise in [Ca2+]c and was similarly blocked by the H1-receptor antagonist, and which may contribute to platelet deposition at sites of inflammation.

The response of human platelets to activated components of the complement system

Many in vivo platelet responses are considered to be mediated, directly or indirectly, by activated components of the complement system. These include the secretion of proteolytic enzymes and the assembly of key enzymes of the coagulation and fibrinolytic pathways. In this review, Peter J. Sims and Therese Wiedmer summarize the known interactions of human platelets with the complement system and discuss the implications of these interactions for platelet hemostatic function within the vasculature.

Inhibition of the complement membrane attack complex by the galactose-specific adhesion of Entamoeba histolytica.

The human complement system is an important early host defense against infection. Entamoeba histolytica activates the complement system but is resistant to killing by complement C5b-9 complexes deposited on the membrane surface. Our aim was to identify components of the amebic plasma membrane that mediate resistance to human complement C5b-9 by screening for neutralizing monoclonal antibodies. A monoclonal antibody was identified that abrogated amebic resistance to C5b-9, and the mAb was shown to recognize the parasites galactose-specific adhesin. The purified adhesin bound to C8 and C9 and conferred C5b-9 resistance to sensitive ameba upon reconstitution; these activities of the adhesin were inhibited by the antiadhesin mAb. The E. histolytica adhesin shared sequence similarities and antigenic cross-reactivity with CD59, a membrane inhibitor of C5b-9 in human blood cells, suggesting both molecular mimicry and shared complement-inhibitory functions.

Caspase-Independent Exposure of Aminophospholipids and Tyrosine Phosphorylation in Bicarbonate Responsive Human Sperm Cells

Abstract Only capacitated sperm cells are able to fertilize egg cells, and this process is triggered by high levels of bicarbonate. Bicarbonate renders the plasma membrane more fluid, which is caused by protein kinase A (PKA)-mediated alterations in the phospholipid (PL) bilayer. We studied exposure of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in human sperm cells. Surface exposure of PS and PE on sperm cell activation in vitro was found to be bicarbonate dependent and restricted to the apical area of the head plasma membrane. The PL scrambling in bicarbonate-triggered human sperm was not related to apoptosis, because the incubated cells did not show any signs of caspases or degeneration of mitochondria or DNA. The PL scramblase (PLSCR) gene family has been implicated in this nonspecific, bidirectional PL movement. A 25-kDa isoform of PLSCR was identified that was homogeneously distributed in human sperm cells. We propose that compartment-dependent activation of PKA is required for the surface exposure of aminophospholipids at the apical plasma membrane of sperm cells. Bicarbonate-induced PL scrambling appears to be an important event in the capacitation process, because the entire intact scrambling sperm subpopulation showed extensive tyrosine phosphorylation, which was absent in the nonscrambling subpopulation. The proportion of live cells with PL scrambling corresponded with that showing capacitation-specific chlortetracyclin staining.

Phospholipid Scramblase 1 Contains a Nonclassical Nuclear Localization Signal with Unique Binding Site in Importin α

Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin α/β. Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS 257GKISKHWTGI266. This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 Å crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin α. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1–4 of importin α, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel nuclear localization signal that presents a distinct motif for binding to importin α.