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

(Received for publication, April 25, 1997, and in revised form, May 19, 1997)

Quansheng Zhou Dagger , Ji Zhao , James G. Stout , Robert A. Luhm , Therese Wiedmer and Peter J. Sims §

From the Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201-2178

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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.


INTRODUCTION

The plasma membrane phospholipids (PL)1 are normally asymmetrically distributed, with phosphatidylcholine (PC) and sphingomyelin located primarily in the outer leaflet, and the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine restricted to the cytoplasmic leaflet (1, 2). An increase in intracellular Ca2+ due to either cell activation, cell injury, or apoptosis causes a rapid bidirectional movement of the plasma membrane PL between leaflets, resulting in exposure of PS and phosphatidylethanolamine at the cell surface (1, 3-5). This exposure of the plasma membrane aminophospholipids has been shown to promote assembly and activation of several key enzymes of the coagulation and complement systems, as well as to accelerate the clearance of injured or apoptotic cells by the reticuloendothelial system, suggesting that Ca2+-induced remodeling of plasma membrane PL is central to both vascular hemostatic and cellular clearance mechanisms (1, 6-9).

We recently reported isolation of a ~37-kDa integral membrane protein from human erythrocytes that when reconstituted into liposomes mediated a Ca2+-dependent, bidirectional scrambling of PL between membrane leaflets mimicking the action of Ca2+ at the endofacial surface of the erythrocyte membrane (10, 11). Evidence for protein(s) in platelet that mediates a similar "PL scramblase" function when incorporated into liposomes has also been reported (12). Here we report the cDNA cloning and deduced structure of the PL scramblase from human erythrocyte and show evidence that this same protein is expressed in human platelet and various other cell lines and tissues where plasma membrane PL scramblase activity has been observed.


EXPERIMENTAL PROCEDURES

Materials

Egg yolk PC, brain PS, and 1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphocholine (NBD-PC) were obtained from Avanti Polar Lipids. Expressed sequence tag (EST) clone with GenBankTM accession number gb AA143025 was obtained through American Type Culture Collection (ATCC 962235). All restriction enzymes and amylose resin were from New England BioLabs, Inc. KlenTaq polymerase was from CLONTECH Laboratories, wheat germ agglutinin Sepharose was from Sigma, isopropyl-beta -D-thiogalactopyranoside was from Eastman Kodak, factor Xa was from Hematologic Technologies, and Bio-Beads SM-2 were from Bio-Rad. N-Octyl-beta -D-glucopyranoside (OG) and Glu-Gly-Arg chloromethyl ketone were from Calbiochem. Sodium dithionite (Na2S2O4, Sigma) was freshly dissolved in 1 M Tris, pH 10, at a concentration of 1 M.

PL Scramblase Isolation and Amino Acid Sequencing

PL scramblase was purified as described previously (10, 11), with the following modifications. The active fraction eluting from Mono S was concentrated and exchanged into 150 mM NaCl, 20 mM Tris, 0.1 mM EGTA, 0.1% Nonidet P-40, pH 7.4, and absorbed against 5 ml of wheat germ agglutinin-Sepharose to remove trace contaminating glycophorins. The breakthrough material was concentrated and exchanged into 20 mM Tris, 0.1 mM EGTA, 0.02% Nonidet P-40, pH 7.4, and subjected to SDS-PAGE under reducing conditions in a 10% NuPAGE gel (Novex, San Diego, CA). The band at ~37 kDa was visualized with 0.1% Brilliant Blue R-250 and excised for amino acid analysis and sequencing (University of Michigan Protein and Carbohydrate Structure Facility). 450 pmol of this protein was subjected to in situ cleavage with 10 mg/ml CNBr in 70% formic acid, the cleaved peptides were extracted into 60% acetonitrile, 10% trifluoroacetic acid, dried in a speed vacuum, and resolved by SDS-PAGE and electroblotted onto sequencing grade polyvinylidene difluoride. Peptides were observed by staining with Coomassie Blue, excised, and subjected to microsequencing using Edman chemistry on a model 494 Applied Biosystems sequencer run with standard cycles, yielding the sequence PAPQPPLNCPPGLEYLSQIDQILIHQQIELLE. This same sequence was partially confirmed in a second preparation of PL scramblase purified from erythrocyte ghosts and subjected to internal peptide sequencing as above. A BLAST homology search revealed 100% identity to the translation product of EST clone gb AA143025 with no significant sequence homology to any protein in available data bases.

Isolation of PL Scramblase cDNA by Plaque Hybridization

The 568-bp insert of EST clone gb AA143025 was labeled with [alpha -32P]dCTP by Random Primed DNA Labeling Kit (Boehringer Mannheim) and used to screen a cDNA library derived from human erythroleukemic cell line K-562 in lambda gt11 (CLONTECH). Escherichia coli strain Y1090r was transformed by K-562 cDNA library (4.86 × 105 pfu) and poured on 27 agarose plates (15-cm diameter, 18,000 plaque-forming unit/plate). Plaques were lifted onto Hybond-N Nylon membranes (Amersham Corp.). After UV-cross-linking and prehybridization in a solution composed of 5 × Denhardt, 5 × SSC, 1% SDS, and 200 µg/ml herring sperm DNA for 3 h at 68 °C, the membranes were hybridized in the same solution containing 5 ng/ml 32P-labeled probe for 16 h at 68 °C. The membranes were washed once with 1 × SSC, 0.1% SDS, then three times with 0.2 × SSC, 0.1% SDS for 20 min at 65 °C, and exposed to x-ray film. Secondary plaque lifts and hybridization were carried out on 32 strongly positive plaques at a density of 50-100 plaques/plate. Single positive and well isolated plaques were picked and amplified. The length of cDNA insert was examined by PCR with lambda gt11 reverse and forward primers. Six clones with cDNA inserts of >1.4 kilobase pairs were selected for DNA sequencing.

DNA Sequencing

DNA was sequenced on an Applied Biosystems DNA Sequencer model 373 Stretch using PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer) and combinations of vector and insert sequence primers.

Cloning of PL Scramblase into pMAL-C2 Expression Vector

To express PL scramblase as a fusion protein with maltose binding protein (MBP), cDNA encoding PL scramblase was cloned into pMAL-C2 (New England BioLabs). PCR was performed on a full-length clone using the primers 5'-TCAGAATTCGGATCCATGGACAAACAAAACTCACAGATG-3' with an EcoRI site before the ATG start codon and 5'-GCTTGCCTGCAGGTCGACCTACCACACTCCTGATTTTTGTTCC-3' with a SalI site after the stop codon. KlenTaq polymerase (CLONTECH) was used to ensure high fidelity amplification. The PCR product was digested with EcoRI and SalI and isolated by electrophoresis on 1% low melting agarose gel and purification with Wizard kit (Promega). The amplified cDNA was cloned into pMAL-C2 vector digested with EcoRI and SalI, immediately 3' of MBP. This construct was amplified in E. coli strain TB1, and the sequence of the cDNA insert of plasmids from single colonies was confirmed.

Expression and Purification of PL Scramblase-MBP Fusion Protein

10 ml of E. coli TB1 transformed with scramblase cDNA-pMAL-C2 were used to inoculate 1 liter of rich LB containing 2 mg/ml glucose, 100 µg/ml ampicillin, and the bacteria were allowed to grow for about 4 h at 37 °C. When A600 reached ~0.5, isopropyl-beta -D-thiogalactopyranoside was added to a final concentration of 0.3 mM. After 2 h of incubation at 37 °C, the cells were centrifuged at 4000 × g for 20 min. The cell pellet was suspended in 50 ml of 20 mM Tris, 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (column buffer), and subjected to a freeze/thaw cycle. After sonication (3 × 30 s on ice) and centrifugation at 43,000 × g for 1 h, the supernatant was applied to 10 ml of amylose resin. The column was washed with 20 volumes of column buffer, and the scramblase-MBP fusion protein eluted with the same buffer containing 10 mM maltose. Digestion of MBP-PL scramblase protein with factor Xa was routinely performed at 1/100 (w/w) ratio of enzyme and monitored by SDS-PAGE. In addition to MBP, the product of this digest is the PL scramblase translation product containing the N-terminal extension Ile-Ser-Glu-Phe-Gly-Phe (codons -6 to -1).

Reconstitution of PL Scramblase or Scramblase Fusion Protein into Proteoliposomes

Reconstitution into proteoliposomes was performed essentially as described previously (10, 11). Briefly, a mixture of PC and PS (9:1 molar ratio) was dried under a stream of nitrogen and resuspended in 100 mM Tris, 100 mM KCl, 0.1 mM EGTA, pH 7.4 (Tris buffer). Protein samples to be reconstituted were added to the liposomes at a final lipid concentration of 4 mg/ml in the presence of 60 mM OG and dialyzed overnight at 4 °C against 200 volumes of Tris buffer containing 1 g/liter Bio-Beads SM-2. To liberate PL scramblase from MBP, the proteoliposomes were incubated for 3 h at room temperature in the presence of 1/40 (w/w) factor Xa. The digestion was terminated by the addition of 100 µM Glu-Gly-Arg chloromethyl ketone. Completeness of the digest was monitored by SDS-PAGE. Following dialysis, the proteoliposomes were labeled in the outer leaflet by the addition of 0.25 mol % fluorescent NBD-PC (in dimethyl sulfoxide; final solvent concentration, 0.25%).

PL Scramblase Activity

PL Scramblase activity was measured as described previously (10, 11). Routinely, proteoliposomes labeled with NBD-PC were incubated for 2 h at 37 °C in Tris buffer in the presence or the absence of 2 mM CaCl2. Proteoliposomes were diluted 25-fold in Tris buffer containing 4 mM EGTA and transferred to a stirred fluorescence cuvette at 23 °C. Initial fluorescence was recorded (SLM Aminco 8000 spectrofluorimeter; excitation, 470 nm; emission, 532 nm), 20 mM dithionite was added, and the fluorescence was continuously monitored for a total of 120 s. The difference in nonquenchable fluorescence observed in the presence versus the absence of CaCl2 was attributed to Ca2+-induced change in NBD-PC located in the outer leaflet (10, 11, 13). Ionized [Ca2+] was calculated using FreeCal version 4.0 software (generously provided by Dr. Lawrence F. Brass, University of Pennsylvania, Philadelphia, PA).

Antibody against PL Scramblase C-terminal Peptide

The peptide CESTGSQEQKSGVW, corresponding to amino acids 306-318 of the predicted open reading frame of PL scramblase with an added N-terminal cysteine, was synthesized and conjugated to keyhole limpet hemocyanin (Protein Core Facility, Blood Research Institute). Antiserum to this protein was raised in rabbit (Cocalico Biologicals, Inc.), and the IgG fraction was isolated on protein A-Sepharose-CL4B (Sigma). Peptide-specific antibody was isolated by affinity chromatography on UltraLink Iodoacetyl beads (Pierce) to which peptide CESTGSQEQKSGVW was conjugated. This affinity-purified antibody (anti-306-318) was used for immunoprecipitation and Western blotting of PL scramblase (see "Results and Discussion").

Immunoprecipitation of PL Scramblase

PL scramblase purified from human erythrocytes was 125I-labeled with Iodogen (Pierce), free iodide removed by gel filtration, and the protein was incubated (4 °C, overnight) with either anti-306-318,or an identical quantity of preimmune rabbit IgG (1 mg/ml in 150 mM NaCl, 10 mM MOPS, 50 mM OG, pH 7.4) or no IgG as control. The IgG was precipitated with protein A-Sepharose and washed exhaustively, and protein bands were resolved by 8-25% SDS-PAGE (Phast System, Pharmacia Biotech Inc.) under reducing conditions. Radioactive bands were visualized by autoradiography. To determine whether antibody to this peptide specifically removed the functional activity associated with the purified erythrocyte PL scramblase protein, the supernatant fractions remaining after immunoprecipitation were reconstituted in liposomes for activity measurements, performed as described above. For these experiments, unlabeled erythrocyte PL scramblase substituted for the 125I-labeled protein.

Western Blot Analysis

2 × 108 washed platelets, 2 × 108 erythrocyte ghost membranes, 0.9 pmol of purified recombinant PL scramblase (obtained by factor Xa digest of the PL scramblase-MBP fusion protein), and 0.3 pmol of PL scramblase purified from human erythrocyte were each denatured by boiling in 40 µl of sample buffer containing 10% SDS, 4%beta -mercaptoethanol, and 1 mM EDTA, and protein bands were resolved by SDS-PAGE. After transfer to nitrocellulose, the blocked membrane was incubated with 1 µg/ml of anti-306-318, and the blot was developed with horseradish preoxidase-conjugated goat anti-rabbit IgG (Sigma) using Chemiluminescence Reagent (DuPont).

Protein Concentrations

Protein concentrations were estimated based upon optical density at 280 nm, using extinction coefficients (M-1 cm-1) of 39,000 (PL scramblase), 64,500 (MBP), and 105,000 (PL scramblase-MBP fusion). PL scramblase contained in human platelet and erythrocyte membranes was estimated by quantitative immunoblotting of the detergent extracts, with reference to known quantities of purified MBP-PL scramblase fusion protein.

Northern Blot Analysis

Human multiple tissue Northern blot and human cancer cell line multiple tissue Northern blot membranes were obtained from CLONTECH. The blots were prehybridized with ExpressHyb (CLONTECH) at 68 °C for 30 min and hybridized with ExpressHyb containing 5 ng/ml 32P-labeled PL scramblase cDNA probe at 68 °C for 1 h, then washed, and exposed to x-ray film. After development, the blots were stripped and hybridized with 32P-labeled beta -actin cDNA probe using identical conditions.


RESULTS AND DISCUSSION

Cloning of PL Scramblase cDNA

PL scramblase was purified from human erythrocyte membranes and cleaved with cyanogen bromide, and Edman degradation was performed on a 12-kDa peptide fragment to obtain 32 residues of peptide sequence (Fig. 1, underlined sequence). This peptide sequence, plus the anticipated methionine residue N-terminal to the predicted site of cyanogen bromide cleavage, was identified in the translation product of a 568-bp EST clone deposited in GenBank by the I.M.A.G.E. Consortium (clone identification number 505141). No other significant matches to this sequence were identified in any protein data base. The EST clone was used to screen a human K-562 leukemic cell cDNA library. Of 32 positive clones identified by plaque hybridization, six clones were sequenced yielding 1445 bp of cDNA (Fig. 1). The open reading frame encodes a protein without a signal sequence that contains 318 residues with a calculated mass of 35.1 kDa, a theoretical pI of 4.8, and a single predicted transmembrane helix near the C terminus (residues Ala291-Gly309), in good agreement with the physical properties observed for the ~37-kDa protein band we tentatively identified as PL scramblase in human erythrocyte membrane (10). Whereas the deduced protein sequence is notable for its high proline (12%) content, homology searching failed to reveal significant concensus to identifiable protein family or domain structures, with the exception of a single potential protein kinase C phosphorylation site (Thr161). The possibility that PL scramblase function is mediated by a phosphoprotein has previously been suggested based on an observed decrease in PL scrambling activity in erythrocytes depleted of ATP (14).


Fig. 1. cDNA and deduced amino acid sequence of PL scramblase. The deduced amino acid sequence of the predicted open reading frame is shown under the nucleotide sequence (GenBankTM accession number AF008445). The 32 residues of peptide sequence that were obtained from cyanogen bromide digest of purified erythrocyte PL scramblase are indicated by single underline. Also indicated are the residues comprising a predicted inside-to-outside transmembrane domain (Ala291-Gly309; double underline) and a protein kinase C phosphorylation site (Thr161; asterisk). See "Experimental Procedures" for details.
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Analysis of the cDNA-derived protein sequence (Tmpred program, ISREC server, University of Lausanne, Epalinges, Switzerland) revealed a strongly preferred (p < 0.01) inside-to-outside orientation of the predicted 19-residue transmembrane helix, consistent with a type II plasma membrane protein. Most of the polypeptide (residues 1-290) thereby extends from the cytoplasmic membrane leaflet, leaving a short exoplasmic tail (residues 310-318). The predicted orientation of this protein is consistent with the anticipated topology of PL scramblase in the erythrocyte membrane, where lipid-mobilizing function is responsive to [Ca2+] only at the endofacial surface of the membrane (3, 5, 10, 11, 15, 16).

To confirm that the cDNA we cloned from the K-562 cDNA library actually encodes the same protein purified as PL scramblase from human erythrocyte membrane, we raised a rabbit antibody against the deduced C terminus predicted from the open reading frame of the cloned cDNA (codons 306-318). As shown in Fig. 2, this antibody precipitated the ~37-kDa red cell protein we tentatively identified as PL scramblase and also absorbed the functional activity detected in this isolated erythrocyte membrane protein fraction. As also evident from Fig. 2 (inset), we often observed the partial proteolysis of 37-kDa PL scramblase to a polypeptide of ~30 kDa. The apparent susceptibility of this protein to proteolytic degradation may account for the reported rapid loss of activity observed in earlier attempts to purify PL scramblase from platelet (12).


Fig. 2. Immunoprecipitation of erythrocyte PL scramblase. PL scramblase purified from human erythrocytes was precipitated with either anti-306-318 IgG (bar 1) or preimmune rabbit IgG (bar 2), and protein remaining in the supernatant was reconstituted into liposomes for measurement of residual PL scramblase activity. Data normalized to PL scramblase activity were measured for identical controls omitting antibody (100%; bar 3). Error bars denote the means ± SD (n = 4). Inset, erythrocyte PL scramblase was labeled with 125I and precipitated with anti-306-318 antibody, and the resulting pellet was analyzed by SDS-PAGE (lane 1). Matched controls incubated with either preimmune rabbit IgG (lane 2) or no IgG (lane 3) served as control. See "Experimental Procedures" for details. Data of single experiment are shown, representative of two so performed.
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Expression and Membrane Reconstitution of Recombinant PL Scramblase

Recombinant PL scramblase was expressed in E. coli as fusion protein with MBP, purified by amylose affinity chromatography, and incorporated into PC/PS liposomes for assay of PL scramblase activity. When incorporated into liposomes, the recombinant protein mediated a Ca2+-dependent transbilayer movement of NBD-PC mimicking the activity of PL scramblase isolated from erythrocyte. PL scramblase activity was observed both for the chimeric MBP-PL scramblase fusion protein (not shown) and for recombinant PL scramblase liberated from MBP through proteolytic digestion with factor Xa (Fig. 3). By contrast, no such activity was observed for control protein consisting of the pMAL-C2 translation product MBP lacking the PL scramblase cDNA insert. The specific PL mobilizing activity of recombinant PL scramblase expressed and purified from E. coli was approximately 50% of that observed for the endogenous protein purified from the erythrocyte membrane, which is likely due to incomplete folding of the recombinant protein. Half-maximal [Ca2+] required for activation was approximately 100-200 µM for recombinant protein purified from E. coli versus ~40 µM for the erythrocyte-derived protein, raising the possibility that altered folding or an unknown post-translational modification in mammalian cells affects the putative Ca2+ binding site (10, 11). In addition to activation by Ca2+, the transbilayer migration of PL in erythrocytes is accelerated upon acidification of the inside leaflet to pH < 6.0 (in absence of Ca2+), a response that is also observed in proteoliposomes containing PL scramblase purified from erythrocyte membranes (11). A similar acid-dependent activation of PL mobilizing function was also exhibited by proteoliposomes incorporating recombinant PL scramblase purified from E. coli (not shown).


Fig. 3. Activity assay of recombinant PL scramblase. Purified PL scramblase-MBP fusion protein (0-43 × 10-11 mol; abscissa) was reconstituted into liposomes (1 µmol of total PL), and MBP was proteolytically removed by incubation with factor Xa in presence of 0.1 mM EGTA. After digest to release MBP, the proteoliposomes were recovered for determination of PL scramblase activity, measured in the absence (open circle ) or the presence (bullet ) of 2 mM CaCl2 as described under "Experimental Procedures." Data are corrected for nonspecific transbilayer migration of NBD-PC probe in identically matched control liposomes containing either MBP or no added protein (<2% NBD-PC sequestered; not shown). Error bars denote the means ± SD (n = 3). Data of single experiment are shown, representative of two so performed. Similar results were also obtained for proteoliposomes containing intact PL scramblase-MBP fusion protein, omitting the factor Xa digest (not shown).
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Platelet PL Scramblase

In addition to the presumed role of PL scramblase in PS exposure following cell injury and upon repeated sickling of SS hemoglobin red cells, the capacity of activated platelets to rapidly mobilize aminophospholipids across the plasma membrane is thought to play a central role in the initiation of thrombin generation required for plasma clotting (17). Whereas incubation with Ca2+ ionophore causes a marked acceleration in transbilayer movement of plasma membrane PL in both platelets and erythrocytes, the apparent rate of transbilayer PL migration in platelet exceeds that in erythrocyte by approximately 10-fold, implying either a higher abundance of PL scramblase or the action of another component in platelet with enhanced PL scrambling function (18, 19). Zwaal and associates recently reported evidence for the existence of protein(s) in platelet with functional properties similar to that of PL scramblase we isolated from erythrocyte (10-12). To determine whether the protein we now identify in the erythrocyte membrane is also found in platelets, we probed platelets with antibody against PL scramblase residues 306-318. As shown in Fig. 4, this antibody blotted a single protein in platelet with similar mobility to the ~37-kDa PL scramblase in erythrocyte. Based on quantitative immunoblotting with anti-306-318, we estimate approximately 104 molecules/cell in platelet versus 103 molecules/cell in erythrocyte, consistent with the increased PL scramblase activity and procoagulant function observed for human platelets versus erythrocytes.


Fig. 4. Immunoblotting of PL scramblase in human erythrocytes and platelets. 2 × 108 platelets (lane 1) and ghost membranes from 2 × 108 erythrocytes (lane 2) were separated by SDS-PAGE, transferred to nitrocellulose, and Western blotted with anti-306-318 antibody as described under "Experimental Procedures." Lane 3 contains 0.9 pmol of factor Xa cleaved recombinant PL scramblase, and lane 4 contains 0.3 pmol of PL scramblase purified from erythrocytes. Data of single experiment are shown, representative of three so performed.
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Tissue Distribution

In addition to platelet and red blood cell, PL scramblase activity has been observed in many other cells, and this Ca2+-induced response is thought to be central to the rapid movement of PS and phosphatidylethanolamine from inner plasma membrane leaflet to the surface of perturbed endothelium and a variety of injured and apoptotic cells (17). The resulting exposure of PS at the cell surface is thought to play a key role in removal of such cells by the reticuloendothelial system, in addition to activation of both the plasma complement and coagulation systems (8, 9, 17). Whereas the molecular mechanism(s) in each circumstance remains unresolved, evidence for a specific platelet membrane protein functioning to accelerate migration of PL between membrane leaflets at increased cytosolic [Ca2+] has been reported (12), similar to the proposed role of PL scramblase in red blood cells (10, 11). It was thus of interest to determine whether mRNA for this protein is expressed in nucleated cells where PL scramblase-like activity has been observed. As shown by Fig. 5, Northern blotting with PL scramblase cDNA revealed transcripts of ~1.6 and ~2.6 kilobases in all tissues and cell lines tested. Some tissue-to-tissue and cell line variability in the relative abundance of these two transcripts is apparent, the significance of which remains to be determined. Also notable was markedly reduced expression in HL-60 and the lymphoma lines Raji and MOLT-4, whereas abundant message was detected in spleen, thymus, and peripheral leukocytes. In addition to the transformed cell lines shown, mRNA for PL scramblase was also confirmed in human umbilical vein endothelial cells (not shown). Whereas these data imply that the same protein identified as mediating accelerated transbilayer flip-flop of the erythrocyte membrane PL also plays a similar role in the plasma membrane of platelets, leukocytes, and other cells, actual confirmation for this role of PL scramblase awaits analysis of a cell line that is selectively deficient in this protein. In Scott syndrome, a bleeding disorder related to an inherited deficiency of plasma membrane PL scramblase function, erythrocytes and other cells deficient in PL scramblase activity were found to contain normal amounts of the PL scramblase protein (11).2 Furthermore, despite the apparent deficiency in Scott syndrome cells of endogenous PL scramblase function, when PL scramblase protein from these cells was purified and reconstituted in proteoliposomes containing exogenous PL, it exhibited normal Ca2+-dependent PL mobilizing activity (11). This suggests that in addition to the known regulation by intracellular [Ca2+], the activity of PL scramblase in the plasma membrane is regulated by other as yet unidentified membrane or cytoplasmic component(s).


Fig. 5. Expression of PL scramblase in multiple human tissues and cancer cell lines. Northern blotting with PL scramblase cDNA is shown for equal amounts of poly(A) RNA obtained from the human tissues indicated (A) and from the following human cancer cell lines (B): promyelocytic leukemia HL-60, epithelial cancer HeLa S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung carcinoma A549, and melanoma G361. Lower panels show results for beta -actin. Blots were developed as detailed under "Experimental Procedures." Data of single experiment are shown. kb, kilobases.
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FOOTNOTES

*   This work was supported in part by Grant R01 HL36946 from the NHLBI, National Institutes of Health (to P. J. S. and T. W.) and a Grant-In-Aid from the American Heart Association (to T. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Recipient of Research Fellowship Award, American Heart Association, Wisconsin Affiliate.
§   To whom correspondence should be addressed: Blood Research Inst., Blood Center of Southeastern Wisconsin, P.O. Box 2178, Milwaukee, WI 53201-2178. Tel.: 414-937-3850; Fax: 414-937-6284; E-mail: peter_s{at}smtpgate.bcsew.edu.
1   The abbreviations used are: PL, phospholipid(s); EST, expressed sequence tag; MBP, maltose binding protein; NBD-PC, 1-oleoyl-2-[6(7nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl-sn-glycero-3-phosphocholine; OG, N-octyl-beta -D-glucopyranoside; PC, phosphatidylcholine; PS, phosphatidylserine; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid.
2   Q. Zhou, J. Zhao, J. G. Stout, P. J. Sims, and T. Wiedmer, unpublished data.

ACKNOWLEDGEMENTS

The excellent technical assistance of Mary J. Blonski and Timothy T. O'Bryan is gratefully acknowledged. The assistance of Dr. Philip C. Andrews in protein sequencing and Trudy Holyst in peptide sequencing is also gratefully acknowledged.


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